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WO2023183657A1 - Analyses de dépistage multiplexés et leurs procédés d'utilisation - Google Patents

Analyses de dépistage multiplexés et leurs procédés d'utilisation Download PDF

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Publication number
WO2023183657A1
WO2023183657A1 PCT/US2023/016468 US2023016468W WO2023183657A1 WO 2023183657 A1 WO2023183657 A1 WO 2023183657A1 US 2023016468 W US2023016468 W US 2023016468W WO 2023183657 A1 WO2023183657 A1 WO 2023183657A1
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sample
analyte
assay
analysis channels
sample reservoir
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Hua Wang
Lu Zhang
Yutong LI
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Ohio State Innovation Foundation
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Ohio State Innovation Foundation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • 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
    • B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/069—Absorbents; Gels to retain a fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0803—Disc shape
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/087—Multiple sequential chambers

Definitions

  • TECHNICAL FIELD This application generally relates to clinical or home potable device(s) using specific binding interaction involving protein or chemicals for rapid and convenient detection, particularly for advanced design(s) enabling multiplexing, along with specifically selected targets for general and specific probing of primary health condition(s) or disease diagnosis of human and animals, as well as other applications in food safety and quality, environmental protection, etc.
  • BACKGROUND The COVID-19 pandemic has made rapid home diagnosis a societal need.
  • Home- testing kits for COVID-19 SARS-CoV-2
  • false positive or false negative results have been a long-lasting challenge from the very beginning, negating its mission.
  • a false negative test result of an infected COVID-19 carrier further provides a false safety assurance, which may facilitate the rapid spread of the infectious agents in the surrounding population and the society.
  • the existing COVID-19 home testing kits are similar to the home pregnancy testing kits, also known as the lateral flow immunochromatographic assay, from the 1990s. When a control band and a targeted detection band both are visible, the test indicates a positive result (infected). If only one band (control) is visible, the test indicates the functionality of the kit but a negative result (uninfected).
  • Detection outcome is directly impacted by a) the detection signal sensitivity limit of the kit by design, b) the amount of virus particles picked up during sampling, such as nose or throat swab, or less common, blood sample, etc., and c) the binding specificity/efficacy between the target for detection and the embedded antibody, affinity agent or receptor, etc.
  • some home testing kits claim detection sensitivity comparable to PCR tests, even PCR tests have significant percentage of false negatives. Even worse, viruses continually mutate, which will also impact the outcome of the existing detection kits or methods.
  • variants may change primary colonization locations.
  • the Omicron variant of COVID-19 differs from the original virus by being primarily located in the upper respiratory tract instead of aggressively invading the lung.
  • additional virus variants may have decreased density in particular tissue location(s), such as nose and throat, which can negatively impact the efficacy of the mainstream sampling and detection outcome from nose or throat swab.
  • Alternatives such as an anal swab has been practiced in certain countries, but privacy has been a major concern of the practice. Mutated virus surface proteins can change, which may decrease the ability in affinity binding with the antibody/receptor embedded on the existing testing strip, thus negate detection efficacy. Accordingly, there is a pressing need for improved diagnostic devices. .
  • multiplexed assay devices can allow for comprehensive, fingerprinting or profiling that can address the above-mentioned challenges and other needs by empowering multiplexed detection with more supporting information for evidence-based decisions. Detection of multiple targets on a single testing strip has been reported previously. But a single strip has very limited capacity in detecting multiple targets, preventing it from serving the purpose of fingerprinting or profiling. Despite of peer-reviewed publications and even clinical web posts by certain healthcare systems, such as the Mayo Clinic, on the efficacy of certain host response indicators in judging bacterial or viral infections, these indicators, usually need blood test by dedicated facility, are rarely checked in general practices, even at major health centers in metropolitan areas.
  • This multiplexed assay devices have the potential to measure a comprehensive panel of targeted parameters, semi-quantitatively, which can probe the overall health condition of the host, ranging from infection by the specific pathogenic agent(s) (for instance, virus or bacteria), the presence of its specific antibody (for instance, IgM specific for SARS-CoV- 2), to host’s response(s) to infection, etc.
  • the comprehensive information not only provides supplemental information or supporting evidence on potential problems and the need for further diagnosis even in the case of negative result on the particular targeted antigen or antibody, but also further senses the detrimental cytokine or septic storm or low host immune function, thus indicating the need for further medical attention.
  • these multiplexed assay devices can also be used to distinguish viral or bacterial infections, or detect secondary bacterial infection following initial viral infection to justify for proper antibiotic treatment. Therefore, they can enhance evidence-based antibiotic stewardship, including but not limiting to potentially replace or supplement the rapid Strep throat test for more comprehensive clinical diagnosis.
  • the multiplexed assay devices can further be used in early detection or probing of other health risks, such as certain cancers, malfunction of the immune system, etc., and applicable for routine assessment for general health indicators, enabling personalized healthcare. This multiplexed assay devices can further be used to address detection needs for risk assessment in food, agricultural production and environmental protection, etc.
  • the multiplexed assay devices may further be used to profile the composition of clinical, food or environmental samples for targeted microbes, toxins etc., for instance, the microbiota of fecal or food samples.
  • multiplexed assay devices that comprise a sample reservoir; and a plurality of analysis channels fluidly connected to the sample reservoir.
  • Each of the analysis channels can comprise a chromatography matrix defining a path for capillary fluid flow from the sample reservoir to an absorbent region; a conjugate region disposed along the path for capillary fluid flow; and a plurality of analyte capture zones disposed along the path for capillary fluid flow between the conjugate region and the absorbent region.
  • the devices described herein can comprise any suitable number of analysis channels.
  • the plurality of analysis channels can comprise at least four, at least six, at least eight, or at least twelve analysis channels. These analysis channels can be arranged in a variety of geometries. For example, in some embodiments, the plurality of analysis channels can radially extend outward from a central sample reservoir. In other embodiments, the plurality of analysis channels can radially extend inward from a peripheral sample reservoir. Each of the analysis channels can comprise any suitable number of analyte capture zones. In some embodiments, each of the analysis channels can comprise at least three, at least four, or at least six analyte capture zones.
  • Each of these analyte capture zones can react to the presence of a different analyte in a sample flowing through the chromatography matrix, so as to permit for the simultaneous detection of a number of analytes of interest.
  • Analyte capture zones can also react to varying concentrations of an analyte in a sample flowing through the chromatography matrix, so as to allow for the quantification of an analytes of interest in a sample.
  • the analyte capture zones can have any suitable shape. For example, in some cases, each of the analyte capture zones is rectangular shaped or circular shaped.
  • the analyte capture zones can be spaced apart and substantially equidistant along the path for capillary fluid flow between the conjugate region and the absorbent region.
  • the assay can be configured, for example, as an enzyme-linked immunosorbent assay (ELISA), a flow cytometry assay, a competitive immunoassay, a noncompetitive immunoassay, a radioimmunoassay, a chemiluminescent immunoassay, a fluorogenic immunoassay, a competition assay, an indirect assay or a sandwich assay, or a colormetric immunoassay.
  • ELISA enzyme-linked immunosorbent assay
  • the conjugate region comprises a plurality of conjugated detector antibodies, wherein each conjugated detector antibody comprises an antibody with a binding affinity an analyte of interest in a sample, wherein the detector antibody is conjugated to a detectable moiety.
  • the conjugated detector antibodies are impregnated within the chromatography matrix within the conjugate region.
  • the detectable moiety of the conjugated detector antibody comprises an enzyme label, a fluorescent label, a radiolabel, a particulate label, a colloidal gold label, a colored latex particles, or a phosphor converting label.
  • the conjugate region can comprise a number of conjugated detector antibodies equal to a number of analyte capture zones disposed along the path for capillary fluid flow between the conjugate region and the absorbent region. In some embodiments, the conjugate region can comprise a number of conjugated detector antibodies equal to one less than a number of analyte capture zones disposed along the path for capillary fluid flow between the conjugate region and the absorbent region.
  • Each analyte capture zone can comprise a capture antibody immobilized on or within the chromatography matrix within the analyte capture zone. In some embodiments, each capture antibody can exhibit a binding affinity for a different analyte of interest in a sample.
  • capture antibodies can exhibit different binding affinities for a single analyte of interest in a sample (e.g., so as to allow for quantification of the concentration of an analyte).
  • at least one of the capture antibodies serves as a control for the assay.
  • the analyte of interest can comprise, for example, a microbial agent, a mutant, variety, and/or derivative of a microbial agent, a host molecule, a biomarker, a health indicator, a toxin, an allergen, a pollutant, an authentic maker ingredient, or a combination thereof.
  • the analyte of interest can comprise CRP, PCT, IL6, or a combination thereof.
  • the presence of one or more of these analytes of interest in a sample can be used differentiate between a bacterial and viral infection in a sample obtained from a generally healthy patient.
  • the analyte of interest can comprise a host biomarker, such as a biomarker for cancer, inflammation, immune function, housekeeping function, and/or a disease (such as diabetes) indicator.
  • the presence of one or more of these analytes of interest in a sample can be used to probe or fingerprint the health status of a subject from whom a sample is obtained.
  • the device can further comprise an actuator (e.g., a handle) operatively coupled to the assay device, wherein actuation of actuator induces flow of a sample from the sample reservoir through the plurality of analysis channels.
  • actuation of the actuator can induce a deformation of the assay device from a planar conformation to a non-planar conformation, wherein the non-planar conformation directs flow of the sample from the sample reservoir through the plurality of analysis channels (e.g., by gravity).
  • the sample reservoir can be segmented so as to accommodate a plurality of samples fluidly isolated from one another.
  • each of the plurality of samples can comprise a biological sample obtained from a different source of a single patient (e.g., so as to allow for simultaneous screening of, for example, a throat swab, a nasal swab, a fecal swab, and/or a blood sample).
  • methods for screening for a plurality of analytes of interest in a sample comprising: providing a multiplexed assay device described herein; loading a sample in the sample reservoir; and allowing the sample to be drawn by capillary fluid flow from the sample reservoir to an absorbent region, wherein reactivity within the plurality of analyte capture zones indicates the presence of one or more analytes in the sample.
  • FIG. 1 is a schematic illustration of a multiplexed assay device (100) that includes a sample reservoir (104); and a plurality of analysis channels (106, four channels)) fluidly connected to the sample reservoir, wherein each of the analysis channels comprises: (i) a chromatography matrix defining a path for capillary fluid flow (114) from the sample reservoir (104) to an absorbent region (112); (ii) a conjugate region (108) disposed along the path for capillary fluid flow (114); and (iii) a plurality of analyte capture zones (110) disposed along the path for capillary fluid flow (114) between the conjugate region (108) and the absorbent region (112).
  • the plurality of analysis channels radially extend outward from a central sample reservoir.
  • Figure 2 is a schematic illustration of a multiplexed assay device that includes six analysis channels fluidly connected to the sample reservoir (as opposed to four as in Figure 1).
  • Figure 3 is a schematic illustration of a multiplexed assay device that includes six analysis channels fluidly connected to the sample reservoir (as opposed to four as in Figure 1).
  • the plurality of analysis channels extend in parallel from an elongated sample reservoir.
  • FIGS 4A-4B are a schematic illustration of a multiplexed assay device (100) that includes a sample reservoir (104); and a plurality of analysis channels (106, four channels)) fluidly connected to the sample reservoir, wherein each of the analysis channels comprises: (i) a chromatography matrix defining a path for capillary fluid flow (114) from the sample reservoir (104) to an absorbent region (112); (ii) a conjugate region (108) disposed along the path for capillary fluid flow (114); and (iii) a plurality of analyte capture zones (110) disposed along the path for capillary fluid flow (114) between the conjugate region (108) and the absorbent region (112).
  • the plurality of analysis channels radially extend outward from a central sample reservoir.
  • the device further comprises an actuator (103) operatively coupled to the assay device, wherein actuation of actuator (116) induces flow of a sample from the sample reservoir through the plurality of analysis channels.
  • actuator operatively coupled to the assay device, wherein actuation of actuator (116) induces flow of a sample from the sample reservoir through the plurality of analysis channels.
  • actuation of actuator induces a deformation of the assay device from a planar conformation (as shown in Figure 4A) to a non-planar conformation (as shown in Figure 4B), wherein the non-planar conformation directs flow of the sample from the sample reservoir through the plurality of analysis channels.
  • this device can provide for the multiplexed detection of host factors (such as cytokines, cancer and other biomarkers) or other targets (such as multiple viruses, bacteria, toxins, etc.) of a sample.
  • Figure 5 is a schematic illustration of a multiplexed assay device that includes eight analysis channels fluidly connected to the sample reservoir (as opposed to four as in Figure 1). Multiplexed detection, semi-quantitatively, can be achieved by dots or bands or blocks (2, 3, 4, 5, etc. dots, bands or blocks on each analysis channels), with or without heads, tails (common terminology describing phenomenon in chromatography or electrophoresis), or combination, on individual analysis channels, with a divergent design (the analysis channels can be 2, 3, 4, ....n).
  • the targets and controls subject to semi-quantitative detection can be located on specific analysis channels of single or multiple positions, or specific locations of multiple strip arms. Multiplexing from a pie design may be achieved exponentially by increasing the number of analysis channels included (such as 2, 3, 4, 5, 6, 8,....n). Not illustrated but also included are fractions of the pie design, such as 1 ⁇ 2, 1/3, 1/4, 1/5, ....1/n, 2/3, 3 ⁇ 4, 2/5, 3/5, 4/5, ....m/n of the pie design.
  • FIGS 6A-6B are a schematic illustration of a multiplexed assay device (100) that includes a sample reservoir (104); and a plurality of analysis channels (106, four channels)) fluidly connected to the sample reservoir, wherein each of the analysis channels comprises: (i) a chromatography matrix defining a path for capillary fluid flow (114) from the sample reservoir (104) to an absorbent region (112); (ii) a conjugate region (108) disposed along the path for capillary fluid flow (114); and (iii) a plurality of analyte capture zones (110) disposed along the path for capillary fluid flow (114) between the conjugate region (108) and the absorbent region (112).
  • the plurality of analysis channels radially extend inward from a peripheral sample reservoir.
  • the device further comprises an actuator (103) operatively coupled to the assay device, wherein actuation of actuator (116) induces flow of a sample from the sample reservoir through the plurality of analysis channels.
  • actuator operatively coupled to the assay device, wherein actuation of actuator (116) induces flow of a sample from the sample reservoir through the plurality of analysis channels.
  • actuation of actuator induces a deformation of the assay device from a planar conformation (as shown in Figure 6A) to a non-planar conformation (as shown in Figure 6B), wherein the non-planar conformation directs flow of the sample from the sample reservoir through the plurality of analysis channels.
  • this device can provide for the multiplexed detection of host factors (such as cytokines, cancer and other biomarkers) or other targets (such as multiple viruses, bacteria, toxins, etc.) of a sample.
  • Figures 7A-7C illustrate another multiplexed assay device that includes twelve analysis channels fluidly connected to the sample reservoir (as opposed to four as in Figure 1).
  • the plurality of analysis channels extend in parallel from an elongated sample reservoir.
  • the device further comprises an actuator (103) operatively coupled to the assay device, wherein actuation of actuator (116) induces flow of a sample from the sample reservoir through the plurality of analysis channels.
  • actuation of actuator induces a deformation of the assay device from a planar conformation (as shown in Figure 7A) to a non-planar conformation (as shown in Figure 7B and 7C), wherein the non-planar conformation directs flow of the sample from the sample reservoir through the plurality of analysis channels.
  • this device can provide for the multiplexed detection of host factors (such as cytokines, cancer and other biomarkers) or other targets (such as multiple viruses, bacteria, toxins, etc.) of a sample.
  • host factors such as cytokines, cancer and other biomarkers
  • targets such as multiple viruses, bacteria, toxins, etc.
  • Figures 8A-8B show another multiplexed assay device.
  • Figure 9 shows assessment results of fecal microbiota of a patient generally in good health but suffered from lower abdominal pain, sought medical attention and was given a dose of oral azithromycin treatment.
  • the data points included Day 0 (D0: baseline, fecal sample before antibiotic treatment), Day 2 (D2, the day after drug treatment), Day 15 (D15, after 13 days of short-term recovery), and Day 45 (D45, after 43 days of long-term recovery).
  • the one dose of oral azithromycin caused broad disturbance of the host gut microbiota (which are of critical importance to host health).
  • FIGS 10A-10B are a wet lab control illustration of rapid differential detection of C Reactive Protein (CRP, one of the host indicators for infection) and E. coli.
  • CRP C Reactive Protein
  • the strips had immobilized antibodies for CRP and E. coli.
  • the testing sample contained CRP but lacked E. coli.
  • FIGS. 11A-11B are a wet lab illustration for pre-detection sample preparation step. A syringe containing filter (Figure 11A) was used to remove particles from food or feces etc. from samples prior to apply the rapid detection.
  • sample reservoir 104 can comprise a first region (120) and one or more additional regions (e.g., a second region 122, a third region 124, etc.) which can be fluidly isolated from one another and each fluidly connected to one or more analysis channels.
  • multiplexed assay devices that comprise a sample reservoir (104) and a plurality of analysis channels (106) fluidly connected to (or fluidly connectable to) the sample reservoir (104).
  • the plurality of analysis channels can be dimensioned and positioned such that assays are performed in each of the analysis channels in parallel (e.g., at approximately the same time).
  • Each of the plurality of analysis channels can comprise a chromatography matrix, such as for example a nitrocellulose membrane, polyvinylidene fluoride membrane, (charge-modified) nylon membrane, polyethersulfone membrane.
  • the chromatography matrix can define a path for capillary fluid flow (114) from the sample reservoir (104) to an absorbent region (112).
  • Each of the plurality of analysis channels can further comprise a conjugate region (108) disposed along the path for capillary fluid flow (114) and a plurality of analyte capture zones (110) disposed along the path for capillary fluid flow (114) between the conjugate region (108) and the absorbent region (112).
  • the conjugate region and/or the absorbent region can be built into chromatography matrix, or they can be adjacent to but separate from and fluidly connected to the chromatography matrix.
  • an analyte capture zone can comprise one or more capture antibodies having an affinity to an analyte, e.g. a protein or peptide.
  • analyte capture zone can be rectangular shaped, circular shaped or any other shape, and where multiple analyte capture zones are present each is spaced apart along chromatography matrix to create discrete analyte capture zones.
  • one of the analyte capture zones can comprise a control analyte capture zone comprising a capture antibody having an affinity to an analyte or antigen that is expected to always be present in a sample from a subject. Control analyte capture zone can therefore be configured to act as a control that indicates the assay is working properly.
  • Capture antibody can have an affinity to IgG of host animals, detectable moiety, or one or multiple protein, peptide, chemical or other ingredients in the test sample.
  • the sample reservoir can comprise a sample receiving area configured to receive a sample, wherein a sample can comprise any sample from a subject, e.g. a human.
  • the sample reservoir can comprise a single region, so as to receive a single sample for analysis.
  • the sample reservoir can be segmented so as to accommodate a plurality of samples fluidly isolated from one another.
  • sample reservoir 104 can comprise a first region (120) and one or more additional regions (e.g., a second region 122, a third region 124, etc.) which can be fluidly isolated from one another and each fluidly connected to one or more analysis channels.
  • the conjugate region can comprise, impregnated thereon, or otherwise releasably attached thereto, a plurality of conjugated detector antibodies, each having conjugated, or otherwise joined thereto, a detectable moiety.
  • the conjugated detector antibodies can be antibodies having an affinity to an analyte of interest captured by one or more capture antibodies present in downstream analyte capture zones.
  • the detectable moiety can comprise any detectable compound that can be suitably conjugated or adjoined to detector antibody.
  • the detectable moiety of conjugated detector antibody can comprise an enzyme label, a fluorescent label, a radiolabel, a particulate label, a colloidal gold label, a colored latex particles, a phosphor converting label, dyes, chromophores, affinity probes, groups with specific reactivity, chemiluminescent moieties, and/or electrochemically detectable moieties.
  • the elements of the device are configured such that the addition or application of a sample to the sample reservoir will cause the movement, flow or wicking of the contents of sample, including any analytes of interest, from the sample reservoir, through the conjugate region, and across the plurality of analyte capture zones present in each of the analysis channels.
  • Flow can be facilitated by the capillary action of the chromatography matrix, which in some embodiments can be enhanced by an absorbent region.
  • an analyte of interest in a sample can migrate from the sample region to the conjugate region where it can interact with conjugated detector antibody.
  • the analyte of interest now with bound conjugated detector antibody, can continue to migrate to an analyte capture zone where it can become bound to a capture antibody. Then, the presence of the analyte of interest can be detected by virtue of detectable moiety conjugated to conjugated detector antibody.
  • the remainder of sample can continue to flow or migrate toward the absorbent region, including for example other analytes and/or a control analyte which can be captured by other capture antibodies present in other analyte capture zones.
  • sample can be mixed with a buffer to, among other things, optimize the sample for migration by capillary action through the device.
  • the elements of the multiplexed assay device can be contained or housed within a cassette or housing (102).
  • a cassette can be configured to secure one or more components of the device in a structure that provides a portable, easy-to-use, and disposable POC device.
  • the cassette can comprise a window or opening to permit access to and visible observation of analyte capture zones.
  • the remainder of device can be enclosed or covered by cassette or can be exposed or uncovered.
  • the multiplexed assay devices described herein can include any number of analysis channels.
  • the multiplexed assay devices described herein can include at least four, at least six, at least eight, or at least twelve analysis channels. These analysis channels can be arrayed in a variety of geometries.
  • the plurality of analysis channels can radially extend outward from a central sample reservoir.
  • the plurality of analysis channels can extend (e.g., in parallel) from an elongated sample reservoir.
  • the plurality of analysis channels can radially extend inward from a peripheral sample reservoir.
  • the device further comprises an actuator (103), such as a handle, operatively coupled to the assay device. Actuation of actuator (116) can induce and/or drive flow of a sample from the sample reservoir through the plurality of analysis channels.
  • actuation of actuator can induce deformation of the assay device from a planar conformation (as shown in Figure 4A) to a non-planar conformation (as shown in Figure 4B), wherein the non-planar conformation directs flow of the sample from the sample reservoir through the plurality of analysis channels (e.g., via gravity, magnetic or electric power, etc.).
  • Similar designs incorporating such features are shown, for example, in Figures 6A-6B, 7A-7C, and 8A-8B. Rapid, portable and accurate detection has always been in great demand. The COVID pandemic has just brought the shortage to the public attention.
  • the rapid diagnostic kits on the market are single strip tests, targeting either specific infectious agents, specific protein(s) (such as the pregnancy hormone HCG human chorionic gonadotrophin, microbial toxins etc.), or their corresponding antibody or antibodies. False negative or positive rates are common, including but not limiting to the rapid home testing kits for COVID-19. Such false detection outcomes complicate decision-making process, causing public health, social and economic losses.
  • a critical challenge in clinical diagnosis, with potentially life-threatening outcome is misdiagnosis of infections, while the symptoms of bacterial infections can easily be confused with viral infections, missing the critical window for prompt drug intervention.
  • the current specimen culturing methods only target several bacterial pathogens, thus false negative results are frequent, when infections are due to microorganisms out of the standard spectrum of detection (such as common pathogens of Staphylococcus, Streptococcus, Enterococcus, E. coli, Salmonella, etc.). Additional organism-specific tests, although still only for limited types of bacterial infections (such as for Lyme disease, etc.) are less commonly offered in local clinics or even medical centers, that the samples often need to be shipped to specific testing center(s) for extended diagnosis.
  • the current clinical guideline and antibiotic stewardship program encourage restricted use of antibiotics, due to the concern of facilitating antibiotic resistance.
  • compositions comprising e.g., the mentioned designs, fraction, multiplication, or transformation of which; the profiling of the multiple microbial agents, host immune indicators, or in combination, etc.; the designs to facilitate sample flow and sample distribution; the supplemental app, etc.
  • Additional assisting components include disposables and reagents for sampling, sample pretreatment and loading etc.
  • special designs and their derivatives including but not limiting to fractions, multiplication, trans-forms etc.
  • single or multi-layer sample channel design(s) or derivatives (fraction, multiplication, transformation, or combination) that enable handling sample(s) of single or multiple origins (such as nose, throat and anus swabs, urine, fecal and blood samples, etc.), or their processed derivatives, with or without dilution (such as serum being diluted 10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 etc.; bacterial or viral lysate; urine or fecal dilutes or lysate, etc. ) or of multiple subjects (Fig 7), based on the purpose of the detection kits.
  • sample(s) of single or multiple origins such as nose, throat and anus swabs, urine, fecal and blood samples, etc.
  • their processed derivatives with or without dilution (such as serum being diluted 10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 etc.; bacterial or viral lysate; urine or fe
  • the samples can further be fractionated into defined volume (such as but not limited to 5 ⁇ l, 10 ⁇ l, 20 ⁇ l, 50 ⁇ l , 100 ⁇ l, 200 ⁇ l, 0.5 ml, 1 ml, etc.), through more details, with or without another top layer in design. Further disclosed herein is the utilization of the multiplexing platform for detection of a panel of microorganisms, host biomarkers, food or environmental risk factors, drug residues, etc., by themselves, or in any combination.
  • the multiplexed platform can further be used to detect or profile multiple targets of interest to public health, food safety, quality, environment assessment, etc., including but not limiting to tumor biomarkers or antigens, cytokines, toxins, allergens, pathogens, drug residues, food ingredients for authentication, or microbial or chemical spoilage agents, environmental pollutants, etc.
  • a supplemental app using the scan function of a mobile device (such as a cell phone or pad) to capture the images, process and calculate the information, and communicate with the established diagnostic program, interpret the test data and display the detection outcome.
  • the multiplexed assay devices described herein can empower exponentially expanded multiplexing detection capacity for ligand-receptor based interaction and colorimetric signal detection mechanism (such as in ELISA, lateral flow immunochromatographic assay, etc.), enabling complex and semi-quantitative fingerprinting or profiling of risk factors, including but not limited to microbial, host, environmental, food factors.
  • the supplemental mobile app program recognizes, processes, interprets the signals and communicates the detection outcome(s).
  • a trained personal may be able to interpret the detection information without the app.
  • the compositions disclosed herein may be used as rapid home or field testing kits for specific purposes, such as for rapid COVID test, various infections, cancer biomarkers, general health profiling, food safety, quality, environmental safety and quality, etc.
  • compositions disclosed herein may also be used for rapid and primary clinical diagnosis in clinics, as portable kits, as a frontend connected to comprehensive diagnosing processor(s), as miniatured ELISA panel, or as part of detection instrument(s) for primary diagnosis, in standard settings or locations lack of onsite standard diagnostic lab capacity Methods of Use
  • the assay devices described herein can serve as a multiplexing rapid detection platform, allowing them to be used to promptly determine the health status of a subject with improved accuracy, including to determine whether a subject is infected by specific or general microbial agent(s) or not, to use multiple instead of single detection parameters, and/or to detect potential microbial agents as well as host responses. These can all provide for improved clinical assessments and outcomes.
  • CRP C Reactive Protein
  • ProCT Procalcitonin
  • a method to rapidly identify the risk factor(s) or etiology agent(s), by simultaneously screening against a panel of potential targets including but not limiting to microorganisms, their mutants and derivatives, toxins, allergens, pollutants, their specific antigen(s), antibody (antibodies), receptor(s), cancer biomarker(s), cytokine(s), immune factor(s), etc., in combination with or without other host biomarkers, enabled by a multiplexing rapid detection platform as described herein.
  • the positive reaction control(s) is (are) located in the nearest, furthest end or both or other specific position of detection strip or strips (arm, circle, spiral, etc.), to serve as the calibration or internal standard for semi-quantification of the titer of the detection target(s).
  • a supplemental app connected with a centralized computer program or data diagnostic center, will process and comprehensively interpret the raw data (positive signals, concentration titer, etc.), and further display the detection outcome, with instruction to the follow-up action(s).
  • the raw image data may also be interpreted by trained personal at the absence of a scanner or the app.
  • Disclosed herein is when mutated microbes escape from effective detection by the antibodies for the parental strains in the existing rapid strip test kits on the market, the infection-triggered host responses will still be captured by the fingerprinting platform, thus providing a signal for further medical attention.
  • the infection-triggered host responses will still be captured by the fingerprinting platform, thus providing a signal for further medical attention.
  • a method to differentiate viral and bacterial infections by distinctive profiles of host responses For instance, the range of values of C reactive protein (CRP) and cytokines are often different between bacterial and viral infection.
  • CRP C reactive protein
  • cytokines are often different between bacterial and viral infection.
  • Such information when used and interpreted comprehensively, will improve evidence-based decision for antibiotic intervention, indicate potential viral infection, cytokine storm, abnormal tumor indicator(s) that need further medical attention.
  • the multiplexed assay devices described herein can be used to detect one or more analytes of interest in a sample.
  • the analytes of interest can comprise a macromolecule, such as a biomacromolecule.
  • a macromolecule refers to a large molecule, typically having a high relative molecular weight, such as a polymer, polysaccharide, protein, peptide, or nucleic acid.
  • the macromolecule can be naturally occurring (i.e., a biomacromolecule) or can be prepared synthetically or semi-synthetically. In certain embodiments, macromolecules have a molecular weight of greater than about 1000 amu (e.g., greater than about 1500 amu, or greater than about 2000 amu).
  • the analytes of interest can comprise an antibody, peptide (natural, modified, or chemically synthesized), protein (e.g., glycoproteins, lipoproteins, or recombinant proteins), polynucleotide (e.g, DNA or RNA), lipid, polysaccharide, pathogen (e.g., bacteria, virus, or fungi, or protozoa), or a combination thereof.
  • the analytes of interest comprise a biomarker for a disease process in a patient.
  • the multiplexed assay devices described herein can be used in clinical and healthcare settings to detect biomarkers (i.e., molecular indicators associated with a particular pathological or physiological state).
  • the multiplexed assay devices described herein can be used to diagnose infections in a patient (e.g., by measuring serum antibody concentrations or detecting antigens).
  • the multiplexed assay devices described herein can be used to diagnose viral infections (e.g., HIV, hepatitis B, hepatitis C, rotavirus, influenza, or West Nile Virus), bacterial infections (e.g., E. coli, Lyme disease, or H. pylori), and parasitic infections (e.g., toxoplasmosis, Chagas disease, or malaria).
  • viral infections e.g., HIV, hepatitis B, hepatitis C, rotavirus, influenza, or West Nile Virus
  • bacterial infections e.g., E. coli, Lyme disease, or H. pylori
  • parasitic infections e.g., toxoplasmosis, Chagas disease, or malaria.
  • the multiplexed assay devices described herein can be used to rapidly screen donated blood for evidence of viral contamination by HIV, hepatitis C, hepatitis B, and HTLV-1 and -2.
  • the multiplexed assay devices described herein can also be used to measure hormone levels.
  • the sensors can be used to measure levels of human chorionic gonadotropin (hCG) (as a test for pregnancy), Luteinizing Hormone (LH) (to determine the time of ovulation), or Thyroid Stimulating Hormone (TSH) (to assess thyroid function).
  • hCG human chorionic gonadotropin
  • LH Luteinizing Hormone
  • the multiplexed assay devices described herein can be used to diagnose or monitor diabetes in a patient, for example, by measuring levels of glycosylated hemoglobin, insulin, or combinations thereof.
  • the multiplexed assay devices described herein can be used to detect protein modifications (e.g., based on a differential charge between the native and modified protein and/or by utilizing recognition elements specific for either the native or modified protein).
  • the multiplexed assay devices described herein can be used to detect proteinaceous toxins, including mycotoxins, venoms, bacterial endotoxins and exotoxins, and cyanotoxins.
  • the multiplexed assay devices described herein could be used to detect botulinum toxin, ricin, tetanus toxin, C. difficile toxin A, C. difficile toxin B, or staphylococcal enterotoxin B (SEB).
  • SEB staphylococcal enterotoxin B
  • the multiplexed assay devices described herein can also be used in other commercial applications.
  • the multiplexed assay devices described herein can be used in the food industry to detect potential food allergens, such as milk, peanuts, walnuts, almonds, and eggs.
  • the multiplexed assay devices described herein can be used to detect and/or measure the levels of proteins of interest in foods, cosmetics, nutraceuticals, pharmaceuticals, and other consumer products.
  • EXAMPLES The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non- critical parameters which can be changed or modified to yield essentially the same results.
  • FIGs 10A-10B show a wet lab control illustration (a proof-of principle experiment) showing the rapid differential detection of C Reactive Protein (CRP, one of the host indicators for infection) and E. coli.
  • CRP C Reactive Protein
  • the strips shown in Figure 10A had immobilized antibodies for CRP and E. coli.
  • the testing sample contained CRP but lacked E. coli.
  • FIG 10B using one sample pad (equivalent to a central sample reservoir in the assay devices described herein), only the two strips with CRP exhibited positive detection signals (pink dots).
  • the strip with E. coli antibody remained to be negative.
  • the concentration of CRP in sample was 10 ⁇ g/ml or 10mg/L.
  • this device can be useful for rapid, affinity binding based rapid detections. In short, this device can allow for advanced, including but not limited to QR-code, bar-code etc. types of rapid detection, profiling and fingerprinting.

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Abstract

Sont divulgués des procédés et dispositifs associés à un appareil de détection multiplexé alimenté de manière exponentielle présentant des caractéristiques semi-quantitatives mobiles et portables, également aptes à constituer une partie clé d'un instrument ou d'un système de service de diagnostic localisé, centralisé ou immobilisé. Lesdits dispositifs d'analyse multiplexés peuvent être utilisés dans une variété de procédés de diagnostic de maladie, d'évaluation d'état de santé général de l'hôte, de surveillance ou d'étude de conditions environnementales, nutritionnelles ou alimentaires. La conception et la sélection de cibles utilisées dans les dispositifs d'analyse multiplexés peuvent surmonter les inconvénients des produits commerciaux existants.
PCT/US2023/016468 2022-03-25 2023-03-27 Analyses de dépistage multiplexés et leurs procédés d'utilisation Ceased WO2023183657A1 (fr)

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