WO2025122781A1 - Flowrate controlled fluidics systems and immunoassay methods using the same - Google Patents

Abstract

A flowrate-controlled microfluidic system, microfluidic devices for on-cartridge bioanalysis, and an immunoassay method are described. The flowrate-controlled microfluidic implements automation code to run an immunoassay workflow with accurate metering. Methods may include one or more fluidics operations or steps defined by the software code, including loading, incubation, washing, and analysis of assay samples at a sensor region, such as an optical sensor region to be analyzed by an optical imaging system or instrument.

Description

FLOWRATE CONTROLLED FLUIDICS SYSTEMS AND IMMUNOASSAY METHODS

USING THE SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This international (PCT) application claims priority to United States Provisional Patent Application No. 63/607,272, filed on December 7, 2023, the content of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] The present subject matter relates to flowrate-controlled microfluidic systems, microfluidic devices for on-cartridge bioanalysis, and immunoassay methods using the same. Systems described herein implement automation code to run an immunoassay workflow with accurate metering. Methods may include one or more fluidics operations or steps defined by the software code, including loading, incubation, washing, and analysis of assay samples at a sensor region, such as an optical sensor region to be analyzed by an optical imaging system or instrument.

BACKGROUND

[0003] Healthcare providers depend on immunodiagnostic (ID) systems and methods to make informed decisions regarding patients’ disease diagnoses and subsequent prognoses. A point-of- care (POC) system for such testing, exhibiting an equal or better performance than sophisticated and expensive laboratory-based systems, would represent an indispensable tool for clinicians in providing prompt and accurate diagnoses and treatment plans.

[0004] For example, cardiovascular disease diagnosis is an area where POC-ID platforms can be helpful, particularly, in early or rapid disease detection and for making a distinction between disease types. To achieve a desired efficacy, a typical immunodiagnostic test workflow requires assay functions such as bio-fluidics sample preparation, metering, loading, washing, and analysis to be performed precisely and in a pre-determined fluid-flow sequence. To accurately manipulate bio-fluidics samples and subsequent assay steps, an active micro-fluidic handling system with point-of-care capabilities is thus required. The systems and methods described herein provides an approach that can execute all assay steps in a precise, accurate, and programmable manner. Additionally, the systems and methods allow for configurations having miniaturized footprints, making it particularly suited for point-of-care applications. BRIEF SUMMARY

[0005] In an aspect, a system for analysis of a liquid sample is provided. The system comprises a liquid reservoir with a first reservoir port and a second reservoir port; a pair of solenoid valves in pneumatic communication with the first port on the liquid reservoir and with the pump, each solenoid valve comprising a member movable between an open position and a closed position; a piezoelectric pump configured for bidirectional fluid flow, the pump comprising a first pump port and a second pump port, the pump in communication with the pair of solenoid valves; a sensor selected from the group consisting of a pressure sensor, a fluid flow sensor and both a pressure sensor and a fluid flow sensor; an electronic board in communication with the pump and the sensors, the electronic board configured to implement software code for control of the pump to cause liquid in the liquid reservoir to flow across the second reservoir port.

[0006] In certain embodiments of the system, the pump is in electrical communication with the pair of solenoid valves.

[0007] In certain embodiments of the system, the first pump port is in communication with a first solenoid valve in the pair of solenoid valves, the first solenoid valve in a normally closed position and movable to an open position.

[0008] In certain embodiments of the system, the second pump port is in communication with a second solenoid valve in the pair of solenoid valves, the second solenoid valve in a normally open position and movable to a closed position.

[0009] In certain embodiments of the system, the sensor is a fluid flow sensor, the fluid flow sensor positioned for fluid communication with liquid that exits or enters the second reservoir port of the liquid reservoir, the fluid flow sensor in communication with the pump.

[0010] In certain embodiments of the system, flow of liquid from or into the liquid reservoir is adjusted in response to a flow rate measurement provided by the fluid flow sensor.

[0011] In certain embodiments, the system comprises a cartridge holder configured to accept a sample cartridge that comprises an optical sensor region that is in communication with the second port of the reservoir through a fluid flow sensor.

[0012] In certain embodiments, the system comprises a valve positioned between the cartridge holder and the liquid reservoir and fluid flow sensor.

[0013] In another aspect, a system for fluidic analysis of an aqueous sample comprises a piezoelectric bidirectional pump configured to generate positive and negative pressures; a pressure sensor; a plurality of solenoid valves configured to toggle between the positive and negative pressures; a pressure-tight fluid reservoir; a fluid flow sensor; a cartridge holder configured to accept a sample cartridge comprising an optical sensor region; an isolation valve; and an electronic board in communication with the fluid flow sensor and the piezoelectric bidirectional pump, wherein the electronic board is configured to implement software code provided by a computer to adjust flow of aqueous sample in response to a flow rate measurement provided by the fluid flow sensor.

[0014] In certain embodiments, the system does not include a pressure reservoir.

[0015] In certain embodiments, implementation of the software code adjusts the flow of aqueous sample to a preset flow rate, or a variety of preset flow rate values orchestrated in a sequence. [0016] In certain embodiments, the isolation valve is configured to prevent fluid backflow or undesired flow in general.

[0017] In certain embodiments, the isolation valve is configured to maintain an aqueous sample essentially undisturbed over the optical sensor region when a sample cartridge is present in the cartridge holder.

[0018] In certain embodiments, the aqueous sample comprises functionalized detectable nanoparticles, such as detectable nanoparticles.

[0019] In certain embodiments, when the fluid volume exceeds a threshold fluid volume, the one or more of the solenoid valves are toggled between open or closed positions, thereby causing the pneumatic pump to alternate between generating positive pressure and negative pressure.

[0020] In another aspect, an immunoassay method comprises providing, in a pressure-tight fluid reservoir, an aqueous sample suspected of containing at least one analyte of interest; executing, via an electronic board, software code provided by a computer, said software code defining a fluidics operation; incubating the aqueous sample over an optical sensor region in a diagnostic cartridge; optionally washing the excess of unwanted materials; analyzing the incubated aqueous sample using an optical imaging system and an algorithm associated with the optical imaging system. In the inventive immunoassay method, executing said software code causes a piezoelectric bidirectional pump to generate an initial positive pressure, thereby causing the aqueous sample to flow from the pressure-tight fluid reservoir and toward the diagnostic cartridge. The aqueous sample flows through a fluid flow sensor and an isolation valve before reaching the diagnostic cartridge, wherein the fluid flow sensor measures a flow rate of the aqueous sample, and the isolation valve prevents backflow of aqueous sample after the leading edge of the aqueous sample has entered the cartridge. The solenoid valves are configured to toggle the piezoelectric bidirectional pump between positive and negative pressures. The fluid flow sensor and the piezoelectric bidirectional pump form a feedback loop. When the fluid volume of the aqueous sample exceeds a maximum threshold value, the solenoid valves are triggered such that the piezoelectric bidirectional pump generates a negative pressure. When the fluid volume of the aqueous sample falls below a minimum threshold value, the solenoid valves are triggered such that the piezoelectric bidirectional pump generates a positive pressure.

[0021] In certain embodiments of the immunoassay method, the fluidics operation defined by the software code defines a volume of the aqueous sample to be delivered.

[0022] In certain embodiments of the immunoassay method, the fluidics operation defined by the software code defines a minimum flow rate and a maximum flow rate for the aqueous sample. [0023] In certain embodiments of the immunoassay method, the fluidics operation defined by the software code defines a fixed flow rate for the aqueous sample.

[0024] In certain embodiments of the immunoassay method, the isolation valve is configured to maintain the aqueous sample essentially undisturbed over the optical sensor region (e.g., the isolation valve is configured to prevent backflow of aqueous sample after the leading edge of the aqueous sample has entered the cartridge).

[0025] In certain embodiments of the immunoassay method, the aqueous sample comprises functionalized detectable particles (e.g., nanoparticles).

[0026] In certain embodiments of the immunoassay method, the aqueous sample comprises functionalized detectable particles and the optical sensor region is functionalized to bind the functionalized detectable particles.

[0027] In certain embodiments, the immunoassay method comprises toggling the solenoid valves to cause the piezoelectric bidirectional pump to alternate between positive pressure generation and negative pressure generation.

[0028] In certain embodiments, the immunoassay method comprises toggling the solenoid valves to cause the piezoelectric bidirectional pump to alternate between positive pressure generation and negative pressure generation, wherein the positive pressure generation and the negative pressure generation moves fluid over the optical sensor region to remove non-specifically bound particles. [0029] In another aspect, a system for fluidic multiplex analysis of aqueous samples comprises a single, or only one, piezoelectric bidirectional pump configured to generate positive and negative pressures; a plurality of solenoid valve sets, wherein each solenoid valve set comprises a plurality of solenoid valves operably connected to a shared pressure regulator configured to toggle between the positive and negative pressures; a plurality of fluid reservoirs, wherein a fluid reservoir is in fluid connection with each pressure regulator; a plurality of fluid flow sensors, wherein a fluid flow sensor is in fluid connection with each fluid reservoir; a plurality of isolation valves, wherein an isolation valve is in fluid connection with each fluid flow sensor; a cartridge holder comprising a fluidics interface comprising a plurality of fluid inputs, each fluid input being in fluid connection with a different isolation valve, and an electronic board in communication with the piezoelectric bidirectional pump, pressure regulators, and fluid flow sensors. In an aspect, the cartridge holder is configured to accept a sample cartridge comprising an optical sensor region and a plurality of lanes for the flow of aqueous sample, each lane being in fluid connection with a different fluid input when the sample cartridge is present in the cartridge holder. In another aspect, the pressure regulators and fluid flow sensors are electronically connected to form a feedback loop. In another aspect, the electronic board is configured to implement software code provided by a computer to adjust flow of aqueous sample in response to a flow rate measurement provided by one or more of the fluid flow sensors and/or in response to a pressure measurement provided by one or more of the pressure regulators.

[0030] In another aspect, described is a system for multiplex analysis of a fluidic sample. The system comprises a cartridge holder and a plurality of sample input arrangements. Each sample input arrangement comprises: a piezoelectric bidirectional pump configured to generate positive and negative pressures, a pressure sensor, a plurality of solenoid valves configured to toggle between the positive and negative pressures, a pressure-tight fluid reservoir, a fluid flow sensor, an isolation valve, and an electronic board in communication with the fluid flow sensor and the piezoelectric bidirectional pump. The electronic board is configured to implement software code provided by a computer to adjust flow of aqueous sample in response to a flow rate measurement provided by the fluid flow sensor. The cartridge holder comprises a fluidics interface comprising a plurality of fluid inputs. The cartridge holder is configured to accept a sample cartridge comprising an optical sensor region and a plurality of lanes for the flow of aqueous sample, each lane configured to be in fluid connection with a different fluid input when the sample cartridge is present in the cartridge holder.

[0031] In another aspect, a cartridge for use in an flowrate controlled immunoassay system is provided. The cartridge comprises a region of interest (e.g., an optical sensor region), a fluid reservoir, detectable particles, and one or more inputs configured to interact with a pneumatic interface. The pneumatic interface is configured to attach to the cartridge, the pneumatic interface being connected to an external pneumatic system. The external pneumatic system comprises a plurality of bidirectional pumps, each in pneumatic communication with a pressure regulator and a set of solenoid valves. Each pressure regulator is associated with its own set of solenoid valves such that each pressure regulator toggles the solenoid valves to provide either a positive or negative pressure from the bidirectional pump in communication therewith. Each grouping of a bidirectional pump, a pressure regulator, set of solenoid valves, and an isolation valve makes up a discrete fluidic assay (e.g., discrete diagnostic lanes).

[0032] In certain embodiments, the cartridge comprises the pneumatic interface. [0033] In certain embodiments, the cartridge comprises a removable pneumatic interface.

[0034] In certain embodiments, the cartridge comprises a portion configured to interact with a pneumatic interface separate from the cartridge.

[0035] In certain embodiments, the cartridge comprises an output, such as a waste output.

[0036] In certain embodiments, the cartridge comprises a removable waste container.

[0037] In certain embodiments, the detectable particles are provided on the cartridge as dried- down (e.g., lyophilized) reagents. In an aspect, the detectable particles reconstitute during operation as fluid flows through the dried-down reagents. Hence, as fluid sample flows through the dried-down reagent, the detectable particles having binding members bound thereto (i.e., functionalized detectable particles) bind to analyte of interest present in the fluid sample prior to reaching the region of interest for optical detection. Reconstitution can be achieved in certain embodiments in situ (i.e., during the course of operation as part of a fluidic immunoassay operation) or in a separate fluid handling step (e.g., prior to the immunoassay operation).

[0038] In an aspect, a diagnostic cartridge is provided. The cartridge comprises a pressure-tight fluid reservoir, one or more diagnostic lanes downstream of the pressure-tight fluid reservoir, an input upstream of the pressure-tight fluid reservoir, the input configured for pressure-tight pneumatic connection, and an output configured to direct fluid downstream of the one or more diagnostic lanes.

[0039] In certain embodiments, the diagnostic cartridge comprises a plurality of diagnostic lanes. [0040] In certain embodiments of the diagnostic cartridge, the one or more diagnostic lanes comprise dried detectable particles.

[0041] In certain embodiments of the diagnostic cartridge, the detectable particles are functionalized nanoparticles.

[0042] In certain embodiments, the diagnostic cartridge comprises a plurality of diagnostic lanes, wherein at least two of the diagnostic lanes comprise different dried detectable particles.

[0043] In certain embodiments, each diagnostic lane comprises a different type of detectable particle. In other words, in the context of a given diagnostic cartridge, the diagnostic lanes all comprise unique detectable particles.

[0044] In certain embodiments, the diagnostic cartridge is configured to test for multiple different analytes within a single provided sample. In such embodiments, a plurality of diagnostic lanes is provided in the diagnostic cartridge, wherein each diagnostic lane tests for a different analyte of interest. In such embodiments, each diagnostic lane comprises a different detectable particle capable of binding a different analyte that may be present in a tested sample. [0045] In certain embodiments of the diagnostic cartridge, the detectable particles are functionalized nanoparticles.

[0046] In certain embodiments of the diagnostic cartridge, the cartridge is configured to removably attach to a pneumatic interface.

[0047] In certain embodiments of the diagnostic cartridge, the cartridge is configured to removably attach to a waste container.

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG. 1 is a schematic view of a single -plex embodiment of the flowrate controlled single- plex system.

[0049] FIG. 2 is a schematic view of a multiplex embodiment of the flowrate controlled single- plex system.

[0050] FIG. 3 is a schematic view of an exemplary embodiment of an integrated cartridge-based fluidics system.

[0051] FIGs. 4A and 4B describe the flow rate (FIG. 4A) and cumulative volume (FIG. 4B) at different points in an exemplary, non-limiting fluidics operation carried out by a fluidics system. [0052] FIGs. 5A and 5B show an output from the successful operation of an exemplary embodiment of a five-plex fluidics system described herein.

DETAILED DESCRIPTION

[0053] Various aspects now will be described more fully hereinafter. Such aspects may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey its scope to those skilled in the art.

[0054] Where a range of values is provided, it is intended that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. For example, if a range of 1 pm to 8 pm is stated, it is intended that 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, and 7 pm are also explicitly disclosed, as well as the range of values greater than or equal to 1 pm and the range of values less than or equal to 8 pm.

[0055] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a “valve” includes a single valve as well as two or more of the same or different valves, reference to a “sensor” includes a single excipient as well as two or more of the same or different excipients, and the like.

[0056] The disjunctive “or” is inclusive, unless otherwise specified. For example, “X or Y” means “X, Y, or both X and Y” unless otherwise specified. [0057] The word "about" when immediately preceding a numerical value means a range of plus or minus 10% of that value, e.g., “about 50” means 45 to 55, “about 25,000” means 22,500 to 27,500, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation. For example, in a list of numerical values such as “about 49, about 50, or about 55,” the term “about 50” means a range extending to less than half the interval(s) between the preceding and subsequent values, e.g., more than 49.5 to less than 52.5. Furthermore, the phrases “less than about” a value or “greater than about” a value should be understood in view of the definition of the term “about” provided herein.

[0058] The systems, devices, and methods of the present disclosure can comprise, consist essentially of, or consist of, the components or steps disclosed.

[0059] All ranges disclosed herein include all subranges contained therein, as well as all discreet values contained therein. Additionally, all ranges disclosed herein are inclusive of their endpoints, unless otherwise specified. For example, “X to Y” means “greater than or equal to X and less than or equal to Y” unless otherwise specified.

[0060] By reserving the right to proviso out or exclude any individual members of any such group, including any sub-ranges or combinations of sub-ranges within the group, that can be claimed according to a range or in any similar manner, less than the full measure of this disclosure can be claimed for any reason. Further, by reserving the right to proviso out or exclude any individual components or groups thereof, or any members of a claimed group, less than the full measure of this disclosure can be claimed for any reason.

[0061] The disclosures of any patent, patent application, or publication referenced in this disclosure are incorporated herein by reference in their entireties in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. This disclosure will govern in the instance that there is any inconsistency between the patents, patent applications and publications cited and this disclosure.

[0062] As used herein to indicate a location of a system component, the direction of fluid flow, or otherwise refer to a point or relative position within a system described herein, the term “downstream” means either at a position further away from a pump or closer to a region of interest than a reference point or system component, or in a direction away from a pump or toward a region of interest. Conversely, “upstream” means either at a position closer to a pump or further from a region of interest than a reference point of system component, or in a direction toward a pump or away from a region of interest. By way of illustration and without limitation, a component B is downstream of a component A if component B is between component A and a region of interest. Also by way of illustration and without limitation, a component C is upstream of component A if it is between component A and a pump.

[0063] For convenience, certain terms employed in the specification, examples and claims are collected here. Unless defined otherwise, all technical and scientific terms used in this disclosure have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0064] As shown in FIG. 1, which depicts an exemplary, non-limiting embodiment, an electronic board 1 is provided in electrical communication with a device (e.g., a computer, server, or mobile device) that contains software code (e.g., automation code), a pneumatic pump 2 having a first pump port 2a and a second pump port 2b, and one or more sensors (e.g., a pressure sensor 4 and/or a fluid flow sensor 6). The software code defines a fluidics operation, which is implemented by the electronic board 1 to cause the pneumatic pump 2 to generate a positive pressure via the second pump port 2b. The first pump port 2a is in direct pneumatic communication with a first solenoid valve 3a, which is normally in a closed position. The second pump port 2b is in direct pneumatic communication with a second solenoid valve 3b, which is normally in an open position. The pair of solenoid valves 3 is in direct pneumatic communication with a fluid reservoir 5 via a fluid reservoir input 5a. A net positive pressure applied upon the fluid reservoir 5 via the fluid reservoir input 5a causes flow of fluid out of the fluid reservoir 5 via a fluid reservoir output 5b. The fluid reservoir output 5b is in direct fluid communication with a fluid flow sensor 6, which is configured to measure a parameter of the fluid flow (e.g., a volume of fluid or a rate of fluid flow). The fluid flows past the fluid flow sensor 6 to reach an isolation valve 7, which is also in electrical communication with the electronic board 1. Based on an interval of time or amount of fluid defined by the software code, the isolation valve 7 is toggled by the electronic board to a closed position to isolate fluid in a substantially undisturbed (e.g., substantially static) state to prevent backflow in a direction back toward the pneumatic pump 2. Downstream of the isolation valve is a fluidics cartridge holder 8 configured to receive a fluidics cartridge 9. When a fluidics cartridge 9 is inserted into or onto the cartridge holder 8, fluid held undisturbed by the isolation valve 7 is allowed to incubate over a region of interest 10. In certain embodiments, the region of interest 10 is an optical sensor region which can be analyzed by an optical instrument (e.g., a spectrophotometer, or other light imaging device). Fluid flows to the region of interest 10 via an input Ila that is in fluid communication with the isolation valve 7. The input Ila may be provided on the cartridge holder 8 or on the fluidics cartridge 9.

Additionally, an output lib is provided either on the cartridge holder 8 or on the fluidics cartridge 9. An optional waste container 12 may be provided in fluid communication with the output lib. [0065] In certain embodiments, such as the one depicted in FIG. 1, a feedback loop is formed between the electronic board 1, the pneumatic pump 2, a pressure sensor 4, and a fluid flow sensor 6. The pneumatic pump 2, pressure sensor 4, and fluid flow sensor 6 are all in electrical communication with the electronic board. In such embodiments, the pressure sensor 4 is preferably positioned between the second pump port 2b and any components downstream of the second pump port 2b, such as one or more solenoid valves (e.g., the second solenoid valve 3b shown in FIG. 1).

[0066] The system comprises a programmable electronic board configured to implement software code, in an embodiment. The software code defines a fluidics operation or cycle of the system. [0067] The software code can define fluidics operations such as minimum flow rate(s), maximum flow rate(s), minimum fluid volume(s), maximum fluid volume(s), wash cycles, incubation times, and the like. For example, the fluidics operation defined by the software code can define one or more of (i) a volume of the aqueous sample to be delivered, and/or (ii) a minimum flow rate, and/or (iii) a maximum flow rate.

[0068] The software code can be stored on a device connected to the electronic board, which implements the software code to carry out a sequence (e.g., one or more operations or cycles) via the system. The device can be connected to the electronic board via a wired or wireless connection.

[0069] Executing the software code causes the pneumatic pump to generate an initial positive pressure, thereby causing the aqueous sample to flow from a fluid reservoir and toward a region of interest (e.g., an optical sensor region), preferably toward a diagnostic cartridge.

[0070] In certain embodiments, the device on which the software code is stored is a computer. In certain embodiments, the device on which the software code is stored is a mobile device, such as a smartphone or a tablet. In certain embodiments, the device on which the software code is stored is a server. In such embodiments, the server may be local or remote to the system.

[0071] In an aspect, the electronic board is in communication with the pneumatic pump and the one or more sensors. In certain embodiments, the electronic board is in communication with the pneumatic pump and a fluid flow sensor. In certain embodiments, the electronic board is in communication with the pneumatic pump and a pressure sensor. In certain embodiments, the electronic board is in communication with the pneumatic pump, a fluid flow sensor, and a pressure sensor.

[0072] In certain embodiments, the electronic board is in communication with the pneumatic pump and one or more sensors, and the electronic board is configured to implement software code for control of the pump to cause liquid in the liquid reservoir to flow across the second reservoir port.

[0073] In certain embodiments, the electronic board is configured to implement software code provided by a computer to adjust flow of aqueous sample in response to a flow rate measurement provided by the fluid flow sensor.

[0074] In certain embodiments, the electronic board is configured to implement software code provided by a computer to adjust flow of aqueous sample in response to a pressure measurement provided by the pressure sensor.

[0075] In certain embodiments, a feedback loop is formed between the electronic board, the pneumatic pump, and one or more sensors. The operation of these components is defined by the software code implemented by the electronic board based on measurements taken by the one or more sensors.

[0076] For example, in certain embodiments, a feedback loop may be formed between the electronic board, the pneumatic pump, and the fluid flow sensor such that, when the fluid volume of the aqueous sample exceeds a maximum threshold value, the solenoid valves are triggered such that the piezoelectric bidirectional pump generates a negative pressure. Conversely, when the fluid volume of the aqueous sample falls below a minimum threshold value, the solenoid valves are triggered such that the piezoelectric bidirectional pump generates a positive pressure. In such embodiments, the fluid flow sensor provides measurements to the electronic board and/or the device storing the software code such that the electronic board can receive instruction from the device in response to the fluid flow sensor measurements.

[0077] In certain embodiments, the electronic board relays the measurements to the device storing the software code.

[0078] In other embodiments, the fluid flow sensor may provide the measurements directly to the device storing the software code.

[0079] In certain embodiments, a feedback loop may be formed between the electronic board, the pneumatic pump, the pressure sensor, and the fluid flow sensor such that, when the fluid volume of the aqueous sample exceeds a maximum threshold value, the solenoid valves are triggered such that the piezoelectric bidirectional pump generates a negative pressure. Conversely, when the fluid volume of the aqueous sample falls below a minimum threshold value, the solenoid valves are triggered such that the piezoelectric bidirectional pump generates a positive pressure. In such embodiments, the pressure sensor measures the pressure provided by the pneumatic pump and provides said pressure measurement to the electronic board and/or the device storing the software code such that the electronic board can receive instruction from the device in response to the pressure sensor and/or fluid flow sensor measurements.

[0080] In certain embodiments, the electronic board relays the pressure sensor and fluid flow sensor measurements to the device storing the software code.

[0081] By way of example and without limitation, in an embodiment of the system, when a fluid volume exceeds a defined maximum, one or more solenoid valves may be triggered to close. Depending on the instructions provided by the software code, the closed solenoid valve(s) may then be toggled to an open position after a defined time interval has elapsed.

[0082] Similarly, by way of example and without limitation, in an embodiment of the system, when a fluid volume exceeds a defined maximum, the isolation valve may be triggered to close. Depending on the instructions provided by the software code, the closed isolation valve may then be toggled to an open position after a defined time interval has elapsed.

[0083] An orchestrated flow program may thus be implemented in which a flow may start and stop by the triggering of solenoid valves via the feedback loop between the electronic board and the flow and/or pressure sensors. The orchestrated flow program may initiate flow of fluid through the system, halt and/or reverse the flow, isolate the flow, and/or resume flow after a period of time or after the fluid flow sensor and/or the pressure sensor provide one or more measurements triggering a further fluidics operation.

[0084] In this way, immunoassays may be performed as cycles. For example and without limitation, the software code may define a first “push” cycle by which a positive pressure is generated to cause flow of sample from the fluid reservoir for a defined period of time or until a defined volume of fluid has flowed (e.g., as measured by the flow rate sensor). The first push cycle could then be followed by a “pull” cycle whereby a net negative pressure is generated to reverse the direction of the fluid flow. Additionally, or alternatively, a cycle may comprise a static or substantially static period whereby the pump does not generate any pressure, either positive or negative. Push, pull, and static cycles can be strung together as defined by the software code to choreograph a sequence defining a fluidic operation or a portion of a fluidic operation.

[0085] For example, a wash cycle is one exemplary portion of a fluidic operation that can be defined by the software code. In an exemplary wash cycle, one or more push cycles and optionally one or more static cycles are followed by one or more pull cycles. Alternating between push and pull cycles (with optional static cycles for optional incubation periods) allows for alternating flow to wash unwanted materials from an aliquot of fluid sample. In cycles involving incubation, it is preferable that said incubation occurs over a region of interest in the system (e.g., over an optical sensor region).

[0086] In an aspect, the pneumatic pump is configured for bidirectional fluid flow and is preferably a pneumatic piezoelectric pump. Such “bidirectional” operability means that the pump can generate both positive and negative pressures, either simultaneously or at different time points.

[0087] Preferably the pneumatic pump has at least two pump ports - e.g., a first pump port and a second pump port.

[0088] A person of ordinary skill in the art will understand “positive” pressure to mean pressure which causes fluid to move in a direction away from the bidirectional pneumatic pump (i.e., to push a fluid sample away from the bidirectional pneumatic pump) and downstream in the system (e.g., in a direction toward an immunoassay cartridge).

[0089] A person of ordinary skill in the art will understand “negative” pressure to mean pressure which causes a net reduction in flowrate or results in a reversal of fluid flow resulting in flow back toward the bidirectional pneumatic pump (i.e., by creating a vacuum or partial vacuum).

[0090] When combined in pneumatic communication with a fluid reservoir, the bidirectional pneumatic pump can thus generate positive flow rates and negative flow rates, where positive flow rate is defined as a net flow in a direction away from the pump and negative flow rate is defined as a net flow in a direction toward the pump.

[0091] The bidirectional pneumatic pump preferably does not require a pressure reservoir to operate. In certain embodiments, the bidirectional pneumatic pump operates via intake of atmospheric air. In other embodiments, the bidirectional pneumatic pump operates uses compressed air.

[0092] In an aspect, the bidirectional (i.e., push-pull) functionality of the pneumatic pump enables operations important to immunoassaying methods without requiring additional components to be included in the system. For example, by alternating between positive and negative pressures, mixing can be achieved without the need for an additional mixing chamber.

[0093] The bidirectional pneumatic pump is provided in electrical communication with an electronic board configured to implement software code that controls the pump. That is, when the software code is run, the pump exerts positive or negative pressure to cause flow of a fluid sample through the system.

[0094] As shown in FIG. 1, which depicts an exemplary, non-limiting embodiment, the pneumatic pump 2 is in electrical communication with the electronic board 1 and the fluid flow sensor 6 to form a feedback loop. The pump 2 is in pneumatic communication with a pair of solenoid valves 3. In the depicted embodiment, the pump 2 has a first pump port 2a and a second pump port 2b, where the first pump port 2a is in direct pneumatic communication with the first solenoid valve 3a, and the second pump port 2b is in direct pneumatic communication with the second solenoid valve 3b. As shown in FIG. 1, each of the first solenoid valve 3a and second solenoid valve 3b.

[0095] In certain embodiments, such as the one depicted in FIG. 1, a pressure sensor 4 is provided, which is in electrical communication with the electronic board 1, pump 2, and fluid flow sensor 6, and is thus part of the feedback loop described above in reference to FIG. 1. In such embodiments, the pressure sensor 4 is preferably positioned between the second pump port 2b and any components downstream of the second pump port 2b, such as one or more solenoid valves (e.g., the second solenoid valve 3b shown in FIG. 1).

[0096] In certain embodiments, two or more solenoid valves are provided in the system. Each solenoid valve has a member that is movable such that the solenoid valve is in either an open position or a closed position.

[0097] In certain embodiments, a pair of solenoid valves - i.e., a first solenoid valve and a second solenoid valve - are provided in the system. The first solenoid valve is in pneumatic communication with the first pump port on the bidirectional pneumatic pump and the second solenoid valve is in pneumatic communication with the second pump port on the bidirectional pneumatic pump. The first solenoid valve is normally in a closed position and is movable to an open position. Conversely, the second solenoid valve is normally in an open position and is movable to a closed position.

[0098] When combined with the bidirectional pneumatic pump - preferably a piezoelectric pump - the solenoid valves can serve as a switch to toggle between the pump’s positive and negative pressures and, therefore, act together as a switch for controlling flowrate.

[0099] The solenoid valves are provided in electrical communication with the bidirectional pneumatic pump, one or more sensors (e.g., a pressure sensor and/or a fluid flow sensor), and the electronic board such that, according to the software code provided implemented by the electronic board, the solenoid valves are toggled between their open and closed (or ‘on’ and “off’) positions to result in a positive or negative pressure being exerted by the bidirectional pneumatic pump. [0100] When, according to the implemented software code and as measured by the one or more sensors, the volume of fluid or the flowrate of the fluid exceeds a defined maximum threshold, the solenoid valves are triggered such that the piezoelectric bidirectional pump generates a negative pressure. [0101] Depending on the extent to which the fluid volume or flowrate exceeds the defined maximum threshold, a negative pressure is generated in place of the positive pressure.

[0102] In other embodiments, depending on the extent to which the fluid volume or flowrate exceeds the defined maximum threshold, the generated negative pressure can be in addition to an exerted positive pressure (i.e., to reduce the positive flowrate) or in place of the positive pressure (i.e., only a negative pressure exerted). Hence, depending on the software code, the system can trigger the solenoid valves such that only one solenoid valve is open at a time, or such that both solenoid valves are open simultaneously.

[0103] As discussed above, FIG. 1 shows one exemplary, non-limiting embodiment of the inventive system having a pair of solenoid valves 3. The first solenoid valve 3a is in direct communication with the first pump port 2a and the second solenoid valve 3b is in direct communication with the second pump port 2b. The first solenoid valve 3a is normally in a closed (or “off’) position, meaning that the negative pressure produced by the pump 2 via the first pump port 2a is not exerted on fluid in the system. The second solenoid valve 3b is normally in an open (or “on”) position, meaning that the positive pressure produced by the pump 2 via the second pump port 2b is exerted on fluid in the system to cause positive flowrate (i.e., flow away from the pump).

[0104] A net positive pressure produced by the pump causes flow of fluid from a fluid reservoir provided in the system.

[0105] In certain embodiments, pressure provided by the pump (and thus flow of liquid from or into the liquid reservoir) is adjusted in response to a volume or flow rate measurement provided by the flow sensor.

[0106] In embodiments, a fluid reservoir is provided downstream of the pump and solenoid valves. Preferably, the fluid reservoir is pressure-tight.

[0107] The fluid reservoir and downstream tubing provides fluid sample, including any optional buffers and/or detectable particles that may bind to analyte(s) of interest.

[0108] When a net positive pressure acts upon the fluid reservoir, positive flow (i.e., away from the pump) occurs, causing flow of fluid provided in the fluid reservoir toward the region of interest via a fluid flow sensor and optionally via an isolation valve provided between the fluid flow sensor and the region of interest.

[0109] The fluid reservoir comprises at least one input and at least one output.

[0110] In certain embodiments, the fluid reservoir has an input in direct pneumatic communication with the solenoid valves and an output in direct pneumatic communication with a fluid flow sensor. [0111] In certain embodiments, a liquid sample (e.g., an aqueous sample) suspected of containing one or more analytes of interest is provided by the fluid reservoir and downstream tubing.

[0112] In certain embodiments, the liquid reservoir tubing contains detectable particles. In certain embodiments, the detectable particles are nanoparticles. In certain embodiments, the detectable particles (e.g., nanoparticles) have binding members bound to them (i.e., the nanoparticles are functionalized) such that the detectable particles bind to an analyte of interest present in a fluid sample.

[0113] As shown in the exemplary, non-limiting embodiment of FIG. 1, the fluid reservoir 5 has a fluid reservoir input 5a and a fluid reservoir output 5b, whereby positive pressure from the pump 2 (permitted by the second solenoid valve 3b being in an open positive) acts upon the fluid reservoir 5 via the fluid reservoir input 5a, causing positive flow of fluid sample from the fluid reservoir output 5b.

[0114] In certain embodiments, the fluid reservoir and downstream tubing provides detectable particles having binding members bound thereto.

[0115] In certain embodiments, the detectable particles are functionalized nanoparticles that are capable of binding to the analyte of interest such that the bound analyte (i.e., analyte plus nanoparticle) can subsequently bind to a functionalized region of interest. In certain embodiments, the flowing fluid sample containing analyte and bound nanoparticles is permitted to incubate at the region of interest to allow for binding at the region of interest. In an aspect, incubating at the region of interest allows for accurate analyte detection. In an aspect, incubation can be achieved effectively and efficiently by including the isolation valve, which maintains the fluid sample at the region of interest in a substantially undisturbed (i.e., static or substantially static) state.

[0116] In certain embodiments, the detectable particles comprise a binding member having binding affinity for the analyte of interest.

[0117] In certain embodiments, the detectable particles comprise a binding member that specifically binds the analyte of interest.

[0118] In certain embodiments, the region of interest comprises a binding member having binding affinity for the analyte of interest. In certain embodiments, the region of interest comprises a binding member that specifically binds the analyte of interest. In certain embodiments, the region of interest comprises a binding member that binds the conjugate formed between the analyte of interest and the detectable particle.

[0119] In certain embodiments, the region of interest is analyzed by an optical system or instrument capable of detecting the detectable particles. [0120] The detectable particles comprise or consist essentially of a metal, preferably a transition metal or a noble metal.

[0121] In certain embodiments, the detectable particles comprise or consist essentially of one or more metals selected from gold, silver, platinum, palladium, iridium, osmium, rhodium, ruthenium and alloys thereof. In certain embodiments, the detectable particles are nanoparticles having at least a plasmonic material incorporated therein (e.g., gold, aluminum, silver or a metamaterial).

[0122] In certain embodiments, the detectable particles consist of a metal selected from gold, silver, platinum, palladium, iridium, osmium, rhodium, and ruthenium. For example, in certain embodiments, the detectable particles comprise or consist essentially of gold, preferably on the surface. In embodiments, the detectable particles consist of gold.

[0123] The detectable particles have an average diameter ranging from about 1 nm to about 1500 nm, or from about 25 nm to about 500 nm, or from about 50 nm to about 250 nm or from 100 to 200 nm.

[0124] In certain embodiments, the detectable particles resonate at a wavelength ranging from about 250 nm to about 1000 nm, or about 300 nm to about 950 nm, or about 350 nm to about 900 nm, or about 400 nm to about 850 nm, or about 450 nm to about 800 nm.

[0125] In certain embodiments, the detectable particles are magnetic.

[0126] In certain embodiments, the detectable particles have a shell-core structure, wherein the core is magnetic and the shell is a transition metal. In embodiments, the core is iron, an oxide of iron, or an iron alloy. In preferred embodiments, the core is iron or iron (II, III) oxide (i.e., Fe Kh). The shell is preferably gold.

[0127] In certain embodiments, the diameter of the magnetic core (i.e., the average magnetic core diameter of a plurality of detectable particles) may be in the range of from about 1 nm to about 300 nm, or from about 25 nm to about 250 nm, or from about 50 nm to about 200 nm, or from about 75 nm to about 150 nm and the thickness of the shell may be in the range of from about 0.5 nm to about 50 nm, or from about 1 nm to about 40 nm, or from about 5 nm to about 30 nm, or from about 10 nm to about 25 nm.

[0128] In certain core-shell embodiments, the diameter of the magnetic core may be in the range of from 0.5 nm to about 60 nm, or from about 1 nm to about 40 nm, or from about 3 nm to about 30 nm, or from about 5 nm to about 25 nm, and the shell may have a thickness in the range of from about 1 nm to about 100 nm, or from about 5 nm to about 80 nm, or from about 5 nm to about 60 nm, or from about 10 nm to about 45 nm. [0129] Optionally, an intermediate layer may be provided between the core and shell of the detectable particles (i.e., the intermediate layer may be provided as a first shell between the core and the outer shell). The intermediate layer may be comprised of silica. The diameter of the magnetic core (e.g., the average magnetic core diameter of a plurality of detectable particles) may be in the range of from about 1 nm to about 300 nm, or from about 25 nm to about 250 nm, or from about 50 nm to about 200 nm, or from about 75 nm to about 150 nm. The thickness of the intermediate layer may be in the range of from about 0.5 nm to about 50 nm, or from about 1 nm to about 40 nm, or from about 5 nm to about 30 nm, or from about 10 nm to about 25 nm. The thickness of the shell may be in the range of from about 0.5 nm to about 50 nm, or from about 1 nm to about 40 nm, or from about 5 nm to about 30 nm, or from about 10 nm to about 25 nm.

The detectable particle may have a diameter (i.e., an average diameter) in the range of from about 25 nm to about 500 nm, or from about 50 nm to about 450 nm, or from about 75 nm to about 350 nm, or from about 100 nm to about 300 nm.

[0130] In other embodiments, the detectable particles do not have a core-shell structure. That is, in such embodiments, the detectable particles consist essentially of a transition metal or alloy thereof. For example, the detectable particles may consist essentially of gold.

[0131] In other embodiments, other optically detectable markers or labels may be used. For example, fluorescent or chemiluminescent particles may be used. In certain non-limiting embodiments, europium beads or quantum dots may be used.

[0132] In an aspect, it is preferable that a fluid sample to be analyzed be allowed to incubate undisturbed for a period of time. For example, it is preferable that the fluid sample be allowed to incubate undisturbed for a period of time over a region of interest, such as over an optical sensor region.

[0133] After fluid has reached the region of interest (e.g., an optical sensor region), it is preferable that the system be configured to minimize-and more preferably prevent-backflow and any undesired downstream flow. Undesired downstream flow might result from, e.g., changes in temperature, hydrostatic pressure, or other atmospheric or environmental variables.

[0134] To achieve this, in certain embodiments of the present system, an isolation valve may be provided to assist with precisely positioning a fluid sample over a region of interest (e.g., an optical sensor region) to incubate during an assay operation. The isolation valve may be, for example, a solenoid diaphragm isolation valve.

[0135] In addition to undesired fluid backflow, the isolation valve may be provided to prevent undesired fluid flow in general. [0136] Thus, certain embodiments comprise an isolation valve configured to maintain a fluid sample (e.g., an aqueous sample) substantially undisturbed over a region of interest (e.g., an optical sensor region) when a cartridge is present in the cartridge holder.

[0137] In such embodiments, the isolation valve is in pneumatic connection with the other components of the system described herein. In embodiments of the inventive system comprising a fluid flow sensor, the isolation valve is located downstream of said fluid flow sensor (i.e., between the fluid flow sensor and the region of interest (e.g., an optical sensor region).

[0138] Optionally, the system may include a waste container that attaches to the cartridge and receives waste generated inside the cartridge during the course of the implemented fluidics operation (i.e., an immunoassay sequence). The waste container can attach directly to the cartridge or directly to the cartridge holder depending on the configuration of the system (i.e., depending on the location of the input and output for the cartridge or cartridge holder). The waste container may be part of the cartridge as a compact compartment.

[0139] Also described is an immunoassay method involving various embodiments of systems described herein. In certain embodiments, the immunoassay method involves providing a fluid sample (e.g., an aqueous sample, whole blood sample, plasma, or extract of liquid or solid tissues) suspected of containing at least one analyte of interest in a fluid reservoir, preferably in a pressure-tight fluid reservoir. Software code defining a fluidics operation is provided by a device in electrical communication with a programmable electronics board. The programmable electronics board is in further electrical communication with a bidirectional pneumatic pump (preferably, a piezoelectric bidirectional pneumatic pump) and one or more sensors. In certain embodiments, the sensors include one or more fluid flow sensors and/or one or more pressure sensors.

[0140] The fluidics operation defined by the software code provides instructions for the electronic board in response to sensor measurements and/or based on defined time intervals to carry out an immunoassay operation. The software code may define one or more of the following: (i) a discrete volume of fluid, (ii) a maximum volume of fluid, (iii) a minimum volume of fluid, (iv) a maximum fluid flow rate, (v) a minimum fluid flow rate, (vi) an average fluid flow rate over a specified time interval, (vii) an average fluid flow rate over a continuous period, (viii) a minimum pressure, (ix) a maximum pressure, (x) one or more time intervals, (xi) a sequence of varying time intervals, (xii) a total time of operation, and other parameters based on time, volume measurements, flow rate measurements, flow rate calculations, and/or pressure measurements. [0141] In embodiments of the method, the software code is executed or implemented by the electronic board to cause the bidirectional pneumatic pump to generate an initial positive pressure, thereby causing the provided fluid sample (e.g., aqueous sample) suspected of containing at least one analyte of interest to flow from the fluid reservoir and toward a region of interest (e.g., an optical sensor region).

[0142] The region of interest (e.g., an optical sensor region) may be provided in a diagnostic cartridge. In certain embodiments, the diagnostic cartridge comprises an optical sensor region which is analyzed by an optical imaging system, such as a spectrophotometer. The optical imaging system uses an algorithm associated with the optical imaging system to provide an analysis of the contents of the fluid sample.

[0143] In such embodiments, the fluid sample is allowed to incubate over the region of interest for a defined period of time.

[0144] In certain embodiments, a wash cycle or operation is performed on the fluid sample to wash away unwanted material, such as excess sample or unbound detectable particles.

[0145] In certain embodiments, the region of interest in the cartridge is functionalized.

[0146] In certain embodiments where the region of interest is functionalized, the sample contains functionalized detectable particles which bind to the functionalized region of interest.

[0147] In certain embodiments a magnetic field is used to induce faster hybridization of the particle bound antibodies with surface bound antibodies. In some embodiments that is done with permanent magnets in others with electromagnets.

[0148] In certain embodiments of the inventive immunoassay method, the optical imaging system is an instrument such as a dark- field optical microscope or dark-field spectrophotometer. In certain embodiments, the optical imaging system is a fluorescent analyzer, such as a fluorospectrometer.

[0149] In certain embodiments, the optical imaging system comprises a magnet configured and/or positioned to magnetically interact with magnetic detectable particles.

[0150] In certain embodiments, a cartridge holder described herein may comprise a magnet configured and/or positioned to magnetically interact with magnetic detectable particles.

[0151] In certain embodiments of the immunoassay method, a magnet is provided under or next to the optical sensor region.

[0152] In another aspect, a system for fluidic multiplex analysis of an aqueous sample is provided. For example, a single fluidic operation may be carried out to detect the presence of two or more different analytes of interest in a fluid sample. Alternatively, multiple assays can be carried out simultaneously on different samples to detect the same analyte of interest across different samples. [0153] The multiplex system comprises, in certain embodiments, a single piezoelectric bidirectional pump configured to generate positive and negative pressures, a plurality of solenoid valve sets, each solenoid valve set comprising a plurality of solenoid valves operably connected to a shared pressure regulator configured to toggle between the positive and negative pressures; a plurality of fluid reservoirs, each fluid reservoir being in fluid connection with each pressure regulator; a plurality of fluid flow sensors, each fluid flow sensor being in fluid connection with a different fluid reservoir; a plurality of isolation valves, each isolation valve being in fluid connection with a different fluid flow sensor; a cartridge holder comprising a fluidics interface comprising a plurality of fluid inputs, each fluid input being in fluid connection with a different isolation valve; and an electronic board in communication with the piezoelectric bidirectional pump, pressure regulators, and fluid flow sensors. The multiplex system is configured to accept a sample cartridge comprising an optical sensor region and a plurality of lanes for the flow of aqueous sample, each lane being in fluid connection with a different fluid input when the sample cartridge is present. The pressure regulators and fluid flow sensors are electronically connected to form a feedback loop. The electronic board is configured to implement software code provided by a computer to adjust flow of aqueous sample in response to a flow rate measurement provided by one or more of the fluid flow sensors and/or in response to a pressure measurement provided by one or more of the pressure regulators. FIG. 2 illustrates an exemplary, non-limiting embodiment of the cartridge of the multiplex system.

[0154] In the exemplary, non-limiting embodiment depicted in FIG. 2, an electronic board 21 is provided in electronic communication with a pneumatic pump 22 having a first pump port 22a and a second pump port 22b. The first pump port 22a and second pump port 22b are in pneumatic communication with a plurality of pairs of solenoid valves 23 (optionally provided as a manifold of solenoid valve pairs). In the depicted embodiment, SV-1 and SV-2 make up a solenoid valve pairing, and so on. The solenoid valves 23 are in pneumatic communication with a plurality of pressure regulators 24. Each depicted solenoid valve pairing is in pneumatic communication with a pressure regulator (e.g., solenoid valve pair SV-1 and SV-2 in pneumatic communication with pressure regulator PR-1). The pressure regulators 24 are further in pneumatic communication with a plurality of sample reservoirs 25, which are in fluid communication with a plurality of fluid flow sensors 26 (optionally provided as a manifold of fluid flow sensors) and a plurality of isolation valves 27 (optionally provided as a manifold of isolation valves).

[0155] In certain embodiments, the sample reservoirs 25 are compartmentalized such that the need for a flow splitter is avoided. That is, a compartmentalized sample reservoir preferably provides, as outputs, discrete lanes of fluid. In FIG. 2, for example, the sample reservoir 25 is compartmentalized such that the flow of fluid therefrom proceeds downstream as five discrete lanes of fluid.

[0156] In other embodiments, a flow splitter may be provided downstream of the sample reservoir 25 to provide discrete lanes of fluid flow.

[0157] The plurality of samples coming in the sample holders may be resulting from the same biological sample and in a preferred embodiment will be in equal, premeasured amounts. The isolation valves 27 are in direct fluid communication with a fluidics interface 28, to which a fluidics cartridge 29 is attached. The fluidics interface 28 and fluidics cartridge 29 can be configured such that the fluidics interface 28 is integrated into the cartridge 29 or such that the fluidics interface 28 is external to the cartridge 29 (e.g., removably attachable), such as part of a cartridge holder (not pictured). An optional waste container (e.g., waste reservoir) 30 may be provided in fluid communication with the cartridge 29. The waste container 30 may be removable or may be integrated into the cartridge 29 or optional cartridge holder. The cartridge 29 has a plurality of lanes, each in fluid communication with a different isolation valve 27. The cartridge also comprises one or more regions of interest (not depicted) as described herein. In certain embodiments, each lane in the cartridge 29 has its own region of interest, specific to an analyte. In other embodiments, there is a single region of interest with which all lanes overlap or otherwise interact or interface.

[0158] The electronic board 21 is also in electronic communication with the plurality of solenoid valves 23, the pressure regulators 24, the fluid flow sensors 26, and the isolation valves 27. A feedback loop may be formed between the fluid flow sensors 26, pressure regulators 25, and pneumatic pump 22 in a manner as described herein.

[0159] In another embodiment, the multiplex system may comprise a cartridge holder and a plurality of sample input arrangements. Each sample input arrangement may comprise, for example, a piezoelectric bidirectional pump configured to generate positive and negative pressures, a pressure sensor, a plurality of solenoid valves configured to toggle between the positive and negative pressures, a pressure-tight fluid reservoir, a flow sensor, an isolation valve, and an electronic board in communication with the flow sensor and the piezoelectric bidirectional pump. In certain embodiments, the electronic board is configured to implement software code provided by a computer to adjust flow of aqueous sample in response to a flow rate measurement provided by the flow sensor. The cartridge holder comprises a fluidics interface comprising a plurality of fluid inputs, wherein the cartridge holder is configured to accept a sample cartridge comprising an optical sensor region and a plurality of lanes for the flow of aqueous sample. Each lane is configured to be in fluid connection with a different fluid input when the sample cartridge is present in the cartridge holder.

[0160] The fluid reservoir of the multiplex system may comprise, in certain embodiments, detectable particles having binding members bound thereto, as described above in other embodiments and aspects. Additionally, the multiplex system is configured for analysis using an optical system or instrument as described above in other embodiments and aspects.

[0161] In an aspect, the present invention also relates to a system for multiplex analysis of a fluidic sample, comprising a cartridge holder and a plurality of sample input arrangement. Each sample input arrangement comprises a piezoelectric bidirectional pump configured to generate positive and negative pressures, a pressure sensor, a plurality of solenoid valves configured to toggle between the positive and negative pressures, a pressure-tight fluid reservoir, a flow sensor, an isolation valve, and an electronic board. The electronic board is in communication with the flow sensor and the piezoelectric bidirectional pump, wherein the electronic board is configured to implement software code provided by a computer to adjust flow of aqueous sample in response to a flow rate measurement provided by the flow sensor. The cartridge holder comprises a fluidics interface comprising a plurality of fluid inputs. The cartridge holder is configured to accept a sample cartridge comprising an optical sensor region and a plurality of lanes for the flow of aqueous sample, each lane configured to be in fluid connection with a different fluid input when the sample cartridge is present in the cartridge holder.

[0162] In certain embodiments, the system further comprises an optical imaging system positioned to analyze the optical sensor region of the sample cartridge.

[0163] In certain embodiments, the system has 2, 3, 4, 5, 6, 7, 8, 9, or 10 sample input arrangements.

[0164] In certain embodiments, the system has 2 sample input arrangements. [0165] In certain embodiments, the system has 3 sample input arrangements. [0166] In certain embodiments, the system has 4 sample input arrangements. [0167] In certain embodiments, the system has 5 sample input arrangements. [0168] In an aspect, the present invention also relates to a system for multiplex analysis of a fluidic sample, comprising a plurality of sample input arrangements. Each sample input arrangement comprises a cartridge holder, a piezoelectric bidirectional pump configured to generate positive and negative pressures, a pressure sensor, a plurality of solenoid valves configured to toggle between the positive and negative pressures, a pressure-tight fluid reservoir, a flow sensor, an isolation valve, and an electronic board. The electronic board is in communication with the flow sensor and the piezoelectric bidirectional pump, wherein the electronic board is configured to implement software code provided by a computer to adjust flow of aqueous sample in response to a flow rate measurement provided by the flow sensor. The cartridge holder comprises a fluidics interface comprising one or more fluid inputs. The cartridge holder is configured to accept a sample cartridge comprising an optical sensor region and one or more lanes for the flow of aqueous sample, each lane configured to be in fluid connection with a different fluid input when the sample cartridge is present in the cartridge holder.

[0169] In certain embodiments, the system further comprises a plurality of optical imaging systems, each positioned to analyze the optical sensor region of a different sample cartridge. [0170] In certain embodiments, the system has 2, 3, 4, 5, 6, 7, 8, 9, or 10 sample input arrangements.

[0171] In certain embodiments, the system has 2 sample input arrangements.

[0172] In certain embodiments, the system has 3 sample input arrangements.

[0173] In certain embodiments, the system has 4 sample input arrangements.

[0174] In certain embodiments, the system has 5 sample input arrangements.

[0175] In an aspect, the present invention also relates to an integrated fluidics cartridge for use in a fluidic system for the detection of one or more analytes of interest. In certain embodiments, the cartridge comprises a region of interest, a fluid reservoir, detectable particles, and one or more inputs configured to interact with a pneumatic interface. The pneumatic interface is configured to attach to the cartridge, the pneumatic interface being connected to an external pneumatic system. [0176] In certain embodiments, the cartridge comprises a portion configured to interact with a pneumatic interface separate from the cartridge.

[0177] In certain embodiments, a cartridge described herein is configured to interface with one or more pumps via a pressure-driven fluid reservoir included on the cartridge.

[0178] The external pneumatic system may comprise, in some embodiments, a plurality of bidirectional pumps, each in pneumatic communication with a fluid reservoir, pressure regulator, and a set of solenoid valves. Each pairing of fluid reservoir and pressure regulator is associated with its own set of solenoid valves such that each pressure regulator toggles the solenoid valves to provide either a positive or negative pressure from the bidirectional pump in communication therewith. Each grouping of a bidirectional pump, a fluid reservoir, a pressure regulator, set of solenoid valves, and an isolation valve makes up a discrete fluidic assay (e.g., discrete diagnostic lanes). In such embodiments, multiplex analysis can be carried out. Each diagnostic lane may be designated for a different analyte of interest.

[0179] In certain embodiments, the cartridge comprises a region of interest, a fluid reservoir, detectable particles, and a pneumatic interface configured to interact with a fluidic system. [0180] In certain embodiments, the cartridge comprises the pneumatic interface. In certain embodiments, the cartridge comprises a removable pneumatic interface.

[0181] In certain embodiments, the cartridge comprises a waste output.

[0182] In certain embodiments, the cartridge comprises a waste container. In certain embodiments, the cartridge comprises a removable waste container.

[0183] In certain embodiments, the cartridge comprises absorbent pads.

[0184] In certain embodiments, the detectable particles are provided on the cartridge as dried- down (e.g., lyophilized) reagents. In an aspect, the detectable particles reconstitute during operation as fluid flows through the dried-down reagents. Hence, as fluid sample flows through the dried-down reagent, the detectable particles having binding members bound thereto (i.e., functionalized detectable particles) bind to analyte of interest present in the fluid sample prior to reaching the region of interest for optical detection.

[0185] For example, the detectable particles may be provided in one or more diagnostic lanes on the cartridge. The lanes may comprise the same or different detectable particles having the same or different binding members bound thereto. Hence, the cartridge may be configured either for multiplex analysis to detect more than one type of analyte of interest, or for simultaneous assaying for the same analyte of interest.

[0186] The detectable particles can thus be pre-packaged on the cartridge for later reconstitution and use.

[0187] Reconstitution of the detectable particles can be achieved in certain embodiments in situ (i.e., during the course of operation as part of a fluidic immunoassay operation) or in a separate fluid handling step (e.g., prior to the immunoassay operation).

[0188] FIG. 3 depicts an exemplary, non-limiting embodiment of the cartridge. The cartridge has a fluid reservoir 31, which is in fluid communication with one or more diagnostic lanes 32, which are in turn in fluid communication with a waste container 33, which can be provided as an integrated part of the cartridge or a removable component. In certain embodiments, the waste container may instead be one or more absorbent pads. Additionally, the fluid reservoir 31 is in pneumatic communication with a pneumatic interface 34, which is configured to be placed in pneumatic communication with a pneumatic pump and/or a plurality of valves. Each of the diagnostic lanes include a detection area (not shown). In certain embodiments, the diagnostic lanes 32 have dried-down detectable particles provided thereon.

[0189] In certain embodiments, the cartridge may comprise an optional reservoir useful for diluting the lyophilized nanoparticles with a wash buffer on the cartridge. Said optional dilution reservoirs 32a are shown in FIG. 3. [0190] Further aspects of the present subject matter will be apparent to persons of ordinary skill in the art based on the following non-limiting Examples.

Example 1

[0191] FIGS. 4A-4B show an output from the successful operation of an exemplary fluidics system. The exemplary system was used to run an IL-6 immunoassay. In this exemplary immunoassay, a 20-pL sample containing assay specific, functionalized nanoparticles was metered and delivered to a pre-functionalized sensor region. Next, the assay was allowed to incubate for eight minutes using a precisely defined fluidics control given by the isolation valve. The sample remained undisturbed over the sensor region. Subsequently, a pre-determined wash operation with multiple push-pull cycles was performed. The turbulent fluidics of the wash operation removed non-specifically bound particles at the sensor region. FIG. 4A shows the flow rate as a function of time during the fluidics operation, including the wash operation. FIG. 4B shows the cumulative volume of fluid pushed through the system at different time points.

[0192] Finally, an optical imaging system and associated algorithm were used to analyze the assay performance by counting the number of particles assayed at the sensor region.

Example 2

[0193] FIGS. 5A-5B show an output from the successful operation of an exemplary five-plex fluidics system. As shown in FIGs. 5A and SB, five lanes were used, which had substantially identical flow rates in corresponding phases or cycles of the fluidics operation.

[0194] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and subcombinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.

Claims

CLAIMS What is claimed:

1 . A system for analysis of a liquid sample, comprising: a liquid reservoir with a first reservoir port and a second reservoir port; a pair of solenoid valves in pneumatic communication with the first port on the liquid reservoir and with the pump, each solenoid valve comprising a member movable between an open position and a closed position; a piezoelectric pump configured for bidirectional fluid flow, the pump comprising a first pump port and a second pump port, the pump in communication with the pair of solenoid valves; a sensor selected from the group consisting of a pressure sensor, a fluid flow sensor and both a pressure sensor and a fluid flow sensor; an electronic board in communication with the pump and the sensors, the electronic board configured to implement software code for control of the pump to cause liquid in the liquid reservoir to flow across the second reservoir port.

2. The system of claim 1 , wherein the pump is in electrical communication with the pair of solenoid valves.

3. The system of claim 1 , wherein the first pump port is in communication with a first solenoid valve in the pair of solenoid valves, the first solenoid valve in a normally closed position and movable to an open position.

4. The system of claim 1 , wherein the second pump port is in communication with a second solenoid valve in the pair of solenoid valves, the second solenoid valve in a normally open position and movable to a closed position.

5. The system of claim 1 , wherein the sensor is a fluid flow sensor, the fluid flow sensor positioned for fluid communication with liquid that exits or enters the second reservoir port of the liquid reservoir, the fluid flow sensor in communication with the pump.

6. The system of claim 5, wherein flow of liquid from or into the liquid reservoir is adjusted in response to a flow rate measurement provided by the flow sensor.

7. The system of claim 1 , further comprising a cartridge holder configured to accept a sample cartridge that comprises an optical sensor region that is in communication with the second port of the reservoir through a flow sensor.

8. The system of claim 7, wherein the system further comprises a valve positioned between the cartridge holder and the liquid reservoir and flow sensor

9. A system for fluidic analysis of an aqueous sample, comprising: a piezoelectric bidirectional pump configured to generate positive and negative pressures; a pressure sensor; a plurality of solenoid valves configured to toggle between the positive and negative pressures; a pressure-tight fluid reservoir; a flow sensor; a cartridge holder configured to accept a sample cartridge comprising an optical sensor region; an isolation valve; and an electronic board in communication with the flow sensor and the piezoelectric bidirectional pump, wherein the electronic board is configured to implement software code provided by a computer to adjust flow of aqueous sample in response to a flow rate measurement provided by the flow sensor.

10. The system of claim 9, wherein the system does not comprise a pressure reservoir.

11. The system of claim 9, wherein implementation of the software code adjusts the flow of aqueous sample to a preset flow rate, or a variety of preset flow rate values orchestrated in a sequence.

12. The system of claim 9, wherein the isolation valve is configured to prevent fluid backflow or undesired flow in general.

13. The system of claim 9, wherein the isolation valve is configured to maintain an aqueous sample essentially undisturbed over the optical sensor region when a sample cartridge is present in the cartridge holder.

14. The system of claim 9, wherein the aqueous sample comprises functionalized nanoparticles.

15. The system of claim 14, wherein the detectable particles are magnetic.

16. The system of claim 14, wherein the optical sensor region is functionalized to bind the functionalized nanoparticles.

17. The system of claim 16, wherein a magnet is provided under or next to the optical sensor region.

18. The system of claim 9, wherein, when the fluid volume exceeds a threshold fluid volume, the one or more of the solenoid valves are toggled between open or closed positions, thereby causing the pneumatic pump to alternate between generating positive pressure and negative pressure.

19. An immunoassay method, comprising: providing, in a pressure-tight fluid reservoir, an aqueous sample suspected of containing at least one analyte of interest; executing, via an electronic board, software code provided by a computer, said software code defining a fluidics operation; incubating the aqueous sample over an optical sensor region in a diagnostic cartridge; washing the excess of unwanted materials analyzing the incubated aqueous sample using an optical imaging system and an algorithm associated with the optical imaging system; wherein executing said software code causes a piezoelectric bidirectional pump to generate an initial positive pressure, thereby causing the aqueous sample to flow from the pressure-tight fluid reservoir and toward the diagnostic cartridge; wherein the aqueous sample flows through a flow sensor and an isolation valve before reaching the diagnostic cartridge, wherein the flow sensor measures a flow rate of the aqueous sample and the isolation valve prevents backflow of aqueous sample after a leading edge of the aqueous sample has entered the cartridge; wherein the solenoid valves are configured to toggle the piezoelectric bidirectional pump between positive and negative pressures; wherein the flow sensor and the piezoelectric bidirectional pump form a feedback loop, wherein: when the fluid volume of the aqueous sample exceeds a maximum threshold value, the solenoid valves are triggered such that the piezoelectric bidirectional pump generates a negative pressure; and when the fluid volume of the aqueous sample falls below a minimum threshold value, the solenoid valves are triggered such that the piezoelectric bidirectional pump generates a positive pressure.

20. The method of claim 19, wherein the fluidics operation defined by the software code defines a volume of the aqueous sample to be delivered.

21. The method of claim 19, wherein the fluidics operation defined by the software code defines a minimum flow rate and a maximum flow rate for the aqueous sample.

22. The method of claim 19, wherein the fluidics operation defined by the software code defines a fixed flow rate for the aqueous sample.

23. The method of claim 19, wherein the isolation valve is configured to maintain the aqueous sample essentially undisturbed over the optical sensor region.

24. The method of claim 19, wherein the aqueous sample comprises functionalized nanoparticles.

25. The method of claim 24, wherein the functionalized nanoparticles are magnetic.

26. The method of claim 24, wherein the optical sensor region is functionalized to bind the functionalized nanoparticles.

27. The method of claim 26, wherein a magnet is provided under or next to the optical sensor region.

28. The method of any one of claims 19-27, further comprising toggling the solenoid valves to cause the piezoelectric bidirectional pump to alternate between positive pressure generation and negative pressure generation.

29. The method of claim 28, wherein the positive pressure generation and the negative pressure generation moves fluid over the optical sensor region to remove non-specifically bound particles.

30. A system for fluidic multiplex analysis of an aqueous sample, comprising: a piezoelectric bidirectional pump configured to generate positive and negative pressures; a plurality of solenoid valve sets, wherein each solenoid valve set comprises a plurality of solenoid valves operably connected to a shared pressure regulator configured to toggle between the positive and negative pressures; a plurality of fluid reservoirs, wherein a fluid reservoir is in fluid connection with each pressure regulator; a plurality of flow sensors, wherein a flow sensor is in fluid connection with each fluid reservoir; a plurality of isolation valves, wherein an isolation valve is in fluid connection with each flow sensor; and a cartridge holder comprising a fluidics interface comprising a plurality of fluid inputs, each fluid input being in fluid connection with a different isolation valve, wherein the cartridge holder is configured to accept a sample cartridge comprising an optical sensor region and a plurality of lanes for the flow of aqueous sample, each lane being in fluid connection with a different fluid input when the sample cartridge is present in the cartridge holder; an electronic board in communication with the piezoelectric bidirectional pump, pressure regulators, and flow sensors, wherein the pressure regulators and flow sensors are electronically connected to form a feedback loop; and wherein the electronic board is configured to implement software code provided by a computer to adjust flow of aqueous sample in response to a flow rate measurement provided by one or more of the flow sensors and/or in response to a pressure measurement provided by one or more of the pressure regulators.

31. A diagnostic cartridge, comprising: a pressure-tight fluid reservoir; one or more diagnostic lanes downstream of the pressure-tight fluid reservoir; an input upstream of the pressure-tight fluid reservoir, the input configured for pressure- tight pneumatic connection; and an output configured to direct fluid downstream of the one or more diagnostic lanes.

32. The diagnostic cartridge of claim 31 , wherein the diagnostic cartridge comprises a plurality of diagnostic lanes.

33. The diagnostic cartridge of claim 31 , wherein the one or more diagnostic lanes comprise dried detectable particles.

34. The diagnostic cartridge of claim 33, wherein the detectable particles are functionalized nanoparticles.

35. The diagnostic cartridge of claim 35, wherein the detectable particles are magnetic.

36. The diagnostic cartridge of claim 31, wherein the diagnostic cartridge comprises a plurality of diagnostic lanes, wherein at least two of the diagnostic lanes comprise different dried detectable particles.

37. The diagnostic cartridge of claim 36, wherein the detectable particles are functionalized nanoparticles.

38. The diagnostic cartridge of claim 31 , wherein the cartridge is configured to removably attach to a pneumatic interface.

39. The diagnostic cartridge of claim 31 , wherein the cartridge is configured to removably attach to a waste container.

40. The diagnostic cartridge of any one of claims 31-39, wherein the cartridge comprises 2-10 diagnostic lanes.

41. The diagnostic cartridge of any one of claims 31-39, wherein the cartridge comprises 5 diagnostic lanes.

42. The diagnostic cartridge of any one of claims 31-39, wherein the cartridge is configured to interface with one or more pneumatic pumps via the pres sure- tight fluid reservoir included on the cartridge.

43. A system for multiplex analysis of a fluidic sample, comprising: a cartridge holder and a plurality of sample input arrangements, wherein each sample input arrangement comprises: a piezoelectric bidirectional pump configured to generate positive and negative pressures, a pressure sensor, a plurality of solenoid valves configured to toggle between the positive and negative pressures, a pressure-tight fluid reservoir, a flow sensor, an isolation valve, and an electronic board in communication with the flow sensor and the piezoelectric bidirectional pump, wherein the electronic board is configured to implement software code provided by a computer to adjust flow of aqueous sample in response to a flow rate measurement provided by the flow sensor; wherein the cartridge holder comprises a fluidics interface comprising a plurality of fluid inputs; and wherein the cartridge holder is configured to accept a sample cartridge comprising an optical sensor region and a plurality of lanes for the flow of aqueous sample, each lane configured to be in fluid connection with a different fluid input when the sample cartridge is present in the cartridge holder.

44. The system of claim 43, comprising 2-10 sample input arrangements.

45. The system of claim 43, comprising 5 sample input arrangements.

46. The system of any one of claims 43-45, further comprising an optical imaging system positioned to analyze the optical sensor region of the sample cartridge.

47. A system for multiplex analysis of a fluidic sample, comprising a plurality of sample input arrangements, wherein each sample input arrangement comprises: a cartridge holder: a piezoelectric bidirectional pump configured to generate positive and negative pressures, a pressure sensor, a plurality of solenoid valves configured to toggle between the positive and negative pressures, a pressure-tight fluid reservoir, a flow sensor, an isolation valve, and an electronic board in communication with the flow sensor and the piezoelectric bidirectional pump, wherein the electronic board is configured to implement software code provided by a computer to adjust flow of aqueous sample in response to a flow rate measurement provided by the flow sensor; wherein the cartridge holder comprises a fluidics interface comprising one or more fluid inputs; and wherein the cartridge holder is configured to accept a sample cartridge comprising an optical sensor region and one or more lanes for the flow of aqueous sample, each lane configured to be in fluid connection with a different fluid input when the sample cartridge is present in the cartridge holder.

48. The system of claim 47, further comprising a plurality of optical imaging systems, each positioned to analyze the optical sensor region of a different sample cartridge.

49. The system of claim 47 or claim 48, comprising 2-10 sample input arrangements.

50. The system of claim 47 or claim 48, comprising 5 sample input arrangements.