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WO2025193544A1 - Dispositifs d'analyse de fluide échantillon, cartouches destinées à être utilisées dans ceux-ci, et leurs procédés d'utilisation - Google Patents

Dispositifs d'analyse de fluide échantillon, cartouches destinées à être utilisées dans ceux-ci, et leurs procédés d'utilisation

Info

Publication number
WO2025193544A1
WO2025193544A1 PCT/US2025/018935 US2025018935W WO2025193544A1 WO 2025193544 A1 WO2025193544 A1 WO 2025193544A1 US 2025018935 W US2025018935 W US 2025018935W WO 2025193544 A1 WO2025193544 A1 WO 2025193544A1
Authority
WO
WIPO (PCT)
Prior art keywords
sample fluid
cartridge
sample
diluent
fluid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/018935
Other languages
English (en)
Inventor
Yang Zhang
Timothy Pletcher
Antti L. VIRTANEN
Jaymin PATEL
Jason Marcus
Barry Bass
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Abbott Laboratories
Original Assignee
Abbott Laboratories
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Abbott Laboratories filed Critical Abbott Laboratories
Publication of WO2025193544A1 publication Critical patent/WO2025193544A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/251—Colorimeters; Construction thereof
    • G01N21/253—Colorimeters; Construction thereof for batch operation, i.e. multisample apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/403—Cells and electrode assemblies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G01N35/00069—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides whereby the sample substrate is of the bio-disk type, i.e. having the format of an optical disk
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N2021/1765—Method using an image detector and processing of image signal
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G01N2035/00099—Characterised by type of test elements
    • G01N2035/00158—Elements containing microarrays, i.e. "biochip"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N2035/1027—General features of the devices
    • G01N2035/1034—Transferring microquantities of liquid
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
    • G01N21/274—Calibration, base line adjustment, drift correction

Definitions

  • cartridges include a calibration fluid reservoir configured to contain a calibration fluid for use with the electrochemical sensors, and in some cases also include the calibration fluid. Some versions of the cartridges include an electrochemical sensor outlet vent.
  • cartridges include a plurality of heating electrodes configured to heat the sample liquid, and/or a temperature-sensing diode.
  • the sample inlet may in some cases be comprised of a tube of a known volume and a capillary stop fluidically connected thereto.
  • the sample inlet includes an air inlet (e.g., comprising a gasket) for gaseously connecting to a sample inlet pressure line.
  • Cartridges include a surface ⁇ treatment (e.g., hydrophobic surface treatment, hydrophilic surface treatment) configured to prevent at least a component of the sample fluid from adhering to the fluidic control subsystem.
  • cartridges include a light-transparent window adjacent to the sample fluid metering chamber configured for optically assessing the volume of the sample fluid from the sample fluid metering chamber.
  • cartridges include a secondary metering chamber, optionally including a gas-permeable membrane.
  • the detection chambers of the plurality are comprised of an elongate structure having an inlet at a proximal end and an outlet at a distal end, and are arranged upright in a detection chamber array.
  • each detection chamber of the plurality contains therein one or more reagent beads comprising a dried reagent.
  • each detection chamber of the plurality comprises a first light-accessible window configured to permit entry of a beam of light at the proximal end and a second light-accessible window configured to permit an exit at of the light at the distal end.
  • the detection chamber array is arranged in a ⁇ staggered pattern or a concentric pattern.
  • sample fluid analysis devices include at least a housing configured to receive a cartridge, a manifold, and an interrogation system configured to analyze the sample fluid.
  • Housings of interest are configured to receive and/or interface with a cartridge of the invention, such as those described above and herein.
  • ⁇ Manifolds of interest include a diluent line fluidically connected to a diluent reservoir and comprising a connector for fluidically connecting to the cartridge (e.g., a microfluidic passage of the cartridge), a diluent fluid metering unit configured to control a metered amount of the diluent fluid supplied to the cartridge via the diluent line, a sample fluid pressure line comprising a connector for gaseously connecting to the cartridge and supplying pressure thereto, and one or ⁇ more valves configured to regulate the pressure supplied by the manifold.
  • the sample fluid pressure line includes a connector for gaseously connecting to the sample inlet and supplying pressure to the sample fluid.
  • the sample fluid pressure line comprises a connector for gaseously connecting to a metering chamber of the cartridge and supplying pressure thereto.
  • the interrogation system is a light ⁇ interrogation system comprising a light source configured to irradiate the plurality of detection chambers, and an optical sensor configured to collect emitted light from the plurality of detection chambers.
  • the interrogation system is an electrochemical interrogation system configured to interface with a plurality of electrochemical sensors in the cartridge.
  • the sample fluid analysis device comprises both a light interrogation system and ⁇ an electrochemical interrogation system.
  • devices include a pump, such as a pump configured to apply positive and/or negative pressure.
  • devices include a diluent reservoir for containing the diluent fluid.
  • devices further comprise a diluent fluid pressure line gaseously connected to the diluent reservoir and the pump for applying pressure to the diluent reservoir.
  • Some embodiments may also include a diluent fluid ⁇ pressure valve operably coupled to the diluent fluid pressure line configured to regulate pressure applied to the diluent reservoir.
  • sample fluid analysis devices include a pressure sensor configured to assess pressure within the manifold.
  • Devices also include a sample inlet pressure valve operably coupled to the sample fluid pressure line and configured to regulate pressure applied to the sample inlet.
  • devices include a sample fluid metering chamber pressure valve operably coupled to the sample fluid pressure line and configured to regulate pressure applied to the sample fluid metering chamber.
  • devices include a secondary ⁇ metering chamber pressure line comprising a connector for gaseously connecting to a secondary metering chamber and supplying pressure thereto, and a secondary metering chamber pressure valve operably coupled to the secondary metering chamber pressure line and configured to regulate pressure applied to the secondary metering chamber.
  • devices include a calibration fluid pressure line configured to apply pressure to a ⁇ calibration fluid reservoir in the cartridge, and a calibration fluid pressure valve operably coupled to the calibration fluid pressure line configured to regulate pressure applied to the calibration fluid reservoir.
  • Devices according to some implementations may also include an electrochemical sensor outlet vent valve configured to regulate venting by an electrochemical sensor outlet vent of the cartridge.
  • devices include an overflow outlet ⁇ vent valve configured to regulate venting by an overflow outlet vent of the cartridge.
  • sample fluid analysis devices of the invention include a housing configured to receive a cartridge, a light interrogation system comprising a light source configured to irradiate the plurality of detection chambers and an optical sensor configured to collect emitted light from the plurality of detection chambers, and a processor operably ⁇ connected to the optical sensor, the light source, and a memory having instructions stored thereon which, when executed by the processor, cause the processor to calculate an absorbance for each detection chamber of the plurality.
  • the housing is configured to receive a cartridge comprising a plurality of detection chambers each comprising a first light-accessible window configured to permit entry of light from the light source, and a ⁇ second light-accessible window configured to permit an exit of the light from the light source to the optical sensor.
  • the subject processor may in some embodiments be configured to calculate an average intensity of incident light from the light source, calculate an average intensity of the emitted light from each detection chamber of the plurality, deactivate the light source and calculate a dark image average intensity of the emitted light from each detection chamber of the ⁇ plurality, and calculate the absorbance for each detection chamber of the plurality based on the average intensity of incident light from the light source, the average intensity of the emitted light from each detection chamber of the plurality, and the dark image average intensity of the emitted light from each detection chamber of the plurality.
  • Embodiments of the devices also include a first pinhole plate configured to be positioned adjacent to the first light-accessible ⁇ windows of the detection chambers of the array, and a second pinhole plate configured to be positioned adjacent to the second light-accessible windows of the detection chambers of the array. Aspects of the invention also include methods.
  • Methods of interest include introducing a sample fluid into a cartridge of the invention (e.g., as described above and herein), inserting the cartridge into a sample fluid analysis system of the invention (e.g., as described above and herein), controlling, via a processor, the amount of diluent applied to the cartridge via the ⁇ diluent line and the amount of pressure applied to the cartridge via the sample fluid pressure line, and interrogating the sample fluid via an interrogation system to analyze the sample fluid.
  • Methods may additionally include calculating an absorbance for each detection chamber of the plurality to analyze the sample fluid.
  • Methods of the invention also include methods of sample fluid dilution.
  • FIG.1A-1C present schematic diagrams of cartridges according to certain embodiments.
  • FIG.2 depicts a flow diagram of a cartridge according to certain embodiments.
  • FIG.3A-3C depict cartridges according to certain embodiments of the invention.
  • FIG.4A-4B depict detection chambers according to certain embodiments of the invention.
  • FIG.5A-5B depict a light interrogation system according to certain embodiments of the invention.
  • FIG.6A-6C present schematic diagrams of a sample fluid analysis device according to certain embodiments.
  • FIG.7A-7E present aspects of light interrogation systems according to embodiments of the invention.
  • FIG.8 presents the fluidic architecture of an experimental device having combiner ⁇ circuit, fluidics, timing, and relay controls.
  • FIG.9 presents the basic architecture of an experimental device with electronics and controls.
  • FIG.10A-10C presents a pump and fluid controls with a closed tube of known volume.
  • FIG.11 presents an evaluation of a pump transfer function.
  • FIG.12 graphically illustrates the results of a pressure pulse experiment.
  • FIG.13 graphically illustrates pump pressure measurements collected over time.
  • FIG.14 graphically illustrates pump pressure pulse measurements collected over time.
  • FIG.15 graphically illustrates pump pressure amplitude measurements collected over time.
  • FIG.16 graphically illustrates pump pressure amplitude measurements collected over time.
  • FIG.17A-17C graphically illustrate pump pressure amplitude measurements collected over time.
  • FIG.18A-18B graphically illustrate pump pressure amplitude measurements collected over time.
  • FIG.19A-19B graphically illustrate pump pressure amplitude measurements collected ⁇ over time.
  • FIG.20A-20B graphically illustrate pump pressure amplitude measurements collected over time.
  • FIG.21 graphically illustrates pump pressure amplitude measurements collected over time.
  • FIG.22 presents a block diagram and method for producing pressure pulses less than 1730Pa.
  • FIG.23A-23B graphically illustrate pump pressure amplitude measurements collected over time.
  • FIG.23C graphically illustrates pressure chamber steady state measurements PWM input to pump from pulse generator.
  • FIG.24A-24B graphically illustrate a signal analysis of Vanalog from a pulse generator.
  • FIG.25 presents a block diagram and method for producing pressure pulses at 50Pa.
  • FIG.26A-26B graphically illustrate pump pressure amplitude measurements collected over time.
  • FIG.27A-27C graphically illustrate filling and exhausting a 3600ul chamber without relay ⁇ control (FIG.27A-27B) and with relay control (FIG.27C).
  • FIG.28 presents a basic architecture of a fluidic system with electronics and controls.
  • FIG.29A-29G depict chamber pressure over time while pulsing.
  • FIG.30A-30B present photographs illustrating droplet sizes.
  • FIG.31A-31B present photographs illustrating droplet sizes.
  • FIG.32 presents a schematic diagram of fluidic architecture for filling cuvettes.
  • FIG.33A-33D present photographs of filling cuvettes.
  • FIG.34A-34B depict an experimental fluidic device.
  • FIG.35 present a photograph illustrating droplet sizes.
  • FIG.36 depict measurement results and findings regarding fluid efficiency.
  • FIG.37 presents a block diagram for pump transfer function for CMP operating range.
  • FIG.38A-38B graphically illustrate pump transfer function data.
  • FIG.39A-39B illustrate expected droplet behavior at a combining point.
  • FIG.40A-40B depict droplet movements.
  • FIG.41 graphically illustrates 200ul droplet movements.
  • FIG.42A-42B depict combining water with tartrazine-stained water.
  • FIG.43A-43C depict air bubbles as estimate of pressure flow from a combining port.
  • FIG.44A-44C depict air bubbles as estimate of pressure flow from a combining port.
  • Sample fluid analysis devices are provided. Devices of interest include a housing configured to receive a cartridge, a manifold, and an interrogation system configured to analyze the sample fluid. Cartridges for use in the subject devices are also provided.
  • Cartridges of ⁇ interest include a plurality of detection chambers and a fluidic control subsystem for supplying a sample fluid to the plurality of detection chambers including a sample inlet configured to receive the sample fluid, a sample fluid metering chamber for supplying a predetermined volume of the sample fluid from the sample inlet, a mixing chamber for homogenizing the sample fluid with a diluent fluid, and a microfluidic passage fluidic fluidically connecting the sample fluid metering ⁇ chamber and the mixing chamber.
  • Methods of using the sample fluid analysis devices and cartridges to analyze a fluidic sample are also provided.
  • aspects of the invention include cartridges.
  • the subject cartridges are configured to provide for dilution(s) of a sample fluid (e.g., blood) for analysis.
  • Devices and cartridges of interest are configured to ⁇ perform a sample fluid dilution in a manner that is suitable for sample fluid analysis by combining the sample fluid (e.g., blood) with a diluent fluid.
  • the dilution is a precision dilution.
  • precision dilution it is meant a dilution that differs negligibly from a target (i.e., intended) dilution.
  • Cartridges of the invention include a fluidic control subsystem for supplying a sample ⁇ fluid to a plurality of detection chambers. Fluidic control subsystems of the invention are configured to provide for the precision dilution of the sample fluid, and may be comprised of ⁇ microfluidic conduits and connections.
  • Microfluidic conduits are channels that are used to convey fluids in the range of nanoliter to microliter volumes. Microfluidic connections allow the transport of fluids in the range of nanoliter to microliter volumes.
  • the fluids may be transported ⁇ from a cartridge containing all or nearly all of the reagents for using the device.
  • the fluids may be transported from bulk reagent containers to the device.
  • the fluids may be transported from both a cartridge based system and from bulk reagent containers.
  • the subject fluidic control subsystems include a sample inlet configured to receive the sample fluid.
  • the sample inlet may in some cases be comprised of a metered chamber where ⁇ sample volume is controlled by the inlet volume.
  • the volume of liquid being received into the cartridge may be known with relative precision (e.g., within a margin of error of ⁇ 10 ⁇ l or less, such as ⁇ 5 ⁇ l or less, such as ⁇ 1 ⁇ l or less).
  • the volume of the sample inlet may in some embodiments range from 2 ⁇ l to 500 ⁇ l, such as 5 ⁇ l to 200 ⁇ l, such as 10 ⁇ l to 400 ⁇ l, such as 15 ⁇ l to 300 ⁇ l, such as 20 ⁇ l to 200 ⁇ ⁇ l, such as 25 ⁇ l to 100 ⁇ l, such as 30 ⁇ l to 50 ⁇ l, and including 35 ⁇ l to 45 ⁇ l.
  • the volume of the sample inlet is 40 ⁇ l.
  • the inlet may be configured to receive the sample fluid, e.g., via pipette.
  • the sample inlet comprises an air inlet for gaseously connecting to a sample fluid pressure line (e.g., described below). Air from the sample fluid pressure line may move the sample fluid from the sample inlet throughout the ⁇ fluidic control subsystem.
  • a sample fluid pressure line e.g., described below. Air from the sample fluid pressure line may move the sample fluid from the sample inlet throughout the ⁇ fluidic control subsystem.
  • embodiments of the sample inlet include no air inlet. In such embodiments, the sample inlet may operate via a passive flow regime.
  • Cartridges of the invention additionally include a sample fluid metering chamber for supplying a predetermined volume of the sample fluid from the sample inlet.
  • the sample fluid metering chamber ⁇ provides a mechanism by which sample fluid from the sample inlet may be apportioned into “droplets” of a known, intended, volume within the microfluidic architecture of the cartridge. Because these droplets have a reliable volume, it may be known in some cases with a high degree of confidence how much sample fluid is being combined with diluent, thereby enabling the precision dilution.
  • the sample fluid metering chamber is comprised of, e.g., a tube having a known volume.
  • the sample fluid metering chamber may in some cases comprise an air inlet for gaseously connecting to a sample fluid pressure line of the sample fluid analysis device of the invention (described in detail below).
  • the volume of the sample fluid ⁇ metering chamber may in some embodiments range from 5 ⁇ l to 500 ⁇ l, such as 10 ⁇ l to 400 ⁇ l, such as 15 ⁇ l to 300 ⁇ l, such as 20 ⁇ l to 200 ⁇ l, and including 25 ⁇ l to 100 ⁇ l.
  • the manner in which the predetermined volume of sample fluid is apportioned may vary.
  • the volume of sample fluid employed for the precision dilution may be controlled by the injection of air into the sample fluid metering chamber (e.g., via a sample fluid pressure line described ⁇ below) in a manner sufficient to form a bubble at the trailing edge of the sample fluid. This may be sufficient to form a droplet of sample fluid.
  • the volume of the sample fluid employed in the dilution may also be controlled by the volume of the chamber. In some such cases, sample fluid fills the sample fluid metering chamber. Once the sample fluid metering chamber is filled, pressure and/or capillary action may be relied on for said fluid to exit the metering chamber and ⁇ combine with the diluent fluid.
  • cartridges are configured for the monitoring of the sample fluid metering chamber, e.g., to ensure that a predetermined amount of sample fluid is being transferred to the microfluidic passage and provide feedback to that end.
  • the sample fluid ⁇ metering chamber comprises a light-accessible window for optical metering of the fluid within the sample fluid metering chamber.
  • fluidic control subsystems include a plurality of metering chambers.
  • fluidic control subsystems include a secondary metering chamber.
  • the secondary metering chamber is “downstream” (defined with respect to ⁇ the direction of fluid flow) of the mixing chamber (described below).
  • the secondary metering chamber comprises a gas-permeable membrane.
  • the secondary metering chamber may be employed to regulate the amount of diluted sample fluid being applied to the detection chambers, e.g., by preventing the build-up of bubbles which then enter the detection chambers.
  • the secondary metering chamber ⁇ may in some cases operate via the same principles as discussed above with respect to the sample fluid metering chamber.
  • the secondary metering chamber includes an air inlet for connecting to a secondary metering chamber pressure line (described below).
  • the subject cartridges also include a mixing chamber for mixing the sample fluid with a diluent fluid.
  • a mixing chamber for mixing the sample fluid with a diluent fluid.
  • Any microfluidic element configured to thoroughly combine (e.g., homogenize) two ⁇ liquids (e.g., sample fluid and diluent) may be employed as the subject mixing chamber.
  • the mixing chamber is selected from a tesla valve, an obstruction mixer and a herringbone mixer.
  • the mixing chamber is comprised of a tesla valve. Tesla valves are described in, e.g., U.S. Patent No.1,329,559; incorporated by reference herein.
  • the mixing chamber is an obstruction mixer comprised of a chamber having obstructions (e.g., rectangular obstructions) positioned therein.
  • the mixing chamber is comprised of a herringbone mixer, e.g., having grooves in one or more sides.
  • the mixing chamber, or a portion of the cartridge proximate to the ⁇ mixing chamber may include a gas-permeable membrane. Said membrane is configured to remove air bubbles that may have formed during the fluid combining process or mixing process.
  • Gas-permeable membranes that may be employed include, but are not limited to polydimethylsiloxane (PDMS) membranes.
  • the mixing chamber comprises an inlet for fluidically connecting to the diluent line (e.g., described below).
  • cartridges include a plurality of mixing chambers.
  • the number of mixing chambers may range, e.g., from 2 to 25.
  • each detection chamber of the plurality is fluidically connected to a respective mixing chamber such that fluid passes through the mixing chamber prior to entering the detection chamber.
  • different dry reagents optionally imprinted on reagent beads, as ⁇ discussed below—are present within each detection chamber, as appropriate for a desired assay (e.g., CMP). Reagent may consequently be dissolved in the diluted sample fluid in the mixing chambers and mixed prior to optical detection.
  • the mixing chambers for use in conjunction with the detection chambers may be any suitable mixing chamber (e.g., tesla valve, obstruction mixer, herringbone mixer).
  • the mixing chambers for use in ⁇ conjunction with the detection chambers are obstruction mixers.
  • Cartridges of the invention further include a microfluidic passage fluidically connecting the sample fluid metering chamber and the mixing chamber.
  • the microfluidic passage is comprised of microfluidic tubing or a microfluidic channel.
  • the dimensions of the microfluidic passage may vary.
  • the microfluidic passage comprises an ⁇ inner diameter ranging from 0.5 mm to 2 mm.
  • the microfluidic passage includes an intersection at which sample fluid is combined with diluent (e.g., from a diluent line, described below) for the precision metering.
  • amounts of the diluent fluid that have been metered as described below in the sample fluid analysis device are combined with the predetermined volume of the sample fluid from the sample inlet.
  • the combination of the ⁇ sample fluid and diluent may be sufficient to create a jet that impinges the back wall of the microfluidic passage, thereby aiding in the mixing of the diluent and the sample fluid.
  • Said predetermined volume is generated in the sample fluid metering chamber.
  • the microfluidic passage includes a capillary stop. Said capillary stop may, in some instances, be located upstream of the intersection at which sample fluid is combined with diluent.
  • fluidic control subsystems instead of diluent fluid being combined with the sample fluid in the microfluidic passage or channel, cartridges are configured such that said fluids are combined directly within the mixing chamber.
  • fluidic control subsystems additionally include a separation filter configured to separate the sample fluid.
  • the separation filter may be configured to separate the sample fluid into one or more components.
  • the separation filter may be a plasma separation filter.
  • the filter is a passive filter.
  • the subject filters may be comprised of any suitable material, e.g., polyethersulfone.
  • the separation filter may in some cases be a vertical flow separation filter, lateral flow separation filter or a composite flow separation filter.
  • the filter is an active filter.
  • the filter is a microfluidic separation circuit configured for use with pump or vacuum pressure.
  • the separation filter is upstream of the sample fluid metering chamber but downstream of the sample inlet.
  • the cartridge is configured to perform separation prior to the combination of the sample fluid with the diluent and the mixing thereof.
  • the separation filter is downstream of the mixing chamber.
  • the cartridge is configured to perform separation after the combination of the ⁇ sample fluid with the diluent and the mixing thereof.
  • the cartridge includes a diluent reservoir.
  • diluent may be housed within the sample fluid analysis devices, in other embodiments the diluent reservoir is present within the cartridge itself.
  • the diluent reservoir may be any suitable reservoir or container (e.g., having rigid or flexible walls) for holding a ⁇ diluent fluid (e.g., a blood diluent fluid).
  • the volume of the diluent reservoir may vary, and can range in some instances from 5 ⁇ l to 1000 ⁇ l, such as 10 ⁇ l to 500 ⁇ l, and including 15 ⁇ l to 250 ⁇ l.
  • cartridges also include a diluent metering chamber, which may operate on the same principle as the sample fluid metering chamber discussed above but with diluent.
  • the dilution rate is the volume ratio of ⁇ the diluent metering chamber to the metering chamber used for the sample fluid. This may be used to calculate the concentration following the dilution.
  • the fluidic control subsystem comprises a surface treatment configured to prevent a component of the sample fluid from adhering to the fluidic control subsystem.
  • the surface treatment is a hydrophilic surface treatment. In ⁇ other cases, the surface treatment is a hydrophobic surface treatment.
  • the surface treatment may be formed using a humectant, i.e., an agent that lowers the total free energy of water and is capable of binding water.
  • Suitable humectants include, e.g., polymeric humectants and non-polymeric humectants.
  • Exemplary polymeric humectants include, but are not limited to, hydroxyethyl acrylate (HEA), 2-hydoxyethyl methacrylate (HEMA), ⁇ dimethacrylamide (DMA), polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene glycol (PEG), di(ethylene glycol)vinyl ether (EO 2 V), cellulose derivatives, and the like and combinations thereof.
  • Exemplary non-polymeric humectants include, but are not limited to, glycerin, urea, propylene glycol, non-polymeric diols, glycerols, and the like.
  • Exemplary hydrophobic surface treatments include, but are not limited to, FluoroPel.
  • the coating is comprised of EDTA.
  • Cartridges of the invention include a plurality of detection chambers. Detection chambers for use in the subject cartridges may be any microfluidic component configured for ⁇ the analysis (e.g., optical analysis) of a sample.
  • Exemplary detection chambers include, but are not limited to, microcuvettes. The number of detection chambers in the plurality may vary.
  • the number of detection chambers in the plurality ranges from 2 to 100, such as 5 to 50, such as 10 to 25 and including 14 to 20.
  • cartridges include 10 or more detection chambers, such as 11 or more detection chambers, such as 12 or more detection ⁇ chambers, such as 13 or more detection chambers, such as 14 or more detection chambers, such as 15 or more detection chambers, such as 16 or more detection chambers, such as 17 or more detection chambers, such as 18 or more detection chambers, such as 19 or more detection chambers, and including 20 or more detection chambers.
  • the shape and size of the detection chambers in the plurality may vary, as desired.
  • the detection ⁇ chambers of the plurality have an elongate structure (e.g., having a length greater than width).
  • the elongate structure may have any convenient cross-sectional shape, where cross-sectional shapes of interest include, but are not limited to rectilinear cross-sectional shapes, e.g., squares, rectangles, trapezoids, triangles, hexagons, etc., curvilinear cross-sectional shapes, e.g., circles, ovals, as well as irregular shapes, e.g., a parabolic bottom portion coupled to a ⁇ planar top portion.
  • the detection chambers of the plurality have a circular cross section. In other embodiments, the detection chambers of the plurality have a square cross section.
  • detection chambers of the plurality have a rectangular cross section.
  • the volume of the detection chambers may also vary.
  • detection chambers of the plurality have a volume ranging from 0.3 ⁇ l to 500 ⁇ l, such as 2 ⁇ l to 300 ⁇ l, ⁇ such as 3 ⁇ l to 200 ⁇ l, such as 4 ⁇ l to 100 ⁇ l and including 5 ⁇ l to 10 ⁇ l.
  • detection chambers of the plurality have a volume ranging from 0.3 ⁇ l to 50 ⁇ l.
  • detection chambers of the plurality have a volume of 5 ⁇ l or more, such as 6 ⁇ l or more, such as 7 ⁇ l or more, such as 8 ⁇ l or more, such as 9 ⁇ l or more, and including 10 ⁇ l or more. In some instances, detection chambers of the plurality have a diameter ranging from 0.1 mm to 20 ⁇ mm, such as 0.5 mm to 15 mm, such as 1 mm to 10 mm, and including 1.5 mm to 2 mm.
  • detection chambers of the plurality have a diameter of 1.5 mm or more, such as 1.6 mm or more, such as 1.7 mm or more, such as 1.8 mm or more, such as 1.9 mm or more, and including 2 mm or more.
  • Adjacent detection chambers of the plurality may be separated by a distance ranging from 1 mm to 10 mm, such as 2 mm to 8 mm, and including 4 mm to 5 mm.
  • Space between detection chambers may in some cases be sufficient to ensure that each one can be interrogated by a beam of light from the illuminator without interfering with its neighbor detection chambers.
  • the detection chambers of the plurality may be arranged in any suitable pattern.
  • the detection chamber array is arranged in a staggered pattern. In other cases, the detection chamber array is arranged in a concentric pattern.
  • the detection chambers in the subject cartridge may be constructed from any suitable material. In some cases, the detection chambers are comprised of a polymeric material that is transparent in a detection wavelength band. In some such cases, the detection chambers of the plurality are ⁇ comprised of polystyrene (PS), PMMA, CoC, or CoP. Detection chambers of the subject cartridges may include an inlet for receiving diluted sample fluid, and an outlet where air and/or excess diluted sample fluid may escape as the detection chambers are being filled.
  • the detection chambers of the plurality are comprised of an elongate structure
  • the detection chambers include an inlet at ⁇ a proximal end of the elongate structure, and an outlet at the distal end of the elongate structure.
  • the cartridge is configured such that the detection chambers are arranged upright (i.e., vertically).
  • the inlets may be arranged at the bottom such that the detection chambers fill with diluted sample fluid from the bottom and ascend via capillary action. In some cases, this arrangement is sufficient to minimize the ⁇ generation of air bubbles when sample fluid fills the detection chambers. In some alternate embodiments, detection chambers fill from the top-down.
  • the detection chambers are light-accessible at certain windows.
  • each detection chamber in the plurality comprises a first light-accessible window configured to permit entry of light, and a second light-accessible window configured to permit an exit of the light from the ⁇ light source.
  • the remainder of the detection chambers may or may not also be light-accessible.
  • the detection chambers are opaque.
  • the outlets of the detection chambers have edges that are not perpendicular to the edges of the detection chamber. In some such embodiments, the outlets ⁇ are beveled. Put another way, at least a portion of the outlet may be chamfered.
  • cartridges include one or more capillary stops (i.e., capillary stop valves).
  • capillary stops halt the flow of liquid in microchannels ⁇ without external intervention using an abrupt change in microchannel geometry.
  • the capillary stop constitutes an abrupt expansion of detection chamber geometry.
  • the capillary stop constitutes an abrupt narrowing of detection chamber geometry.
  • microfluidic channels are molded between detection chambers for transferring sample fluid and waste.
  • cartridges include an overflow reservoir ⁇ fluidically connected to each detection chamber in the plurality.
  • Said overflow reservoir may constitute a void in the cartridge into which diluted sample fluid may flow if/when said fluid surpasses the capillary stop.
  • cartridges may include an overflow outlet vent gaseously connected to each capillary stop.
  • Said overflow outlet vent may constitute a microfluidic channel gaseously connected to each capillary stop that vents to the surrounding environment.
  • at least a subset of the detection chambers in the plurality comprise one or more reagent spheres/beads comprising a dried reagent.
  • the dried reagent may be, ⁇ e.g., lyophilized or printed reagents.
  • the reagent beads may have a diameter, width and/or length from about 0.1 ⁇ m to about 35 ⁇ m, from about 0.1 ⁇ m to about 20 ⁇ ⁇ m, or from about 0.1 ⁇ m to about 10 ⁇ m.
  • the reagent beads may be coated with a reagent capable of binding a target antigen in a sample.
  • the reagent may comprise an antibody, an antibody fragment, an ionophore, an enzyme, a set of enzymes, a peptide with a cleavable detectable moiety, an optical marker dye identifying a type of assay bead, and/or combinations thereof.
  • the cartridge is pre-filled with reagents and is ready to use.
  • all or a portion of the reagents may be present.
  • the devices may need to be stored in appropriate conditions to preserve the reactivity of the reagents. For example, depending on the reagents present, the devices may need to be stored in a refrigerator or a freezer before use. When the reagents are not sensitive to room temperature, the devices may ⁇ be stored at room temperature.
  • the one or more dry reagents may in certain embodiments comprise a one or more non-fluorescent or fluorescent dyes such as Eosin, Methylene Blue, Acridine Orange (also referred to as “Basic Orange 15” or “ACO”), or Astrazon Orange (also referred to as "AO” or Basic Orange 21), a component to bind to nucleic DNA in cells (e.g., blood cells such as ⁇ WBCs), an anticoagulant, an antibody, an antibody fragment, an ionophore, an enzyme, a set of enzymes, a peptide with a cleavable detectable moiety, a substrate, an optical marker dye identifying a type of assay bead, and/or combinations thereof.
  • a non-fluorescent or fluorescent dyes such as Eosin, Methylene Blue, Acridine Orange (also referred to as “Basic Orange 15" or “ACO”), or Astrazon Orange (also referred to as "AO” or Basic Orange 21)
  • cartridges are designed for assaying clinical chemistry panels in a blood sample.
  • the clinical chemistry panels refer to groups of tests that are routinely ordered ⁇ to determine a subject’s general health status.
  • the clinical chemistry panels include metabolic panels.
  • the clinical chemistry panels help evaluate, for example, the body's electrolyte balance and/or the status of several major body organs.
  • the assays are performed on a blood sample, usually drawn from a vein.
  • Examples of clinical chemistry panels that may be detected by assays of the present disclosure include, but are not limited to, ⁇ basic metabolic panel (BMP), comprehensive metabolic panel (CMP), electrolyte panel, lipid panel, liver panel, renal panel, and thyroid function panel.
  • the basic metabolic panel (BMP) includes 8 tests, all of which are found in the CMP.
  • the BMP provides information about the current health of kidneys and respiratory system as well as electrolyte and acid/base balance and level of blood glucose.
  • the CMP measurement is used for liver and kidney health, level of ⁇ blood glucose, acid/base balance in blood, fluid and electrolyte balance, and important blood proteins.
  • the CMP measures glucose, calcium, total amount of albumin and globulins, bilirubin, BUN (blood urea nitrogen), creatinine, albumin, sodium, potassium, bicarbonate, chloride, alkaline phosphatase (ALP), alanine transaminase (ALT), and aspartate aminotransferase (AST).
  • BUN blood urea nitrogen
  • creatinine, albumin, sodium, potassium, bicarbonate chloride
  • ALP alkaline phosphatase
  • ALT alanine transaminase
  • AST aspartate aminotransferase
  • the electrolyte panel is used to detect a problem with the body’s fluid ⁇ and electrolyte balance.
  • the electrolyte panel measures the blood levels of carbon dioxide, chloride, potassium, and sodium.
  • the lipid panel measures the amount of cholesterol and other fats in blood, such as total cholesterol, LDL (low-density lipoprotein), HDL (high-density lipoprotein), and triglycerides.
  • the liver panel (hepatic function panel) is ⁇ used to screen for, detect, evaluate, and monitor acute and chronic liver inflammation (hepatitis), liver disease and/or damage.
  • the liver panel measures different enzymes, proteins, and other substances made by liver.
  • the liver panel includes albumin, total protein, ALP, ALT, AST, gamma-glutamyl transferase (GGT), bilirubin, Lactate dehydrogenase (LD), Prothrombin time (PT).
  • the renal panel includes tests such as ⁇ albumin, creatinine, BUN, eGFR to evaluate kidney function.
  • the thyroid Function Panel is used to evaluate thyroid gland function and to help diagnose thyroid disorders.
  • the thyroid function panel measure thyroid hormone such as thyroxine (T4), triiodothyronine (T3), and thyroid stimulating hormone (TSH).
  • T4 thyroxine
  • T3 triiodothyronine
  • TSH thyroid stimulating hormone
  • ⁇ in which the TSH level is low usually indicates that the thyroid is producing too much thyroid hormone (hyperthyroidism).
  • TSH and low free T4 (FT4) or free T4 index (FTI) indicates primary hypothyroidism due to disease in the thyroid gland.
  • a low TSH and low FT4 or FTI indicate hypothyroidism due to a problem involving the pituitary gland.
  • a low TSH with an elevated FT4 or FTI is found in individuals who have hyperthyroidism.
  • cartridge 110a An embodiment of the subject cartridge is shown as cartridge 110a in FIG.1A.
  • cartridge 110a includes sample inlet 115, separation filter 114, sample fluid ⁇ metering chamber 116, microfluidic passage 118, mixing chamber 117, detection chambers 111a-111n (where n may represent any suitable number of detection chambers, e.g., microcuvettes) and overflow reservoir 119. Also shown are multiple capillary stops C.
  • pressure may be supplied to sample inlet 115 (e.g., via a sample inlet pressure line) to advance the sample fluid to the separation ⁇ filter 114 and sample fluid metering chamber 116.
  • Intervals of pressure applied to the sample fluid in sample fluid metering chamber 116 generates predetermined volumes of the sample fluid, which then advances to microfluidic passage 118 where it is combined with a metered amount of diluent (e.g., from a diluent line) at an intersection.
  • the combined liquid is mixed in mixing chamber 117, and distributed to detection chambers 111a-111n for optical analysis.
  • Capillary stops C associated with detection chambers 111a-111n are connected to overflow reservoir 119 that receives excess fluid or air. Additional details regarding the interface of cartridge 110a with a sample fluid analysis device of the invention are provided below with respect to FIG.6A. Another embodiment of the subject cartridge is depicted as cartridge 110b in FIG.1B.
  • cartridges are configured for electrochemical analysis. ⁇ Electrochemical analysis is performed by utilizing a working electrode that detects an electrical signal generated by an electroactive species generated by the presence of an analyte in the sample.
  • the detected electrical signal may be quantitated to determine the presence or concentration of the analyte in the sample as the electrical signal is proportional to the amount of analyte present in the sample.
  • Electrochemical detection may involve amperometry, ⁇ coulometry, potentiometry, voltametery, impedance, or a combination thereof.
  • the electrochemical species may be generated by action of an analyte-specific enzyme on the analyte.
  • the electrochemical species may be generated by action of an enzyme on a substrate.
  • the enzyme is not specific to the analyte. Rather, the enzyme is conjugated to a binding member that specifically binds to the ⁇ analyte.
  • redox mediators may be included in order to amplify the electrical signal generated by the electrochemical species.
  • Analyte specific enzymes and redox mediators are well known and may be selected based on the desired sensitivity and/or specificity.
  • cartridges additionally include an electrochemical sensor for ⁇ determining one or more target analytes, e.g., as part of an electrochemical assay.
  • the size and shape of the electrodes required for detection of the electrochemical species can be determined empirically or can be based on the literature.
  • the electrodes may be similar to those disclosed in U.S. Patent No.5,200,051, which is herein incorporated by reference in its entirety.
  • the material of the electrodes may be any ⁇ material conducive to electrochemical sensing.
  • Exemplary electrode materials include carbon, platinum, gold, silver, rhodium, iridium, ruthenium, mercury, palladium, and osmium.
  • the working electrode may be a made from silver and the reference electrode may be silver/silver halide (e.g. silver chloride).
  • the working electrode (and optional reference electrode) may ⁇ be covered with a selectively permeable layer.
  • the selectively permeable layer may substantially exclude molecules with a molecular weight of about 120 kDa or more while allowing the free permeation of molecules with a molecular weight of about 50 kDa or less.
  • interfering electroactive species having a molecular weight above a desired threshold may effectively be excluded from interacting with the working electrode surface by employing a selectively permeable silane layer described in U.S. Patent No.5,200,051.
  • a permselective layer allows lower molecular weight ⁇ electroactive species, like dioxygen and hydrogen peroxide, to undergo a redox reaction with the underlying electrode surface.
  • Such a perselective layer may be especially useful in amperometric measurement.
  • a polymeric material having functional groups and chemical properties conducive to the further incorporation of certain ionophoric compounds ⁇ may be used as a semipermeable ion-sensitive film which is established on the working electrode of the analyte detection chip.
  • the development of a potential at the electrode-film interface depends on the charge density, established at equilibrium, of some preselected ionic species. The identity of such ionic species is determined by the choice of the ionophore incorporated in the semipermeable film.
  • An enzyme which is, in turn, immobilized in the ⁇ biolayers described herein catalyzes the conversion of a particular analyte, present in the sample, to the preselected ionic species.
  • the enzyme may not be immobilized in the biolayers but rather brought in proximity to the analyte by the DMF electrodes transporting a droplet containing the enzyme to a sample droplet and fusion of the two droplets.
  • the systems, devices, and methods of the present disclosure may include ⁇ electrodes that are used for detection of an analytes, such as, ions, e.g., Na2+, K+, Ca2+, and the like.
  • the electrodes may be covered with an ion-selective membrane.
  • the electrochemical sensor includes a base sensor or sensing electrode such as an amperometric electrode or potentiometric electrode on a substantially ⁇ planar chip/substrate where the sensing electrode is positioned in an auxiliary conduit for receiving a sample fluid mixed with a reagent.
  • the microfabricated sensor chip(s) comprises a plastic, polyester, polyimide, or silicon planar substrate, a plastic, polyester, polyimide, or silicon non-planar substrate, a transparent plastic, polyester, polyimide, or silicon substrate, a printed circuit board (PCB), and the like.
  • PCB printed circuit board
  • cartridges include digitally programmable active electronics capable of configuring the electrochemical sensing elements for potentiometric, coulometric, or amperometric signals.
  • cartridges include digitally programmable active electronics capable of ⁇ multiplexing electrochemical sensing elements for the potentiometric, coulometric or amperometric signals.
  • the digitally programmable active electronics are capable of reducing the number of signal interconnects needed to interface with instrument control electronics.
  • cartridges according to such embodiments of the invention may be more compact and more components can be included.
  • cartridges of the invention enable both a CBC and CMP panel to be performed on a single cartridge.
  • the one or more electrochemical sensors comprise reagents imprinted thereon.
  • the electrochemical sensors may be printed with appropriate dry reagents as needed for each analyte type.
  • Reagents used to amend biological samples or fluid within the cartridge may include antibody-enzyme conjugate, magnetic beads coated with capture antibodies, or blocking agents that prevent either specific or non-specific binding ⁇ reactions among assay compounds.
  • the reagent can be preferentially dissolved and concentrated within a predetermined region of the segment. This is achieved through control of the position and movement of the segment. Thus, for example, if only a portion of a segment, such as the leading edge, is reciprocated over the reagent, then a high local concentration of the reagent can be achieved close to the leading ⁇ edge. Alternatively, if a homogenous distribution of the reagent is desired, for example if a known concentration of a reagent is required for a quantitative analysis, then further reciprocation of the sample or fluid will result in mixing and an even distribution.
  • the electrochemical sensors employed may vary.
  • a ⁇ non-conducting substrate having a planar top and bottom surface may be used as a base for the electrochemical sensor chip.
  • a conducting layer may be deposited on the substrate by conventional means, e.g., screen printing, or micro-fabrication technique known to those of skill in the art to form at least one component (e.g., an amperometric electrode).
  • the electrochemical sensor chip may also comprise an electrical connection that connects each component of the conducting layer to one or more conductive pins such as a temporary (make and break) electrical connector.
  • the cartridge comprises an array of 5-10 ⁇ m noble metal disks, ⁇ e.g., 7 ⁇ m noble metal disks, on 15 ⁇ m centers.
  • the electrodes have a working area of about 130,000 to 300,000 sq ⁇ m (i.e., a microelectrode), the volume of sample directly over the electrodes may be about 0.1-0.3 ⁇ l, and the volume of the sample over the sensor chip may be 1-3 ⁇ l.
  • cartridges additionally include a calibration fluid reservoir configured to contain a calibration fluid for use with the electrochemical sensors.
  • Calibration fluid may be employed to obtain a calibration reference measurement using the electrochemical sensors discussed above.
  • Calibration fluid may include any fluid having a known composition that may be used to ⁇ calibrate electrodes in an electrochemical assay.
  • the calibration fluid is a plasma or plasma-like substance.
  • cartridges also include an electrochemical sensor outlet vent valve configured to ⁇ regulate venting by an electrochemical sensor outlet vent of the cartridge.
  • the calibration fluid may be drained into an electrochemical sensor outlet vent.
  • Cartridge 110c in FIG.1C is configured for optical sensing and electrochemical sensing. The components of cartridge 110c are the same as those described above with respect to FIG. ⁇ 1A, with the addition of calibration fluid reservoir 112, electrochemical sensors 113, overflow outlet vent 120 and electrochemical sensor outlet vent 121.
  • FIG.2 presents a schematic diagram for an embodiment of the cartridge according to certain embodiments of the invention.
  • sample fluid e.g., whole blood
  • Plasma is separated from the blood sample through a ⁇ separation filter 204.
  • the plasma is then metered in sample fluid metering chamber 205.
  • Diluent from built-in diluent reservoir/pouch 201 is metered in a diluent fluid metering chamber 203. Metered plasma and diluent are mixed downstream in a mixing chamber 206 for low Reynolds numbers, such as a Tesla valve or a Herringbone mixer. The diluted plasma may then be aliquoted in detection chambers 207 (i.e., microcuvettes). A waste reservoir is also ⁇ included to receive excess fluid.
  • a capillary restriction also referred to as a restriction capillary
  • a capillary restriction is a portion of a tube having a narrow internal diameter configured to restrict the flow of fluid moving therethrough.
  • cartridges include a single capillary restriction.
  • cartridges include a capillary restriction upstream of the plurality of detection chambers , i.e., such that diluted sample fluid passes through the capillary restriction prior to reaching any one of the detection chambers . In some cases, this is sufficient ⁇ to allow diluted sample fluid to accumulate downstream of the capillary restriction so that a single pressure pulse may be used to fill all detection chambers at once.
  • cartridges include a plurality of capillary restrictions, such as where the number of capillary restrictions ranges from 2 to 25. In such cases, cartridges may include a capillary restriction associated with each detection chamber, e.g., near the inlet.
  • cartridges include one or more temperature-regulating elements configured to maintain the temperature of the cartridge during analysis. In some instances, the temperature regulating elements are configured to maintain the temperature of the sample fluid at approximately human body temperature (e.g., ranging from 36°C to 38°C). In embodiments, cartridges include one or more heating electrodes configured to heat the sample liquid.
  • the one or more heating electrodes are comprised of titanium tungsten (TiW).
  • Cartridges according to some embodiments may also include a temperature-sensing element configured to provide feedback for the heating electrodes.
  • cartridges may include one or more temperature-sensing diodes.
  • FIG.3A depicts a cartridge 300 according to certain embodiments of the invention.
  • ⁇ Cartridge 300 includes detection chambers 301 arranged upright and in a staggered pattern.
  • diluted plasma tank 302 where, in embodiments, diluted plasma may either be introduced (e.g., via pipette), or accumulate following dilution.
  • FIG.3B presents an exemplary microfluidic configuration for filling ⁇ detection chambers 301 according to one embodiment of the invention.
  • capillary restriction 304a is upstream of the inlets of the detection chambers 301.
  • Capillary restriction 304a allows diluted sample fluid to accumulate upstream of capillary restriction 304a so that all of the detection chambers 301 may be filled at once, e.g., using one “push”.
  • FIG.3C depicts an alternative configuration involving a plurality of capillary restrictions 304a-304e.
  • FIG.4A depicts detection chambers 410 and 420 of a cartridge according to certain embodiments of the invention. Diluted sample fluid 403 is depicted entering the detection chambers at inlets 411, which then mixes with reagents dried on reagent beads 402. Capillary stops 412 are shown at the outlets of detection chambers 410 and 420. Excess fluid passing ⁇ through capillary stops 412 may be collected in overflow reservoir 413. Gas may also escape through overflow outlet vent 404.
  • Incident light 401 passes through first light-accessible windows 414 of detection chambers 410 and 420, irradiates the fluid therein, and exits through second light-accessible windows 415. This light is subsequently collected for analysis.
  • the detection chambers are configured to minimize overfilling via a ⁇ difference between a capillary pressure of the detection chamber inlet and the detection chamber outlet.
  • the capillary pressures across the capillary stop meniscuses can be calculated with the Young-Laplace equation, as follows: ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ where ⁇ is surface tension of the sample fluid (water 0.0726 N/m at 20°C); ⁇ is the contact angle of the sample fluid; and ⁇ is the radius of the capillary channel.
  • ⁇ surface tension of the sample fluid (water 0.0726 N/m at 20°C); ⁇ is the contact angle of the sample fluid; and ⁇ is the radius of the capillary channel.
  • an inlet radius 0.8 mm has capillary pressure 363 N/m 2 (Pa), and outlet radius 0.3 ⁇ mm, 968 N/m 2 (Pa).
  • FIG.4B presents an embodiment of an outlet 412 according to one embodiment.
  • Digital microfluidics uses the principles of emulsion science to create fluid-fluid dispersion into channels (principally water-in-oil emulsion). It allows the production of monodisperse drops/bubbles or with a very low polydispersity. Digital microfluidics is based upon the micromanipulation of discontinuous fluid droplets within a reconfigurable network. Complex instructions can be programmed by combining the basic operations of droplet formation, translocation, splitting, mixing and merging. ⁇ Digital microfluidics operates on discrete volumes of fluids that can be manipulated by binary electrical signals.
  • a microfluidic operation may be defined as a set of repeated basic operations, i.e., moving one unit of fluid over one unit of distance.
  • Droplets may be formed using surface tension properties of the liquid. Actuation of a droplet is based on the presence of electrostatic forces generated by electrodes placed ⁇ beneath the bottom surface on which the droplet is located. Different types of electrostatic forces can be used to control the shape and motion of the droplets.
  • One technique that can be used to create the foregoing electrostatic forces is based on dielectrophoresis which relies on the difference of electrical permittivities between the droplet and surrounding medium and may utilize high frequency AC electric fields.
  • the cartridge disclosed herein include a DMF region and an analyte detection region which may overlap or be spatially segregated.
  • the DMF region may be ⁇ used to transfer droplets for analysis to a detection region where the droplets are analyzed electrochemically.
  • Electrochemical analysis is performed by utilizing a working electrode that detects an electrical signal generated by an electroactive species generated by the presence of an analyte in the sample.
  • the detected electrical signal may be quantitated to determine the presence or concentration of the analyte in the sample as the electrical signal is proportional to ⁇ the amount of analyte present in the sample.
  • Electrochemical detection may involve amperometry, coulometry, potentiometry, voltammetry, impedance, or a combination thereof.
  • the electrochemical species may be generated by action of an analyte-specific enzyme on the analyte.
  • the electrochemical species may be generated by action of an enzyme on a substrate.
  • the enzyme is ⁇ not specific to the analyte. Rather, the enzyme is conjugated to a binding member that specifically binds to the analyte.
  • redox mediators may be included in order to amplify the electrical signal generated by the electrochemical species.
  • Analyte specific enzymes and redox mediators are well known and may be selected based on the desired sensitivity and/or specificity.
  • Electrodes for detection of an electrochemical species may be provided in numerous configurations. Such electrodes may be separate from the DMF electrodes or may be DMF electrodes that have been modified into electrodes for electrochemical sensing. Exemplary configurations of analyte detection chips containing DMF electrodes and electrodes for electrochemical sensing are further described below.
  • the cartridge may be manufactured from plastic (e.g., thermoplastic, such as, Cyclo Olefin Polymer or Cyclo Olefin Copolymer), metal, paper, glass, and the like or a combination thereof. If metal material is used for the cartridge, the metal may be non-magnetic, i.e., not include substantial amount of iron.
  • a paper material may include a non-wettable coating, e.g., a wax coating.
  • the cartridge may be substantially opaque or substantially ⁇ transparent.
  • the housing may comprise therein the functional elements of the device (e.g., manifold, etc.), described in greater detail below.
  • Embodiments of devices, and in particular, embodiments of housings of devices may have any ⁇ convenient shape and size, and such may vary, e.g., based on an intended application environment or use environment for the system or, for example, a desired throughput of the system. That is, it should be understood that the housing may have a variety of shapes depending on particular design considerations.
  • embodiments of devices may be utilized in a plurality of different contexts, including, but not limited to, home environments, point of care environments, ambulance environments, emergency room environments, doctor’s office environments, pharmacy environments, small clinics or pop-up clinics, hospital lab environments or core lab environments. Accordingly, the size and shape of ⁇ embodiments of systems may be determined to suit the desired environment.
  • An aspect of the desired environment for use of embodiments of systems of the present disclosure is the number of sample analyses, e.g., determining one or more analyte levels across a number of samples, that can be performed in a fixed amount of time and/or the number of sample analyses that can be performed concurrently.
  • the device in certain home environments, it ⁇ may be desired that the device be configured to run only one sample analysis at a time, whereas in certain core lab environments, it may be desired that the system be configured to run one, two, three, four, five, six, seven, eight, nine, ten, tens, hundreds or thousands or more sample analyses concurrently.
  • the size and/or shape of an embodiment of a device may be dictated by ⁇ the number of sample analyses that can be performed concurrently by an embodiment of the device.
  • embodiments capable of performing a plurality of sample analyses concurrently may be configured to receive and concurrently hold a plurality of cartridges and/or a plurality of sample collection devices.
  • Embodiments of housings may be configured to hold 1 or more cartridges concurrently, such as 1 cartridge, 2 cartridges, 3 cartridges, 4 cartridges, 5 ⁇ cartridges, 6 cartridges, 7 cartridges, 8 cartridges, 9 cartridges, 10 cartridges, 20 cartridges, 30 cartridges, 40 cartridges, 50 cartridges, 100 cartridges or 500 or more cartridges.
  • Embodiments of devices may be configured to receive one or more samples concurrently, such as 1 sample, 2 samples, 3 samples, 4 samples, 5 samples, 6 samples, 7 samples, 8 samples, 9 samples, 10 samples, 20 samples, 30 samples, 40 samples, 50 samples, 100 samples or 500 ⁇ or more samples.
  • sample fluid analysis devices and cartridges may be configured to perform one or more sample analyses (i.e., determining level(s) of one or more analytes of one or more samples) concurrently, such 1 sample analysis at a time, 2 sample analyses concurrently, 3 sample analyses concurrently, 4 sample analyses concurrently, 5 sample analyses concurrently, 6 sample analyses concurrently, 7 sample analyses ⁇ concurrently, 8 sample analyses concurrently, 9 sample analyses concurrently, 10 sample analyses concurrently, 20 sample analyses concurrently, 30 sample analyses concurrently, 40 sample analyses concurrently, 50 sample analyses concurrently, 100 sample analyses concurrently, or 500 or more sample analyses concurrently.
  • sample analyses i.e., determining level(s) of one or more analytes of one or more samples
  • polymeric materials include acrylonitrile butadiene styrene (ABS), polylactic acid (PLA), acrylic styrene acrylonitrile (ASA), ⁇ polyethylene terephthalate (PET), glycol-modified polyethylene terephthalate (PETG), polyaryletherketones (PAEK), polyetherimides (PEI), polypolycarbonate (PC), polypropylene, (PP), aliphatic polyamides (PPA), polyoxymethylene (POM), polymethyl methacrylate (PMMA), polybutylene terephthalate (PBT), polyphenylsulfone (PPSU), polyether ether ketone (PEEK), and nylon as well as composites and hybrids thereof.
  • ABS acrylonitrile butadiene styrene
  • PLA polylactic acid
  • ASA acrylic styrene acrylonitrile
  • PET polyethylene terephthalate
  • PET glycol-modified polyethylene terephthalate
  • Embodiments of devices may be configured to be substantially sealed such that contents of samples and/or contents of cartridges are disposed of in one or more waste disposal locations of the and are not otherwise emitted from, i.e., leaked out of, the device.
  • the device is substantially sealed such that substances cannot enter the primary device and/or cartridges and/or other aspects of systems other than through dedicated entry ⁇ points, such as a substance entering the sample fluid analysis device from a cartridge via the cartridge interface, for example.
  • the system may be configured for self- cleaning of certain aspects of the system and in some cases one or more cartridge types may be configured to facilitate such self-cleaning functionality. Housings of interest are configured to receive a cartridge.
  • different types of cartridges and the associated receptacles of the cartridge interface comprise other physical or mechanical ⁇ features, such as pins or other keying techniques, that prevent cartridges other than a specific type of cartridge from being loaded into a dedicated cartridge receptacle.
  • a specific type of cartridge may be shaped with a chamfered cross section such that a receptacle of the cartridge interface cannot receive the cartridge unless it also comprises a similar chamfered cross section.
  • the housing may be configured to interface with the cartridge when said cartridge is received therein.
  • interface it is meant connect in a functional (e.g., mechanical) and signal-communicating relationship with the cartridge.
  • Cartridge interfaces may comprise at least one receptacle and are configured to functionally interconnect the sample fluid analysis device with the cartridge, i.e., such that one or more contents of the cartridge are made functionally ⁇ available to the sample fluid analysis device in connection with performing sample analysis. Therefore, cartridge interfaces may comprise, for example, one or more mechanical interconnections with the cartridge (e.g., gears, push rods, other mechanical engagement features, etc.), electrical interconnections with the cartridge (e.g., wired or wireless connections), fluidic interconnections (e.g., tubing or other fluidic paths) with the cartridge or ⁇ any other interconnection necessary for the contents of the cartridge to be functionally available to the sample fluid analysis device.
  • mechanical interconnections with the cartridge e.g., gears, push rods, other mechanical engagement features, etc.
  • electrical interconnections with the cartridge e.g., wired or wireless connections
  • fluidic interconnections e.g., tubing or other fluidic paths
  • devices of the invention may be configured to cause the various functions that will be described in greater detail below to be carried out within the cartridge (e.g., sample fluid metering, sample fluid separation, mixing, etc.).
  • devices of the invention may be configured to emit to and receive signals ⁇ (e.g., electrical signals, optical signals/light) from the cartridge for interrogating a sample fluid within the cartridge.
  • the housing is configured to mechanically hold or fix the cartridge in place.
  • the cartridge and the housing are configured to utilize spring loading to hold the cartridge in place.
  • the housing may comprise a cartridge interface with a receptacle with flexible members, e.g., tabs, configured to grip or press into the sides of a cartridge as the cartridge is loaded into the housing.
  • the housing and a cartridge are configured to utilize gravity or magnetic interactions or other mechanical, e.g., ⁇ spring, interactions or the like to hold the cartridge in place.
  • the cartridge may be latched or otherwise fixed or locked into place, e.g., by a user or by a robotic feature.
  • any convenient number of latches or locks may be provided.
  • the housing and the cartridge are configured to utilize a press-fit engagement between the cartridge and the housing.
  • the physical interfaces i.e., the physical interface of the housing and the ⁇ physical interface of a cartridge, may be removably coupled to one another by incorporating any of a variety of releasably engaging mechanisms, e.g., snap, slide, magnetic, Velcro, clasp, hook, hinge, lock, latch, etc.
  • the physical interfaces, as well as the overall housing of the aspects of the system may be form fitted to provide a close fit for sturdy coupling, as well as to provide other functional features, e.g., portability, when coupled as a single unit.
  • the housing may be configured to enable add-on capability.
  • the housing may be configured to enable adding one or more additional cartridge interfaces such that the sample fluid analysis device can interface with different types of cartridges and/or more than one cartridge simultaneously. That is, embodiments of systems of the present disclosure are modular at least insofar as the housing is configured to add on the ⁇ capacity to receive different and/or additional cartridges, e.g., to add one or more additional cartridge interfaces to the sample fluid analysis device.
  • a cartridge interface of a housing may be reconfigurable such that, upon reconfiguration, it is capable of receiving different types of cartridges.
  • the cartridge interface may be configured to receive an adaptor that allows different or additional types of cartridges to interface with the ⁇ housing when the adaptor is used.
  • embodiments of housing are used in conjunction with one or more cartridges, i.e., the housing may be configured to interface with one or more cartridges. It should be understood that the housing and the one or more cartridges are removably coupled to one another. Therefore, in this disclosure, references to the cartridge(s) removably coupled ⁇ to the housing; references of the sample fluid analysis device removably coupled to the cartridge(s); references to the housing and cartridge(s) removably coupled; and references to one or more cartridges being loaded into, or received by, the housing, or similar phrases, are used interchangeably.
  • a single cartridge may be loaded into the housing, and in other cases, more than one cartridge may be loaded into the housing.
  • Loading a cartridge into the housing may comprise inserting the cartridge into the housing, i.e., such that one face of the cartridge is completely or substantially exposed to the housing.
  • causing a sample fluid analysis device to interface with a cartridge comprises applying a connector or interface between an aspect of the cartridge and an aspect of the housing.
  • Such connector or interface may comprise any convenient mechanical and/or ⁇ electrical and/or fluidic interconnections or any other functional interconnections such that the contents of the cartridge are made available to the housing in connection with performing sample analysis.
  • devices may be configured to indicate a required cartridge type for performing a specific sample analysis, e.g., for determining a level of a specific analyte in a ⁇ specific type of sample.
  • the system may comprise a display unit for displaying a type of cartridge that is required to be loaded into the sample fluid analysis device for performing a specific type of sample analysis.
  • the system may be configured to provide an indication that a cartridge is loaded into the housing, or, in some cases, that a specific type of cartridge is loaded into the housing.
  • the device ⁇ may comprise an indicator element; e.g., the housing or a cartridge interface thereof, may comprise an indicator element configured to indicate whether a cartridge is loaded into the housing and/or the type of cartridge that is loaded into the sample fluid analysis device and/or a status of the cartridge loaded into the housing (e.g., a level or some resource present within the cartridge) and/or any other information pertinent to conducting sample analysis using the ⁇ device.
  • Indicator elements may comprise one or more indication lights, such as LEDs, or display interfaces, for example.
  • Any convenient technique may be utilized in order to identify the presence and/or the type of cartridge loaded into a sample fluid analysis device, including, for example, mechanical techniques (e.g., keying or a unique pin structure associated with different types of cartridges) or electronic techniques (e.g., digital encodings stored on a non- ⁇ volatile memory, RFID techniques or other wireless identifiers, magnetic encodings, etc.) or optical techniques, such as bar codes, 2D bar codes or other optical identifiers capable of being read by a camera present on the sample fluid analysis device, for example, or combinations thereof.
  • mechanical techniques e.g., keying or a unique pin structure associated with different types of cartridges
  • electronic techniques e.g., digital encodings stored on a non- ⁇ volatile memory, RFID techniques or other wireless identifiers, magnetic encodings, etc.
  • optical techniques such as bar codes, 2D bar codes or other optical identifiers capable of being read by a camera present on the sample fluid analysis device, for example, or combinations
  • Embodiments of systems of the invention may utilize any convenient technique for ⁇ identifying whether a cartridge is loaded into the sample fluid analysis device and/or the type of cartridge that has been loaded into the sample fluid analysis device, such as, for example, one or more mechanical/physical indicators (e.g., a specific pattern or pins, tabs or the like may be present on a cartridge and an opposing pattern of such element may be present on the receptacle of the cartridge interface of the sample fluid analysis device), or one or more ⁇ electrical indicators (e.g., an indicator electrode, magnetic encoding, software identifier utilized in conjunction with a processor and memory of the system, wireless communication, RFID identification or the like), or one or more optical indications (e.g., a bar code or a 2D bar code and a camera configured, in conjunction with a processor and memory of the system to identify the presence of a cartridge and/or the type of cartridge loaded into the sample fluid analysis device).
  • one or more mechanical/physical indicators e.g., a specific pattern or pins, tab
  • Devices of the invention include a manifold.
  • the term “manifold” is used herein in its conventional sense to refer to a series of tubing and/or chambers that branch to form several ⁇ inlets and/or outlets.
  • the subject manifold is configured to interface with the cartridge in such a manner that liquid and/or gas may be provided to the cartridge so that, e.g., metering, mixing, etc., can be carried out within the cartridge.
  • Manifolds of the invention include a diluent line fluidically connected to a diluent reservoir and comprising a connector for fluidically connecting to a mixing chamber of the cartridge.
  • the diluent line is a channel ⁇ manufactured within the manifold.
  • the diluent line is comprised of tubing.
  • the tubing may be comprised of any suitable material, including but not limited to polyether ether ketone (PEEK), polytetrafluoroethylene (PTFE), Tygon, fluorinated ethylene-propylene (FEP), ethylene tetrafluoroethylene (ETFE), polypropylene (PP), polyethylene (PE), and the like, as well as combinations thereof.
  • PEEK polyether ether ketone
  • PTFE polytetrafluoroethylene
  • FEP fluorinated ethylene-propylene
  • ETFE ethylene tetrafluoroethylene
  • PP polypropylene
  • PE polyethylene
  • the dimensions of the diluent line may vary. For example, ⁇ in some instances, the diluent line comprises an inner diameter ranging from 0.5 mm to 2 mm. In certain cases, the diluent line comprises an outer diameter ranging from 1 mm to 3 mm.
  • the connector may
  • Exemplary connectors include, e.g., quick disconnect connectors, threaded connectors, luer connectors, multiport connectors, tri clamp fittings, and puncture and seal sterile fittings.
  • Suitable quick disconnect connectors include, but are not limited to, snap- ⁇ type (ball-latching) connectors, bayonet connectors, threaded connectors, non-latching connectors, single-shutoff connectors, double-shutoff connectors, non-shutoff connectors, dry break connectors, roller lock connectors, pin lock connectors, ring lock connectors, and cam lock connectors.
  • the connector is comprised of a gasket material suitable for forming an air-tight seal with the inlet of the mixing chamber of the cartridge.
  • the gasket ⁇ material may vary as desired and can include, but is not limited to, butyl rubber, nitrile rubber, neoprene, styrene-butadiene rubber, and the like.
  • the diluent reservoir may be any suitable reservoir or container (e.g., having rigid or flexible walls) for holding a diluent fluid (e.g., a blood diluent fluid).
  • the volume of the diluent reservoir may vary, and can range in some instances from 5 mL to 500 mL, such as 10 mL to ⁇ 250 mL, such as 15 mL to 100 mL.
  • the diluent reservoir is contained within the sample fluid analysis device.
  • the diluent reservoir is contained within the cartridge.
  • the diluent reservoir may have a sealed output to prevent evaporation to the environment.
  • Manifolds of the invention additionally include a diluent fluid metering unit for controlling ⁇ a metered amount of the diluent fluid supplied to the mixing chamber via the diluent line.
  • a “metered amount” it is meant a precise, known, volume of the diluent fluid supplied to the cartridge at a precise, known, time. A value may be considered “precise” if it exists within an acceptable tolerance.
  • sample fluid analysis devices of the invention may be configured to deliver a particular volume of diluent fluid to the cartridge using pressure pulses having a width measured in time.
  • volumes of diluent fluid delivered to the cartridge vary between pulses by ⁇ l ⁇ l or less, such as ⁇ 0.1 ⁇ l or less, such as ⁇ 0.01 ⁇ l or less, and including ⁇ 0.001 ⁇ l or less.
  • pulse widths ⁇ vary between pulses by ⁇ 1 ms or less, such as ⁇ 0.1 ms or less, such as ⁇ 0.01 ms or less, and including ⁇ 0.001 ⁇ l or less.
  • the diluent fluid metering unit comprises a flow meter.
  • pressure control is implemented as a pressure feedback circuit. This method has been measured to show ⁇ pressure control to 1% of control loop setpoint in the range of 40 Pa to 32000 Pa.
  • the flow meter of the subject manifold may be configured to monitor the transport of fluid through the diluent line and provide feedback (e.g., to a pump) so that said transport may be adjusted, if necessary.
  • Flow meters that may be employed can include, but are not limited to Coriolis flow meters, differential pressure meters, electromagnetic flow meters, multiphase flow meters, ⁇ ultrasonic flow meters, and vortex flow meters.
  • the flow meter is a Coriolis flow meter.
  • the diluent fluid metering unit comprises a metering pressure line gaseously connected to a diluent fluid metering chamber fluidically connected to the diluent line.
  • the diluent fluid metering chamber may operate via the same principle as the sample fluid metering chamber.
  • diluent fluid enters the diluent ⁇ fluid metering chamber having a certain volume, and pulses from the metering pressure line may be used to cause fluid to enter the cartridge via the diluent line.
  • Manifolds according to some embodiments of the invention also include a sample fluid pressure line comprising a connector for gaseously connecting to the cartridge and supplying pressure thereto.
  • a sample fluid pressure line comprising a connector for gaseously connecting to the cartridge and supplying pressure thereto.
  • gaseously connect it is meant couple in a manner sufficient to permit the ⁇ passage of a gas.
  • gaseously coupled elements are essentially air-tight.
  • the sample pressure line is a channel manufactured within the manifold.
  • the sample inlet pressure line is comprised of tubing.
  • the tubing may be comprised of any suitable material, including but not limited to polyether ether ketone (PEEK), polytetrafluoroethylene (PTFE), Tygon, fluorinated ethylene-propylene (FEP), ethylene ⁇ tetrafluoroethylene (ETFE), polypropylene (PP), polyethylene (PE), and the like, as well as combinations thereof.
  • PEEK polyether ether ketone
  • PTFE polytetrafluoroethylene
  • FEP fluorinated ethylene-propylene
  • ETFE ethylene ⁇ tetrafluoroethylene
  • PP polypropylene
  • PE polyethylene
  • the dimensions of the sample inlet pressure line may vary.
  • the sample inlet pressure line comprises an inner diameter ranging from 0.5 mm to 2 mm.
  • the diluent line comprises an outer diameter ranging from 1 mm to 3 mm.
  • the connector may also vary.
  • Exemplary connectors include, e.g., quick disconnect ⁇ connectors, threaded connectors, luer connectors, multiport connectors, tri clamp fittings, and puncture and seal sterile fittings.
  • Suitable quick disconnect connectors include, but are not limited to, snap-type (ball-latching) connectors, bayonet connectors, threaded connectors, non- latching connectors, single-shutoff connectors, double-shutoff connectors, non-shutoff connectors, dry break connectors, roller lock connectors, pin lock connectors, ring lock connectors, and cam lock connectors.
  • the connector is comprised of a gasket material suitable for forming an air-tight seal with the inlet of the mixing chamber of the cartridge.
  • the gasket material may vary as desired and can include, but is not limited to, butyl ⁇ rubber, nitrile rubber, neoprene, styrene-butadiene rubber, and the like.
  • the sample fluid pressure line may be configured to gaseously connect to one or more corresponding components of the cartridge. In some cases, the sample fluid pressure line is configured to gaseously connect to a sample inlet of the cartridge. In such cases, pressure from the sample fluid pressure line may be used to move sample fluid within the sample inlet through ⁇ other parts of the cartridge (e.g., to the sample fluid metering chamber). In other cases, the sample fluid pressure line is configured to gaseously connect to the sample fluid metering chamber of the cartridge.
  • the sample fluid pressure line may be used to apportion the predetermined volume of sample fluid in the metering chamber, e.g., by creating a bubble at the trailing edge of the fluid within the metering chamber at certain intervals, as ⁇ described above.
  • the sample fluid pressure line does not gaseously connect to the sample inlet, said sample inlet may operate via a completely passive flow regime.
  • the sample fluid pressure line is configured to gaseously connect to the sample inlet and the sample fluid metering chamber.
  • the sample fluid pressure line is branched (i.e., forked) such that the sample fluid pressure line can gaseously connect to both ⁇ the sample inlet and the sample fluid metering chamber.
  • devices may include multiple (e.g., 2) different sample fluid pressure lines: one to connect to the sample inlet, and another to connect to the metering chamber. Portions of the sample fluid pressure line(s) configured to gaseously connect to the sample inlet may in ⁇ some cases be referred to herein as the “sample inlet pressure line”, and portions of the pressure line(s) configured to connect to the sample fluid metering chamber may be referred herein as the ”sample fluid metering chamber pressure line”.
  • devices include a sample inlet pressure valve operably coupled to the sample fluid pressure line and configured to regulate pressure applied to the ⁇ sample inlet.
  • the valve may vary, as desired.
  • the valve comprises an actuator (e.g., magnetic actuator, electrostatic actuator, piezoelectric actuator, thermal actuator, etc.) for opening/closing the fluid path. The functioning of the actuator may be controlled, e.g., by a processor.
  • devices include a sample fluid metering chamber pressure ⁇ valve operably coupled to the sample fluid pressure line and configured to regulate pressure applied to the sample fluid metering chamber. The valve may vary, as desired.
  • the valve comprises an actuator (e.g., magnetic actuator, electrostatic actuator, piezoelectric actuator, thermal actuator, etc.) for opening/closing the fluid path.
  • the functioning of the actuator may be controlled, e.g., by a processor.
  • devices are configured for feedback with respect to the metering of sample fluid within the sample fluid metering chamber of the cartridge.
  • the feedback may be ⁇ employed to determine, e.g., if the predetermined volume of the sample fluid was correctly metered in the chamber. This may be particularly important due to efficiency of plasma separation at different hematocrits.
  • Said feedback may be used by, e.g., the pump to adjust pressure pulses such that the correct volume is obtained for a desired dilution.
  • the feedback is optical feedback.
  • devices include a light source configured ⁇ to irradiate the metering chamber through a light-accessible window (e.g., described above) of the cartridge, and an optical sensor configured to receive light from the sample fluid metering chamber. Any of the light sources and/or sensors described herein may be adapted for use in the optical metering of the metering chamber.
  • the optical sensor may be operably connected to the pump, e.g., such that if a deviation in volume of sample fluid is detected, the activity of the ⁇ pump may be adjusted accordingly.
  • devices and cartridges may be configured for measuring and estimating flow from meniscus tracking during the combining process (e.g., at the microfluidic passage).
  • devices and cartridges are configured to provide feedback for the filling of the detection chambers, and respond if they are not filled (e.g., by ⁇ activating the pump).
  • devices and cartridges are configured to provide bubble detection in the microfluidic passage and downstream of the microfluidic passage, and adjust parameters to reduce instances of such bubbles.
  • devices include a mechanical positioning apparatus for gaseously connecting the diluent line to the microfluidic passage, and the sample fluid pressure line to the ⁇ cartridge.
  • devices of the invention are configured to automatically gaseously connect the relevant lines to the appropriate location on the cartridge, e.g., without any manual intervention.
  • embodiments of the present devices include a pump.
  • devices include a single pump.
  • devices include a plurality of pumps, such as 2 ⁇ or more pumps, and including three or more pumps.
  • the pump(s) of the invention may be configured to apply positive pressure and/or negative (i.e., vacuum) pressure.
  • the pump is a positive displacement pump.
  • a “positive displacement pump” refers to pumps that move fluid by trapping a fixed amount of fluid and forcing (displacing) that trapped volume out of the device, where such pumps may operate with a series of working ⁇ cycles, each cycle trapping a certain volume of fluid and moving the fluid mechanically through the pump and into a fluidic system.
  • Positive displacement pumps that may be employed include, but are not limited to: rotary-type positive displacement pumps, such as peristaltic pumps, internal gear pumps, screw pumps, shuttle block pumps, flexible vane or sliding vane pumps, circumferential piston pumps, flexible impeller pumps, helical twisted roots pumps or liquid-ring pumps; reciprocating-type positive displacement pumps, such as piston pumps, plunger pumps or diaphragm pumps; and linear-type positive displacement pumps, such as rope pumps and chain pumps.
  • the positive displacement pump ⁇ includes a pump selected from the group consisting of a peristaltic pump, gear pump and a diaphragm pump.
  • the positive displacement pump is a peristaltic pump.
  • systems include a vacuum pump configured to apply negative pressure.
  • devices include an adjustable variable resistor such as a potentiometer configured to regulate/control activity of the pump.
  • the pressure applied by the pump to may ⁇ vary in some cases, the pressure ranges from 50 Pa to 30000 Pa, such as 200 Pa to 2000 Pa, such as 300 Pa to 1000 Pa, such as 350 Pa to 800 Pa, and including 400 Pa to 600 Pa.
  • Embodiments of the subject devices also include a pressure sensor, e.g., configured to assess pressure within the manifold.
  • the pressure sensor may, in some instances, be operably connected to the pump.
  • the pressure sensor may be employed to ⁇ provide feedback for the pump, e.g., to ensure that an indicated pressure is being applied in order to achieve a particular precision dilution.
  • manifolds include a diluent fluid pressure line gaseously connected to the diluent reservoir and the pump for applying pressure to the diluent reservoir.
  • the diluent fluid pressure line is a channel manufactured within the manifold. In other embodiments, diluent fluid pressure line is comprised of tubing.
  • the tubing may be comprised of any suitable material, including but not limited to polyether ether ketone (PEEK), polytetrafluoroethylene (PTFE), Tygon, fluorinated ethylene-propylene (FEP), ethylene tetrafluoroethylene (ETFE), polypropylene (PP), polyethylene (PE), and the like, as well as ⁇ combinations thereof.
  • PEEK polyether ether ketone
  • PTFE polytetrafluoroethylene
  • FEP fluorinated ethylene-propylene
  • ETFE ethylene tetrafluoroethylene
  • PP polypropylene
  • PE polyethylene
  • the dimensions of the diluent fluid pressure line may vary.
  • the diluent fluid pressure line comprises an inner diameter ranging from 0.5 mm to 2 mm.
  • the diluent fluid pressure line comprises an outer diameter ranging from 1 mm to 3 mm.
  • devices include a diluent fluid pressure valve operably coupled to the diluent fluid pressure line configured to regulate pressure applied to the ⁇ diluent reservoir.
  • the valve may vary, as desired.
  • the valve comprises an actuator (e.g., magnetic actuator, electrostatic actuator, piezoelectric actuator, thermal actuator, etc.) for opening/closing the fluid path.
  • the functioning of the actuator may be controlled, e.g., by a processor.
  • the manifolds further include a calibration fluid pressure line configured to apply pressure to a calibration fluid reservoir in the cartridge.
  • the calibration fluid pressure line is a channel manufactured within the manifold.
  • calibration fluid pressure line is comprised of tubing.
  • the tubing may be comprised of any suitable material, including but not limited to polyether ether ketone (PEEK), polytetrafluoroethylene (PTFE), Tygon, fluorinated ethylene-propylene (FEP), ethylene tetrafluoroethylene (ETFE), polypropylene (PP), polyethylene (PE), and the like, as well as combinations thereof.
  • PEEK polyether ether ketone
  • PTFE polytetrafluoroethylene
  • FEP fluorinated ethylene-propylene
  • ETFE ethylene tetrafluoroethylene
  • PP polypropylene
  • PE polyethylene
  • the dimensions of the calibration fluid pressure line may vary.
  • the calibration fluid pressure line comprises an inner diameter ranging from 0.5 mm to 2 mm.
  • the diluent line comprises an outer diameter ranging from 1 mm to 3 mm.
  • devices include a calibration fluid pressure valve operably coupled to the calibration fluid pressure line configured to regulate pressure applied to the calibration fluid.
  • the valve may vary, as desired.
  • the valve comprises an ⁇ actuator (e.g., magnetic actuator, electrostatic actuator, piezoelectric actuator, thermal actuator, etc.) for opening/closing the fluid path.
  • the functioning of the actuator may be controlled, e.g., by a processor.
  • manifolds include an electrochemical sensor outlet vent valve configured to regulate venting by an electrochemical sensor outlet vent of the cartridge.
  • manifolds also include an overflow outlet vent valve configured to regulate ⁇ venting by an overflow outlet vent of the cartridge.
  • sample fluid analysis devices of the invention additionally include a processor operably connected to at least the pump and/or the diluent fluid metering unit (e.g., flow meter).
  • processors of interest are configured to control the amount of diluent applied to the cartridge via the diluent line, and control the pressure applied to the sample fluid metering ⁇ chamber.
  • the processor may enable precision dilutions of the sample fluid by controlling the volume of sample fluid and volume of diluent fluid that is combined. Dilutions achieved using the present devices may vary.
  • devices may be configured to achieve a low range dilution of a biological sample, e.g., dilutions that are less than about 50:1 (v/v diluent: sample fluid).
  • the devices and methods are adapted for high ⁇ range dilution, e.g., dilutions of about 50:1 or greater (v/v diluent: sample fluid), typically from 50:1 to 50,000:1.
  • devices are configured to prepare the sample fluid dilution at a dilution ratio ranging from 1:1 to 100:1.
  • the control of the pump may be executed by a specifically programmed processor, which may include one or more processors, such as one or more digital signal processors (DSPs), configurable ⁇ microprocessors, application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), or other equivalent integrated or discrete logic circuitry.
  • DSPs digital signal processors
  • ASICs application specific integrated circuits
  • FPGAs field programmable gate arrays
  • a combination of computing devices e.g., a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration in at least partial data connectivity may implement one or more of the ⁇ features described herein.
  • FIG.6A presents a schematic diagram of a sample fluid analysis device according to certain embodiments of the invention.
  • the sample fluid analysis device comprises a manifold 600a, which comprises pump 601, pressure sensor 602, diluent reservoir 603, flow meter 604 and diluent fluid pressure line 605a.
  • FIG.6A further depicts cartridge 610a and its constitutive elements (described above with respect to FIG.1A).
  • Manifold 600a also includes diluent line 605b connected to diluent reservoir 603 configured to connect to microfluidic passage 118 of cartridge 110a.
  • Manifold 600a additionally includes flow meter 604 ⁇ for controlling a metered amount of diluent fluid supplied to microfluidic passage 118 via diluent line 605b.
  • sample inlet pressure line 607 for gaseously connecting to sample inlet 115 of cartridge 110a and supplying pressure to the sample fluid.
  • Manifold 600a further includes sample fluid metering chamber pressure line 606 for gaseously connecting to a sample fluid metering chamber 116 of cartridge 110a configured to supply a predetermined ⁇ volume of the sample fluid from sample inlet 115 to microfluidic passage 118.
  • Sample inlet pressure line 607 and sample fluid metering pressure line 606 may together be considered a sample fluid pressure line of the invention, which branches so that sample inlet pressure line 607 and sample fluid metering pressure line 606 are configured to gaseously connect to sample inlet 115 and sample fluid metering chamber 116, respectively.
  • the device of FIG.6A also ⁇ includes valves configured to regulate pressure.
  • Diluent fluid pressure valve V1 is operably coupled to diluent fluid pressure line 605a configured to regulate pressure applied to diluent reservoir 603.
  • Sample fluid metering chamber pressure valve V2 is operably coupled to sample fluid metering chamber pressure line 606 and configured to regulate pressure applied to the sample fluid metering chamber 116 of cartridge 110a.
  • Sample fluid pressure valve V3 is ⁇ operably coupled to sample inlet pressure line 607 and configured to regulate pressure applied the sample inlet 115.
  • Sample inlet 115 of cartridge 110a is configured to receive the sample fluid, and is a metered chamber where sample volume is controlled by the inlet volume.
  • V3 is open, V2 is closed, and V1 is closed.
  • the device is ⁇ configured to apply pressure to sample inlet 115 through V3 moving sample fluid (e.g., whole blood) through separation filter 114 (e.g., plasma separation filter) into sample fluid metering chamber 116 that consists of a chamber or a tube.
  • This step can be timed and depend upon the capillary stop at the outlet of the sample fluid metering chamber to prevent flow of separated plasma into the tube location where it is combined with the diluent.
  • ⁇ sample fluid metering chamber filling can be monitored optically and controlled using optical feedback.
  • the device is configured to close V3.
  • V1 is then opened to apply pressure to diluent reservoir 603.
  • Diluent may be passed through diluent line 605b to an intersection of microfluidic passage 118 where it is combined with, e.g., separated plasma from separation filter 114. It is at this intersection that the two fluids are combined. Diluent volume is controlled ⁇ by flow meter 604. When a predetermined volume of diluent is dispensed, V1 is closed. V2 is then opened and a pressure is applied for a fixed period of time to combine a portion of the separated sample fluid (e.g., plasma) with the dispensed diluent. The device may be configured to repeat these steps until all of the plasma and the diluent are combined.
  • V1 When a predetermined volume of diluent is dispensed, V1 is closed. V2 is then opened and a pressure is applied for a fixed period of time to combine a portion of the separated sample fluid (e.g., plasma) with the dispensed diluent.
  • the device may be configured to
  • the device is configured to control volume of the sample fluid by the injection of air into the sample fluid metering chamber 116 such that a bubble is formed at the trailing edge of the sample fluid. This could also be monitored optically if more precision is needed.
  • the combined fluids are then subject to mixing in mixing chamber 117 configured to homogenize the two fluids, which may ⁇ then be distributed to detection chambers 111a-111n.
  • FIG.6B presents a schematic diagram of a sample fluid analysis device according to alternative embodiments of the invention.
  • FIG.6B includes the same elements as those described above with respect to FIG.6A, with the addition of secondary metering chamber pressure line 608 of manifold 600b gaseously connecting to secondary metering chamber 120 ⁇ of cartridge 110b (described above with respect to FIG.1B), and a secondary metering chamber pressure valve V4 operably coupled to secondary metering chamber pressure line 608 and configured to regulate pressure applied to secondary metering chamber 120.
  • Sample inlet 115 of cartridge 110b is configured to receive the sample fluid, and is a metered chamber where sample volume is controlled by the inlet volume. After the cartridge ⁇ 110b is placed in the device, V3 is open, V2 is closed, and V1 is closed.
  • the device is configured to apply pressure to sample inlet 115 through V3 into sample fluid metering chamber 116 comprised of a chamber or a tube. This can be timed and depend upon the capillary stop C at the outlet of the precision sample fluid metering chamber 116 to prevent flow of sample fluid into the location where it is combined with the diluent. Alternatively, sample fluid metering ⁇ chamber filling can be monitored optically and controlled using optical feedback.
  • V3 is closed and V1 is then opened to apply pressure to diluent reservoir 603. Diluent is passed through diluent line 605b to the intersection in microfluidic passage 118 where it is combined with the sample fluid. Diluent volume is controlled by flow meter 604.
  • V5 is closed, V3 is closed, V2 is closed, and V1 is closed, while V6 and V7 are open.
  • Pump 601 is then used to transfer calibration fluid needed as the electrochemical measurement reference sample from the calibration fluid reservoir 112 to ⁇ electrochemical sensors 113. Metering is performed by using a liquid sensing electrode at the end of electrochemical sensors 113. When the calibration reference measurement is complete, the remaining calibration fluid is then pumped into electrochemical overflow vent 121. Once the sample fluid is in position, the V7 valve is closed. This permits the fluidic controls to subsequently perform the sample preparation for the optical sensing assays. Upon initiation of ⁇ optical sensing analysis, V3 and V5 are open; V2, V1, V6, and V7 are closed.
  • sample fluid metering chamber 116 comprised of a chamber or a tube. This step can be timed and depend upon the capillary stop C at the outlet of the sample fluid metering chamber 116 to prevent flow of separated sample fluid into the location where it is combined with the ⁇ diluent. Alternatively, sample fluid metering chamber 116 filling can be monitored optically and controlled using optical feedback.
  • V3 is closed, and V1 is then opened to apply pressure to diluent reservoir 603. Diluent is passed through microfluidic passage 118 to the intersection where it is combined with the separated plasma.
  • Diluent volume is dispensed into a microfluidic passage 118 and is controlled by an flow meter 604.
  • V1 is closed.
  • V2 is then opened and a pressure is applied for a fixed period of time to combine a portion of the sample fluid with the dispensed diluent. These steps can be repeated until all of the sample fluid and diluent are combined.
  • Volume control of the sample fluid is controlled by the injection of air into the sample fluid metering chamber 116 such that a bubble is formed at the trailing edge of the sample fluid. This could also be monitored optically if ⁇ more precision is needed.
  • Sample fluid analysis devices of the invention additionally include interrogation systems.
  • the interrogation system may be any system configured to determine a property/characteristic ⁇ of a fluidic sample or an analyte present therein. In some cases, the interrogation system is configured to determine the property/characteristic of the fluidic sample or an analyte present therein via an optical protocol, an electrochemical protocol or both.
  • the interrogation system is a light interrogation system comprising a light source configured to irradiate a plurality of detection chambers of the cartridge, and an optical sensor configured to ⁇ collect emitted light from the plurality of detection chambers.
  • a light source configured to irradiate a plurality of detection chambers of the cartridge, and an optical sensor configured to ⁇ collect emitted light from the plurality of detection chambers.
  • Any convenient light source may be employed as the light source described herein.
  • the light source is a laser.
  • the laser may be any convenient laser, such as a continuous wave laser.
  • devices include a plurality of light sources. In some such cases, the ⁇ plurality of light sources are positioned in an array.
  • the spectra of the light source(s) may lie in any predetermined region of the electromagnetic spectrum detectable using photosensitive arrays, with or without specialized treatments to extend the effective ranges of wavelengths detectable by such arrays.
  • the predetermined wavelength or wavelength band is in the infrared spectrum.
  • the predetermined wavelength or ⁇ wavelength band is in the ultraviolet spectrum.
  • the predetermined wavelength or wavelength band is in the visible spectrum.
  • the light source is comprised of one or more light emitting diodes (LEDs).
  • the light source is comprised of an array of LEDs. The number of LEDs in the array may vary.
  • the light source may be comprised of one or more LED emitters ⁇ including but not limited to e.g., two or more LED emitters, three or more LED emitters, four or more LED emitters, one LED emitter, two LED emitters, three LED emitters, four LED emitters, etc.
  • the number of LEDs ranges from 4 to 20, such as 5 to 15, and including 6 to 8.
  • an illumination component containing four LED emitters may contain two ⁇ pairs of identical LEDs or one pair of LEDs of a first wavelength and a second pair of LEDs of a second wavelength.
  • any useful arrangement of the LED emitters may find use in the light source including but not limited to e.g., linear arrangement, staggered arrangement, arrayed (e.g., “checker-board”) arrangement, and the like.
  • Useful LED emitters of the subject disclosure will vary, e.g., based on the ⁇ particular assay to be performed by the device the optical, electrical or physical constraints of the device and the like.
  • LED emitters may include but are not limited to e.g., LED emitters with a peak minimum wavelength ( ⁇ ) in nanometers (nm) of between 350 and 750 nm, including but not limited to e.g., between 350 and 450, between 350 and 400, between 400 and 450, between 450 and 550, between 450 and 500, between 500 and 550, between 550 and 650, between 550 and 600, between 600 and 650, between 650 and 750, between 650 and 700, between 700 and 750, about 400 nm, about 580 nm, about 470 nm, about 628 nm, about 528 nm, about 674 nm, ⁇ and the like.
  • ⁇ peak minimum wavelength
  • light sources contain two LED emitters of different wavelengths where the distance between the different wavelengths will vary and may range from 5 nm to 300 nm or more including but not limited to e.g., at least 5 nm apart, at least 10 nm apart, at least 15 nm apart, at least 20 nm apart, at least 25 nm apart, at least 30 nm apart, at least 35 ⁇ nm apart, at least 40 nm apart, at least 45 nm apart, at least 50 nm apart, at least 55 nm apart, at least 60 nm apart, at least 65 nm apart, at least 70 nm apart, at least 75 nm apart, at least 80 nm apart, at least 85 nm apart, at least 90 nm apart, at least 95 nm apart, at least 100 nm apart, at least 105 nm apart, at least 110 nm apart, at least 115 nm apart, at least 120 nm apart, at least 125 nm apart
  • LED emitters of the subject disclosure may be capable of being toggled (i.e., capable being turned on and off, including turned on/off repeatedly).
  • the wiring circuitry of light sources having two or more LED emitters is configured or the programing controlling such light sources is configured such that only one LED emitter may be toggled on at a time.
  • the toggling of LED emitters of an optic block includes a time period where neither LED emitter of the optic block is toggled on.
  • such a time period where neither LED emitter of the optic block is toggled on is between toggling the toggling off of a first emitter and the toggling on of a second emitter.
  • one or more of the LEDs in the array is configured to emit light at a wavelength ranging from 400 nm to 410 nm. In some cases, one or more of the LEDs in the array is configured to emit light at a wavelength ranging from 460 nm to 470 nm. In some cases, one or more of the LEDs in the array is configured to emit light at a wavelength ranging from 600 nm to 610 nm.
  • one or more of the LEDs in the array is configured to emit light at a wavelength ranging from 850 nm to 860 nm.
  • the LED array comprises LEDs configured to emit light at 405 nm, 467 nm, 550 nm, 600 nm, and 850 nm. Each LED is turned on independently or simultaneously to illuminate the correct wavelength for ⁇ each assay.
  • the LEDs are edge-lit micro-light emitting diodes (LEDs).
  • the light source composed of a light guide plate and a plurality of multiple- wavelength LEDs configured to emit different wavelengths of light (e.g., such as the wavelengths discussed above).
  • the LEDs are edge-lit micro LEDs.
  • devices may include an edge-lit LED illuminator composed of a light guide plate ⁇ (LGP) and a plurality of multiple-wavelength LEDs.
  • LGP light guide plate
  • the LGP is engraved with reflective patterns such as dot or V-type trunking to reflect/refract the light beam towards the first pinhole plate.
  • LGPs may be comprised of any suitable material, including but not limited to, cyclo olefin polymer (CoP), cyclic olefin copolymer (CoC), poly(methyl methacrylate) (PMMA) and polystyrene (PS).
  • the light source is or comprises an arc lamp.
  • ⁇ Light sources according to certain embodiments may also include one or more optical adjustment components.
  • the optical adjustment component is located between the light source and the detection chambers, and may include any device that is capable of changing the spatial width of irradiation or some other characteristic of irradiation from the light source, such as for example, irradiation direction, wavelength, beam width, beam ⁇ intensity and focal spot.
  • Optical adjustment protocols may include any convenient device which adjusts one or more characteristics of the light source, including but not limited to lenses, mirrors, filters, fiber optics, wavelength separators, pinholes, slits, collimating protocols and combinations thereof.
  • devices of interest include one or more focusing lenses.
  • the focusing lens in one example, may be a de-magnifying lens.
  • devices of interest include fiber optics.
  • devices include a tapered mixing rod configured to integrate incident light from the light source (e.g., LED array) and generate a uniform output (total reflection) in a smaller area.
  • devices of the invention include an optional diffuser configured to improve light uniformity.
  • devices include a folding mirror.
  • the optical sensor of the light interrogation system may also vary.
  • Sensors of interest may include, but are not limited to, optical sensors or detectors, such as active-pixel sensors (APSs), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes ⁇ (PMTs), phototransistors, quantum dot photoconductors or photodiodes and combinations thereof, among other detectors.
  • APSs active-pixel sensors
  • CCDs charge-coupled devices
  • ICCDs intensified charge-coupled devices
  • PMTs photomultiplier tubes ⁇
  • phototransistors quantum dot photoconductors or photodiodes and combinations thereof, among other detectors.
  • the collected light is measured with a charge-coupled device (CCD), semiconductor charge-coupled devices (CCD), active pixel sensors (APS), complementary metal-oxide semiconductor (CMOS) image sensors or N-type metal-oxide semiconductor (NMOS) image sensors.
  • the optical sensor is a multi-dimensional array of pixels that form part of a high-resolution photosensitive array.
  • high-resolution refers to a ⁇ resolution that equals or exceeds the resolution of standard lens-based optical microscopes.
  • the resolution of a standard lens-based optical microscopes is defined as the shortest distance between two points on a specimen that can still be distinguished by the observer or camera system as separate entities.
  • Pixels per inch (PPI) or pixels per centimeter (PPCM) are measurements of the pixel density of the optical sensor.
  • the resolution of the optical sensor is ⁇ the count of pixels that contribute to the final image and is typically measured in megapixels (meaning millions of pixels).
  • a photosensitive array comprising 1280 x 720 pixels has 921,600 pixels or less than 1 mega pixel resolution
  • a photosensitive array comprising 1920 x 1080 pixels has 2,073,600 pixels or about 2.1 mega pixel resolution.
  • Micro-fabrication techniques e.g., photolithography and plasma deposition
  • CCDs have advantages for contact optical microscopy applications, including the ability to detect light over an exposed surface.
  • full-frame architecture may be used to maximize the proportion of the chip available for imaging, but requires an external shutter to prevent image smearing during readout; whereas frame-transfer architecture avoids ⁇ image smearing, but in the process requires a masked, non-photosensitive area of the parallel register of about the same size as the photosensitive area of the parallel register, with the result that the imaging integrated circuit has about half the photosensitive area of a full-frame architecture.
  • CMOS devices have alternative advantages for these applications, including less expensive fabrication, signal processing by electronic elements embedded in individual pixels, ⁇ and the ability to read out independently-addressed pixel values individually without sequential transfer.
  • thinned back-side illuminated arrays are used; though previously requiring expensive and complex fabrication methods, these may be fabricated cheaply using bonded wafer processes such as those that use silicon-on-insulator substrates with a buried oxide layer as an etch-stop to yield a uniformly optimally thinned light-absorbing ⁇ back layer (see as an example, U.S. Patent No.7,425,460, which is incorporated herein by reference).
  • the multi-dimensional array of pixels may be light sensors or photodetectors formed of ⁇ semiconductor materials used in very-large-scale or larger integrated circuits.
  • the defining property of a semiconductor material is that it can be doped with impurities that alter its electronic properties in a controllable way; in some embodiments, the array is formed substantially of a crystalline inorganic solid such as silicon; and in other embodiments the array is formed substantially of a compound semiconductor comprised of elements of at least two ⁇ different species.
  • the compound semiconductor may be comprised of elements in groups 13- 15 (old groups III-V), for example of elements from group 13 (old group III, boron, aluminum, gallium, indium) and from group 15 (old group V, nitrogen, phosphorus, arsenic, antimony, bismuth).
  • the range of possible formulae for the compound semiconductor may include binary (two elements, e.g., gallium (III) arsenide (GaAs)), ternary (three elements, e.g., indium gallium ⁇ arsenide (InGaAs)), and quaternary (four elements, e.g., aluminum gallium indium phosphide (AllnGaP)) alloys.
  • the array of pixels are light sensors or photodetectors such as PD(s), e.g., a silicon photo PIN diode(s) having an undoped intrinsic semiconductor region sandwiched between a p-type semiconductor region and an n-type semiconductor region.
  • the spectral response of the multi-dimensional array of pixels may be in the range of 300 nm to 1000 nm. This provides the capability to cover a wide spectrum of LED wavelengths.
  • the signals from the optical sensor provide information for each pixel of the image, which information includes, or can be derived to include, intensity, wavelength, and optical ⁇ density. Intensity values may be assigned an arbitrary scale of, for example, 0 units to 4095 units ("IVlJs").
  • Optical density is a measure of the amount of light absorbed relative to the amount of light transmitted through a medium; e.g., the higher the "OD” value, the greater the amount of light absorbed during transmission.
  • OD may be quantitatively described in optical density units ("OD") or fractions thereof; e.g., a MilliOD is a 1/1000 th of an OD.
  • One "OD” unit ⁇ decreases light intensity by 90%.
  • “OD” or “MilliOD” as a quantitative value can be used for images acquired or derived by transmission light, for example, the transmission blue light.
  • the information from the optical sensor is separated into multiple channels, for example, three channels, which provides particular utility for determining a four part LDC.
  • a first ⁇ of the three channels may be directed toward information relating to light emitted from the sample at a first wavelength (e.g., 540 nm, which appears green).
  • a second channel may be directed toward information relating to light emitted from the sample at a second wavelength (e.g., 660 nm, which appears red).
  • a third channel may be directed toward information relating to light passing through the sample at a third wavelength (e.g., 413 nm, which is used to determine blue optical density "OD").
  • Additional channels can be implemented to gather information at different wavelengths and/or transmission values. That information, in turn, can be used to evaluate additional constituents within the sample and/or to increase the accuracy of the analysis.
  • a fourth and a fifth channel can be added.
  • the fourth channel can be directed toward information relating to light ⁇ passing through the sample at a fourth wavelength (e.g., 540 nm), which is used to determine green OD
  • the fifth channel can be directed toward information relating to light passing through the sample at a fifth wavelength (e.g., 660 nm), which is used to determine red OD.
  • a fourth wavelength e.g., 540 nm
  • the fifth channel can be directed toward information relating to light passing through the sample at a fifth wavelength (e.g., 660 nm), which is used to determine red OD.
  • FIG.7A depicts components of a light interrogation system according to certain ⁇ embodiments of the invention.
  • a cartridge with detection chambers 701 having inlets 705 and outlets 706 is included in a system comprising pinhole plates 703 and 702 (described in greater detail below).
  • an optical sensor 704 which in the embodiment of FIG.7A is a CMOS image sensor which covers the top of the detection chamber array behind pinhole plate 703 to measure the transmitted light power of all the ⁇ detection chambers 701 simultaneously. Collected beams project hologram images on the CMOS image sensor.
  • the pinhole images 708 form “super photodiodes” to measure the light intensity of all cuvettes.
  • the pixel summation of the pinhole image is the transmittance light power of each cuvette and is used in absorbance calculation.
  • the optical sensor is an optical spectrometer configured to measure ⁇ properties over a portion or portions of the electromagnetic spectrum.
  • the optical spectrometer is a miniaturized optical spectrometer. Such miniaturized optical spectrometers are described in, e.g., U.S. Patent Application Publication No.2017/0010154.
  • devices include one or more optical filters configured to permit the collection of certain wavelengths of light. Various optical filters may be employed depending on ⁇ the requirements for analyzing a particular analyte. In some cases, one or more bandpass filters are included.
  • one or more low pass filters are included.
  • one or more high pass filters are included.
  • the one or more optical filters are comprised of some combination of bandpass filters, low pass filters and high pass filters.
  • the one or more spectral filters are configured to remove the ⁇ sample's and consumable plastic's auto-fluorescence.
  • an illumination filter as described herein may be characterized in having a particular combination of CWL and FWHM, including e.g., combinations of the CWL and the FWHM described above.
  • an illumination filter of the subject disclosure may be characterized as having a 409 nm CWL and a 65 nm FWHM, 583 ⁇ nm CWL and a 22 nm FWHM, 475 nm CWL and a 36 nm FWHM, 638 nm CWL and a 24 nm FWHM, 535 nm CWL and a 18 nm FWHM, 690 nm CWL and a 25 nm FWHM, and the like.
  • the devices and systems described herein may include an optical component external to the housing where such external components are useful in, e.g., making calibration measurements and/or measurements used in determining proper functioning of the ⁇ system or device.
  • an external optical component may include a dark target, wherein a “dark target” is a darkly colored (i.e., black) component of the system or device from which a “dark measurement” may be made. Such dark measurements may be employed for calibration according to the methods as described herein. Any convenient and appropriate optically dark element may be employed as a dark target including but not limited to e.g., black ⁇ polycarbonate, black plastic, and the like.
  • the dark target may contain a specific angle or be mounted at a specific angle to further enhance a dark measurement by preventing stray light, including e.g., reflected light, from returning to the optics block during a dark target measurement.
  • the device of the present disclosure includes a microspot array ⁇ for detecting the analyte selected from the group consisting of: drugs of abuse, classes of cytokines, and inflammatory markers, such as hCG, K, Na, Cl, Ca, Mg, pH, pO 2 , pCO 2 , glucose, urea, creatinine, lactate, CKMB, TnI, TnT, BNP, NTproBNP, proBNP, TSH, D-dimer, PSA, PTH, NGAL, galectin-3, AST, ALT, albumin, phosphate, and ALP.
  • drugs of abuse classes of cytokines
  • inflammatory markers such as hCG, K, Na, Cl, Ca, Mg, pH, p
  • the microarray comprises: at least one electrochemical sensor; and an optically readable microspot array comprising an entry port for receiving said test sample into a holding chamber; a first conduit comprising said at least one electrochemical sensor; and a second conduit comprising ⁇ said optically readable microspot array, optionally comprising a qualitative or semi-quantitative test for said analyte.
  • the microspot array optionally comprises at least one spot that is a quantum dot or a nanoparticle having properties of fluorescence or phosphorescence singly or in combination with magnetic or paramagnetic properties.
  • the microspot array preferably comprises a plurality of spots in a plurality of columns and a plurality of rows, and may ⁇ comprise a plurality of spots that are substantially circular and have a diameter in a range of about 10 ⁇ m to about 500 ⁇ m.
  • the microspot array preferably comprises at least one immobilized antibody.
  • the at least one immobilized antibody is preferably configured to bind with said analyte selected from the group consisting of: D-dimer, HSA, inflammatory and traumatic disease markers such as C-reactive protein, NGAL, D-dimer, TNFa, TGFb, IL-1 and ⁇ IL6; metabolic disease indicators such as HSA, liver enzymes ALP, ALT, AST, bilirubin, vitamine-D, vitamine-B12, and folic acid; protein hormones such as TSH, hCG, LH, FSH, prolactin, insulin, and somatostatin; cardiac disease markers such as cardiac troponins, CK- MB, BNP and Galectin-3; tumor markers such as PSA, CEA, AFP, CA125, VEGF, BrCa1, and HER2/neu; hormones such as T3, T4, DHEA, estrogen, and progesterone; infectious diseases ⁇ organisms and/or plasma antibodies against infectious disease organisms such as Hepatitis B,
  • light interrogation systems of the invention include one or more pinhole plates.
  • Such light interrogation systems are described in U.S. Provisional Patent Application No.63/563,780 (Atty. Dkt. No. ADDV-153PRV), filed on March 11, 2024, the ⁇ disclosure of which is incorporated by reference herein in its entirety.
  • light interrogation systems include a first pinhole plate comprising one or more pinholes.
  • the first pinhole plate is comprised of a planar surface having pinholes located therein.
  • the first pinhole plate may be constructed from any suitable material.
  • plates include one or more metals including, for example, aluminum, titanium, brass, ⁇ iron, lead, nickel, steel (e.g., ⁇ stainless steel), copper, tin as well as combinations and alloys thereof.
  • the first pinhole plate includes a polymeric material, such as a plastic material.
  • the first pinhole plate includes one or more rigid plastic materials such as, for example, polycarbonates, polyvinyl chloride (PVC), polyurethanes, polyethers, polyamides, polyimides, among other polymeric plastic materials.
  • the pinhole plate is comprised of polyoxymethylene. The number of pinholes in the first pinhole plate may vary.
  • the first pinhole plate includes a single pinhole.
  • the first pinhole plate includes a plurality of pinholes.
  • the number of pinholes ranges from ⁇ 2 to 1000, such as 2 to 100, such as 5 to 50, such as 10 to 25 and including 14 to 20.
  • the pinholes of the plurality may be arranged in an array.
  • array of pinholes it is meant a particular arrangement of pinholes that is organized according to a certain pattern or principle.
  • pinholes of the array are arranged in rows and columns.
  • pinholes of the array are ⁇ arranged in a staggered pattern.
  • pinholes of the array are arranged in a concentric pattern.
  • the dimensions of the pinholes in the first pinhole plate may vary.
  • the pinholes range in diameter from 0.1 mm to 5 mm, such as 0.2 mm to 4 mm, such as 0.3 mm to 3 mm, such as 0.4 mm to 2 mm, and including 0.5 mm to 1 mm.
  • pinholes have ⁇ a diameter of 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm or 1.0 mm.
  • adjacent pinholes in the plurality may be separated by any suitable distance, where the distance is measured between geometric centers of the pinholes.
  • adjacent pinholes are separated a distance ranging from 0.5 mm to 10 mm, such as 1 mm to 7 mm, such as 1.5 mm to 5 mm, and including 2 mm to 2.5 mm.
  • adjacent pinholes are separated by a distance of 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm or 2.5 mm.
  • the height/thickness of the first pinhole plate may likewise vary.
  • the pinhole plate has a height ranging from 1 mm to 50 mm, such as 2 mm to 40 mm, such as 3 mm to 30 mm, such as 4 mm to 25 mm and including 20 mm to 25 mm.
  • the first pinhole plate has a thickness of 20 mm, 21 mm, 22 mm, 23 mm or 24 mm.
  • the ⁇ pinhole diameter and the plate thickness is sufficient to narrow collimated light of chief ray angle 1°-3° and minimize the stray light and cross-talk signal to the neighbor detection chambers.
  • Pinholes of the subject first pinhole plates are each configured for optical alignment with a detection chamber of a plurality of detection chambers (e.g., microcuvettes).
  • ⁇ the pinholes are sized and positioned within the plate so that detection chambers employed in conjunction with the subject systems (e.g., in conjunction with a removable cartridge that is inserted into the system, as described in further detail below) are in optical alignment with the pinholes.
  • optical alignment it is meant that one or more pinholes and detection chambers, when such chambers are present in the system, share an optical axis such that passes through ⁇ the pinholes to (or from, as appropriate) their corresponding detection chambers.
  • pinholes may be sized relative to the detection chambers in such a manner to reduce the negative effects of stray light and associated optical density (OD) variance. Suitable sizes may include but are not limited to those presented above.
  • pinholes may be matched to a particular array of detection chambers (e.g., in a cartridge) such that the first pinhole plate and detection chambers are arranged according to the same pattern or principle.
  • both the detection chambers and pinholes may be arranged in rows and columns, in a staggered pattern, or concentrically.
  • Light interrogation systems of the invention also include a second pinhole plate.
  • Second pinhole plates of interest include one or more pinholes each optically aligned with a pinhole of the first pinhole plate and configured for optical alignment with a detection chamber of the plurality.
  • the second pinhole plate is comprised of a planar surface having pinholes located therein.
  • the second pinhole plate may be constructed from any suitable material.
  • plates include one or more metals including, for example, aluminum, titanium, brass, ⁇ iron, lead, nickel, steel (e.g., stainless steel), copper, tin as well as combinations and alloys thereof.
  • the second pinhole plate includes a polymeric material, such as a plastic material.
  • the second pinhole plate includes one or more rigid plastic materials such as, for example, polycarbonates, polyvinyl chloride (PVC), polyurethanes, ⁇ polyethers, polyamides, polyimides, among other polymeric plastic materials.
  • the pinhole plate is comprised of polyoxymethylene. The number of pinholes in the second pinhole plate may vary.
  • the second pinhole plate includes a single pinhole.
  • the second pinhole plate includes a plurality of pinholes.
  • the number of pinholes ranges from 2 to 1000, such as 2 to 100, such as 2 to 50, such as 10 to 25 ⁇ and including 14 to 20.
  • the number of pinholes in both the first and second pinhole plates ranges from 2 to 50.
  • the pinholes of the plurality may be arranged in an array according to the same principle or pattern as the first pinhole plate. In certain cases, pinholes of the array are arranged in rows and columns. In other embodiments, pinholes of the array are arranged in a ⁇ staggered pattern.
  • pinholes of the array are arranged in a concentric pattern.
  • the dimensions of the pinholes in the second pinhole plate may vary.
  • the pinholes range in diameter from 0.1 mm to 5 mm, such as 0.2 mm to 4 mm, such as 0.3 mm to 3 mm, such as 0.4 mm to 2 mm, and including 0.5 mm to 1 mm.
  • pinholes ⁇ have a diameter of 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm or 1.0 mm.
  • adjacent pinholes in the plurality may be separated by any suitable distance, where the distance is measured between geometric centers of the pinholes.
  • adjacent pinholes are separated a distance ranging from 0.5 mm to 10 mm, such as 1 mm to 7 mm, such as 1.5 mm to 5 mm, and including 2 mm ⁇ to 2.5 mm.
  • adjacent pinholes are separated by a distance of 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm or 2.5 mm.
  • adjacent pinholes the first and second pinhole plates are separated by a distance ranging from 2 mm to 2.5 mm.
  • the height/thickness of the second pinhole plate may likewise vary.
  • the pinhole plate has a height ranging from 1 mm to 50 mm, such as 2 mm to 40 mm, such as 3 mm to 30 mm, such as 4 mm to 25 mm and including 20 mm to 25 mm.
  • the second pinhole plate has a thickness of 20 mm, 21 mm, 22 mm, 23 mm or 24 mm.
  • second pinhole plates of the invention include a light trap pocket configured to prevent stray emission light from the ⁇ light source from being collected by the optical sensor.
  • the light trap pocked may be comprised of a void in the interior of the second pinhole plate which is configured to absorb stray rays of light.
  • the pinhole diameter and the plate thickness is sufficient to narrow collimated light of chief ray angle 1°-3° and minimize the stray light and cross-talk signal to the neighbor detection chambers.
  • the dimensions of the first and second ⁇ pinhole plates may be the same or different.
  • pinholes of the first pinhole plate have a larger diameter than pinholes of the second pinhole plate.
  • pinholes of the second pinhole plate have a larger diameter than pinholes of the first pinhole plate.
  • the pinhole image forms “super photodiodes” to measure the light intensity of all cuvettes.
  • the pixel summation of the pinhole image is the transmittance light power of each ⁇ cuvette and is used in absorbance calculation.
  • Pinholes of the subject second pinhole plates are each configured for optical alignment with a detection chamber of a plurality of detection chambers (e.g., microcuvettes).
  • the pinholes are sized and positioned so that detection chambers employed in conjunction with the subject systems (e.g., in conjunction with a removable cartridge that is ⁇ inserted into the system, as described in further detail below) are in optical alignment with the pinholes.
  • pinholes may be sized relative to the detection chambers in such a manner to reduce the negative effects of stray light and associated optical density (OD) variance. Suitable sizes may include but are not limited to those presented above.
  • the first and second pinhole plates are components of a cartridge that are received by a ⁇ system of the invention rather than the system per se.
  • FIG.5A-5B depict a light interrogation system having pinhole plates according to certain embodiments of the invention.
  • FIG.5A depicts a top view of a light interrogation system showing a second pinhole plate 503 having pinholes through which detection chambers 501 may be irradiated.
  • FIG.5B presents a profile view of the same light interrogation system.
  • the system includes first pinhole plate 502 positioned adjacent to the first light-accessible windows of the detection chambers 501 near inlets 505, and second pinhole plate 503 positioned adjacent to the second light-accessible windows of the detection chambers 501 near outlets 506. Also shown is optical sensor 504.
  • FIG.7B depicts components of a light interrogation system according to certain ⁇ embodiments of the invention.
  • FIG.7B includes the same elements as those described above with respect to FIG.7A.
  • micro-LED array 711, tapered optical mixing rod 712, diffuser 713, folding mirror 714, and condenser lens 715 are included.
  • FIG.7C depicts components of a light interrogation system according to certain embodiments of the invention.
  • FIG.7C includes the same elements as those described above with respect to FIG.7A, with the addition of light guide plate (LGP) 716 and edge-lit ⁇ micro LEDs 717.
  • FIG.7D depicts micro-spectrometer 718 configured to receive light from the detection chambers 701.
  • FIG.7D also represents an embodiment of the detection chambers where inlet 705 is located at the top of each detection chamber such that sample fluid fills from the top town.
  • Outlet 706 is likewise located on the top of the detection chambers.
  • a processor is operably connected to an optical sensor, a light source, ⁇ and a memory having instructions stored thereon which, when executed by the processor, cause the processor to calculate an absorbance from the cartridge.
  • cartridges of the invention include a plurality of detection chambers. Accordingly, the processor may be configured to calculate an absorbance for each detection chamber of the plurality.
  • FIG.7E depicts a pinhole image 750. Region 751 is for intensity ⁇ measurement.
  • calculating the absorbance includes calculating an average intensity of incident light from the light source.
  • the device may be configured to, for example, measure the average intensity of the first pinhole plate for each cuvette before a cartridge is loaded into the device.
  • the processor may additionally be configured to calculate an average intensity of the ⁇ emitted light from each detection chamber of the plurality. Such can include measuring light intensity again after a cartridge comprising sample fluid has been inserted into the device.
  • the processor may be additionally configured to deactivate the light source and calculate a dark image average intensity of the emitted light from each detection chamber of the plurality.
  • the processor may calculate the absorbance for each detection chamber of the ⁇ plurality based on the average intensity of incident light from the light source, the average intensity of the emitted light from each detection chamber of the plurality, and the dark image average intensity of the emitted light from each detection chamber of the plurality.
  • the processor is configured to calculate the absorbance for each detection chamber as follows: ⁇ ⁇ ⁇ ⁇ where ⁇ is the absorbance, ⁇ ⁇ is the average intensity of incident light from the light source, ⁇ ⁇ is the average intensity of the emitted light from each detection chamber of the ⁇ plurality, and ⁇ ⁇ is the dark image average intensity of the emitted light from each detection chamber of the plurality.
  • the processor is configured to repeat the calculation of the absorbance for each detection chamber until an absorbance change is measured.
  • the absorbance measured above is considered starting absorbance at a first time point.
  • the processor may repeat the absorbance calculation until a significant absorbance change is ⁇ measured at a later time point.
  • the processor is configured to calculate an analyte concentration based on a rate of change of the absorbance. For example, in certain versions, the processor is configured to calculate the rate of change as follows: ⁇ where ⁇ is the rate of change, " ⁇ is the absorbance calculated at a time point # $ when the significant absorbance change is measured, and " ⁇ is a first absorbance calculated at a time point # % .
  • the absorbance of the single or multiple wavelengths is read directly ⁇ from the sensor (e.g., spectrometer) for absorbance calculation.
  • An alternative is to capture the entire absorbance spectrum, e.g., 340nm to 850 nm. In some cases, this provides more data than a single wavelength to calculate the concentration of the product of the reaction.
  • the devices and methods of present disclosure may employ analyses using imaging or signal analysis, algorithms for assisting image or signal analysis, and cutoffs.
  • the analyzing ⁇ performed e.g., to extract one or more density distribution feature values, fluorescent intensity, will vary and may include where the density distribution feature is or is not based on a color feature of the image. As such, density distribution feature values may be color feature values or non-color feature values.
  • Color feature values will generally depend on image information extracted from one or more color channels of the image which is influenced by the color ⁇ staining of the specimen.
  • Non-color feature values may be derived from image information extracted from the overall image or a portion thereof regardless of color mode (e.g., color, grayscale, binary, etc.) of the image, or one or more color channels, but is generally not influenced by any color staining of the specimen.
  • Density distribution feature values extracted from density distribution features will be ⁇ indicative, either alone or in combination, of the density distribution of cells of the specimen and/or whether the image or region (ROI) analyzed contains a morphology assessment area or sample detection region.
  • one or more morphology assessment area(s) of a specimen may be identified based on one or more extracted density distribution feature values.
  • density distribution features analyzed in the subject methods will include those ⁇ density distribution features that may be automatically extracted from digital images and analyzed to identify one or more morphology assessment area(s) of a specimen that can be used in an assessment performed by an automated digital cell morphology analyzer.
  • useful numbers of individual density distribution feature values of such combinations will vary and may range from 2 to 20 or more, including but not limited to 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 2 to 20, 3 to 20, 4 to 20, 5 to 20, ⁇ 6 to 20, 7 to 20, 8 to 20, 2 to 15, 3 to 15, 4 to 15, 5 to 15, 6 to 15, 7 to 15, 8 to 15, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, etc.
  • Combinations of multiple density distribution feature values may include those derived exclusively from non-color features, exclusively from color features, or a combination of color and non-color features.
  • Useful density distribution features of a specimen will vary and may include but are not limited to e.g., the density of cells within an image or an ROI, the variation in the density of cells within an image or ROI, the variation in the density of cells between images or ROIs, the size of cells within an image or an ROI, the variation in the size of cells within an image or ROI, the variation in the size of cells between images or ROIs, the shape of cells within an image or an ⁇ ROI, the variation in the shape of cells within an image or ROI, the variation in the shape of cells between images or ROIs, the pallor of cells within an image or an ROI, the variation in the pallor of cells within an image or ROI, the variation in the pallor of cells between images or ROIs, the color of cells within an image or an ROI, the variation in the color of cells within an image or an ROI, the variation in the color of cells between images or ROIs
  • useful density distribution features include but are not limited to e.g., a cell count, a coefficient of variation (CV) index for a cell count, a cell size, a CV index for a cell size, an index defining a cell shape, a CV for an index for a cell shape, an index defining central pallor of the cell, an index defining cell color, a count of overlapping cells, and combinations thereof.
  • Extracting density distribution feature values from an image or a ROI, according to the subject methods may include one more image processing steps which may employ one or more image processing algorithms.
  • Useful image processing steps may include but are not limited to e.g., splitting channels of a multichannel image, generating one or more image masks (e.g., a foreground mask, a color mask, a threshold mask, a combined mask (e.g., a combined ⁇ color and foreground mask), etc.), space filing or hole closing (e.g., hole closing in a generated mask), noise filtering, segmentation (e.g., cell segmentation), and the like.
  • Image processing steps generally include the processing of digital images, which may vary and may be in binary (e.g., black and white), grayscale or color formats. Images of various formats may further be converted between formats, as desired, by suitable image processing ⁇ algorithms.
  • a color image may be “split” into individual color channels to produce individual grayscale images for each color channel.
  • a red, green and blue image (RGB) image may be split into individual red, green and blue channels to produce a grayscale image of the red channel, a grayscale image of the green channel and a grayscale image of the blue channel.
  • Color images may be converted between color spaces and split into any convenient and appropriate color channels of a particular color space including but not limited to e.g., RGB color space, CMYK color space, HSV color space, CIE color space, Lab color space, CIELUV color space, YCbCr color space, and the like.
  • digital color images may be processed as color images (i.e., as multichannel images) or may be converted or split into two or more individual color channels prior to processing.
  • any number of the resulting split images may be used in further processing steps including but not limited to all the split images (i.e., all the individual channels of the image) or only one of the split images (i.e., ⁇ only one of the individual channels of the image) or one or more, including but not limited to two or more, three or more, two, three, etc. of the split images (i.e., the individual channels of the image).
  • image segmentation may make use of one or more of threshold based segmentation, edge based segmentation and region based segmentation.
  • Specific image segmentation methods include but are not limited to thresholding methods, clustering ⁇ methods, compression-based methods, histogram-based methods, edge detection methods, dual clustering methods, region-growing methods, partial differential equation-based methods (e.g., parametric methods, level set methods, fast marching methods, etc.), variational methods, graph partitioning methods (e.g., Markov Random Fields methods), watershed transformation methods, model based segmentation methods, multi-scale segmentation methods, semi-automatic segmentation methods, trainable segmentation methods, and the like.
  • Other digital image processing image transformations that may find use in the described methods include but are not limited to e.g., point processing transformations (e.g., negative ⁇ transform, log transform, inverse log transform, nth root transform, nth power transform, gamma correction, contrast transforms (e.g., contrast stretching), window center correction, histogram equalization, etc.), filtering (i.e., neighbor) transformations (e.g., mean filters, Gaussian filters, median filters, image gradient filters, Laplacian filters, normalized cross correlation (NCC) filters, etc.), and the like.
  • point processing transformations e.g., negative ⁇ transform, log transform, inverse log transform, nth root transform, nth power transform, gamma correction, contrast transforms (e.g., contrast stretching), window center correction, histogram equalization, etc.
  • filtering i.e., neighbor transformations
  • mean filters e.g., Gaussian filters, median filters, image gradient filters, Laplacia
  • the device receives power, i.e., electrical power that is distributed throughout the device, i.e., via wired connections.
  • the device is configured to receive power via an external source, such as, for example, a wall outlet or the like, via an electrical cord and plug.
  • the device comprises a power supply unit configured to modulate electrical power received from an external source into a configuration capable of being utilized by the components of the device, e.g., one or more rectifier circuits, transformers or the like.
  • the device comprises a battery unit ⁇ such that the device does not need to be tethered to an external power source, e.g., a wall outlet.
  • the battery unit may comprise a battery that is a onetime use battery or a rechargeable battery.
  • the battery may be recharged using any convenient protocol, including, but not limited to, wireless charging protocols such as inductive charging.
  • the device may have a battery life ranging from 0.1 hours to ⁇ 120 days, from 14-30 days, from eight hours to 30 days, from eight hours to 12 days, from 12 hours to 24 hours, from 0.5 to ten hours.
  • Some embodiments are configured to receive power from both an external source as well as a battery unit.
  • one or more cartridges may comprise a dedicated power source; however, more typically cartridges receive power via the sample fluid analysis device, e.g., via a wired electrical connection to the sample ⁇ fluid analysis device.
  • embodiments of the invention include one or more processors or controllers and associated memories operably coupled thereto. Processors of the invention may be used to carry out various functions of the device, e.g., as described in greater detail above.
  • memories operably coupled to the processor may comprise instructions ⁇ stored thereon, which when executed by the one or more processors or controllers, cause the one or more processors or controllers of the sample fluid analysis device to control one or more aspects of the sample analysis performed by the device.
  • the one or more memories are located within the device. In other cases, one or more memories are present on or within the cartridge. In such cases, the instructions on these memories may be executed by the processor when the cartridge is received within the housing of the device.
  • Control units/processors of embodiments of the sample fluid analysis device (and/or one or more cartridges) may be configured to, for example, control internal timing, perform various ⁇ algorithms, result calculations and to operate the hardware components, e.g., mechanical components, of the device, including, e.g., controlling interfacing between the housing and one or more cartridges removably coupled to the housing. Any convenient processor and memory may be used in embodiments of the subject systems, including embodiments of sample fluid analysis device or cartridges.
  • the processor may comprise a general purpose processor or a controller or microcontroller or other processor configured to control aspects of the system, or combinations thereof.
  • the processor and memory are operably connected to each other. Such operable connection may take any convenient form such that instructions and data may be ⁇ obtained by the processor by any convenient input technique, such as via a wired or wireless network connection, Bluetooth® connections, shared memory, a bus or any other functionally similar communication protocol.
  • systems according to some embodiments may include a display and operator input device. Operator input devices may, for example, be a keyboard, mouse, a ⁇ touchscreen, a keypad or the like.
  • embodiments of the sample fluid analysis device include, and in some cases, embodiments of a cartridge may also include, a processing module comprising one or more processors, which have access to one or more memories having instructions stored thereon for controlling aspects of the system, i.e., the sample fluid analysis device and cartridge(s), to perform sample analysis.
  • the processing ⁇ module may include an operating system, a graphical user interface (GUI) controller, a system memory, memory storage devices, input-output controllers, cache memory, a data backup unit and many other aspects.
  • GUI graphical user interface
  • Processors may be commercially available processors or maybe one or more other processors that are or will become available.
  • the processor executes the operating system and the operating system interfaces with firmware and hardware in a well- ⁇ known manner, and facilitates the processor in coordinating and executing the functions of various computer programs that may be written in a variety of programming languages, such as Java, Perl, C++, other high-level or low-level languages, as well as combinations thereof, as is known in the art.
  • the operating system typically in cooperation with the processor, coordinates and executes functions of the other components of the processing module.
  • the operating ⁇ system also provides scheduling, input-output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques.
  • the processor may be any suitable analog or digital system.
  • the processor includes analog electronics which provide feedback control, such as for example negative feedback control.
  • the memory of the processing module may be any of a variety of known or future memory storage devices. Examples include any commonly available random-access memory ⁇ (RAM), magnetic medium such as a resident hard disk or tape, an optical medium such as a read and write compact disc, flash memory devices, or other memory storage device.
  • RAM random-access memory
  • the memory storage device may be any of a variety of known or future devices, including a compact disc drive, a tape drive, a removable hard disc drive, or a diskette drive.
  • Such types of memory storage devices typically read from, and/or write to, a program storage medium such ⁇ as, respectively, a compact disc, magnetic tape, removable hard disc, or floppy diskette. Any of these program storage media, or others now in use or that may later be developed, may be considered a computer program product.
  • program storage media typically store a computer software program and/or data.
  • Computer software programs also called computer control logic
  • typically are stored in system memory and/or the program ⁇ storage device used in conjunction with the memory storage device.
  • a computer program product is described having a computer usable medium having control logic (computer software program, including program code) stored therein.
  • the control logic when executed by a processor, causes the processor to perform functions described herein, including, for example, controlling aspects of a sample fluid ⁇ dilution.
  • some functions are implemented primarily in hardware using, for example, a hardware state machine. Implementation of the hardware state machine so as to perform the functions described herein will be apparent to those skilled in the relevant arts. Further details regarding computer-controlled systems are provided below.
  • Devices may further include, for example, a communication connector unit (e.g., a ⁇ universal serial bus (USB) connector and associated circuitry) to communicate any relevant data, e.g., results of a specimen analysis, to a remote device, such as a personal computer, laptop, PDA, cellular phone, smartphone, set-top box, etc.
  • a communication connector unit e.g., a ⁇ universal serial bus (USB) connector and associated circuitry
  • USB universal serial bus
  • the communication connector may be of any of the following ⁇ technologies, or family of technologies (but not limited thereto): USB, FireWire, SPI, SDIO, RS- 232 port, or any other suitable electrical connector to allow data communication between the sample fluid analysis device and a remote device.
  • the communication connector unit provides the capability to communicate with a remote device having an appropriate interface to operatively couple with the communication connector.
  • the communication ⁇ connector is configured to communicate with a smartphone, such as an iPhone or Samsung Galaxy or the like.
  • more than one communication connector unit may be implemented on the system, e.g., multiple communication units on the sample fluid analysis device and/or one or more cartridges.
  • the term “communication connector” is used in this disclosure to represent any variety of connection interfaces, e.g., male or female connection interfaces. Using USB as an example, the communication connector may be any of the variety of USB plugs or USB receptacles/ports.
  • USB receptacles are typically located on computer ⁇ and other devices
  • a corresponding USB plug used as a communication connector will enable the sample fluid analysis device, cartridge, sample collection device or other aspect of a system of the present disclosure, as applicable, to be plugged directly into the USB receptacle, avoiding the use of cables.
  • the appropriate USB receptacle may be used on an aspect of the system to enable communication using a USB cable (similar to many other ⁇ devices such as digital cameras, smartphones, smartwatches, etc.).
  • the communication connector unit may in some instances implement a wireless technology, in which case the connection interfaces would be corresponding transmitters, receivers, and/or transceivers.
  • Various functional features may be performed using the communication connector unit.
  • the communication connector may be used to transfer data from the system to a remote device.
  • a remote device may store the data and/or further process the data and/or combine the data with other additional information.
  • the data may include more than just analyte measurements and may also include such things as user settings/preferences, logged data, rate of change of analyte level, and/or the exceeding of a threshold analyte level, etc.
  • the sample fluid analysis device may be configured to store and/or further process and/or combine such data with other additional information.
  • a remote device may also communicate data, e.g., raw data or any additional data (e.g., further processed data), via a separate communication channel (wired or wirelessly) to a second remote device, e.g., at a physician’s office, hospital, or third-party site, depending on ⁇ the application environment of the system.
  • the second remote device may be, for example, a personal computer, laptop, PDA, smartphone, set-top box, etc.
  • data may be transferred from the sample fluid analysis device to a user’s personal computer, stored therein, and then transmitted to a distant server at a hospital via an internet connection on the personal computer.
  • a physician at the hospital may then access and review the data on the server.
  • the sample fluid analysis device may be configured to receive a program update from a remote device via the communication connector unit.
  • the communication connector unit is coupled to the housing of the sample fluid analysis device.
  • the communication connector is not required to be on the sample fluid analysis device, in some instances, the communication connector may be ⁇ included as part of the sample fluid analysis device so that each additional cartridge does not require the additional cost of a communication connector. If the sample fluid analysis device includes a communication connector of a first technology (e.g., USB plug), then additional cartridges have the option of including additional capabilities such as, for example, a new wireless communication protocol.
  • a remote device such as that described above, includes a network interface which connects it to a network (e.g., the internet).
  • a user interface application ⁇ operated by the remote device may provide a user with the option to view data on a monitor, to store data on storage media (e.g., CD-ROM, memory card, etc.), further analyze and/or manipulate data, transmit data to another device), and/or print out data such as charts, reports, etc., on a printer.
  • Remote devices may also include a network interface (e.g., network interface card (NIC), modem, router, RF front end, etc.) used to connect the remote device to a network.
  • NIC network interface card
  • NIC network interface card
  • a sample fluid analysis device may couple via a USB connection to the remote device which may be a personal computer or laptop connected to the internet using a wireless interface.
  • a sample fluid analysis device may couple via a micro USB connection to a remote device which is a smartphone having an RF front end to access a mobile network.
  • User interface applications provide a user interface for using the ⁇ network connection of the remote device, e.g., to forward data to a physician, hospital, health provider, and/or other third party located at a second remote device on network. Appropriate action may then be taken by the receiving party at the second remote device.
  • devices include a display unit coupled to its housing. Display units may be configured to include a display and/or a display port for coupling a monitor to the ⁇ system.
  • the display unit may display aspects of results of sample analysis determined using aspects of the system, which may include any desired analytical result that the system is configured to determine, such as, for example, analyte concentration, rate of change of analyte concentration, and/or the exceeding of a threshold analyte concentration.
  • the display unit may be configured to include a dot-matrix display. In other aspects, ⁇ other display types, such as liquid-crystal displays (LCD), plasma displays, light-emitting diode (LED) displays, or seven-segment displays, among others, may alternatively be used.
  • the display may be monochromatic (e.g., black and white) or polychromatic (i.e., having a range of colors).
  • the display unit can be configured to provide an alphanumeric display, a graphical display, a video display, an audio display, a auditory or vibratory output or combinations ⁇ thereof.
  • the display unit can also be configured to provide, for example, information related to a sample analysis, such as a current analyte concentration, as well as predictive aspects, such as predictive analyte concentrations, such as trending information.
  • a display unit can be configured to include a touchscreen display where a user may enter information or commands via the display area using, for example, a ⁇ stylus, finger, or any other suitable input device, such as, for example, where the touchscreen is configured as a user interface in an icon or motion driven environment, for example.
  • a system of the present disclosure including a touch screen may include the same functions and basic design as a system of the present disclosure without a touchscreen.
  • a touchscreen system would include a larger display unit compared to the display unit of a system without a touchscreen in order to accommodate the extra area required for any touchscreen buttons that may be used.
  • the device does not have a display (i.e., is display-less).
  • the device may include input elements coupled to its housing that enable the user to make entries, selections, etc. (In certain instances, a cartridge may also include input elements coupled to its housing.)
  • a touchscreen may be employed with or without input elements.
  • Methods of ⁇ interest include supplying a predetermined volume of a sample fluid to a microfluidic passage, supplying a metered amount of a diluent fluid to the microfluidic passage, and mixing the sample fluid and the diluent fluid to prepare the sample fluid dilution.
  • supplying the metered amount of the diluent fluid to the microfluidic passage comprises applying pressure to diluent fluid in a diluent fluid reservoir via a pump (such as those described above).
  • the ⁇ pressure applied by the pump to may vary in some cases, the pressure ranges from 50 Pa to 30000 Pa, such as 200 Pa to 2000 Pa, such as 300 Pa to 1000 Pa, such as 350 Pa to 800 Pa, and including 400 Pa to 600 Pa.
  • the pressure supplied by the pump may be controlled by supplying a voltage to the pump.
  • Voltages may range, e.g., depending on a desired pressure.
  • methods include applying a voltage ranging from 10 mV to 200 mV, such as 20 ⁇ mV to 150 mV, such as 50 mV to 100 mV and including 55 mV to 90 mV.
  • the voltages are applied to the pump via a potentiometer.
  • the pump is configured to apply pressure in pulses.
  • Pulses may range in length, e.g., from 5 ms to 5000 ms, such as 10 ms to 1000 ms, such as 20 ms to 900 ms, such as 30 ms to 800 ms, such as 40 ms to 700 ms, and including 50 ms to 600 ms.
  • Methods according to some embodiments also include supplying the predetermined volume of the sample fluid via a sample fluid metering chamber (such as those described above). Methods may include applying pressure to the sample fluid metering chamber via the pump, in some cases in accordance with the ⁇ aforementioned parameters.
  • the predetermined volume of the sample fluid may vary according to the desired dilution, and can range from 2 ⁇ l to 500 ⁇ l, such as 3 ⁇ l to 300 ⁇ l, such as 5 ⁇ l to 200 ⁇ l, such as 8 ⁇ l to 100 ⁇ l, such as 9 ⁇ l to 50 ⁇ l and including 10 ⁇ l to 40 ⁇ l.
  • Methods of interest may be used to prepare a sample fluid dilution at a dilution ratio ranging from 1:1 to100:1.
  • methods include introducing the sample fluid into a cartridge of the invention.
  • suitable cartridges include a plurality of detection chambers, and a fluidic control subsystem for supplying a sample fluid to the plurality of detection chambers.
  • the fluidic control subsystem includes a sample inlet configured to receive the sample fluid, a sample fluid metering chamber for supplying a predetermined volume of the ⁇ sample fluid from the sample inlet, a mixing chamber for homogenizing the sample fluid with a diluent fluid, and a microfluidic passage fluidically connecting the mixing chamber and the mixing chamber.
  • Methods additionally include inserting the cartridge into a sample fluid analysis device.
  • suitable sample fluid analysis devices include a housing configured to receive the cartridge and a manifold.
  • the sample fluid e.g., blood
  • methods include introducing the sample fluid into the cartridge before the cartridge is inserted into the device.
  • methods include introducing the sample fluid into the cartridge after the cartridge is inserted into the device.
  • Methods of interest additionally include controlling the amount of diluent applied to the cartridge via the diluent line, ⁇ and the amount of pressure applied to the cartridge (e.g., sample fluid metering chamber, sample inlet).
  • Methods subsequently involve interrogating (e.g., optically and/or electrochemically interrogating) the sample fluid via an interrogation device.
  • methods include introducing the sample fluid into a cartridge comprising a plurality of detection chambers each comprising a first light-accessible window configured to permit entry of light, and a second light-accessible window configured to permit an exit of the light.
  • Methods may additionally include inserting the cartridge into a sample fluid analysis device of the invention (e.g., described above).
  • methods include irradiating ⁇ the plurality of detection chambers using the light source, and calculating an absorbance for each detection chamber of the plurality to analyze the sample fluid (e.g., using one or more of the equations/algorithms described above).
  • one or more cartridges are typically used and consumed in connection with performing a single sample analysis of a sample, e.g., determining an analyte level of a sample. That is, typically one or more cartridges will be loaded into the sample fluid analysis device prior to initiating sample ⁇ analysis by the system and will remain present, e.g., latched or locked or otherwise loaded into place, in the sample fluid analysis device until the system has completed the desired sample analysis, after which its contents may be depleted, or otherwise consumed, and the cartridge needs to be removed.
  • a single cartridge may be utilized in connection with more than one sample analysis.
  • the diluent fluid is a simulant of plasma without the presence of the analyte.
  • the diluent fluid is aqueous based and includes electrolytes, buffers and proteins typically found in plasma at high concentration, e.g., ⁇ albumin and immunoglobulins.
  • the diluent fluid may also include lysing agents, stabilizers, and antibacterial agents, which are well-known in the clinical biochemical arts.
  • sample refers to fluid sample containing or suspected of containing an analyte of interest.
  • the sample may be derived from any suitable source.
  • the sample may comprise a liquid, fluent particulate solid, ⁇ or fluid suspension of solid particles.
  • the sample may be processed prior to the analysis described herein. For example, the sample may be separated or purified from its source prior to analysis; however, in certain embodiments, an unprocessed sample containing the analyte may be assayed directly.
  • the source of the analyte molecule may be synthetic (e.g., produced in a laboratory), the environment (e.g., air, soil, fluid samples e.g., water ⁇ supplies, etc.), an animal, e.g., a mammal, a plant, or any combination thereof.
  • the source of an analyte is a human bodily substance (e.g., bodily fluid, blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, lacrimal fluid, lymph fluid, amniotic fluid, interstitial fluid, lung lavage, cerebrospinal fluid, feces, tissue, organ, or the like).
  • Tissues may include, but are not limited to skeletal muscle tissue, liver tissue, lung tissue, kidney tissue, ⁇ myocardial tissue, brain tissue, bone marrow, cervix tissue, skin, etc.
  • the sample may be a liquid sample or a liquid extract of a solid sample.
  • the source of the sample may be an organ or tissue, such as a biopsy sample, which may be solubilized by tissue disintegration/cell lysis. A wide range of volumes of the fluid sample may be analyzed.
  • the sample volume may be about 0.5 nL, about 1 nL, about 3 nL, about 0.01 ⁇ L, about 0.1 ⁇ L, about 1 ⁇ L, about 5 ⁇ L, about 10 ⁇ L, about 50 ⁇ L, about 100 ⁇ L, about 1 mL, about 5 mL, about 10 mL, or the like.
  • the volume of the fluid sample is between about 0.01 ⁇ L and about 10 mL, between about 0.01 ⁇ L and about 1 mL, between about 0.01 ⁇ L and about 100 ⁇ L, between about 0.1 ⁇ L and about 10 ⁇ L, between about 1 ⁇ L and about 100 ⁇ L, between about 10 ⁇ L and about 100 ⁇ L, or between about 10 ⁇ L and about 75 ⁇ L.
  • the sample may undergo pre-analytical processing. Pre-analytical processing may offer additional functionality such as nonspecific protein removal and/or ⁇ effective yet cheaply implementable mixing functionality.
  • pre-analytical processing may include the use of electrokinetic trapping, AC electrokinetics, surface acoustic waves, isotachophoresis, dielectrophoresis, electrophoresis, or other pre-concentration techniques known in the art.
  • the fluid sample may be concentrated prior to use in an assay.
  • the source of an analyte molecule is a human body fluid (e.g., blood, serum)
  • the fluid may be concentrated by precipitation, evaporation, filtration, centrifugation, or a combination thereof.
  • a fluid sample may be concentrated about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold, or greater, prior to use.
  • a sample of the present disclosure is whole blood.
  • Samples for hematology are typically whole blood.
  • the whole blood sample consists of red blood cells, white blood cells, and platelets suspended in a protective yellow liquid known as plasma.
  • samples for immunoassays and clinical chemistry assays are typically serum or plasma.
  • the whole blood sample is obtained from a subject.
  • the subject is a living subject, including an animal and a human.
  • a needle is inserted into a vein to collect a sample of blood for testing.
  • a sample of the present disclosure is capillary blood.
  • capillary blood or “capillary sample” refers to a blood sample collected by pricking the skin. Capillary blood is generally obtained by pricking a finger in adults and a heel in infants and small children. Capillaries are tiny blood vessels near the surface of the skin. Capillary ⁇ plasma typically contains higher concentrations of proteins, calcium and chloride, and lower levels of potassium, sodium, and urea nitrogen compared to venous plasma.
  • a sample of the present disclosure is plasma.
  • plasma refers to the colorless fluid part of blood, lymph, or milk, in which corpuscles or fat globules are suspended.
  • plasma is the blood's liquid component and is made up of water, proteins, waste products, minerals, clotting factors, immunoglobulins, carbon dioxide and hormones.
  • the method for separating plasma from blood is well known in the art.
  • plasma is produced when whole blood is collected in tubes that are ⁇ treated with an anticoagulant. The blood does not clot in the plasma tube, thereby the cells are removed by centrifugation. The supernatant, designated plasma is carefully removed from the cell pellet using a Pasteur pipette.
  • a sample of the present disclosure is serum.
  • serum refers to the watery, clear portion of an animal fluid or plant sap.
  • blood serum refers to an amber-colored, protein-rich liquid that separates out when blood coagulates.
  • serum includes, but not limited to, blood serum, serous (or serosal) fluid secreted by the serous glands, and plant sap.
  • the method for separating serum from blood is well known in the art.
  • the blood serum is collected after whole blood is allowed to clot. The clot is removed by centrifugation, ⁇ and the resulting supernatant, designated serum, is carefully removed using a Pasteur pipette.
  • providing a sample comprises transferring the sample from a sample collection vessel to the inlet(s) via a sponge stick sampler.
  • the providing a sample comprises transferring the sample from a sample collection vessel to the inlet(s) via a Vacutainer®.
  • a sample collection device is used to collect a ⁇ sample from a subject and provide the sample to the device of the present disclosure.
  • the sample collection device is inserted directly into the device to provide a sample.
  • a sample in a sample collection vessel is poured into the inlet of the device.
  • one or more sample collection devices are, not limited to, syringes, sterile containers, standard urine collection vessels, sponge stick samplers, microsampling ⁇ devices, micro-needles, or other minimally invasive pain-free blood collection devices; blood collection tube(s); lancets; capillary blood collection tubes; other single fingertip-prick blood collection devices, 16-gauge or other size needles, or the like.
  • a number of devices are presently available for collecting, handling and storage of whole blood or other fluids.
  • blood collection devices include such as micro sampling devices, ⁇ micro-needles, or other minimally invasive pain-free blood collection devices; blood collection tube(s); lancets; capillary blood collection tubes; other single fingertip-prick blood collection devices and the like.
  • the blood collection device includes a phlebotomy needle connected through tubing to one end of a sample pouch. The tubing is connected to the ⁇ opposite end of the sample pouch and communicates the sample pouch with a blood bag. With this device, blood from the subject passes through the first tubing, the sample pouch and then through the second tubing into the blood bag. When the blood bag is full, the tubing closest to the bag is clamped off. This is described in U.S.
  • the collected blood in the sample pouch is transported to a sample holding chamber of the system of the present disclosure.
  • the blood collection device may comprise an integrated double- ended needle which is well known in the art.
  • U.S. Patent No.5,086,780, ⁇ incorporated by reference herein, which discusses a blood collection device comprising a double-ended needle which is sheathed before use and safely re-sheathed after use, thereby reducing the risk of accidental needle wounds and resultant infections to a minimum.
  • This blood collection device also serves as a holder of blood collection tubes during sample taking, with the collection tubes being easily inserted and removed through the rear bore opening of ⁇ the device.
  • a sample collection device of the present disclosure is a capillary collection device.
  • the capillary collection device is, but not limited to, a lancing ⁇ device/lancet device and a finger stick.
  • the lancing device is for obtaining a blood sample from a finger or at an alternate site of a subject. Exemplary lancing devices are described in U.S. Patent No.8,152,826 and U.S. Patent No.8,556,827; which are incorporated herein by reference.
  • the lancing device described in U.S. Patent No.8,556,827 ⁇ comprises a lancet and a torsion spring coupled to the lancet through a lancet holder.
  • the torsion spring includes inner, middle, and outer rings which are concentrically configured, a plurality of activation spring arms which connect the middle and outer rings and a plurality of return spring arms which connect the inner and middle rings.
  • the plurality of activation and return spring arms can be independently transformed between energized and de-energized ⁇ states using a single, button-shaped mechanism. Rotation of the mechanism is used to energize the activation and return spring arms.
  • a blood sample from a subject is drawn by medical laboratory scientists, medical practitioners, some emergency medical technicians, paramedics, phlebotomists, and other nursing staff. The blood sample is then collected into an evacuated ⁇ tube.
  • one or more evacuated tubes containing blood samples are transported to the system of the present disclosure.
  • the tubes contain a variety of additives or none at all.
  • whole blood sample needs to be mixed with EDTA, which chelates calcium to prevent it clotting, unless the clotting time is the test to be measured, in which case citrates are used.
  • EDTA chelates calcium to prevent it clotting
  • citrates are used.
  • the majority of biochemistry tests are performed on serum, and, consequently, either a plain tube or a clotting accelerator is used.
  • some assays may also require whole blood but are interfered with by EDTA and in this case Lithium Heparin is a suitable alternative. Procedures for sample collection by ⁇ phlebotomists are well known in the art.
  • a sample of the present disclosure is a cerebrospinal fluid.
  • cerebrospinal fluid CSF
  • cerebrospinal fluid refers to a clear fluid that surrounds and protects the brain and spinal cord.
  • the analysis for cerebrospinal fluid may look for proteins, sugar (glucose), and other substances.
  • the method for collecting cerebrospinal fluid is well known in the art.
  • cerebrospinal fluid is usually obtained through a lumbar puncture (spinal tap). During the procedure, a needle is inserted usually between the 3rd and 4th lumbar vertebrae and the CSF fluid is collected for testing.
  • a sample of the present disclosure is saliva.
  • saliva refers to watery liquid secreted into the mouth by glands, providing lubrication ⁇ for chewing and swallowing, and aiding digestion.
  • Saliva consists of 99% water and 1% protein and salts. The method of collecting saliva is well known in the art.
  • saliva sample can be refrigerated for up to a week before it needs to be added to the stabilizing fluid in the tube.
  • a sample of the present disclosure is urine.
  • urine refers to a watery, typically yellowish fluid stored in the bladder and discharged through the urethra.
  • Urine is one of the body's chief means of eliminating excess water and salt, and also contains nitrogen compounds such as urea and other waste substances removed from the blood by the kidneys. Collecting a urine sample is well known in the art. In exemplary embodiments, either a "first-catch" or a "mid-stream" sample of urine is collected in a ⁇ completely sterile container. The first-catch urine sample is the first part of the urine that comes out. The mid-stream urine is for reducing the risk of the sample being contaminated with bacteria from hands, or the skin around the urethra or the tube that carries urine out of the body. In some embodiments, the collected urine sample may be stored in a fridge at 4 °C less than 24 hours in a sealed plastic bag.
  • the urine sample is used for ⁇ infections such as urinary tract infection (UTI), some sexually transmitted infections (STIs) such as chlamydia in men, or kidney damage, such as ACR test.
  • a sample of the present disclosure is interstitial fluid.
  • ISF interstitial fluid
  • lymph tissue fluid
  • tissue fluid refers to clear fluid that occupies the space between the cells in the body or fluid found in the spaces around cells. It ⁇ comes from substances that leak out of blood capillaries. Interstitial fluid helps bring oxygen and nutrients to cells and to remove waste products from them. As new interstitial fluid is made, it replaces older fluid, which drains towards lymph vessels.
  • ISF interstitial fluid
  • a sample of the present disclosure is intestinal fluid.
  • Intestinal fluid or gastrointestinal fluid contains, for example electrolytes, bile salts, lipids and lipid ⁇ digestion products, cholesterol, proteins, enzymes plus other components and may also vary depending upon the anatomical location (stomach vs small intestine vs colon).
  • the method of collecting intestinal fluid samples is well known in the art.
  • the intestinal fluid can be collected through a nasojejunal tube and be made into capsules using the freeze- dried powder method.
  • a sample of the present disclosure is a sample collected from nasal swabs.
  • a sample of the present disclosure is a sample collected from throat swabs.
  • a sample of the present disclosure is a sample collected from vaginal swabs. Nasal swabs, throat swabs, and vaginal swabs are well known in the art.
  • a sample includes respiratory specimen.
  • the respiratory specimen includes, but not is limited to, nasal swab, throat swab, sputum, tracheal/bronchial secretion, and bronchial lavage fluid.
  • respiratory sampling includes upper respiratory materials and lower respiratory secretions.
  • the upper respiratory materials comprise nasal swab, throat swab, and the like.
  • ⁇ the lower respiratory secretions comprise sputum, tracheal/bronchial secretion, bronchoalveolar lavage fluid, and the like.
  • the sputum is collected by well known process in the art.
  • tracheal/bronchial secretion is collected by inserting suction catheter as deeply as possible and aspirating secretion, which is well known in the art.
  • bronchoalveolar lavage fluid is collected by use of bronchoscopy, which is well known in the art.
  • a sample includes any tissue obtained from a subject.
  • a sample includes any cell obtained from a subject.
  • the subject is any living subject including a human.
  • tissues may include, but are not limited to skeletal muscle tissue, liver tissue, heart tissue, lung tissue, pancreas tissue, adipose tissue, stomach tissue, gastrointestinal tract tissue, colon tissue, kidney tissue, myocardial tissue, brain tissue, breast tissue, nerve tissue, bone marrow, cervix tissue, skin, etc.
  • cells may include, but are not limited to skeletal muscle cells, liver cells, heart cells, lung cells, pancreas cells, adipose cells, stomach cells, gastrointestinal tract cells, colon cells, kidney cells, myocardial cells, brain cells, breast cells, nerve cells, bone marrow cells, cervix cells, skin cells, etc.
  • the sample is tumor or cancer cells.
  • the sample includes, but is not limited to, brain cancer cells, liver cancer cells, pancreas cancer cells, lung cancer cells, breast cancer cells, kidney cancer cells, metastatic cancer cells, ovarian cancer cells, colorectal cancer cells, bladder cancer cells, thyroid cancer cells, lymphoma cells, cervical cancer cells, gynecologic cancer cells, head and neck cancer cells, ⁇ mesothelioma cells, myeloma cells, skin cancer cells, prostate cancer cells, uterine cancer cells, vaginal and vulvar cancer cells, and the like.
  • the source of the sample may be an organ or tissue, such as a biopsy sample, which may be solubilized by tissue disintegration/cell lysis.
  • a sample may be processed prior to performing immunoassay on the sample.
  • the sample may be concentrated, ⁇ diluted, purified, amplified, etc.
  • one or more analytes in a sample may be measured, detected, or assessed by a device of the present disclosure.
  • the sample may be any test sample containing or suspected of containing an analyte.
  • analyte As used herein, "analyte”, “target analyte”, and “analyte” are used interchangeably and refer to the analyte being measured in the devices ⁇ disclosed herein. Examples of analytes provided herein are for illustrative purposes and are not intended to limit the scope of the present disclosure.
  • Blood cells and blood cell types that may be detecting by the systems, devices and methods disclosed herein include, without limitation, red blood cells, hemoglobin, white ⁇ blood cells (including neutrophils, lymphocytes, monocytes, eosinophils, and basophils), platelets, reticulocytes, and nucleated red blood cells.
  • Various measurements of different blood components may be performed, including, but not limited to, cell count, cell size, cell complexity, granularity, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration.
  • the above disclosed ⁇ measurements may be performed using stain independent methods in the absence of histological staining.
  • the analyte may be a pathogen, a prion protein, a cancer cell, a blood component, or a biomolecule.
  • the pathogen is, but not limited to, a virus, a bacterium, a fungus, or a protozoan.
  • ⁇ the prion protein may arise from a sporadic prion disease, a genetic prion disease, or an acquired prion disease.
  • the cancer cell may be a cancer cell from a tumor or a circulating tumor cell.
  • the blood component may be red blood cells, white blood cells, platelets, or proteins found in the blood.
  • a biomolecule may be a metabolite, a macromolecule, a protein, or a chemical compound. Any combination of analytes ⁇ may be measured by the assays of the methods and systems of the present disclosure.
  • assays of the present disclosure can be used to determine the presence or absence of an analyte in a sample or measure the amount of an analyte in a sample to identify or assess a disease or condition.
  • Measurements of an analyte can be used, for example, but not by way of limitation, determine the likelihood of developing a disease or condition; diagnose, identify, or classify a disease or condition; estimate prognosis; determine the extent of a disease or condition; determine appropriate treatment; predict response of a disease or condition to treatment; monitor response of a disease or condition to treatment; ⁇ determine treatment efficacy; and identify recurrence of a disease or condition.
  • the analytes/properties to which the sensors respond may be selected from among particles (e.g., blood cells or microparticles), human chorionic gonadotropin, pH, partial pressure, CO2, partial pressure O2, glucose, lactate, creatinine, urea, sodium, potassium, chloride, calcium, magnesium, phosphate, hematocrit, prothrombin time (PT), activated partial ⁇ thromboblastin time (APTT), activated clotting time (ACT), D-dimer, prostate-specific antigen (PSA), creatine kinaseME (CKMB), brain natriuretic peptide (BNP), troponin I (Tni), cardiac troponin (cTni), human chorionic gonadotrophin, troponin T, troponin C, myoglobin, neutrophil gelatinase-associated lipocalin (NGAL), galectin-3, prostate-specific antigen (PSA), parathyroid hormone (PTH), galectin-3, aspartate amino
  • an optical sensor is configured to convert light received from cells within a portion of the imaging chamber to an output signal
  • a processor connected to the optical sensor is configured to convert the output signal to a number count or percentage for each type of cell in the blood sample.
  • a differential blood cell count is a ⁇ measurement of a number or percentage of each type of cell (e.g., white blood cells (WBCs)) that is in a whole blood sample.
  • WBCs white blood cells
  • Cell types include erythrocytes and leukocytes and platelets. Imaging can distinguish various types of leukocytes including neutrophils, lymphocytes, granulocytes, eosinophils, basophils and monocytes.
  • CBC complete blood count
  • reticulocyte counts reticulocyte counts
  • LDC leukocyte differential count
  • WBCs white blood cells
  • a differential blood cell count includes: (i) identifying the cells, for example white blood cells, within the sample residing within the chamber; (ii) quantitatively analyzing at least some of the ⁇ identified cells within the image relative to one or more predetermined quantitatively determinable features; and (iii) identifying at least one type of cell from the identified cells using the quantitatively determinable features.
  • the algorithm utilizes a set of identifying features, each of which features is distinguishable from the other features and each of which is quantitatively determinable from an image of the sample.
  • Each WBC can be characterized by the presence or absence of certain identifying features, and/or by quantitative information associated with certain features.
  • the present invention is described herein in ⁇ terms of an exemplary set of identifying features that can be used to selectively identify and distinguish WBCs. This set is not inclusive of all possible features, and therefore the present invention is not limited to this particular set.
  • an exemplary set of identifying features includes those entitled: Cell, Nucleus, number of Lobes, Cell Area, Nucleus ⁇ Area Ratio of Large Granules, Ratio of Nucleus, Red-Green Ratio, Nucleus Shape, Cell Shape, Nucleus Brightness, Cytoplasm Brightness, Average Cell Absorption at a Given Wavelength, Nucleus Texture, Cytoplasm Texture, Cell Absorption Texture at a Given Wavelength, Nucleus Hollowness, and Cytoplasm Hollowness; each of which is described in U.S. Patent Publication No.2012/0034647, which is incorporated herein by reference.
  • certain ⁇ features directly provide information about a particular cell (e.g., Nucleus Shape).
  • a feature e.g., Cell Area
  • the identifying features are based on quantifiable characteristics such as light intensity, light color. OD, area, and relative position (e.g., shape).
  • the colors may be ⁇ created by one or more fluorescent colorants admixed with the sample, which upon excitation, produce fluorescent light emission at particular wavelengths associated with particular colors.
  • ACO is a fluorescent dye that, when mixed with a whole blood sample, selectively stains constituents within the sample; e.g., white blood cells, platelets, reticulocytes, and nucleated red blood cells.
  • the ACO permeates through the respective WBC and stains its DNA and RNA
  • the color(s) emitted by the dye within the WBC arc a function of a number of factors, including: the quantity of RNA ⁇ and DNA within the dye, the concentration of the dye in the constituent, and the pH of the constituent.
  • the present invention is not limited to using ACO, and other dyes (e.g., Astrazon Orange) may be used in place of ACO or in combination with ACO.
  • one or more analytes may be a cell such as a circulating tumor cell.
  • the analyte is a biological cell (e.g., mammalian, avian, reptilian, other vertebrate, insect, yeast, bacterial, cell, etc.).
  • the analyte may be an infectious agent, such as a bacterium (e.g., Mycobacterium tuberculosis, Staphylococcus aureus, Shigella dysenteriae, Escherichia coli O157:H7, Campylobacter jejuni, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella O8, and Salmonella enteritidis), virus ⁇ (e.g., retroviruses (such as HIV), herpesviruses, adenoviruses, lentiviruses, Filoviruses (e.g., West Nile, Ebola, and Zika viruses), hepatitis viruses (e.g., A, B, C, D, and
  • one or more analytes are tumor or cancer cells.
  • a cancer cell may be directly detected, e.g., a nucleic acid or an antigen specific to the ⁇ cancer cell is detected.
  • the presence of a cancer cell may be detected by a change or mutation in a nucleic acid sequence of the cancer cell, including, but not limited to, a SNP, an insertion, a deletion, a chromosome translocation, or gene amplification.
  • a cancer cell may be detected by detecting the presence of tumor or cancer markers associated with the cancer cell.
  • a cancer cell may be detected by detecting the ⁇ expression of receptors associated with a cancer cell.
  • a cancer cell may be indirectly detected, e.g., metabolic markers associated with the cancer cell can indicate the presence of the cancer cell.
  • types of cancer cells that may be detected by assays of the present disclosure include, but are not limited to, carcinoma cells, leukemia cells, lymphoma cells, ⁇ myeloma cells, sarcoma cells, central nervous system cancer cells, and mesothelioma cells.
  • Bone Cancer includes Ewing Sarcoma and Osteosarcoma and Malignant Fibrous Histiocytoma
  • Brain Tumors Breast Cancer, Cervical cancer, Colorectal Cancer, Endometrial Cancer (Uterine Cancer), Esophageal Cancer, Head and Neck Cancer, Hepatocellular (Liver) Cancer, Hodgkin Lymphoma, Kidney (Renal ⁇ Cell) Cancer, gynecologic cancer cells, vaginal and vulvar cancer cells, Leukemia, Lung Cancer (Non-Small Cell, Small Cell, Pleuropulmonary Blastoma, Pulmonary Inflammatory Myofibroblastic Tumor, and Tracheobronchial Tumor), Lymphoma, Melanoma, Multiple Myeloma/Plasma Cell Neoplasms, Neuroblastoma, Non-Hodgkin Lymphoma, Ovarian Cancer, Pancreatic Cancer, Prostate Cancer, Skin Cancer, Testi
  • Markers ⁇ of cancer include, but are not limited to, ALK gene rearrangements and overexpression, Alpha- fetoprotein (AFP), B-cell immunoglobulin gene rearrangement, BCL2 gene rearrangement, Beta-2-microglobulin (B2M), Beta-human chorionic gonadotropin (Beta-hCG), Bladder Tumor Antigen (BTA), BRCA1 and BRCA2 gene mutations, BCR-ABL fusion gene (Philadelphia chromosome), RAF V600 mutations, C-kit/CD117, CA15-3/CA27.29, CA19-9, CA-125, CA ⁇ 27.29, Calcitonin, Carcinoembryonic antigen (CEA), CD19, CD20, CD22, CD25, CD30, CD33, Chromogranin A (CgA), Chromosome 17p deletion, Chromosomes 3, 7, 17, and 9p21, Circulating tumor cells of epithelial origin (CELLSEARCH), Cytokeratin fragment
  • types of cancer cells that may be detected by assays of the present disclosure include gastric cancer cells (e.g., HGC-27 cells); non-small cell lung cancer (NSCLC) cells, colorectal cancer cells (e.g., DLD-1 cells), H23 lung adenocarcinoma cells, Ramos cells, ⁇ T-cell acute lymphoblastic leukemia (T-ALL) cells, CCRF-CEM cells, acute myeloid leukemia (AML) cells (e.g., HL60 cells), small-cell lung cancer (SCLC) cells (e.g., NCI-H69 cells), human glioblastoma cells (e.g., U118-MG cells), prostate cancer cells (e.g., PC-3 cells), HER-2- overexpressing human breast cancer cells (e.g., SK-BR-3 cells), pancreatic cancer cells (e.g., Mia-PaCa-2)).
  • gastric cancer cells e.g., HGC-27 cells
  • one or more analytes is a virus.
  • the virus is directly detected, e.g., a nucleic acid or an antigen specific to the virus is detected.
  • the virus is indirectly detected, e.g., detection of anti-virus antibodies produced by a subject can indicate the presence of a virus, or the presence of a virus induces hemagglutination in blood.
  • viruses that may be detected by the assays of the ⁇ present disclosure include animal, plant, fungal and bacterial viruses.
  • viruses that may be detected by the assays of the present disclosure include those which impact animals, especially mammals, in particular humans and domestic animals.
  • viruses that may be detected by the assays of the present disclosure include, but are not limited to, Papovaviruses, e.g. polyoma virus and SV40; Poxviruses, e.g. vaccinia virus ⁇ and variola (smallpox); Adenoviruses, e.g., human adenovirus; Herpesviruses, e. g. Human Herpes Simplex types I and II; Parvoviruses, e.g. adeno associated virus (AAV); Reoviruses, e.g., rotavirus and reovirus of humans; Picornaviruses, e.g.
  • Papovaviruses e.g. polyoma virus and SV40
  • Poxviruses e.g. vaccinia virus ⁇ and variola (smallpox)
  • Adenoviruses e.g., human adenovirus
  • Herpesviruses e
  • poliovirus including the alpha viruses (group A), e.g. Sindbis virus and Semliki forest virus (SFV) and the flaviviruses (group B), e.g. dengue virus, yellow fever virus and the St. Louis encephalitis virus; Retroviruses, e. g. lentiviruses, HIV I and II, Rous sarcoma virus (RSV), and mouse leukemia viruses; Rhabdoviruses, e.g. vesicular stomatitis virus (VSV) and rabies virus; Paramyxoviruses, e.g.
  • mumps virus measles virus and Sendai virus
  • Arena viruses e.g., lassa ⁇ virus
  • Bunyaviruses e.g., bunyawere (encephalitis)
  • Coronaviruses e.g. common cold, GI distress viruses
  • Orthomyxovirus e.g., influenza
  • Caliciviruses e.g., Norwak virus, Hepatitis E virus
  • Filoviruses e.g., ebola virus and Marburg virus
  • Astroviruses e.g. astrovirus, among others.
  • viruses include, but are not limited to, Sin Nombre virus, influenza (especially H5N1 influenza), Herpes Simplex Virus (HSV1 and HSV-2), Coxsackie virus, ⁇ Human immunodeficiency virus (I and II), Andes virus, Dengue virus, Epstein-Barr virus (mononucleosis), Variola (smallpox) and other pox viruses, West Nile virus, hepatitis viruses (e.g., A, B, C, D, and E), HPV, SARS-CoV-2 (COVID-19), CMV, Parvovirus B19, Chlamydia, Gonorrhea, Zika Virus, Chikungunya Virus, Babesia, Malaria, and Usutu virus.
  • one or more analytes may be a bacterium.
  • the ⁇ bacterium is directly detected, e.g., a nucleic acid or an antigen specific to the bacterium is detected.
  • the bacterium is indirectly detected, e.g., detection of anti-bacteria antibodies produced by a subject can indicate the presence of bacteria, or the presence of bacterial enzyme activity products can indicate the presence of bacteria.
  • bacteria that may be detected by assays of the present disclosure include, but are not limited to, ⁇ Achromobacter denitrificans, Achromobacter xylosoxidans, Acinetobacter baumannii, Acinetobacter calcoaceticus, Actinomyces israelii, Aerococcus christensenii, Aeromonas hydrophile, Aeromonas sobria, Aggregatibacter actinomycetemcomitans, Alcaligenes faecalis, Alistipes onderdonkii, Anaerococcus vaginalis, Anaeroglobus geminatus, Arcanobacterium haemolyticum, Arcanobacterium pyogenes, Arthrobacter cumminsii, Atopobium vaginae, ⁇ Bacillus anthracis, Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus sphae
  • one or more analytes may be a fungus.
  • the ⁇ fungus is directly detected, e.g., a nucleic acid or an antigen specific to the fungus is detected.
  • the fungus is indirectly detected, e.g., a cell wall component of a fungus released into the blood can indicate the presence of the fungus.
  • fungi that may be detected by assays of the present disclosure include, but are not limited to, fungi from a fungal genera selected from the group consisting of Candida, Aspergillus, Rhyzopus, ⁇ Cryptococcus, Histoplasma, Pneumocystis, Stachybotrys, Sporothrix, Trichophyton, Microsporum, Blastomyces, Mucoromycotina, Coccidioides, Exserohilum, Cladosporium, Coccoides, Encephalitozoon, Encephalitozoon, Fusarium, Lichtheimia, Mortierella, Malassezia, Prototheca, Pythium, Rhodotorula, Fusarium, Thielaviopsis, Verticillium, Magnaporthe, Sclerotinia, Ustilago, Rhizoctonia, Puccinia, Armillaria, Botrytis, Blumeria, Mycosphaerella, ⁇
  • fungal species that can be detected by the assays of the present disclosure include, but are not limited to, Candida albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. auris; Cryptococcus neoformans and C. gattii; Coccidioides immitis and C. posadasii; Histoplasma capsulatum; Blastomyces dermatitidis; and Pneumocystis jirovecii. ⁇
  • one or more analytes may be a protozoa.
  • the protozoan is directly detected, e.g., a nucleic acid or an antigen specific to the protozoan is detected. In other cases, the protozoan is indirectly detected, e.g., a metabolic product of the protozoan can indicate the presence of the protozoan.
  • classes of protozoa that may be detected by assays of the present disclosure include, but are not limited to, ⁇ Plasmodium (malaria), Leishmania (leishmaniasis), Trypanosoma (sleeping sickness and Chagas disease), Cryptosporidium, Giardia, Toxoplasma, Babesia, Balantidium and Entamoeba.
  • protozoa that can be detected by the assays of the present disclosure include, but are not limited to, Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, Plasmodium vivax, Leishmania donovani, Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma gondii and Babesia microti.
  • one or more analytes may be a prion protein.
  • the prion is directly detected, e.g., a nucleic acid or an antigen specific to the prion is ⁇ detected.
  • the presence of prions or potential for prion formation is detected by identifying a mutation in a nucleic acid sequence.
  • the presence of structures formed by prions can indicate the presence of prions.
  • the prion is indirectly detected, e.g., biochemical changes induced by prion formation can indicate the presence of prions.
  • prions are amplified prior to detection using methods such as protein ⁇ misfolding cyclic amplification (PMCA) or real-time quaking-induced conversion (RT-QUIC).
  • Exemplary prion proteins include, but are not limited to, Scrapie (Sheep and goats), transmissible mink encephalopathy (TME), chronic wasting disease (CWD) in mule deer and elk, bovine spongiform encephalopathy (BSE) cattle, feline spongiform encephalopathy (FSE) in cats, exotic ungulate encephalopathy (EUE), Kuru in humans, Creutzfeldt-Jakob disease ⁇ (CJD) in humans, Fatal familial insomnia (FFI) in humans and Gerstmann-Strässler-Scheinker syndrome (GSS) in humans.
  • Scrapie Sheep and goats
  • TAE transmissible mink encephalopathy
  • CWD chronic wasting disease
  • BSE bovine spongiform encephalopathy
  • FSE feline spongiform encephalopathy
  • EUE exotic ungulate encephalopathy
  • Kuru Kuru in humans
  • one or more analytes measured by the assays of the methods and systems of the present disclosure may be a blood component.
  • blood components that may be detected by assays of the present disclosure include, but are not ⁇ limited to, red blood cells, hemoglobin, white blood cells (including neutrophils, lymphocytes, monocytes, eosinophils, and basophils), platelets, reticulocytes, and nucleated red blood cells.
  • Various measurements of different blood components may be performed, including, but not limited to, cell count, cell size, cell complexity, granularity, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration.
  • one or more analytes may be a biomolecule.
  • biomolecules include macromolecules such as, for example, proteins, lipids, and carbohydrates.
  • the analyte may be hormones, antibodies, growth factors, cytokines, electrolytes (e.g., sodium, potassium, and chloride), enzymes (e.g., alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and amylase), ⁇ receptors (e.g., neural, hormonal, nutrient, and cell surface receptors) or their ligands, cancer markers (e.g., PSA, TNF-alpha), markers of myocardial infarction (e.g., troponin, creatine kinase, and the like), toxins, drugs (e.g., therapeutic drugs, drugs of addiction), metabolic agents (e.g., including vitamins and minerals), metabolic products (e.g., glucose, urea
  • Non-limiting embodiments of protein analytes ⁇ include peptides, polypeptides, protein fragments, protein complexes, fusion proteins, recombinant proteins, phosphoproteins, glycoproteins, lipoproteins, and the like.
  • the analyte may be a post-translationally modified protein (e.g., phosphorylated, methylated, glycosylated protein).
  • the analyte is a nucleic acid.
  • the analyte is a protein or a small molecule.
  • a non-limiting list of analytes that may be analyzed by the devices presented herein include A ⁇ 42 amyloid beta-protein, fetuin-A, tau, secretogranin II, prion protein, Alpha- ⁇ synuclein, tau protein, neurofilament light chain, parkin, PTEN induced putative kinase 1, DJ-1, leucine-rich repeat kinase 2, mutated ATP13A2, Apo H, ceruloplasmin, Peroxisome proliferator- activated receptor gamma coactivator-1 alpha (PGC-1 ⁇ ), transthyretin, Vitamin D-binding Protein, proapoptotic kinase R (PKR) and its phosphorylated PKR (pPKR), CXCL13, IL-12p40, CXCL13, IL-8, Dkk-3 (semen), p14 endocan fragment, Serum, ACE2, autoantibody to CD25, ⁇ hTERT, CAI25 (MUC 16),
  • Exemplary targets of nucleic acid aptamers that may be measured in a sample such as an environmental sample, a biological sample obtained from a patient or subject in need using the subject devices include: drugs of abuse (e.g.
  • protein biomarkers including, but not limited to, Nucleolin, nuclear factor-kB essential modulator (NEMO), CD-30, protein tyrosine kinase 7 (PTK7), vascular endothelial growth factor (VEGF), MUC1 glycoform, immunoglobulin ⁇ ⁇ Heavy Chains (IGHM), Immunoglobulin E, ⁇ v ⁇ 3 integrin, ⁇ -thrombin, HIV gp120, NF- ⁇ B, E2F transcription factor, HER3, Plasminogen activator inhibitor, Tenascin C,CXCL12/SDF-1, prostate specific membrane antigen (PSMA), gastric cancer cells, HGC-27); cells (including, but not limited to, non-small cell lung cancer (NSCLC), colorectal cancer cells, (DLD-1), H23 lung adenocarcinoma cells, Ramos cells, T-cell acute lymphoblastic leukemia (T-ALL) cells, CCRF- ⁇ CEM, acute myeloid leukemia
  • Exemplary targets of protein or peptide aptamers that may be measured in a sample obtained from a patient or subject in need using the subject devices include, but are not limited to: HBV core capsid protein, CDK2, E2F transcription factor, Thymidylate synthase, Ras, EB1, and Receptor for Advanced Glycated End products (RAGE).
  • HBV core capsid protein CDK2, E2F transcription factor, Thymidylate synthase, Ras, EB1, and Receptor for Advanced Glycated End products (RAGE).
  • a biological sample e.g., human blood sample
  • preparation/processing may include the following steps: i) isolation of total nucleic acid that contains a target nucleic acid from the sample, ii) optionally, enrichment of the target nucleic acid, iii) amplification of the target nucleic acid, and iv) processing of the amplified ⁇ target nucleic acid.
  • steps can be performed manually, automatically, or by a combination thereof.
  • the analyte is not amplified (i.e., the copy number of the analyte is not increased) prior to the measurement of the analyte.
  • the analyte is DNA or RNA
  • the analyte is not replicated to increase copy numbers of the analyte.
  • methods involve the use of one or more reference standards for quantifying an analyte.
  • the reference standards may be employed to establish standard curves ⁇ for interpolation and/or extrapolation of the analyte concentrations.
  • a system of the present disclosure may include reference standards that vary in terms of concentration level.
  • the device may include one or more reference standards with either a high concentration level, a medium concentration level, or a low concentration level.
  • concentration ranges for the reference standard include but are not limited to, for example: about 10 fg/mL, about 20 fg/mL, about 50 fg/mL, about 75 fg/mL, about 100 fg/mL, about 150 fg/mL, about 200 fg/mL, about 250 fg/mL, about 500 fg/mL, about 750 fg/mL, about 1000 fg/mL, about 10 pg/mL, about 20 pg/mL, about 50 pg/mL, about 75 pg/mL, about 100 pg/mL, about 150 pg/mL, about 200 pg/mL, about 250 pg/mL, about 500 pg/m/m
  • a system of the present disclosure optionally includes quality control components (for example, sensitivity panels, calibrators, and positive controls). Preparation of quality control reagents is well-known in the art and is described on insert sheets ⁇ for a variety of immunodiagnostic products. Sensitivity panel members optionally are used to establish assay performance characteristics, and further optionally are useful indicators of the integrity of the device reagents, and the standardization of assays.
  • a system of the present disclosure can also optionally include other reagents required to conduct a diagnostic assay or facilitate quality control evaluations, such as buffers, salts, enzymes, enzyme co-factors, substrates, detection reagents, and the like.
  • the device can additionally include one or more other controls.
  • One or more of the components of the device can be lyophilized, in which case the device can further comprise reagents suitable for the reconstitution of the lyophilized components.
  • One or more of the components may be in liquid form.
  • the various components of the device optionally are provided in suitable containers as necessary.
  • the device further can include containers for holding or storing a sample (e.g., a container or cartridge for a urine, saliva, plasma, cerebrospinal fluid, or serum sample, or appropriate container for storing, transporting or processing tissue so as to create a tissue aspirate).
  • a sample e.g., a container or cartridge for a urine, saliva, plasma, cerebrospinal fluid, or serum sample, or appropriate container for storing, transporting or processing tissue so as to create a tissue aspirate.
  • the device ⁇ optionally also can contain reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents or the test sample.
  • analyzing the sample fluid comprises analyzing nucleic acids of the sample fluid.
  • Nucleic acid testing can include, but is not limited to, polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), real time quantitative PCR (RT-qPCR), ⁇ isothermal PCR, thermocycle based PCR, hot-start PCR, loop-mediation isothermal amplification (LAMP), recombinase polymerase amplification (RPA), nucleic acid lateral flow immunoassay (NAFLIA), helicase dependent amplification (HAD), rolling circle amplification (RCA), nicking enzyme amplification reaction (NEAR), CRISPR-Cas detection methods (e.g., SHERLOCK (specific high-sensitivity enzymatic reporter unlocking), DETECTR (DNA ⁇ Endonuclease Targeted CRISPR Trans Reporter), and HOLMES (one-Hour Low-cost Multipurpose highly Efficient System), nucleic acid hybridization detection and probes (e.g., dot- blot, Southern blot, in situ hybridization, sequence specific probe
  • ⁇ amplification is used for increasing the amount of nucleic acid for performing the assay and in the process of detecting and identifying nucleic acid sequences.
  • the amplification is performed in PCR and RT-PCR.
  • analyzing the sample fluid comprises an immunoassay (IA).
  • An immunoassay generally comprises contacting an antigen with an antibody specific for the antigen to form an antibody-antigen complex and detecting the antibody-antigen complex.
  • the antibody-antigen complex is an antibody-analyte complex.
  • the analyte is an antigen.
  • an antigen that may be bound by an antibody includes, but is not limited to, proteins, peptides, polysaccharides, lipids, or nucleic acids. Cartridges may be designed to perform various types of immunoassays.
  • the immunoassay may be a labelled immunoassay. In labelled immunoassays, ⁇ the antibody-analyte complex may be detected using a detectably labeled antibody.
  • Detectable labels may be selected from a variety of such labels known in the art, but normally are radioisotopes, fluorophores, enzymes (e.g., horseradish peroxidase), or other moieties or compounds which either emit a detectable signal (e.g., radioactivity, fluorescence, color) or emit a detectable signal after exposure of the label to its substrate.
  • Additional labels can include, but ⁇ are not limited to, DNA probes and reporters, electrochemiluminescent tags, and magnetic particles.
  • the immunoassay may be an unlabeled immunoassay.
  • Unlabeled ⁇ immunoassays are performed without labels and include, but are not limited to, techniques such as immunodiffusion and nephelometry.
  • the immunoassay may be a heterogeneous immunoassay.
  • the ⁇ immunoassay may be a homogeneous immunoassay. Homogeneous immunoassays do not require separation of the antibody-analyte complex from the other components of the immunoassay prior to analysis.
  • the immunoassay may be a competitive immunoassay. In competitive immunoassays, the analyte competes with a specific quantity of labeled antigen for ⁇ the antibody. In other embodiments, the immunoassay may be a noncompetitive immunoassay.
  • Antibodies and antigens of an immunoassay may be arranged in a variety of configurations. In some embodiments, the antibodies and antigens of the immunoassay are in solution. In other embodiments, either the antibody or the antigen is bound to a solid surface. In ⁇ yet another embodiment, the antibody or the antigen from the sample is bound to a solid surface. In some embodiments, the antibody is labelled. In some embodiments, the antigen is labelled. In some embodiments, more than one antibody may be used to detect the analyte. In other embodiments, two or more antibodies may bind to the same antigen.
  • two or more antibodies may bind to different epitopes of the same antigen. In other embodiments, two or more antibodies may bind to different antigens of an analyte. In other embodiments, a first antibody binds to an antigen, and a second antibody binds to the first antibody. In other embodiments, two antibodies compete to bind an antigen. In some ⁇ embodiments, a known amount of an identifiable antigen or analyte competes with the antigen or analyte for binding with an antibody. Any suitable immunoassay may be utilized.
  • immunoassay examples include, but are not limited to, immunoassay, such as sandwich immunoassay (e.g., monoclonal-polyclonal sandwich immunoassays), enzyme detection, such as enzyme ⁇ immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) (e.g., direct, indirect, competitive, and sandwich ELISA), competitive inhibition immunoassay (e.g., forward and reverse), enzyme multiplied immunoassay technique (EMIT), a competitive binding assay, bioluminescence resonance energy transfer (BRET), one-step antibody detection assay, homogeneous assay, heterogeneous assay, capture on the fly assay, and the like.
  • sandwich immunoassay e.g., monoclonal-polyclonal sandwich immunoassays
  • enzyme detection such as enzyme ⁇ immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) (e.g., direct, indirect, competitive, and sandwich ELISA)
  • immunoassays may be used to detect nucleic acid sequences. Once a desired degree of target nucleic acid sequence amplification is achieved, the amplification product can be detected using an immunoassay.
  • an immunoassay can be performed to capture target amplified nucleic acid sequences using the tag incorporated into the amplified target nucleic acid.
  • a ⁇ capture object such as, a bead, e.g., a magnetic bead
  • a ⁇ capture object include a binding member of a specific binding pair and captures the amplified target nucleic acid via interaction of the member of the binding pair with the other member of the binding pair, which other member that has been introduced into the amplified target nucleic acid during amplification.
  • the capture object is not coated with a nucleic acid that can bind to the amplified target nucleic acid.
  • Immunoassays of the present methods and devices may be analyzed using various methods to detect the antibody-analyte complex.
  • assays for measuring biomolecules or clinical chemistry panels in a sample include enzymatic methods by using enzymes to react with analyte, such as electrolytes, CO 2 , serum creatinine, blood urea nitrogen, and detect reaction product.
  • analyte such as electrolytes, CO 2 , serum creatinine, blood urea nitrogen, and detect reaction product.
  • the assays for measuring biomolecules or clinical chemistry panels in a ⁇ sample include chemical reaction methods, which are similar to enzymatic methods but with chemical reagents and using spectrophotometry.
  • the assays for measuring biomolecules or clinical chemistry panels in a sample include changes in pH level.
  • the assays for measuring biomolecules or clinical chemistry panels in a sample include the use of nephelometry. Nephelometry is used to measure the amount of turbidity or cloudiness by measuring scattered light and can be used in combination with immunoassays.
  • the assays for measuring biomolecules or clinical chemistry panels in a sample include the use of photometry, which measures absorbed light ⁇ (UV, visible, IR) to determine amount of an analyte in a solution or liquid.
  • the assays for measuring biomolecules or clinical chemistry panels in a sample include coagulation assays. In coagulation assays, reagents are added to blood to measure coagulating/clotting time. In yet other embodiments, the assays for measuring biomolecules or clinical chemistry panels in a sample include the use of electrophoresis.
  • systems include one or more computers for complete automation or partial automation.
  • systems include a computer operably connected to a memory having ⁇ instructions stored thereon which, when executed cause the computer to carry out one or more methods of the invention (e.g., discussed above).
  • the computer may be configured to cause a device of the invention to supply a predetermined volume of a sample fluid to a microfluidic passage, supply a metered amount of a diluent fluid to the microfluidic passage, and mix the sample fluid and the diluent fluid to prepare the sample fluid dilution.
  • the computer may be configured to calculate an absorbance for each detection chamber of the plurality to analyze the sample fluid. As discussed above, this can include calculating an average intensity of incident light from the light source, calculating an average intensity of the emitted light from each detection chamber of the plurality, deactivating the light source and calculate a dark image average intensity of the emitted light from each ⁇ detection chamber of the plurality, and calculating the absorbance for each detection chamber of the plurality based on the average intensity of incident light from the light source, the average intensity of the emitted light from each detection chamber of the plurality, and the dark image average intensity of the emitted light from each detection chamber of the plurality.
  • Systems may include a display and operator input device.
  • Operator input devices may, ⁇ for example, be a keyboard, mouse, or the like.
  • the processing module includes a processor which has access to a memory having instructions stored thereon for performing the steps of the subject methods.
  • the processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, memory storage devices, and input-output controllers, cache memory, a data backup unit, and many other devices.
  • GUI graphical user interface
  • the processor may be ⁇ a commercially available processor, or it may be one of other processors that are or will become available.
  • the processor executes the operating system and the operating system interfaces with firmware and hardware in a well-known manner, and facilitates the processor in coordinating and executing the functions of various computer programs that may be written in a variety of programming languages, such as Java, Perl, C++, Python, other high level or low level languages, as well as combinations thereof, as is known in the art.
  • the operating system typically in cooperation with the processor, coordinates and executes functions of the other components of the computer.
  • the operating system also provides scheduling, input-output ⁇ control, file and data management, memory management, and communication control and related services, all in accordance with known techniques.
  • the processor includes analog electronics which provide feedback control, such as for example negative feedback control.
  • the system memory may be any of a variety of known or future memory storage ⁇ devices. Examples include any commonly available random access memory (RAM), magnetic medium such as a resident hard disk or tape, an optical medium such as a read and write compact disc, flash memory devices, or other memory storage device.
  • RAM random access memory
  • the memory storage device may be any of a variety of known or future devices, including a compact disk drive, a tape drive, or a diskette drive.
  • Such types of memory storage devices typically read from, ⁇ and/or write to, a program storage medium (not shown) such as a compact disk. Any of these program storage media, or others now in use or that may later be developed, may be considered a computer program product. As will be appreciated, these program storage media typically store a computer software program and/or data.
  • Computer software programs typically are stored in system memory and/or the program ⁇ storage device used in conjunction with the memory storage device.
  • a computer program product is described comprising a computer usable medium having control logic (computer software program, including program code) stored therein.
  • the control logic when executed by the processor the computer, causes the processor to perform functions described herein.
  • some functions are ⁇ implemented primarily in hardware using, for example, a hardware state machine. Implementation of the hardware state machine so as to perform the functions described herein will be apparent to those skilled in the relevant arts.
  • Memory may be any suitable device in which the processor can store and retrieve data, such as magnetic, optical, or solid-state storage devices (including magnetic or optical disks or ⁇ tape or RAM, or any other suitable device, either fixed or portable).
  • the processor may include a general-purpose digital microprocessor suitably programmed from a computer readable medium carrying necessary program code. Programming can be provided remotely to processor through a communication channel, or previously saved in a computer program product such as memory or some other portable or fixed computer readable storage medium ⁇ using any of those devices in connection with memory.
  • a magnetic or optical disk may carry the programming, and can be read by a disk writer/reader.
  • Systems of the invention also include programming, e.g., in the form of computer program products, algorithms for use in practicing the methods as described above.
  • Programming according to the present invention can be recorded on computer readable media, e.g., any medium that can be read and accessed directly by a computer.
  • Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; portable flash drive; ⁇ and hybrids of these categories such as magnetic/optical storage media.
  • the processor may also have access to a communication channel to communicate with a user at a remote location.
  • remote location By remote location is meant the user is not directly in contact with the system and relays input information to an input manager from an external device, such as a computer connected to a Wide Area Network (“WAN”), telephone network, satellite network, or ⁇ any other suitable communication channel, including a mobile telephone (i.e., smartphone).
  • WAN Wide Area Network
  • systems according to the present disclosure may be configured to include a communication interface.
  • the communication interface includes a receiver and/or transmitter for communicating with a network and/or another device.
  • the communication interface can be configured for wired or wireless communication, including, ⁇ but not limited to, radio frequency (RF) communication (e.g., Radio-Frequency Identification (RFID), Zigbee communication protocols, Wi-Fi, infrared, wireless Universal Serial Bus (USB), Ultra Wide Band (UWB), Bluetooth® communication protocols, and cellular communication, such as code division multiple access (CDMA) or Global System for Mobile communications (GSM).
  • RF radio frequency
  • the communication interface is configured to include one or more communication ports, e.g., physical ports or interfaces such as a USB port, a USB-C port, an RS-232 port, or any other suitable electrical connection port to allow data communication between the subject systems and other external devices such as a computer terminal (for example, at a physician’s office or in hospital environment) that is configured for similar ⁇ complementary data communication.
  • the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol to enable the subject systems to communicate with other devices such as computer terminals and/or networks, communication enabled mobile telephones, personal digital ⁇ assistants, or any other communication devices which the user may use in conjunction.
  • the communication interface is configured to provide a connection for data transfer utilizing Internet Protocol (IP) through a cell phone network, Short Message Service (SMS), wireless connection to a personal computer (PC) on a Local Area Network (LAN) which is connected to the internet, or Wi-Fi connection to the internet at a Wi-Fi hotspot.
  • IP Internet Protocol
  • SMS Short Message Service
  • PC personal computer
  • LAN Local Area Network
  • Wi-Fi connection to the internet at a Wi-Fi hotspot.
  • the subject systems are configured to wirelessly communicate with a server device via the communication interface, e.g., using a common standard such as 802.11 or Bluetooth ® RF protocol, or an IrDA infrared protocol.
  • the server device may be another portable device, such as a smart phone, Personal Digital Assistant (PDA) or notebook computer; or a larger device such as a desktop computer, appliance, etc.
  • PDA Personal Digital Assistant
  • the server device has a display, such as a liquid crystal display (LCD), as well as an input device, such as buttons, a keyboard, mouse or touch-screen.
  • the communication interface is configured to automatically or ⁇ semi-automatically communicate data stored in the subject systems, e.g., in an optional data storage unit, with a network or server device using one or more of the communication protocols and/or mechanisms described above.
  • Output controllers may include controllers for any of a variety of known display devices for presenting information to a user, whether a human or a machine, whether local or remote. If ⁇ one of the display devices provides visual information, this information typically may be logically and/or physically organized as an array of picture elements.
  • a graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing graphical input and output interfaces between the system and a user, and for processing user inputs.
  • the functional elements of the computer may communicate with each other via system ⁇ bus. Some of these communications may be accomplished in alternative embodiments using network or other types of remote communications.
  • the output manager may also provide information generated by the processing module to a user at a remote location, e.g., over the Internet, phone or satellite network, in accordance with known techniques.
  • the presentation of data by the output manager may be implemented in accordance with a variety of known ⁇ techniques.
  • data may include SQL, HTML or XML documents, email or other files, or data in other forms.
  • the data may include Internet URL addresses so that a user may retrieve additional SQL, HTML, XML, or other documents or data from remote sources.
  • the one or more platforms present in the subject systems may be any type of known computer platform or a type to be developed in the future, although they typically will be of a class of ⁇ computer commonly referred to as servers. However, they may also be a main-frame computer, a workstation, or other computer type. They may be connected via any known or future type of cabling or other communication system including wireless systems, either networked or otherwise. They may be co-located or they may be physically separated. Various operating systems may be employed on any of the computer platforms, possibly depending on the type ⁇ and/or make of computer platform chosen.
  • kits include one or more sample analysis devices of the invention.
  • kits may include 1 or more sample analysis devices, such as 2 or more sample analysis devices, such as 3 or more sample analysis devices, and including 5 or more sample analysis devices.
  • kits may include one or more cartridges of the invention.
  • kits may include a number of cartridges ranging from 1 to 100, such as 2 to 50 and including 3 to 10.
  • components of the subject kits are provided in packaging, such as sealed packaging.
  • the sealed packaging is sterile packaging.
  • the subject kits may further include (in some embodiments) instructions for carrying out methods of the invention, e.g., performing a sample fluid dilution. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit.
  • FIG.8 presents a combiner circuit, fluidics, timing, and relay controls for use in addressing the above questions.
  • the fluidic system includes normally open (NO) ports to be sealed to prevent flow backwards in channel, and a pump that will be operated at different pressure values for each flow control channel.
  • reservoirs one for diluent, and ⁇ one for dye/plasma. The volume of the reservoirs was controlled with pipetting. Three relays were included for venting to the atmosphere. These were needed for changing pump pressure and different fluid manipulation requirements.
  • Relay 1, 2, and 3 are control signals to open and close the fluidic values shown in FIG.8.
  • FIG.9 presents the same basic architecture but with electronics and controls.
  • V_dac is the output of the D/A converter that is part of the Engineering control processor and was used to set the operating pressure of the pump.
  • V_Ppump is the signal that is proportional to the measured pressure at the pump and was measured by pump ⁇ interface electronics.
  • DIO 1, 2, and 3 are digital signals generated at the pump control processor and used to control the state of the fluidic valves.
  • V_Ptube is the voltage that is proportional to the tube pressure. First, pressure as a function of voltage applied to the pump was measured.
  • FIG.10A presents the architecture of a system used to make this measurement.
  • FIG.11 Voltages are applied to ⁇ a function generator (e.g., 10-turn potentiometer, see FIG.10B), and resulting pressures (V_Ptube) were measured and results are shown in FIG.11. As shown in FIG.11, the result is nearly linear, but a better curve was fit with a polynomial. A limited range was at Vin ⁇ 50mV, and there seemed to be a threshold at 50 mV. The pressure voltage reading was spot checked with a voltimeter. Subsequently, the ability of the system to generate pressure pulses was ⁇ assessed. Relay pulsing was conducted at 10ms with a 10% duty cycle. An added potentiometer for pump control via Vanalog is shown in FIG.10C.
  • a function generator e.g., 10-turn potentiometer, see FIG.10B
  • V_Ptube resulting pressures
  • FIG.12 A resulting graph of pressure (Pa) vs. time (count @ 10 ⁇ s) is shown in FIG.12. It was noted that flow information can be calculated from pressure measurement and knowledge of tube volume, as depicted in FIG.13.
  • the pressure pulse (Pa) was plotted as a function of time (seconds).
  • the ⁇ result is shown in FIG.14.
  • the rising edge of the graph is related to changing pressure from atmosphere; i.e.101500Pa to 101500+600Pa.
  • the rising edge is associated with the pump increasing pressure in combined tubes following initial transfer of air.
  • a later measurement with longer pulse indicates time constant of about 100ms to reach full amplitude.
  • the falling edge is due to pump not keeping up with filling additional tubing.
  • this method demonstrates good timing from relay valves.
  • 0.3V was applied (1Hz, 80% duty cycle @ relay). Results are shown in FIG.15.
  • 100ms is needed to reach full pump pressure.
  • Pump pressure amplitude was increased from 1830Pa to 5200Pa, and the pulse on-time was increased to 800ms. It took 100ms to raise pressure to 5200Pa from 3000Pa. Accordingly, it was shown that pump variation is about 1% pulse-to-pulse and day to ⁇ day.
  • Minimum working voltage for the pump is approximately 0.1V which corresponds to pressure equal to 1730Pa.
  • flow rates can be estimated from leading edge of pump pressure measurement and knowledge of tubing volume.
  • the pulses (e.g., 20ms pulses) were suitable to produce pulse-to-pulse uniformity differing by ⁇ 1%.
  • a pressure chamber was used as means of producing more uniform pressure pulses that, while applied demonstrate ⁇ 1-2%CV variation on a pressure pulse to pressure pulse basis.
  • the additional pressure chamber volume also can be used to modulate the pressure using pulse width modulation. Vanalog is modulated for low pressure experiments using a separate pulse generator. No feedback was used to control pressure. Results were obtained at 600Pa using a 3600ul chamber and a 50ul tube (10Hz, 20ms), ⁇ and are shown in FIG.23A-23B. As shown in FIG.23A-23B, successful uniformity was obtained at 600Pa.
  • FIG.23C Pressure chamber steady state measurements PWM input to pump from pulse generator are shown in FIG.23C.
  • the variation shown in FIG.23C is due to PWM input to pump (30% duty cycle).
  • a signal analysis of Vanalog from the pulse generator is shown in FIG.24A-24B.
  • pulse width precision shows no error; sampled at ⁇ 100kHz, (10usec per sample).
  • pulse amplitude (filtered) measures at 0.25%.
  • Example 4 The amount of time for the pressure chamber to be filled and emptied to change operating pressure was investigated. The results of filling and exhausting 3600ul chamber without relay control are shown in FIG.27A-27B. The results of exhausting a 3600ul tube with relay control are shown in FIG.27C. The results show that, at a working pressure of 5000Pa, ⁇ about 260ms is needed to change operating pressure given that pressure chamber first emptied to atmosphere through a relay control, and that 3600ul chamber pressurized to 500. Switching from lower pressure to higher pressure would not require chamber to be emptied. This would save ⁇ 40ms.
  • Example 4 The system depicted in FIG.28 was constructed. As shown in FIG.28, manual controls are included. For example, there is an on/off control for each relay, a start sequence momentary switch, a pressure up/down gauge (voltage meter at Vanalog or Vchamber), and a pump on/off switch. Also included is an additional relay for the pressure chamber. This can be ⁇ used for faster switching between pressures.
  • FIG.29C-29E shows a more detailed view of pressure change in the pressure chamber while switching. As indicated by the arrow, the pressure ⁇ change reflects filling of the tube when the relay is opened. The larger the ratio of volumes; i.e. chamber volume/tube volume, the smaller the pressure drop.
  • Example 5 The efficiency of transporting fluid through 1/16 th tube was assessed. The collection tube was tared, pipette transfer efficiency was measured five times, and 1/16 th tube transfer efficiency was measured ( ⁇ 500ul of tubing with fluoropel). Volumes of 200ul, 100ul, 50ul, 20ul, 10ul, and 5ul were used. Pressure of ⁇ 400Pa was employed with 500ms pulsing. The movement of liquid transfer through the tube was recorded via camera. Efficiency results are recorded in Table 1, below: ⁇ Table 1: Resulting photographs are shown in FIG.30A-30B and FIG.31A-31B. As shown in FIG. 30A, assuming 2mm 2 tube ID, this sample should measure 6-7ul (as measured by weight, ⁇ 6.7mg).
  • Example 6 The fluidic device depicted schematically in FIG.32 was constructed that can be used to fill microcuvettes. There is ⁇ 225ul total volume past capillary stop at input. A series capillary stop is needed to prevent backflow.
  • FIG.33A-33D show photographs depicting a progression as the lines are filled with fluid. Flow restriction prevents sample from flowing back into tube toward inlet.
  • Example 7 The experimental device of FIG.34A was created to demonstrate the splitting of droplets. The left-most inlet is for introducing a diluent. The middle inlet is where splitting and dyeing takes place. On the right, an outlet is used for mixing an homogenizing the sample. Liquid was then split to a known volume.
  • Droplet position was estimated with leading edge meniscus.
  • the droplet length in a 1/16 th tube was ⁇ measured for an initial volume (Table 3).
  • the mass of liquid collected at the outlet was also measured.
  • Table 2 ⁇ Table 3 Mean flows for without restriction were less than 1.5%. Fluid transfer efficiency (collected mass/1/16th tube volume) was greater than 98.4%.
  • the square tube was designed ⁇ as 2mm x 2mm with 4mm 2 area. Measured area was about 4.23mm 2 based upon optical measurement of length.
  • Example 9 A system similar to the one shown in FIG.34A was used to assess droplet behavior at a combining point. Expected droplet behavior is shown in FIG.39A-39B. As shown in FIG.39A, droplet height and contact angle should be visible during pulsing into air. The tube walls and a ⁇ ruler can be used to estimate volume. As depicted in FIG.39B, dye can be pulsed into liquid within the channel. Pulse pressure needs to be high enough to displace large droplet volume.
  • FIG.44A-44C air bubbles were used as an estimate of pressure flow from combining port.
  • a video recording was obtained. Representative frames from the video at approximately 0, 12 and ⁇ 25 seconds are shown in FIG.43A, FIG.43B, and FIG.43C, respectively.
  • This video was captured while rinsing the experimental combining tube. Stained water was completely emptied from the combining port and can be seen on the tube floor at the onset of the video. The port continued to be pressurized during the rinsing procedure. This created air bubbles in the main flow. These bubbles could be measured to estimate combining port flow.
  • Example 10 The following steps were performed with a light interrogation system comprising first and second pinhole plates: 1. Measure the average intensity (I incident ) of the illuminator pinhole region of the ⁇ image for each cuvette before a cuvette array is loaded at exposure Tincident e.g. 100us. 2. Run sample preparation steps, fill each cuvette, and wait for a specific time for reaction with reagents to generate color changes. 3. Load the cuvette array and record the average intensity of a cuvette at a ⁇ sequence of longer exposure time e.g.1ms, 10, 100ms, 500ms. Find the intensity close to 80% of the well capacity of the pinhole region: Iabs_i, and record the corresponding exposure time: T abs_i. 4.
  • Example 11 An assessment of the optical interrogation system of FIG.7A was carried out using a simulation and a bench test. It was found that a super photodiode of ⁇ 1mm has 6-9x10 8 ⁇ electrons well capacity, increasing SNR and reducing the cv% at high OD measurement. Both simulation and bench test showed that cv% is less than 0.5% up to 1.8 OD. Using a multiple- exposure algorithm, the measurement range extended to 4 OD at low cv%.

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Abstract

L'invention concerne des dispositifs d'analyse de fluide échantillon. Des dispositifs d'intérêt comprennent un logement conçu pour recevoir une cartouche, un collecteur, et un système d'interrogation conçu pour analyser le fluide échantillon. L'invention concerne également des cartouches destinées à être utilisées dans les dispositifs sujets. Des cartouches d'intérêt comprennent une pluralité de chambres de détection et un sous-système de commande fluidique pour fournir un fluide échantillon à la pluralité de chambres de détection comprenant une entrée d'échantillon conçue pour recevoir le fluide échantillon, une chambre de dosage de fluide échantillon pour fournir un volume prédéterminé du fluide échantillon à partir de l'entrée d'échantillon, une chambre de mélange pour homogénéiser le fluide échantillon avec un fluide diluant, et un passage microfluidique assurant une communication fluidique entre la chambre de dosage de fluide échantillon et la chambre de mélange. L'invention concerne également des procédés d'utilisation des dispositifs d'analyse de fluide échantillon et des cartouches pour analyser un échantillon fluidique.
PCT/US2025/018935 2024-03-11 2025-03-07 Dispositifs d'analyse de fluide échantillon, cartouches destinées à être utilisées dans ceux-ci, et leurs procédés d'utilisation Pending WO2025193544A1 (fr)

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US202463563861P 2024-03-11 2024-03-11
US63/563,861 2024-03-11
US202463566072P 2024-03-15 2024-03-15
US63/566,072 2024-03-15

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WO2025193544A1 true WO2025193544A1 (fr) 2025-09-18

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PCT/US2025/018935 Pending WO2025193544A1 (fr) 2024-03-11 2025-03-07 Dispositifs d'analyse de fluide échantillon, cartouches destinées à être utilisées dans ceux-ci, et leurs procédés d'utilisation

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