Diagnostic Assays Using Magnetic Particles

Abstract

The present invention relates to an apparatus and method for the determination of an analyte in a biological sample by immunoassay incorporating magnetic capture of beads on a sensor, which can be used in the point-of-care diagnostic field.

Description

[Technical field]

[0001] The present invention relates to an apparatus and method for the determination of an analyte in a biological sample by immunoassay incorporating magnetic capture of beads on a sensor, which can be used in the point-of-care diagnostic field. [Background technology]

[0002] Diagnostic tests such as immunoassays are often used to detect specific analytes in a sample. For example, pairs of antibodies that can bind to the analyte to form a sandwich detectable by an enzyme or label on one or more of the antibodies are well known and available for a wide range of different analytes of interest. Antibodies against specific biomarkers such as testosterone or cortisol can be used to test the levels of these substances in saliva, blood or urine samples. The presence of the analyte is then determined using, for example, electrochemical measurements or optical measurements such as fluorescence. Many electrochemical measurement techniques are known to those skilled in the art, such as electrochemical impedance spectroscopy, differential pulse voltammetry, square wave voltammetry, cyclic voltammetry, chronoamperometry, open circuit potential measurements and chronopotentiometry.

[0003] Point-of-care detection allows diagnostic testing to be easily and quickly delivered to subjects, allowing better and faster clinical decisions to be made.However, it is difficult to integrate diagnostic testing into point-of-care devices or systems.Preparing samples for immunoassays can require the mixing of multiple solutions and reagents, with precise control of volume and mixing time.Furthermore, the device is ideally automated to eliminate the need for medical professionals to be present.

[0004] Existing liquid handling devices typically flow multiple liquids (such as sample liquids, reagents or wash buffers) in the same flow direction across a measurement chamber, reaction zone or other detection means (i.e., different liquids flow sequentially through the same conduits and parts of the device). This can create problems with contamination because when the next liquid or reagent is added, some of the liquid or reagent involved in the previous step may still be present in the conduit, measurement chamber, reaction zone or other detection means. This contamination can reduce the accuracy of the diagnostic assay.

[0005] Existing liquid handling devices that flow multiple liquids in the same flow direction through a measurement chamber, reaction zone or other detection means are unable to provide rapid, precise and controllable cessation of a reaction and / or biological reaction in the measurement chamber, reaction zone or other detection means because the continuous linear flow of multiple reagents in the same direction does not clear previous liquids or analytes from the measurement chamber, reaction zone or other detection means quickly enough. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there is a need to provide improved immunoassay techniques that allow for more accurate detection of analytes of interest and improved signal-to-noise ratios. [Means for solving the problem]

[0007] This Summary introduces concepts that are described in more detail in the Detailed Description, and is not intended to identify essential features of the claimed subject matter, nor should it be used to limit the scope of the claimed subject matter.

[0008] The present invention provides a method for measuring an analyte of interest in a biological sample, comprising the steps of: The biological sample is a magnetically susceptible bead conjugated to a first antibody or an antigen-binding portion thereof capable of binding to the analyte of interest; and A second antibody or antigen-binding portion thereof capable of binding to the analyte of interest conjugated to an enzyme. and combining with a composition comprising holding the magnetically susceptible beads on the electrodes using a magnetic field; contacting the magnetically susceptible beads with a substrate of the enzyme, where the enzyme substrate is converted by the enzyme into an electroactive molecule; and obtaining an electrochemical measurement using the electrode.

[0009] The present invention also provides a method for measuring one or more analytes of interest in a biological sample, comprising the steps of: The biological sample is magnetically sensitive beads conjugated to one or more antibodies or antigen-binding portions thereof, each antibody or antigen-binding portion thereof being capable of binding to one of the analytes of interest; and one or more second antibodies or antigen-binding portions thereof conjugated to an enzyme, each second antibody or antigen-binding portion thereof being capable of binding to one of the analytes of interest; and combining with a composition comprising holding the magnetically susceptible beads on the electrodes using a magnetic field; contacting the magnetically susceptible beads with one or more substrates of the enzyme, where the enzyme substrate is converted by the enzyme into an electroactive molecule; and obtaining an electrochemical measurement using the electrode.

[0010] In some embodiments, the method is capable of detecting at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different analytes of interest. In some embodiments, each magnetically susceptible bead is conjugated to a plurality of different antibodies or antigen-binding portions thereof, each antibody or antigen-binding portion thereof being capable of binding to one of the analytes of interest. In some embodiments, the magnetically susceptible bead comprises a plurality of different magnetically susceptible bead sets, each magnetically susceptible bead set being conjugated to a different antibody or antigen-binding portion thereof, each antibody or antigen-binding portion thereof being capable of binding to one of the analytes of interest.

[0011] In some embodiments, the enzyme substrate is converted by the enzyme to a soluble electroactive molecule at the electrode, hi some embodiments, the enzyme substrate is converted by the enzyme to an electroactive molecule precipitated onto a magnetically susceptible bead.

[0012] In some embodiments, the method comprises: The method further includes incubating the combined biological sample and composition so that the one or more first antibodies and / or the one or more second antibodies bind to the analyte of interest.

[0013] In some embodiments, the method comprises: holding the magnetically susceptible beads in a fixed position using a magnetic field; Washing the magnetically sensitive beads Further includes:

[0014] In some embodiments, the fixed location is located at an electrode, hi some embodiments, the fixed location is a location spatially separated from said electrode, optionally, the spatially separated location is a blank or non-functional electrode.

[0015] In some embodiments, the method comprises: holding the magnetically susceptible beads in a first position using a magnetic field; washing the magnetically susceptible beads by modulating the magnetic field such that the magnetically susceptible beads are retained in a second position; Further includes:

[0016] In some embodiments, the magnetic field is generated by a stationary magnet. In some embodiments, the magnetic field is generated by an electromagnet. In some embodiments, the magnetic field is modulated by physically actuating a magnet. In some embodiments, the magnetic field is modulated by controlling the current in an electromagnet.

[0017] In some embodiments, the magnetic field is located within a microfluidic device having a flow conduit, and the magnetic field is modulated by actuating a magnet in a direction perpendicular to the direction of flow in the flow conduit. In some embodiments, the magnetic field is located within a microfluidic device having a flow conduit, and the magnetic field is modulated by controlling a current in an electromagnet.

[0018] In some embodiments, the magnetically susceptible beads are moved between the first and second positions at least 20 times, such as at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, or at least 19 times. In some embodiments, the magnetically susceptible beads are moved between the first and second positions 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, or 20 times, preferably 12 times.

[0019] In some embodiments, the magnetic field is modulated such that the magnetically susceptible beads are periodically moved between the first and second positions. In some embodiments, the period during which the magnetically susceptible beads are in the first and second positions is about 0.01 seconds to about 5 seconds, about 0.05 seconds to about 5 seconds, about 0.1 seconds to about 5 seconds, about 0.2 seconds to about 5 seconds, about 0.3 seconds to about 5 seconds, about 0.4 seconds to about 5 seconds, about 0.5 seconds to about 5 seconds, about 1 second to about 5 seconds, about 2 seconds to about 5 seconds, about 3 seconds to about 5 seconds, about 4 seconds to about 5 seconds, about 0.01 seconds to about 4 seconds, about 0.05 seconds to about 4 seconds, about 0.1 seconds to about 4 seconds, about 0.2 seconds to about 4 ...4 seconds to about 5 seconds, about 0.4 seconds to about 5 seconds, about 0.4 seconds to about 5 seconds, about 0.4 seconds to about 5 seconds, about 0.4 seconds to about 5 seconds, about 0.4 seconds to about 5 seconds, about 0.4 seconds to about 5 seconds, about 0.4 seconds to about 5 seconds, about 0.4 seconds to about 5 seconds, about 0.4 seconds to about 5 seconds, about 0.4 seconds to about 5 seconds Approximately 4 seconds, approximately 0.4 seconds to approximately 4 seconds, approximately 0.5 seconds to approximately 4 seconds, approximately 1 second to approximately 4 seconds, approximately 2 seconds to approximately 4 seconds, approximately 3 seconds to approximately 4 seconds, approximately 0.01 seconds to approximately 3 seconds, approximately 0.05 seconds to approximately 3 seconds, approximately 0.1 seconds to approximately 3 seconds, approximately 0.2 seconds to approximately 3 seconds, approximately 0.3 seconds Approximately 3 seconds, approximately 0.4 seconds to approximately 3 seconds, approximately 0.5 seconds to approximately 3 seconds, approximately 1 second to approximately 3 seconds, approximately 2 seconds to approximately 3 seconds, approximately 0.01 seconds to approximately 2 seconds, approximately 0.05 seconds to approximately 2 seconds, approximately 0.1 seconds to approximately 2 seconds, approximately 0.2 seconds to approximately 2 seconds, approximately 0.3 seconds to approximately 2 seconds, approximately 0.4 seconds ~2 seconds, 0.5 seconds~2 seconds, 1 second~2 seconds, 0.01 seconds~1 second, 0.05 seconds~1 second, 0.1 seconds~1 second, 0.2 seconds~1 second, 0.3 seconds~1 second, 0.4 seconds~1 second, 0.5 seconds~1 second, approx. 0.01 seconds to approximately 0.5 seconds, approximately 0.05 seconds to approximately 0.5 seconds, approximately 0.1 seconds to approximately 0.5 seconds, approximately 0.2 seconds to approximately 0.5 seconds, approximately 0.3 seconds to approximately 0.5 seconds, approximately 0.4 seconds to approximately 0.5 seconds, approximately 0.01 seconds to approximately 0.4 seconds, approximately 0.05 seconds to approximately 0.4 seconds, about 0.1 seconds to about 0.4 seconds, about 0.2 seconds to about 0.4 seconds, about 0.3 seconds to about 0.4 seconds, about 0.01 seconds to about 0.3 seconds, about 0.05 seconds to about 0.3 seconds, about 0.1 seconds to about 0.3 seconds, about 0.2 seconds to about 0.3 seconds, about 0.01 seconds to about 0.2 seconds, about 0.05 seconds to about 0.2 seconds, about 0.1 seconds to about 0.2 seconds, about 0.01 seconds to about 0.1 seconds, about 0.05 seconds to about 0.1 seconds, about 0.01 seconds to about 0.05 seconds, preferably about 3 seconds to about 5 seconds, and more preferably about 4 seconds.

[0020] In some embodiments, the magnetically susceptible beads are washed with a washing solution and / or air. In some embodiments, the magnetically susceptible beads are washed consecutively and separately with both a washing solution and air. In some embodiments, the magnetically susceptible beads are washed alternately with a washing solution and air at least twice, at least three times, at least four times, or at least five times, and preferably the magnetically susceptible beads are washed alternately with a washing solution and air twice.

[0021] In some embodiments, the biological sample is diluted prior to combination with the magnetically susceptible beads, hi some embodiments, the electrodes are carbon ink electrodes.

[0022] In some embodiments, the analyte of interest is brain natriuretic peptide or N-terminal pro-BNP. In some embodiments, the analyte of interest is cardiac troponin or cardiac troponin subunit I (cTnI).

[0023] In some embodiments, the one or more analytes of interest are selected from the list consisting of brain natriuretic peptide, N-terminal pro-BNP, cardiac troponin, and cardiac troponin subunit I (cTnI). In some embodiments, the one or more analytes of interest are N-terminal pro-BNP and cardiac troponin subunit I (cTnI).

[0024] In some embodiments, the enzyme is horseradish peroxidase (HRP). In some embodiments, the enzyme is alkaline phosphatase (ALP). In some embodiments, at least one of the one or more second antibodies or antigen-binding portions thereof is conjugated to horseradish peroxidase (HRP) and at least one of the one or more second antibodies or antigen-binding portions thereof is conjugated to alkaline phosphatase (ALP).

[0025] In some embodiments, the substrate for the enzyme is selected from the list consisting of 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS), o-phenylenediamine dihydrochloride (OPD), para-nitrophenyl phosphate (PNPP), and BCIP / NBT (a combination of BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (nitroblue tetrazolium)).

[0026] In some embodiments, the product of the enzymatic reaction is precipitated, and optionally the product is precipitated onto magnetically susceptible beads. In some embodiments, the substrate is 3,3',5,5'-tetramethylbenzidine (TMB), and the product of the enzymatic reaction is precipitated, and optionally the product is precipitated onto magnetically susceptible beads. In some embodiments, the substrate is BCIP / NBT (a combination of BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (nitro blue tetrazolium)), and the product of the enzymatic reaction is precipitated, and optionally the product is precipitated onto magnetically susceptible beads.

[0027] In some embodiments, the electrochemical measurement indicates the concentration or amount of the analyte of interest. In some embodiments, the concentration or amount of the analyte of interest is determined by comparison to a reference solution.

[0028] In some embodiments, the electrochemical measurement is an amperometric, voltammetric, potentiometric, impedimetric, or electrochemical impedance spectroscopy measurement, preferably a chronoamperometric measurement. In some embodiments, the electrochemical measurement is differential pulse voltammetry (DPV).

[0029] In some embodiments, the method of measurement is a sandwich immunoassay. In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the beads) are retained on the surface of the electrode, measured by weight.

[0030] The present invention also provides a method for measuring an analyte of interest in a biological sample, comprising the steps of: The biological sample is a magnetically susceptible bead conjugated to a first antibody or an antigen-binding portion thereof capable of binding to the analyte of interest; and A second antibody or antigen-binding portion thereof capable of binding to the analyte of interest conjugated to an enzyme. and combining with a composition comprising holding the magnetically susceptible beads on the electrodes using a magnetic field; contacting the magnetically susceptible beads with a substrate of the enzyme, where the enzyme substrate is converted by the enzyme into a precipitated electroactive molecule; and obtaining an electrochemical measurement using the electrode.

[0031] The present invention also provides a method for measuring an analyte of interest in a biological sample, comprising the steps of: The biological sample is magnetically sensitive beads conjugated to one or more antibodies or antigen-binding portions thereof, each antibody or antigen-binding portion thereof being capable of binding to one of the analytes of interest; and one or more second antibodies or antigen-binding portions thereof conjugated to an enzyme, each second antibody or antigen-binding portion thereof being capable of binding to one of the analytes of interest; and combining with a composition comprising holding the magnetically susceptible beads on the electrodes using a magnetic field; contacting the magnetically susceptible beads with one or more substrates of the enzyme, where the enzyme substrate is converted by the enzyme into a precipitated electroactive molecule; and obtaining an electrochemical measurement using the electrode.

[0032] In some embodiments, the method can detect at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different analytes of interest. In some embodiments, the electroactive molecules are precipitated on magnetically susceptible beads. In some embodiments, the one or more analytes of interest are N-terminal pro-BNP and cardiac troponin subunit I (cTnI). In some embodiments, the enzyme is horseradish peroxidase (HRP). In some embodiments, the enzyme is alkaline phosphatase (ALP). In some embodiments, at least one of the one or more second antibodies or antigen-binding portions thereof is conjugated to horseradish peroxidase (HRP), and at least one of the one or more second antibodies or antigen-binding portions thereof is conjugated to alkaline phosphatase (ALP).

[0033] In some embodiments, the substrate for the enzyme is selected from the list consisting of 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS), o-phenylenediamine dihydrochloride (OPD), paranitrophenyl phosphate (PNPP) and BCIP / NBT (a combination of BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (nitroblue tetrazolium)).

[0034] In some embodiments, the magnetically susceptible beads and biological sample are incubated at a temperature between 10°C and 50°C, optionally between 15°C and 45°C, further optionally between 20°C and 40°C, further optionally between 20°C and 30°C, further optionally between 25°C and 35°C, further optionally at 25°C, further optionally at 30°C, and further optionally at 40°C.

[0035] The present invention also provides a composition comprising a magnetically susceptible bead conjugated to a first antibody or antigen-binding portion thereof capable of binding to an analyte of interest; a second antibody or antigen-binding portion thereof capable of binding to the analyte of interest conjugated to an enzyme; Also provided is a composition comprising:

[0036] The present invention also provides a composition comprising magnetically sensitive beads conjugated to one or more antibodies or antigen-binding portions thereof, each antibody or antigen-binding portion thereof capable of binding to an analyte of interest; one or more second antibodies or antigen-binding portions thereof conjugated to an enzyme, each of the second antibodies or antigen-binding portions thereof being capable of binding to one of the analytes of interest; Also provided is a composition comprising:

[0037] In some embodiments of the compositions of the invention, each magnetically susceptible bead is conjugated to a plurality of different antibodies or antigen-binding portions thereof, each antibody or antigen-binding portion thereof being capable of binding to one of the analytes of interest. In some embodiments of the compositions of the invention, the magnetically susceptible bead comprises a plurality of different magnetically susceptible bead sets, each magnetically susceptible bead set being conjugated to a different antibody or antigen-binding portion thereof, each antibody or antigen-binding portion thereof being capable of binding to one of the analytes of interest.

[0038] In some embodiments of the compositions of the present invention, the compositions can detect at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 different analytes of interest. In some embodiments of the compositions of the present invention, the analyte of interest is brain natriuretic peptide or N-terminal pro-BNP. In some embodiments of the compositions of the present invention, the analyte of interest is cardiac troponin or cardiac troponin subunit I (cTnI). In some embodiments of the compositions of the present invention, the one or more analytes of interest are N-terminal pro-BNP and cardiac troponin subunit I (cTnI). In some embodiments of the compositions of the present invention, the enzyme is horseradish peroxidase (HRP) or alkaline phosphatase (ALP). In some embodiments of the compositions of the present invention, at least one of the one or more second antibodies or antigen-binding portions thereof is conjugated to horseradish peroxidase (HRP), and at least one of the one or more second antibodies or antigen-binding portions thereof is conjugated to alkaline phosphatase (ALP).

[0039] In some embodiments of the composition of the present invention, the substrate for the enzyme is selected from the list consisting of 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS), o-phenylenediamine dihydrochloride (OPD), paranitrophenyl phosphate (PNPP) and BCIP / NBT (a combination of BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (nitroblue tetrazolium)).

[0040] The present invention also relates to a kit for carrying out the method according to the invention, comprising: Magnetically sensitive beads; an immunoassay device comprising an electrode; a magnet positioned adjacent to the chip to hold the magnetically susceptible beads adjacent to the electrodes; A kit comprising:

[0041] In some embodiments of the kit of the present invention, the kit further comprises a means for holding the magnetically susceptible beads in a separate location spatially separated from the electrode. In some embodiments of the kit of the present invention, the means for holding the magnetically susceptible beads in a separate location spatially separated from the electrode is the same magnet as that used to hold the magnetically susceptible beads in close proximity to the electrode. In some embodiments of the kit of the present invention, the means for holding the magnetically susceptible beads in a separate location spatially separated from the electrode is a second magnet configured to hold the magnetically susceptible beads in a separate location spatially separated from the electrode. In some embodiments of the kit of the present invention, the magnet is a permanent magnet or an electromagnet.

[0042] In some embodiments of the kit of the present invention, the kit further comprises a means for holding the magnetically susceptible beads in a separate location spatially separated from the electrode. In some embodiments of the kit of the present invention, the means for holding the magnetically susceptible beads in a separate location spatially separated from the electrode is the same magnet as that used to hold the magnetically susceptible beads in close proximity to the electrode. In some embodiments of the kit of the present invention, the means for holding the magnetically susceptible beads in a separate location spatially separated from the electrode is a second magnet configured to hold the magnetically susceptible beads in a separate location spatially separated from the electrode. In some embodiments of the kit of the present invention, the magnet is a permanent magnet or an electromagnet.

[0043] The present invention also provides a method for measuring an analyte of interest in a biological sample, comprising the steps of: The biological sample is a magnetically susceptible bead conjugated to a first antibody or an antigen-binding portion thereof capable of binding to the analyte of interest; and A second antibody or antigen-binding portion thereof capable of binding to the analyte of interest conjugated to an enzyme. and combining with a composition comprising holding the magnetically susceptible beads on the electrodes using a magnetic field; contacting the magnetically susceptible beads with a substrate of the enzyme, where the enzyme substrate is converted by the enzyme into a precipitated electroactive molecule; obtaining electrochemical measurements using said electrodes, wherein a biological sample and a composition are combined at a location spatially separated from said electrodes, and optionally, the spatially separated location is a blank or non-functional electrode; A method is also provided, including:

[0044] The present invention also provides a method for measuring an analyte of interest in a biological sample, comprising the steps of: The biological sample is magnetically sensitive beads conjugated to one or more antibodies or antigen-binding portions thereof, each antibody or antigen-binding portion thereof being capable of binding to one of the analytes of interest; and one or more second antibodies or antigen-binding portions thereof conjugated to an enzyme, each second antibody or antigen-binding portion thereof being capable of binding to one of the analytes of interest; and combining with a composition comprising holding the magnetically susceptible beads on the electrodes using a magnetic field; contacting the magnetically susceptible beads with one or more substrates of the enzyme, where the enzyme substrate is converted by the enzyme into a precipitated electroactive molecule; obtaining electrochemical measurements using said electrodes, wherein a biological sample and a composition are combined at a location spatially separated from said electrodes, and optionally, the spatially separated location is a blank or non-functional electrode; A method is also provided, including:

[0045] In some embodiments, the magnetically susceptible beads are contacted with one or more substrates of the enzyme at a location spatially separated from the electrode. In some embodiments, the magnetically susceptible beads are washed with a washing solution and / or air at a location spatially separated from the electrode. In some embodiments, the magnetically susceptible beads are washed with both a washing solution and air consecutively and separately at a location spatially separated from the electrode.

[0046] In some embodiments, the magnetically sensitive beads are alternately washed with washing solution and air at least two times, at least three times, at least four times, or at least five times at a position spatially separated from the electrode, and preferably the magnetically sensitive beads are alternately washed with washing solution and air twice at a position spatially separated from the electrode. In some embodiments, the biological sample and the composition are combined at a position spatially separated from the electrode, and then the magnetically sensitive beads are moved to the electrode to obtain electrochemical measurements. In some embodiments, the magnetically sensitive beads are contacted with one or more substrates of the enzyme at a position spatially separated from the electrode, and then the magnetically sensitive beads are moved to the electrode to obtain electrochemical measurements.

[0047] In some embodiments, magnetically susceptible beads are contacted with one or more substrates of the enzyme at a location spatially separated from the electrode, and the magnetically susceptible beads are then moved to the electrode and an electrochemical measurement is obtained.

[0048] In some embodiments, the biological sample and composition are combined at a location spatially separated from the electrodes, and / or the magnetically susceptible beads are held at a location spatially separated from the electrodes and washed with a wash solution and / or air, and then the magnetically susceptible beads are moved to the electrodes to obtain electrochemical measurements, optionally the spatially separated locations for combining the biological sample and composition and the spatially separated locations for washing are the same.

[0049] In some embodiments, the biological sample and composition are combined at a location spatially separated from the electrodes, the magnetically susceptible beads are contacted with one or more substrates for the enzyme at a location spatially separated from the electrodes, and / or the magnetically susceptible beads are held at a location spatially separated from the electrodes and washed with a wash solution and / or air, and then the magnetically susceptible beads are moved to the electrodes to obtain electrochemical measurements, optionally, the spatially separated locations for combining the biological sample and composition, the spatially separated locations for washing, and / or the spatially separated locations for contacting the magnetically susceptible beads with one or more substrates for the enzyme are the same.

[0050] In some embodiments of the invention, a first antibody or antigen-binding portion thereof capable of binding to the analyte of interest binds to the same portion or epitope of the analyte of interest as a second antibody or antigen-binding portion thereof capable of binding to the analyte of interest. In a preferred embodiment of the invention, a first antibody or antigen-binding portion thereof capable of binding to the analyte of interest binds to a first portion or epitope of the analyte of interest, and a second antibody or antigen-binding portion thereof capable of binding to the analyte of interest binds to a second portion or epitope of the analyte of interest. In some embodiments, the first portion or epitope of the analyte of interest is distant from the second portion or epitope of the analyte of interest, thereby reducing the steric hindrance of having two antibodies or antigen-binding portions thereof that bind to the same analyte of interest.

[0051] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0052] [Figure 1] A shows a signal comparison with and without magnet activation after lowering onto the electrode, and B shows a visualization of beads dispersed on an electrode with and without magnet activation after lowering the magnetic particles. [Diagram 2]FIG. 13 shows an evaluation of the implementation of the actuation method during the wash process for the bead assay (chronoamperometric current after 60 seconds) for samples containing 0 ng / L analyte. [Diagram 3] FIG. 13 shows an assessment of the possible reduction in wash volume when using the magnet actuation method during the wash process for a bead assay (chronoamperometric current after 60 seconds) between samples containing 0 pg / ml and 100 pg / ml of analyte. [Figure 4] FIG. 13 shows the improvement in signal to noise using the magnet actuation method (chronoamperometric current after 60 seconds) between samples containing 0 ng / L and 50 ng / L of analyte. [Diagram 5] FIG. 13 shows bead resuspension using an air-liquid interface (chronoamperometric current after 60 seconds) for a sample containing 100 ng / L analyte. [Figure 6] 1 shows Differential Pulse Voltammetry (DPV) measurements of troponin at concentrations of 0 ng / ml and 50 ng / ml in troponin-free serum. Peak heights in nA were recorded for N=16 in each group. [Figure 7] Figure 1 shows plots of signal to noise (S / N) ratios for two concentrations of troponin, 0 and 50 ng / ml, N=16 per group. [Figure 8] FIG. 1 illustrates an isometric view of an exemplary liquid treatment device. [Figure 9] FIG. 9 is an exploded view showing components of the liquid treatment device of FIG. [Figure 10] 9 is a top cross-sectional view of the liquid treatment device shown in FIG. 8. [Figure 11] FIG. 9 is an isometric underside view of the first rigid layer of the liquid treatment device shown in FIG. [Figure 12] FIG. 9 is a top view of a fluidic layer that can be implemented in the liquid treatment device shown in FIG. [Figure 13] FIG. 13 is a bottom view of the alternative fluidic layer shown in FIG. 12. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] Suitable microfluidic devices According to a first aspect of the present disclosure, the method of the present invention can be performed on a liquid treatment device comprising a first rigid layer and a second rigid layer, a fluidic layer disposed between the first rigid layer and the second rigid layer, the fluidic layer being formed from an elastomer, the fluidic layer comprising a network of channels, and a fluidic network comprising a plurality of conduits, the plurality of conduits being at least partially defined by the network of channels in the fluidic layer.

[0054] The use of channels in an elastomeric layer improves sealing of the fluidic network regardless of the bonding process (e.g., pressure-sensitive adhesive (PSA) tape, laser welding, etc.) used to seal the network. This is because the elastomeric layer acts as a flexible layer when sealed to another layer. Furthermore, by providing channels in a flexible elastomeric layer, the channels can be compressed to provide valves in the liquid handling device. Thus, the flow of liquid in the liquid handling device can be controlled by compressing the channels in the elastomeric layer, thereby closing valves in the liquid handling device. The use of a single layer for the network of channels also simplifies the construction of the liquid handling device.

[0055] The liquid treatment device may further comprise a plurality of valves, each of which may be configured to close a corresponding one of the plurality of conduits, the valves allowing for controlling the flow of liquid within the liquid treatment device.

[0056] Each of the plurality of valves may include a deformable valve region disposed in the fluidic layer. Each deformable valve region may be deformable to a deformed state in which a corresponding one of the plurality of conduits is blocked. Providing the deformable valve regions in the fluidic layer simplifies construction of the liquid treatment device because the fluidic layer implements both the conduits of the device and the valves of the device.

[0057] The fluidic layer may include a first surface configured to face the first rigid layer and a second surface configured to face the second rigid layer. At least a portion of the network of channels may be provided on the second surface. Each deformable valve region may include a depression on the first surface of the fluidic layer. The depression may be aligned with a corresponding channel of at least a portion of the network of channels provided on the second surface. By providing a depression on the first surface of the fluidic layer, a volume of material that needs to be deformed to close each valve of the liquid handling device is reduced. This reduces the force required to close each valve.

[0058] A subset of the network of channels may be provided on the first side. Providing channels on both sides of the fluidic layer means that the available area for providing respective binding regions around the channels is increased, which is particularly important in view of the limited area available on the fluidic layer due to the small size of the point-of-care device. Providing channels on the first side also allows the channels of the fluidic layer to cross, which means that more complex networks of channels can be implemented.

[0059] The first rigid layer can include a plurality of openings. Each deformable valve region can be accessible through one of the plurality of openings. Providing openings in the rigid layer means that the liquid handling device has a rigid housing while allowing the valves to be actuated by applying an external force (e.g., from an actuator of the analytical device).

[0060] The liquid treatment device may further comprise a plurality of openings extending through at least a portion of the thickness of the fluidic layer. Each of the plurality of openings may be in fluid communication with one of the plurality of conduits. The plurality of openings may include a first plurality of openings and a second plurality of openings. The second plurality of openings may be different from the first plurality of openings. The plurality of openings allows fluid in the fluidic layer (i.e., either liquid or air provided from a pneumatic supply system) to communicate with fluidic components in other layers.

[0061] The liquid treatment device may further comprise a plurality of ports configured to provide a seal against the pneumatic interface. Each of the plurality of ports may include a protrusion protruding from a surface of the fluidic layer and a respective opening of the first plurality of openings. Each opening of the first plurality of openings may extend through the protrusion. Implementing the ports in the elastomeric fluidic layer allows the ports to form a seal with the pneumatic interface. This is because the fluidic layer acts as a flexible layer when a force is applied to the port by the pneumatic interface (e.g., a pneumatic actuator of a pneumatic supply system). Providing the ports in the same fluidic layer as the network of channels also simplifies the construction of the liquid treatment device. Fluid communication between the ports and the conduits allows for the application of pneumatic pressure through the ports to move liquid within the conduits.

[0062] Each protrusion may have a frusto-conical shape. The frusto-conical shape of the protrusion aids in the formation of a seal between the port and the pneumatic interface because the frusto-conical shape narrows the cross-section of the protrusion as it increases in height from the surface. In other words, due to the sloping walls provided by the frusto-conical shape, the frusto-conical shape provides less material at the top of the protrusion than at the base of the protrusion. The reduced cross-section at the top of the protrusion means that less material needs to be deformed by the pneumatic interface to provide a seal around the port. Less material to deform means a lesser amount of force needs to be applied to compress the port.

[0063] Each of the first plurality of openings can have a diameter that increases as the height above the surface of the fluid layer increases, further reducing the amount of material at the top of the protrusion, resulting in a lower force being required to deform the port.

[0064] Each protrusion may include an annular rim around the open end of the protrusion. The annular rim may define an area of ​​minimum cross-sectional area of ​​the protrusion. The annular rim further reduces the amount of material at the top of the protrusion, which means that less force is required to deform the protrusion.

[0065] One or more of the plurality of ports may further comprise a plurality of support ribs. Each of the plurality of support ribs may extend between the protrusion and a surface of the fluidic layer from which the protrusion protrudes. The support ribs help prevent excessive deformation of the port when a force is applied to the port by the pneumatic interface.

[0066] The first rigid layer can include a plurality of openings. Each port can be accessible through one of the plurality of openings. Providing openings in the rigid layer means that the liquid handling device has a rigid housing, while allowing the application of air pressure to the ports using an external pneumatic interface (e.g., the pneumatic actuator of the analytical device).

[0067] Each of the plurality of ports can be in fluid communication with one of the plurality of conduits through a corresponding trough in the second rigid layer. The trough prevents liquid from reaching the port that connects to the pneumatic interface. Thus, the trough prevents liquid from reaching the pneumatic interface, particularly during aspiration of liquid. Such liquid may be capable of contaminating or damaging the pneumatic interface (e.g., in the analytical device). In particular, liquid drawn from a channel of the fluidic layer during aspiration pools at the bottom of the trough and does not reach the port. Thus, liquid drawn from the channel is not drawn through the port into the pneumatic interface.

[0068] The liquid treatment device may further comprise at least one liquid storage capsule disposed over two of the second plurality of openings. Disposing the liquid storage capsule over the openings allows the fluidic network to interface with the liquid storage capsule. This also allows the capsule to deform to conform to the openings to form the openings in the capsule.

[0069] The fluidic layer may include one or more chambers, each of which is in fluid communication with one of the conduits. As a result of providing the chambers in the fluidic layer, the construction of the liquid treatment device is simplified. In particular, by providing the fluidic layer with one or more chambers, the functionality of the fluidic layer is expanded.

[0070] The fluid layer may include a protrusion extending from a surface of the fluid layer. The protrusion may include a plurality of cavities. Each of the one or more chambers may be at least partially defined by a corresponding one of the plurality of cavities. Providing a protrusion extending from a surface of the fluid layer means that the volume of the chamber is not limited by the thickness of the fluid layer. Thus, an increased chamber volume may be provided.

[0071] The liquid treatment device may further comprise a sealing film. The plurality of conduits may be defined by a network of channels in the fluidic layer and the sealing film. The flexibility of the elastomeric fluidic layer aids in the channels being sealed by the sealing film.

[0072] Each channel comprises a groove provided in the surface. Each channel therefore has an open cross-section. In other words, the cross-section of each channel is not sealed. Each conduit (i) comprises a channel that is sealed (e.g., by a sealing layer), thereby providing a closed cross-section, or (ii) comprises a hole or tunnel that extends at least partially through the body.

[0073] 8 is an isometric view of a liquid handling device in the form of a diagnostic cartridge 100 (eg, a microfluidic cartridge). The cartridge 100 comprises several components, as can be seen in an exploded view shown in FIG.

[0074] Specifically, the cartridge 100 includes a first portion 200 and a second portion 500, each formed from a rigid material. In use (i.e., when the cartridge 100 is in the orientation shown in FIG. 8), the first portion 200 is an upper portion and the second portion 500 is a lower portion. Together, the first portion 200 and the second portion 500 define a housing for the cartridge 100. Specifically, the first portion 200 includes a rigid surface 250 that defines an upper surface of the cartridge 100. Similarly, the second portion 500 includes a rigid surface 570 (as best shown in FIG. 8) that defines a lower surface of the cartridge 100. Returning to FIG. 9, it can be seen that the first portion 200 further includes a sidewall 252 joined to the rigid surface 250, and the second portion 500 further includes a sidewall 572 joined to the rigid surface 570. The side wall 252 of the first portion 200 and the side wall 572 of the second portion 500 cooperate together to define a side wall of the cartridge 100 .

[0075] The cartridge 100 further comprises a fluidic layer 300 arranged in a housing defined by the first part 200 and the second part 500. In particular, the fluidic layer 300 is arranged between the rigid surface 250 of the first part 200 and the rigid surface 570 of the second part 500. The fluidic layer 300 is thus arranged between a first rigid layer in the form of the rigid surface 250 and a second rigid layer in the form of the rigid surface 570. The fluidic layer 300 is formed from an elastomeric material, such as a thermoplastic elastomer (TPE), for example a silicon-based TPE or styrene-ethylene-butylene-styrene (SEBS), polydimethylsiloxane (PDMS), or liquid silicone rubber (LSR).

[0076] As described in more detail below with reference to FIG. 12, the first surface 308 of the fluidic layer 300 includes a plurality of valve regions 302. The cartridge 100 is received in an analytical device that includes an actuator that applies a force to the valve regions 302 of the fluidic layer 300 to close one or more conduits in the cartridge 100. The properties of the material used for the fluidic layer 300 depend on the available force that can be applied by the actuator to the valve regions 302 of the fluidic layer 300. Two properties that are important are the hardness of the material and the relaxation time of the material (i.e., the time it takes for the material to return to its original form after deformation). Examples of suitable materials include the elastomeric materials listed above. In some implementations, the fluidic layer 300 may be a medical grade material to prevent reaction of the fluidic layer 300 with reagents used in the diagnostic test or assay.

[0077] As described in more detail below, the fluidic layer 300 includes a network of channels 304 disposed (at least in part) in a second surface 310 of the fluidic layer 300 opposite the first surface 308. The cartridge 100 also includes a fluidic network including a plurality of conduits defined at least in part by the network of channels 304 in the fluidic layer 300. Specifically, the conduits are defined by (i) the network of channels 304 in the fluidic layer 300, (ii) a sealing layer 400 (shown in FIG. 9 ) configured to seal the channels 304 in the second surface 310 of the fluidic layer 300, and, optionally, (iii) a sealing layer (not shown) configured to seal any channels 304 of the network disposed in the first surface 308.

[0078] Providing the channels 304 in the elastomeric fluid layer 300 improves sealing of the fluid layer regardless of the bonding process (e.g., pressure sensitive adhesive tape, laser welding, etc.) used to seal the network of channels 304. This is because the elastomeric fluid layer 300 acts as a flexible layer (e.g., sealing layer 400) when sealed against another layer. Furthermore, the use of an elastomeric material for the fluid layer 300 means that the channels 304 can be compressed to close off the respective conduits. This means that a single layer can be utilized to implement the channels 304 and valves (i.e., valve region 302), thereby providing a simple cartridge construction.

[0079] Returning to the exploded view shown in FIG. 9 , it can be seen that the cartridge 100 further comprises a label 110 arranged to cover at least a portion of the rigid surface 250 of the first part 200, a plurality of liquid storage capsules 120 arranged within the cartridge 100 between the fluidic layer 300 and the first surface 250, and a sealing tape 130 arranged to seal one or more chambers 332 within the fluidic layer 300.

[0080] 9 also shows that cartridge 100 further comprises a flow cell strip 140 including a plurality of openings 142, each of which partially defines a corresponding measurement chamber 610 of cartridge 100, a sensor strip 150 comprising a plurality of sensors, each sensor in fluid communication with a respective measurement chamber 610, and a pair of absorbent waste pads 160, each of which is positioned to fit within a corresponding waste chamber provided in second portion 500. In some embodiments, flow cell strip 140 is not present and openings 142 that partially define measurement chambers 610 are instead provided in an alternative sealing layer.

[0081] 8 and 9, the first portion 200 comprises a receptacle in the form of a cylinder 202 configured to receive a portion of a liquid storage container, such as a blood collection tube (e.g., a Vacutainer® blood collection tube manufactured by Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Blood collection tubes typically contain a volume of liquid (e.g., blood) and a headspace that contains a volume of gas.

[0082] The disclosed assay methods are not limited to biological samples derived from a particular source. The disclosed assay methods can also be performed from any other suitable receptacle that contains a biological sample, such as a capillary blood sample, a plasma sample, or a blood sample from a subject.

[0083] fluid layer In one embodiment of the fluidic layer shown in Figures 12 and 13, the channels 304 are provided on both the first surface 308 and the second surface 310 of the fluidic layer 300. It is difficult to configure a network of fluidic channels in a limited space. Point-of-care devices are designed to be small, which limits the area available on the fluidic layer 300 to lay channels with their respective bonding areas (i.e. for bonding to the sealing layer 400) around them. By implementing the channels 304 on both surfaces 308, 310 of the fluidic layer 300, for example, the channels 304 used to transport air (e.g. for cleaning the measurement chamber or for transferring liquid from the liquid storage capsule 120) can be moved to the first surface 308 without affecting the flow of liquid. Providing the channels 364 on the first surface 308 also allows the channels 304 of the fluidic layer 300 to cross, which means that a more complex network of channels 304 can be implemented.

[0084] In the implementation of the fluidic layer 300, the valve region 302 is still provided on the first surface 308 of the fluidic layer 300. Thus, the channels 304 in the first surface 308 are either channels 304 that do not pass under the valve region 302, or channels 304 that have a first portion in the first surface 308 and a second portion in the second surface 310. For example, the second portion of the channel 304 may be a portion of the channel 304 that passes under the valve region 302. These two portions of the channel 304 may be connected by a vertical or inclined conduit that extends through the thickness of the fluidic layer 300.

[0085] 12 and 13 show various examples of channels 304 having portions in both surfaces 308, 310. For example, the channel 304a in Figs. 12 and 13 includes a first portion 382a disposed on the second surface 310, a second portion 382b disposed on the first surface 308, and a third portion 382c disposed on the second surface 310. The first portion 382a extends between a point on the first trough 514a (shown in Fig. 9) and the first through-hole 300 of the fluidic layer 384a. The second portion 382b extends between the first through-hole 384a and the second through-hole 384b in the fluidic layer. The third portion 382c extends between the second through-hole 384b and an opening 386 over which the liquid storage capsule 120 may be placed when the cartridge 100 with the fluidic layer 300 is assembled. By providing portions of channel 304a on both surfaces 308, 310, channel 304b can intersect channel 304a (as shown in Figures 12 and 13).

[0086] Liquid Storage Capsule FIG. 10 also illustrates the arrangement of multiple liquid storage capsules 120 within the cartridge 100. In particular, the liquid storage capsules 120 are sealed to the fluidic layer 300 using a sealing tape 180. FIG. 10 illustrates that the sealing tape 180 includes openings that allow features (i.e., pneumatic port 312 and protrusion 330 defining chamber 332) to protrude above the fluidic layer 300. As shown in FIG. 10, each liquid storage capsule 120 includes an inlet chamber 122, a main chamber 124 for storing a liquid, such as a liquid reagent, and an outlet chamber 126. A sealing layer (e.g., a sealing foil) is used to seal the chambers 122, 124, 126 of each liquid storage capsule 120. The inlet chamber 122 and the outlet chamber 126 each include a corresponding recess 128a, 128b in the upper surface of the chamber.

[0087] The liquid storage capsule 120 shown in Figure 10 includes two smaller liquid storage capsules 120a and two larger liquid storage capsules 120b. The smaller liquid storage capsules 120a are aligned such that the recesses 128a, 120b of the smaller storage capsules 128a are all in a straight line. Each of the larger liquid storage capsules 120b is positioned perpendicular to the corresponding smaller liquid storage capsule 120a such that the larger liquid storage capsules 120b are parallel to each other.

[0088] As will be explained in more detail below, each of the liquid storage capsules 120 is positioned over two openings 350 of the fluidic layer 300. Specifically, the inlet chamber 122 of the liquid storage capsule 120 covers a first one of the openings 350, while the outlet chamber 126 of the liquid storage capsule 120 covers a second one of the openings 350. When a force is applied to the recesses 128a, 128b of the liquid storage capsule 120, the material of the liquid storage capsule 120 deforms to fit each of the openings 350. When sufficient force is applied, the deformation of the liquid storage capsule 120 to fit the openings 350 causes the rupture of a sealing layer (e.g., foil) used to seal the capsule 120.

[0089] In alternative embodiments, the inlet chamber 122 and the outlet chamber 126 may not include the recess 128. Instead, a force may be applied directly to a portion of the inlet chamber 122 and the outlet chamber 126 to cause the liquid storage capsule 120 to deform.

[0090] Figure 11 is a bottom isometric view of the first portion 200. As shown in Figure 11, the first portion 200 includes an actuatable portion 240 (e.g., an actuatable platform) that is actuatable from a first position in which the actuatable portion 240 does not deform the liquid storage capsule 120 to a second position in which the actuatable portion 240 deforms the liquid storage capsule 120.

[0091] The actuatable portion 240 is U-shaped so that it can deform towards each of the liquid storage capsules 120. The U-shape of the actuatable portion 240 also allows the actuatable portion 240 to pass around a protrusion 330 extending from the first surface 308 of the fluid layer 300.

[0092] As shown in Fig. 11, the lower surface of the actuatable portion 240 includes four pairs of protrusions 242. Each pair of protrusions 242 extends toward the liquid storage capsule 120 and is aligned with one of the recesses 128a, 128b of the liquid storage capsule 120. Thus, when the actuatable portion 240 moves to the second position, the protrusions 242 engage with the recesses 128a, 128b of the capsule 120. In an alternative embodiment, the liquid storage capsule 120 may not include the recesses 128a, 128b, in which case the protrusions 242 may engage with a portion of the inlet and outlet chambers 122, 126 of each liquid storage capsule 120 (e.g., a flat or dome-shaped upper surface of the inlet and outlet chambers 122, 126).

[0093] The underside of the actuatable portion 240 also includes four recessed areas 244. Each recessed area 244 is located between two protrusions 242. Each recessed area 244 is configured to accommodate the main chamber 124 of its corresponding liquid storage capsule 120 when the actuatable portion 240 is in the second position. This means that the main chamber 124 is not deformed by the actuatable portion 240 when the actuatable portion 240 is in the second position.

[0094] Assuming that the projections 242 extend from a single actuatable portion 240, actuation of the actuatable portion 242 to the second position causes simultaneous deformation of each of the multiple capsules 120. As a result, all of the capsules 120 in the cartridge 100 can be punctured using a single movement of the actuatable portion 240.

[0095] In an alternative embodiment, the actuable portion 240 may include two sets of protrusions 242, i.e., a first set of protrusions each extending a first distance toward the recess 128 of the liquid storage capsule 120, and a second set of protrusions each extending a second distance toward the recess 128 of the liquid storage capsule 120, the second distance being smaller than the first distance.

[0096] This alternative embodiment allows for the puncturing of the liquid-storage capsules 120 in two stages. When the actuatable portion 240 is moved to the second position (as described above), the capsules 120 aligned with the first set of protrusions are punctured first. However, to puncture the capsules 120 aligned with the second set of protrusions, the actuatable portion 240 is actuated beyond the second position to a third position (because the second set of protrusions is shorter). Thus, this alternative embodiment allows for liquid (e.g., liquid reagent) to be released from some capsules before other capsules are punctured.

[0097] Thus, for example, a fluidic workflow step involving liquid reagents stored in the first and second capsules can be completed before releasing liquid reagents from the third and fourth capsules (e.g., if the liquid reagents in the third and fourth capsules are required at a later stage in the fluidic workflow).To further alternate the release of liquid from the capsules 120, additional sets of protrusions extending different distances from the actuatable portion 240 can be implemented.

[0098] Devices that allow bidirectional flow Immunoassays rely on the delivery of liquid in a controlled manner. The volume of liquid delivered and the time of interaction are important for the success and reproducibility of the assay. In addition, heterogeneous immunoassays require washing steps to remove unbound antibodies, unbound antigens and enzyme tags from the detection surface. Reagents can be trapped in the liquid flow paths and then interact in non-specific reactions. This can increase background signals that reduce assay sensitivity, dynamic range and accuracy. Assay performance can be significantly improved by adding potentially cross-reacting reagents using different flow paths and / or different liquid flow directions.

[0099] The configuration of the liquid handling device providing bidirectional flow allows for rapid, precise and controllable cessation of reactions and / or biological interactions in the measurement chamber. The use of conduits with different flow directions also results in reduced contamination of each liquid during the different method steps (i.e. reduced contamination of sample liquids in washing steps). This may not be easily achievable with known fluid handling devices such as conventional microfluidic devices.

[0100] In one aspect, a liquid handling device may comprise a sample chamber for receiving a sample, a measurement chamber for performing one or more measurements on the sample, the measurement chamber including a reaction zone, a first liquid reagent chamber, a sample chamber conduit fluidly connecting the sample chamber to the measurement chamber, a sample chamber conduit valve for opening and closing the sample chamber conduit, a first liquid reagent chamber conduit fluidly connecting the first liquid reagent chamber to the measurement chamber in an alternating flow direction relative to the sample chamber conduit, and a first liquid reagent chamber conduit valve for opening and closing the first liquid reagent chamber conduit.

[0101] The flow direction of the first liquid reagent chamber conduit to the measurement chamber may be at least 90 degrees to the flow direction of the sample chamber conduit to the measurement chamber. In one embodiment, the flow direction of the first liquid reagent chamber conduit to the measurement chamber is opposite to the flow direction of the sample chamber conduit to the measurement chamber. In some embodiments, the opposite flow direction is equivalent to a second flow direction that is 180 degrees to the first flow direction in the same horizontal plane of the device.

[0102] In some embodiments, the device further comprises a second liquid reagent chamber, a second liquid reagent chamber conduit fluidly connecting the second liquid reagent chamber to the measurement chamber in an alternating flow direction relative to the sample chamber conduit, and a second liquid reagent chamber conduit valve for opening and closing the second liquid reagent chamber conduit.

[0103] In some embodiments, the second liquid reagent chamber conduit fluidly connects to the measurement chamber in alternating orientation with respect to both the sample chamber conduit and the first liquid reagent chamber conduit.

[0104] In some embodiments, the second liquid reagent chamber conduit is fluidly connected to the first liquid reagent chamber conduit, thereby providing a combination conduit that fluidly connects both the first and second liquid reagent chambers to the measurement chamber. In some embodiments, the flow direction of the combination conduit to the measurement chamber is at least 90 degrees relative to the flow direction of the sample chamber conduit to the measurement chamber.

[0105] In some embodiments, the flow direction of the combination conduit to the measurement chamber is opposite to the flow direction of the sample chamber conduit to the measurement chamber, hi some embodiments, the opposite flow direction is equivalent to a second flow direction that is 180 degrees to the first flow direction in the same horizontal plane of the device.

[0106] In some embodiments, the flow direction of the second liquid reagent chamber conduit to the measurement chamber is at least 90 degrees to the flow direction of the sample chamber conduit and / or the first liquid chamber conduit to the measurement chamber.

[0107] In some embodiments, the flow direction of the second liquid reagent chamber conduit to the measurement chamber is opposite to the flow direction of the sample chamber conduit and / or the first liquid chamber conduit to the measurement chamber, in some embodiments, the opposite flow direction is equivalent to a second flow direction that is 180 degrees to the first flow direction in the same horizontal plane of the device.

[0108] In some embodiments, the reaction zone comprises one or more electrodes. In some embodiments, the one or more electrodes comprise one or more electrodes selected from the list of a counter electrode, a reference electrode, and a working electrode. In some embodiments, the one or more electrodes comprise at least one working electrode.

[0109] In some embodiments, the device comprises two or more measurement chambers, each fluidly connected to the sample chamber and each fluidly connected to a first liquid reagent chamber, and the device comprises a corresponding number of sample chamber conduit valves and / or first liquid reagent chamber valves for independently controlling the flow of sample liquid and / or first liquid reagent to each measurement chamber.

[0110] In some embodiments, the device further comprises a second liquid reagent chamber, each of the measurement chambers being fluidly connected to the second liquid reagent chamber, and the device comprising a corresponding number of second liquid reagent chamber conduit valves for independently controlling the flow of the second liquid reagent to each measurement chamber.

[0111] In some embodiments, the second liquid reagent chamber conduit is fluidly connected to the first liquid reagent chamber conduit, thereby providing one or more combination conduits fluidly connecting both the first and second liquid reagent chambers to each measurement chamber.

[0112] In some embodiments, the flow of any one or more of the sample liquid, the first liquid reagent and / or the second liquid reagent into each measurement chamber can be independently controlled to adjust the residence time of each liquid in each measurement chamber. In one embodiment, the flow of the sample liquid into each of the measurement chambers can be independently controlled to adjust the residence time of the sample liquid in each of the measurement chambers. In one embodiment, the flow of the first liquid reagent into each of the measurement chambers can be independently controlled to adjust the residence time of the first liquid reagent in each of the measurement chambers. In one embodiment, the flow of the second liquid reagent into each of the measurement chambers can be independently controlled to adjust the residence time of the second liquid reagent in each of the measurement chambers.

[0113] In some embodiments, the flow of one or more of the sample liquid, the first liquid reagent and / or the second liquid reagent is controlled such that the residence time of each liquid is a predetermined period of time. In one embodiment, the flow of the sample liquid is controlled such that the residence time of the sample liquid is a predetermined period of time. In one embodiment, the flow of the first liquid reagent is controlled such that the residence time of the first liquid reagent is a predetermined period of time. In one embodiment, the flow of the second liquid reagent is controlled such that the residence time of the second liquid reagent is a predetermined period of time.

[0114] In some embodiments, the device further comprises a mixing zone located between the sample chamber and the measurement chamber, the mixing zone being fluidly connected to both the sample chamber and the measurement chamber.

[0115] In some embodiments, the mixing zone comprises a mixing chamber, which is fluidly connected to the sample chamber conduit and the measurement chamber by a mixing chamber conduit.

[0116] In some embodiments, the device further comprises a third liquid reagent chamber, a third liquid reagent chamber conduit fluidly connecting the third liquid reagent chamber to the mixing zone, optionally the third liquid reagent chamber conduit connecting to the mixing zone in an alternating flow direction relative to the sample chamber conduit, and a third liquid reagent chamber conduit valve for opening and closing the third liquid reagent chamber conduit.

[0117] In some embodiments, the flow of the third liquid reagent into the mixing zone can be independently controlled to regulate the residence time of the third liquid reagent in the mixing zone, hi some embodiments, the flow of the third liquid reagent is controlled such that the residence time of the third liquid reagent is a predetermined period of time.

[0118] In some aspects of the invention, one or more of the first liquid reagent chamber, the second liquid reagent chamber, and the third liquid reagent chamber may be referred to as an auxiliary chamber. In one embodiment, the first liquid reagent chamber is referred to as the auxiliary chamber. In one embodiment, the second liquid reagent chamber is referred to as the auxiliary chamber. In one embodiment, the third liquid reagent chamber is referred to as the auxiliary chamber.

[0119] In one aspect, a method of performing a diagnostic assay may include sequentially moving liquid from a sample chamber to a measurement chamber and moving a first liquid reagent into the measurement chamber in an alternating flow direction, the method including filling the sample chamber with sample liquid, moving the sample liquid from the sample chamber to the measurement chamber, retaining the sample liquid in the measurement chamber for a predetermined period of time, moving the first liquid reagent from the first liquid reagent chamber into the measurement chamber in an alternating flow direction relative to the sample chamber liquid, and performing the measurement, optionally wherein the first liquid reagent is retained in the measurement chamber for the predetermined period of time.

[0120] In some methods, the first liquid reagent is removed from the measurement chamber before the measurement is taken.

[0121] In some embodiments, the method further comprises moving liquid from the second liquid reagent chamber to the measurement chamber in an alternating flow direction relative to the sample liquid.

[0122] In one aspect, a method of performing a diagnostic assay can include sequentially moving liquid from a sample chamber to a measurement chamber and moving a first liquid reagent and a second liquid reagent to the measurement chamber in alternating flow directions, the method including filling the sample chamber with sample liquid, moving the sample liquid from the sample chamber to the measurement chamber, retaining the sample liquid in the measurement chamber for a predetermined period of time, moving the first liquid reagent from the first liquid reagent chamber into the measurement chamber in an alternating flow direction relative to the sample liquid, moving the second liquid reagent from the second liquid reagent chamber into the measurement chamber in an alternating flow direction relative to the sample liquid, and performing the measurement, optionally wherein the first liquid and the second liquid reagent are each retained in the measurement chamber for a predetermined period of time.

[0123] In some methods, the second liquid reagent is removed from the measurement chamber before the measurement is taken.

[0124] In one aspect, a method of performing a diagnostic assay can include sequentially moving liquid from a sample chamber to a measurement chamber and moving a first liquid reagent and a second liquid reagent to the measurement chamber in alternating flow directions, the method including filling the sample chamber with sample liquid, moving the sample liquid from the sample chamber to the measurement chamber, retaining the sample liquid in the measurement chamber for a predetermined period of time, moving the first liquid reagent from the first liquid reagent chamber into the measurement chamber in alternating flow directions relative to the sample liquid, moving the second liquid reagent from the second liquid reagent chamber into the measurement chamber in alternating flow directions relative to the sample liquid, moving a further volume of the first liquid reagent from the first liquid reagent chamber to the measurement chamber in alternating flow directions relative to the sample liquid, and performing the measurement, optionally wherein the first liquid and the second liquid reagent are each retained in the measurement chamber for a predetermined period of time.

[0125] In some methods, the flow direction of the first liquid reagent and / or the second liquid reagent is at least 90 degrees to the flow direction of the sample liquid into the measurement chamber, and preferably the flow direction of the first liquid reagent and / or the second liquid reagent is opposite to the flow direction of the sample liquid.

[0126] In some embodiments, the method further comprises mixing the sample liquid with one or more additional reagents prior to moving the sample liquid into the measurement chamber.

[0127] In some methods, the sample liquid is mixed with a third liquid reagent from a third liquid reagent chamber in a mixing zone.

[0128] Methods of the Invention The invention also provides a method for carrying out any of the methods of the invention on any of the devices of the invention described above.

[0129] In some embodiments, the first liquid reagent is any liquid composition suitable for use as a washing liquid, such as a washing buffer, in an immunoassay. In some embodiments, the first liquid reagent is a liquid that includes one or more reagents selected from the list of pH buffers (e.g., PBS, Tris, carbonate / bicarbonate, HEPES, MOPS, MES), salt solutions (e.g., NaCl, KCl, MgCl2), detergents (e.g., Tween20, Tween80, Triton-X, CHAPS) and stabilizers / blocking agents (e.g., BSA, casein).

[0130] In some embodiments, the first liquid reagent is Tris-buffered saline (TBS) and phosphate-buffered saline (PBS) containing 0.05% (v / v) Tween® 20.

[0131] In some embodiments, the second liquid reagent is a detection reagent for use in immunoassay.In some embodiments, the second liquid reagent comprises one or more reagents selected from DAB (3,3'-diaminobenzidine), metal-enhanced DAB, AEC (3-amino-9-ethylcarbazole), BCIP (5-bromo-4-chloro-3-indolyl phosphate), NBT (nitro blue tetrazolium chloride), TMB (3,3',5,5'-tetramethylbenzidine), ELF (enzyme-labeled fluorescence) and OPD (phenylenediamine dihydrochloride), and preferably, the second liquid reagent comprises 3,3',5,5'-tetramethylbenzidine (TMB).

[0132] In some embodiments, the predetermined time period is between 1 and 180 seconds (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 109, 102, 104, 105, 106, 107, 7, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 1 01, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179 or 180 seconds).

[0133] In some embodiments, the predetermined time period is between 1 and 60 seconds (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 seconds).

[0134] In some embodiments, the predetermined period of time is between 10 and 30 seconds (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 seconds).

[0135] In some embodiments, the predetermined time period is between 60 and 180 seconds (e.g., 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 22,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179 or 180 seconds).

[0136] In one aspect, a cartridge for a microfluidic system is provided, in which reagents are integrated and stored in the cartridge in reservoirs sealed from flowing into the microfluidic device until directed by operation. This allows for long-term storage of the cartridge containing the reagents while protecting the reagents and the microfluidic device from contamination and degradation. Advantages of the devices described herein include valves in microfluidic systems with simple structural geometries, allowing for cost-effective manufacturing of valve features and components. Another advantage is the very small volume, suitable for the smaller volumes of fluids utilized in microfluidic devices, compared to any non-integrated off-device valves.

[0137] In one aspect, the liquid handling device may comprise a sample chamber for receiving a sample, a measurement chamber for performing one or more measurements on the sample, the measurement chamber comprising a reaction zone, a first liquid reagent chamber fluidly connected to the measurement chamber in an alternating flow direction relative to the sample chamber, a variable pressure source conduit for connecting the measurement chamber to a variable pressure source, a sample chamber conduit fluidly connecting the sample chamber to the measurement chamber, a sample chamber conduit valve for opening and closing the sample chamber conduit, a respective measurement chamber conduit for each measurement chamber, each respective measurement chamber conduit fluidly connecting a respective measurement chamber to the measurement chamber, and a respective measurement chamber conduit valve for opening and closing each respective measurement chamber conduit.

[0138] The liquid handling device allows the transfer of the first or second liquid reagent to the measurement chamber in alternating flow directions relative to the sample liquid. This configuration allows the transfer of liquid reagents (such as buffers or detection reagents) to the measurement chamber through separate conduits through which the sample liquid did not previously flow. This configuration allows for rapid, precise and controllable stopping of reactions and / or biological interactions in the measurement chamber. The use of conduits with different flow directions also results in reduced contamination of each liquid during the different method steps (i.e. reduced contamination of the sample liquid in washing steps). This may not be easily achievable with known fluid handling devices such as conventional microfluidic devices.

[0139] The liquid handling device allows for the transfer of sample from the sample chamber to the measurement chamber by lowering the pressure in the measurement chamber relative to the sample chamber. By controlling the pressure change in the measurement chamber, the volume of sample transferred into the measurement chamber can be precisely controlled. In the measurement chamber, the sample liquid may react or mix with a reagent. The device allows for the sample to be held in the measurement chamber for as long as necessary, for example for the duration required to complete the reaction with the reagent. This may not be easily achievable with known fluid handling devices, such as conventional microfluidic devices.

[0140] The sample may be held in the measurement chamber while a measurement is performed, for example as part of a diagnostic test such as an immunoassay. Again, the volume of sample transferred into the measurement chamber and its residence time in the measurement chamber may be precisely controlled.

[0141] The liquid treatment device may or may not be provided with a variable pressure source, i.e. the variable pressure source may be integrated into the liquid treatment device, but is preferably reversibly connected to the liquid treatment device and therefore provided separately.

[0142] A variable pressure source is a pressure source that can apply or provide both positive and negative pressure changes. For example, the variable pressure source may be a syringe and may be controlled by a stepper motor. Other variable pressure sources and methods of controlling a variable pressure source are known to those skilled in the art.

[0143] A liquid handling device is not limited to having only one measurement chamber or only one variable pressure source.

[0144] The measurement chambers may be configured to receive fluid from the sample chambers when the sample chamber conduit valves are open and a negative pressure change is applied to the measurement chamber or chambers.

[0145] The reagent chamber can store reagents, such as an antibody or antigen-binding portion thereof or protein solution, an antibody or antigen-binding portion thereof or protein powder, a buffer, an enzyme substrate such as 3,3',5,5'-tetramethylbenzidine "TMB", etc., for mixing or reacting with the sample to facilitate a measurement on the sample in the measurement chamber, e.g., to perform a diagnostic test on the sample.

[0146] The reagents in the reagent chambers can be easily mixed with the sample by controlling pressure changes in the liquid handling device. By providing a measurement chamber surrounded by one or more reagent chambers, the device facilitates complex mixing or washing operations, such as operations having multiple steps each of which requires precise volume control and timing that cannot be easily achieved using known fluid handling devices.

[0147] The one or more measurement chambers may comprise a first measurement chamber for performing a first measurement on a sample and a second measurement chamber for performing a second measurement on the sample. Thus, the liquid handling device may comprise a first measurement chamber conduit fluidly connecting the first measurement chamber to the sample chamber or the mixing chamber, a second measurement chamber conduit fluidly connecting the second measurement chamber to the sample chamber or the mixing chamber, a first measurement chamber conduit valve for opening and closing the first measurement chamber conduit, and a second measurement chamber conduit valve for opening and closing the second measurement chamber conduit.

[0148] Thus, a single liquid handling device may be configured to receive only one sample in the sample chamber yet perform multiple measurements or diagnostic tests to determine multiple properties of the sample.

[0149] The one or more reagent chambers may comprise one or more first dedicated reagent chambers for reagents used only for the diagnostic test performed in the first measurement chamber, one or more second dedicated reagent chambers for reagents used only for the diagnostic test performed in the second measurement chamber, and one or more shared reagent chambers for reagents used for the diagnostic test measured in both the first and second measurement chambers.

[0150] Typically, each separate measurement chamber requires its own separate source of reagent, but a more compact liquid handling device can be provided by providing a shared reagent chamber that provides reagents, such as buffers, common to two separate diagnostic tests or measurements. The same dedicated reagent chamber stores reagents, such as specific antibodies or proteins, that can be selectively mixed with the sample for a particular diagnostic test or measurement, providing a wider range of functionality for the device.

[0151] A liquid handling device comprising one or more reagent chambers may further comprise a mixing chamber for mixing the sample with a reagent from one of the one or more reagent chambers. Accordingly, the device also comprises a mixing chamber conduit fluidly connecting the mixing chamber to the measurement chamber, and a mixing chamber conduit valve for opening and closing the mixing chamber conduit.

[0152] Once the reagent is combined with the sample, the resulting combination can be shuttled (moved back and forth) between the measurement chamber and the mixing chamber to accelerate mixing of the reagent and sample (homogenize the reagent and sample) or to accelerate dissolution of the reagent in the sample or other liquid.

[0153] The liquid handling device may further comprise a waste chamber and a waste chamber conduit, the waste chamber conduit fluidly connecting the waste chamber to the measurement chamber and / or the mixing chamber.

[0154] The waste chamber may be used to safely store excess sample and / or reagents, for example, after a measurement has been performed on the sample using the liquid handling device. Additionally, the sample can be over-supplied to the mixing chamber and then transferred in precise amounts to another chamber, such as a measurement chamber, while the excess sample is drained into the waste chamber. The precisely measured sample can then be transferred to a different chamber with a precisely known volume.

[0155] The liquid handling device may further comprise a waste chamber conduit valve for opening and closing the waste chamber conduit. Alternatively, the waste chamber conduit may fluidly connect the waste chamber to the mixing chamber via the measurement chamber. Thus, the sample may be transferred directly from the measurement chamber to the waste chamber after the measurement has been performed.

[0156] At least one of the one or more measurement chambers can include a plurality of electrodes. The plurality of electrodes may be for performing electrochemical measurements. Alternatively or additionally, at least one of the one or more measurement chambers may include an element for performing optical measurements, such as a window.

[0157] Each conduit valve may be a pinch valve. The pinch valves may be operated by an external actuator that selectively applies pressure to the pinch valves to open and close them. Optionally, the conduit valves may be arranged in a circular array such that they may be operated by an actuator having a circular array of actuating elements. A pinch valve is a valve that uses a pinching effect to impede the flow of a fluid.

[0158] The conduit valves of the above-mentioned devices may be configured such that only one valve is open at any given time. The conduit valves of the above-mentioned devices may be closed by default.

[0159] The chambers of the liquid handling device may, but are not required to, include gas exchange holes to allow air or any other ambient gas to enter or exit each chamber to balance pressure changes resulting from liquid (such as a sample or reagent) entering the respective chamber.

[0160] The liquid handling devices can be fabricated from conventional materials known to those skilled in the art, such as acrylic, glass, silicon, or polydimethylsiloxane (PDMS), using conventional methods such as chemical etching, laser etching, routing or molding.

[0161] The pressure change may be applied via a variable pressure source conduit of the liquid handling device and may be applied using a variable pressure source, such as a syringe or any other means suitable for applying positive and negative pressure changes, connected to the variable pressure source conduit. The variable pressure source conduit may be connected to a measurement chamber or a mixing chamber. Alternatively, the variable pressure source conduit may be connected to another suitable part of the device to allow precise control of pressure changes throughout the device.

[0162] In a method of operating a liquid handling device, the liquid handling device comprises one or more reagent chambers as described above, and the method may further include opening a reagent chamber conduit valve corresponding to one of the one or more reagent chambers, reducing the pressure in the mixing chamber for one of the one or more reagent chambers, and closing the reagent chamber conduit valve corresponding to one of the one or more reagent chambers.

[0163] Thus, reagents can be transferred from the reagent chamber to the mixing chamber. The method may further include increasing the pressure in the mixing chamber relative to one of the one or more reagent chambers to transfer a liquid in the mixing chamber, such as a sample, to one of the one or more reagent chambers prior to reducing the pressure in the mixing chamber relative to one of the one or more reagent chambers. Thus, if one of the one or more reagent chambers contains a dry or powdered reagent, the liquid in the mixing chamber may be used to suspend or dissolve the reagent and then transfer it to the measurement chamber.

[0164] Where the liquid treatment device comprises a mixing chamber as described above, a method of operating the liquid treatment device may further comprise opening the mixing chamber conduit valve, increasing the pressure in the measurement chamber relative to the mixing chamber, decreasing the pressure in the measurement chamber relative to the mixing chamber, and closing the mixing chamber conduit valve.

[0165] Thus, a mixture, such as a mixture of sample and reagent, can be shuttled between the measurement chamber and the mixing chamber, or between one or more reagent chambers and the mixing chamber, to accelerate mixing of the reagent and sample (e.g., homogenization of the reagent and sample) or to accelerate dissolution of the reagent in the sample.

[0166] The method may further include repeating increasing the pressure in the mixing chamber and decreasing the pressure in the mixing chamber one or more times before closing the mixing chamber conduit valve.

[0167] Where the liquid treatment device comprises a waste chamber, a waste chamber conduit and a waste chamber conduit valve as described above, a method of operating the liquid treatment device includes closing one of the respective measurement chamber conduit valves, opening the waste chamber conduit valve, increasing pressure in the measurement chamber relative to the waste chamber and closing the waste chamber conduit valve. The method may further include closing one of the respective measurement chamber conduit valves.

[0168] Thus, liquid in the measurement chamber may be transferred to a waste chamber where it may be safely stored, for example after the liquid handling device has been used to perform a measurement on a sample.

[0169] Where the liquid handling device comprises a waste chamber and a waste chamber conduit fluidly connecting the waste chamber to the mixing chamber via the measurement chamber, the method of operating the liquid handling device may further include increasing the pressure in the mixing chamber relative to the waste chamber after performing measurements on the sample.

[0170] Thus, liquid in the mixing chamber can be transferred to a waste chamber where it can be safely stored, for example after the liquid handling device has been used to perform a measurement on a sample.

[0171] Where the one or more measurement chambers include multiple electrodes, the method of operating a liquid treatment device may further include performing an electrochemical measurement on the sample using the multiple electrodes.

[0172] Where each conduit valve of the liquid treatment device is a pinch valve, the method of operating a liquid treatment device may further comprise opening and closing at least one of the pinch valves by operating an actuator. The pin valves may be configured to be only one open at a time (i.e. only one pinch valve open at any one time).

[0173] As will be appreciated, the above methods can be performed in many different orders or multiple times in combination with one another as required for a given diagnostic test. The order of each method is not limited to the order in which the features are presented above, and one method need not be completed before another method is started. For example, a method may be performed in which the sample and reagents are mixed after being introduced into the measurement chamber, but before at least a portion of the sample is transferred to the measurement chamber.

[0174] In another aspect, a method of performing a diagnostic test using a liquid handling device as described above comprises loading a sample into a sample chamber and performing one or more of the methods described above. Optionally, the liquid handling device comprises one or more reagent chambers, each of the one or more reagent chambers comprising a respective reagent for the diagnostic test.

[0175] In another aspect, a method of operating a liquid handling device can include opening a third liquid reagent chamber conduit valve and increasing or decreasing the pressure in the mixing chamber by a predetermined amount relative to the third liquid reagent chamber, thereby enabling the transfer of a metered volume of liquid between the mixing chamber and the third liquid reagent chamber. Thus, the liquid handling device comprises a mixing chamber, a third liquid reagent chamber, a third liquid reagent chamber conduit fluidly connecting the third liquid reagent chamber to the mixing chamber, and a third liquid reagent chamber conduit valve for opening and closing the third liquid reagent chamber conduit. The use of a predetermined pressure change enables the transfer of a precise volume of liquid.

[0176] Increasing or decreasing the pressure in the measurement chamber or mixing chamber relative to the auxiliary chamber by a predetermined amount may include applying a predetermined pressure change for a predetermined period of time.

[0177] Increasing the pressure in the mixing chamber allows a metered volume of liquid to be transferred from the mixing chamber to the auxiliary chamber. Decreasing the pressure in the mixing chamber allows a metered volume of liquid to be transferred from the auxiliary chamber to the mixing chamber.

[0178] In another aspect, a computer program may include computer-executable instructions that, when executed by a system, cause the system to perform any of the methods described above.

[0179] In another aspect, the system may include a processor configured to execute a computer program including computer-executable instructions that, when executed by the system, cause the system to perform any of the methods described above.

[0180] The system may be a point-of-care system or a diagnostic system and / or for performing a diagnostic test on a sample.

[0181] The system may further comprise one or more of a variable pressure source configured to connect to a liquid treatment device, a variable pressure source controller to control the variable pressure source, an actuator configured to selectively open and close each of the plurality of pinch valves, and a liquid treatment device as described above. The processor may be configured to control the variable pressure source controller to control the variable pressure source according to any of the methods described above. The system may further comprise a memory for storing a computer program.

[0182] In some embodiments, the alternate flow direction may be at least 90 degrees relative to the first flow direction when measured in the same horizontal plane of the device. In some embodiments, the alternate flow direction may be between 90 and 180 degrees relative to the first flow direction when measured in the same horizontal plane of the device. In some embodiments, the alternate flow direction may be between 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 1, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179 or 180 degrees.

[0183] How to Conduct an Immunoassay The present invention is applicable to a method of performing immunoassays using a sensor that includes a means for magnetically holding beads on the sensor surface. In some embodiments, the present invention may be utilized in one or more of the following areas: immunosensors, most notably in the context of point-of-care testing, electrochemical immunoassays, whole blood immunoassays, and disposable cartridge-based immunoassays. As will be appreciated by those skilled in the art, the general concepts disclosed herein are applicable to many immunoassay methods and platforms.

[0184] The methods of the present invention are applicable to a variety of biological sample types (e.g., blood, plasma, serum, urine, interstitial fluid, and cerebrospinal fluid). The present invention is applicable to a variety of immunoassays, including both sandwich and competitive immunoassays.

[0185] Sandwich immunoassay Immunoassays are often used to detect specific analytes in a sample. For example, pairs of antibodies that can bind to the analyte to form a sandwich detectable by an enzyme or particle label on one of the antibodies are well known and available for a wide range of different analytes of interest. For example, antibodies against specific biomarkers such as testosterone or cortisol can be used to test the levels of these substances in saliva, blood or urine samples.

[0186] The presence of the antibody-analyte sandwich can be detected by a variety of means, including the use of electrochemical measurements. Some assays have been reported that utilize enzyme substrates to amplify the assay signal, thus improving assay sensitivity by producing an insoluble precipitate that is detectable by electrochemical measurements.

[0187] Many assays for biomarkers or analytes of interest have been performed on electrode devices. A typical sandwich assay uses a primary capture antibody or its antigen-binding portion and a secondary signal antibody or its antigen-binding portion to bind to different sites on the analyte. Traditional sandwich assays rely on immobilization of the capture antibody or its antigen-binding portion and removal of excess signal antibody or its antigen-binding portion before a read is taken. The signal generated is proportional to the amount of sandwiched analyte. There is no target analyte, which results in only signal noise. Sandwich assays are used, for example, in pregnancy test devices.

[0188] In some embodiments of the invention, a biological sample, such as a whole blood sample, is collected and then modified by adding a reagent comprising magnetically susceptible beads to the biological sample. The beads preferably comprise an antibody or antigen-binding portion thereof against the analyte of interest immobilized on their outer surface.

[0189] The magnetically sensitive bead concentration utilized may vary according to the assay configuration. In some embodiments, the sample is modified with magnetically sensitive beads to provide a lysing bead concentration of at least 5 μg per μl of sample, for example at least 10 μg per μl of sample, or at least 15 μg per μl of sample. Once the sample is mixed with the magnetically sensitive beads, preferably containing an antibody or antigen-binding portion thereof immobilized on its outer surface against the analyte of interest, it is possible to perform an immunoassay, for example an electrochemical immunoassay, on the modified sample to determine the concentration of the analyte. In some embodiments of the invention, at least about 10,000 beads are used for each assay. In some embodiments, the lysing bead volume is less than about 1% of the total sample assay volume, and optionally less than about 0.1% of the total sample assay volume.

[0190] In the measurement step, in some embodiments, when the sandwich is formed between the analyte-specific antibody or its antigen-binding portion immobilized on the bead, the analyte and the signal antibody, a magnetic field is applied to draw the bead to a position where it can be washed.Then, while the beads (e.g., substantially all the beads in the sample) are held in a fixed position in the cartridge (optionally on the electrode or optionally in a position spatially separated from the electrode), the sample is washed into a waste chamber, and then the sandwich on the magnetically sensitive bead is exposed to a substrate that can react with an enzyme to form a product that can be electrochemically detected.One exemplary format is an electrochemical enzyme-linked immunosorbent assay.

[0191] Biological samples In the present invention, the sample liquid may be any suitable biological sample that contains the diagnostic biomarker of interest. In some embodiments, the biological sample may be a whole blood sample, a serum sample, a plasma sample, a saliva sample, a biopsy sample (such as a healthy tissue sample or a tumor sample), a urine sample, a semen sample, a tear sample, a sputum sample, a sweat sample, a mucous membrane sample, a fecal sample, a gastric fluid sample, an intraperitoneal fluid sample, an amniotic fluid sample, a cyst fluid sample, an ascites sample, a spinal fluid sample or a synovial fluid sample, although a whole blood sample is particularly preferred. In a preferred embodiment of the present invention, the biological sample is a whole blood sample. The method may include a step of obtaining or providing a biological sample, or alternatively, the sample may already be obtained from the subject, for example by ex vivo methods.

[0192] Biological samples obtained from subjects can be stored until needed. Suitable storage methods include freezing biological samples immediately after collection, within 2 hours of collection, or up to 2 weeks after collection. Keeping at -80°C can be used for long-term storage. One or more suitable preservatives may be added, or the sample may be collected in a tube that contains one or more suitable preservatives. Preferably, the sample is analyzed immediately (or as soon as possible) after collection.

[0193] The method of the present invention may include additional steps performed on the biological sample. The sample liquid is considered to represent the biomarker status of the biomarker or analyte of interest in different patient disease states. Thus, the method of the present invention can use quantitative data regarding the biomarker or analyte of interest to determine the presence or absence or severity of different disease states. The method disclosed herein is particularly useful for the analysis of human blood.

[0194] The sample may be additionally processed prior to determining the status of the biomarker or analyte of interest. The sample may be concentrated (e.g., to increase the concentration of the biomarker or analyte of interest being quantified), centrifuged, or diluted. In other embodiments, the sample does not undergo any pretreatment and is used unprocessed (such as whole blood).

[0195] In some embodiments of the present invention, biological samples may be fractionated or enriched for specific biomarkers prior to detection and quantification (i.e., measurement). The fractionation or enrichment step may be any suitable pretreatment method step to increase the concentration of the biomarker of interest in the sample. For example, the fractionation and / or enrichment step may include centrifugation and / or filtration to remove cells or undesired analytes or fractions from the sample, or to increase the concentration of the biomarker of interest in a particular blood fraction. Such methods are known to those skilled in the art and may be used to enrich a sample for any biomarker of interest.

[0196] The method of the present invention may be performed on one test sample from a subject. Alternatively, multiple test samples may be taken from a subject, for example at least two, at least three, at least four or at least five samples from the subject. Each sample may be subjected to a single assay to quantify one of the biomarker panel members, or alternatively the sample may be tested for all of the biomarkers being quantified. Each sample may be subjected to a separate analysis using the method of the present invention, or alternatively the method may include multiple samples from a single subject undergoing diagnosis.

[0197] "Sample(s)", "one or more samples", sample liquid, biological sample, or "sample(s) of interest" are terms used interchangeably in the singular or plural, are not intended to be limited to any particular quantity, and as used herein may be any molecule or substance about which a user desires to gather information. A sample may become larger or smaller in size, volume, or content during the performance of an assay (e.g., by expansion, dilution, fractionation, or partitioning). Thus, a sample may be amplified and / or fractionated one or more times during the performance of an assay. In some embodiments, the sample contains or is suspected to contain a biomarker or analyte of interest.

[0198] As used herein, "liquid" is any aqueous or lipophilic phase that can flow freely.Liquid may further comprise one or more reagents, reaction components or samples of interest selected from cells (including any eukaryotic or prokaryotic cells, including but not limited to cells selected from human, animal, plant, fungus, bacteria, virus, protozoa, yeast, mold, algae, rickettsia and prion), proteins, peptides, antibodies, nucleic acid sequences, oligonucleotide probes, polymerase enzymes, buffers, dNTPs, organic and inorganic chemicals, and fluorescent dyes.

[0199] The embodiments are not limited to the microfluidic scale, other larger scale applications are envisioned as well.

[0200] Magnetic capture In some embodiments of the invention, the immunosensor includes an electrode and has a magnet, e.g., a permanent magnet or an electromagnet, positioned adjacent to (preferably below) the sensor. The magnetic immunosensor of the invention provides a magnetic field of greater than about 0.1 Tesla (e.g., 0.1 Tesla, 0.2 Tesla, 0.3 Tesla, 0.4 Tesla, or 0.5 Tesla, preferably 0.4 Tesla) that can attract and hold magnetically susceptible beads ranging from about 0.05 mm to about 5 mm in the region of the electrode.

[0201] The magnetic field can be measured, for example, as the magnetic field on a substantially flat surface area of ​​the magnet. Those skilled in the art will recognize that permanent magnets can include ferrite or aluminum nickel cobalt (AINiCo) magnets, which typically exhibit magnetic fields of 0.1 to 1 Tesla. Other high-field permanent magnets constructed from alloys of rare earth elements (e.g., neodymium alloys and samarium cobalt (SmCo) alloys) exhibit magnetic fields of greater than 1 Tesla, for example greater than 1.2 Tesla, or greater than 1.4 Tesla.

[0202] In some embodiments, the magnetic field can be modulated by physically actuating a permanent magnet or electromagnet between one or more positions located at different physical distances from the desired location (e.g., a sensor, or a spatially separated location for washing purposes). For example, the magnetic field can be modulated by physically actuating a permanent magnet or electromagnet between two positions, a first position away from the desired location (e.g., a sensor, or a spatially separated location for washing purposes) and a second position close to the desired location (e.g., a sensor, or a spatially separated location for washing purposes), where in the second position the magnet provides a magnetic field large enough to capture the magnetically susceptible beads at the desired location (e.g., a sensor, or a spatially separated location for washing purposes).

[0203] In another embodiment, the magnet comprises an electromagnet in which a magnetic field is generated by the flow of an electric current. The electric current may be provided by an analytical device into which a cartridge comprising the sensor is inserted and with which the sensor is in electrical contact.

[0204] Magnetically sensitive beads In some embodiments of the present invention, a biological sample, such as a blood sample, is mixed with magnetically sensitive beads. Magnetically sensitive beads may be made of any material known in the art that is susceptible to movement by a magnet (e.g., a permanent magnet or an electromagnet) that is utilized in or in conjunction with the immunosensor cartridge of the present invention. Thus, the terms "magnetic" and "magnetically sensitive" with respect to beads can be used interchangeably.

[0205] In some embodiments of the invention, the beads comprise a magnetic core, preferably coated completely or partially with a coating material. The magnetic core may comprise a ferromagnetic, paramagnetic or superparamagnetic material. In a preferred embodiment, the magnetically susceptible beads comprise a ferrite core and an outer polymer coating. In a preferred embodiment, the magnetically susceptible beads are Dynabeads®. Dynabeads® magnetic beads are uniform, non-porous, superparamagnetic, monodisperse and highly cross-linked polystyrene microspheres consisting of a uniform dispersion of magnetic material throughout the bead. The magnetic material within Dynabeads® magnetic beads consists of a mixture of maghemite (gamma-Fe2O3) and magnetite (Fe3O4). The iron content (Fe) of the beads is 12% by weight for Dynabeads® magnetic beads M-280 and 20% by weight for Dynabeads® magnetic beads M-450. Dynabeads® magnetic beads are coated with a thin polystyrene shell that encases the magnetic material, preventing any leakage from the beads or trapping of ligands inside the beads. The shell also protects the target from exposure to iron while providing a defined surface area for the adsorption or binding of various molecules.

[0206] The magnetic core may include one or more of Fe, Co, Mn, Ni, metals containing one or more of these elements, ordered alloys of these elements, crystals of these elements, magnetic oxide structures such as ferrites, and combinations thereof. In other embodiments, the magnetic core may be composed of magnetite, maghemite, or divalent metal ferrites, with the metal being, for example, Cu, Fe, Ni, Co, Mn, Mg, or Zn, or combinations of these materials.

[0207] Materials suitable for coating include synthetic and biological polymers, copolymers and polymer blends, as well as inorganic materials. Polymeric materials may include various combinations of acrylate, siloxane, styrene, acetate, alkylene glycol, alkylene, alkylene oxide, parylene, lactic acid, and glycolic acid polymers. Biopolymeric materials include starch or similar carbohydrates. Inorganic coating materials may include any combination of metals, metal alloys, and ceramics. Examples of ceramic materials may include hydroxyapatite, silicon carbide, carboxylates, sulfonates, phosphates, ferrites, phosphonates, and oxides of Group IV elements of the Periodic Table of Elements.

[0208] In other embodiments of the invention, the magnetic beads may be formed from a non-magnetic substrate, for example a material selected from the group consisting of polystyrene, polyacrylic acid and dextran, on which is disposed a suitable magnetic coating.

[0209] Taking into account the dispersibility requirements of the magnetically sensitive beads, any suitable size of magnetically sensitive beads that can be positioned with the magnet of the present invention may be utilized. In a preferred embodiment, at least 50% (measured by weight) of the magnetically sensitive beads in an assay are retained on the electrode surface (or on the spatially separated washing area surface). In some embodiments, at least 60%, 70%, 80%, 90% or 95% (measured by weight) of the magnetically sensitive beads in a given sample are retained on the electrode surface (or on the spatially separated washing area surface). As used herein, the term "spatially separated" or "spatially separated position" refers to any suitable area where the magnetically sensitive beads can be retained (e.g., for washing purposes) that does not substantially overlap with the electrode or detection area.

[0210] In some exemplary embodiments, the average particle size of the magnetically susceptible beads is from about 0.01 μm to about 10 μm, from about 0.05 μm to about 10 μm, from about 0.1 μm to about 10 μm, from about 0.2 μm to about 10 μm, from about 0.5 μm to about 10 μm, from about 0.8 μm to about 10 μm, from about 1 μm to about 10 μm, from about 2 μm to about 10 μm, Approximately 5μm to approximately 10μm, approximately 0.01μm to approximately 5μm, approximately 0.05μm to approximately 5μm, approximately 0.1μm to approximately 5μm, approximately 0.2μm to approximately 5μm, approximately 0.5μm ~about 5μm, about 0.8μm to about 5μm, about 1μm to about 5μm, about 2μm to about 5μm, about 0.01μm to about 2μm, about 0.05μm to about 2μm, about 0. 1 μm to about 2 μm, about 0.2 μm to about 2 μm, about 0.5 μm to about 2 μm, about 0.8 μm to about 2 μm, about 1 μm to about 2 μm, about 0.05 μm to about 1 μm m, about 0.1 μm to about 1 μm, about 0.2 μm to about 1 μm, about 0.5 μm to about 1 μm, about 0.8 μm to about 1 μm, about 0.05 μm to about 0.8 μm, about 0 The average particle size of the magnetically susceptible beads may range from about 0.1 μm to about 0.8 μm, about 0.2 μm to about 0.8 μm, about 0.5 μm to about 0.8 μm, about 0.05 μm to about 0.5 μm, about 0.1 μm to about 0.5 μm, about 0.2 μm to about 0.5 μm, about 0.05 μm to about 0.2 μm, about 0.1 μm to about 0.2 μm, about 0.05 μm to about 0.1 μm. Preferably, the magnetically susceptible beads have an average particle size of about 1 μm.

[0211] As used herein, the term "average particle size" refers to the average longest dimension of a particle, e.g., a bead, e.g., the diameter of a spherical particle, as determined by methods well known in the art. The particle size distribution of the magnetically susceptible beads is preferably unimodal, although beads having a multimodal distribution may also be used. While the use of spherical magnetically susceptible beads is preferred, in other embodiments, other bead shapes and structures, e.g., ellipsoidal, subspherical, cylindrical and other irregularly shaped particles, are within the meaning of the terms "bead" and "microparticle" as used herein.

[0212] Commercial sources of magnetically sensitive bead preparations include Invitrogen (Carlsbad, CA, USA) by Life Technologies, Ademtech (Pessac, France), Chemicell GmbH (Berlin, Germany), Bangs Laboratories, Inc.® (Fishers, IN) and Seradyn, Inc. (Indianapolis, IN). Many of the commercially available products incorporate surface functionalization that can be utilized to immobilize antibodies (e.g., IgG) on the bead surface. Bead functionalization can include carboxyl, amino or streptavidin modified magnetically sensitive beads.

[0213] The magnetically susceptible beads are preferably coated with an antibody or an antigen-binding portion thereof against an analyte that is a cardiovascular marker, such as, inter alia, cardiac troponin I, troponin T, troponin complex, proBNP, NT-proBNP, human chorionic gonadotropin, BNP, creatine kinase, creatine kinase subunit M, creatine kinase subunit B, creatine kinase MB (CK-MB), myoglobin, myosin light chain or modified fragments thereof. In a preferred embodiment, the cardiovascular marker is troponin I or troponin T. In another preferred embodiment, the cardiovascular marker is proBNP, NT-proBNP.

[0214] In addition, other symptom markers may be utilized. Further exemplary analytes include, but are not limited to, β-HCG, TSH, Ultra hTSH II, TT3, TT4, FT3, FT4, myeloperoxidase, D-dimer, CRP, NGAL, PSA, LH, FSH, galectin-3, prolactin, progesterone, estradiol, DHEA-S, AFP, CA125 II, CA125, CA15-3, CA19-9, CA19-9XR, CEA, thyroxine (T4), triiodothyronine (T3), T-uptake, Tg, anti-Tg, anti-TPO, ferritin, cortisol, insulin, HBsAg, HCV Ag / Ab combo, HCV core Ag, anti-HCV, AUSAB (anti-HBs), CORE, CORE-M, SHBG, iPTH, theophylline, sirolimus, tacrolimus, anti-HAV, anti-HAV IgM, HAVAB, HAVAB-M, HAVAB-M2.0, HAVAB-G, HAVAB2.0, HAVAB2.0 Quant, IgM, CMV IgM, CMV IgG, a-2-microglobulin, digitoxin, HBe, anti-HBe, HBeAg, HIVl / 2gO, HIV Ag / Ab combo, testosterone, SCC, vitamin B12, folate, syphilis, anti-HBc, Ibella IgG, Ibella IgM, homocysteine, MPO, cytomegalovirus (CMV) IgG avidity, toxo Ig avidity, toxo IgG, toxo IgM, C-peptide, vitamin D, HTLV I / II, total Includes hCG, progesterone, estradiol, prolactin, myomatin, tPSA, fPSA, carbamazepine (CBZ), digoxin, gentamicin, NAPA, phenytoin, phenobarbital, valproic acid, vancomycin, procaine, quinidine, tobramycin, methamphetamine (METH), amphetamine (AMPH), barbiturates, benzodiazepines, cannabis, cocaine, methadone, opiates, PCP, acetaminophen, ethanol, salicylates, tricyclics, holoTc, anti-CCP, HbAlc, barbs-U.In certain embodiments of the invention, the antibody or antigen-binding portion thereof is directed against an analyte of low abundance in a sample. The above abbreviations are familiar to those skilled in the art.

[0215] The magnetic immunosensor and method of the present invention also includes a second antibody or antigen-binding portion thereof, which is preferably an enzyme-linked antibody or antigen-binding portion thereof, also referred to herein as a signal antibody or antigen-binding portion thereof. In some embodiments, the enzyme-linked antibody or antigen-binding portion thereof is in the form of a liquid reagent, which may also include the magnetically susceptible beads utilized in the present invention, as described below. Both the bead-immobilized antibody and the enzyme-linked antibody may be monoclonal, polyclonal, fragments thereof, and combinations thereof. Furthermore, one or more of the antibodies may be labeled with a variety of labels, including radioactive labels, enzymes, chromophores, fluorophores, chemiluminescent species, ionophores, electroactive species, and others known in the immunoassay field. When the second antibody or antigen-binding portion thereof is labeled with an enzyme, ALP, horseradish peroxidase (HRP), or glucose oxidase are preferred. In other embodiments, the analyte is labeled with fluorescein, ferrocene, p-aminophenol, or derivatives thereof. In a preferred embodiment, the second antibody or antigen-binding portion thereof is enzymatically linked to horseradish peroxidase (HRP).

[0216] In some embodiments, the magnetically sensitive beads are mixed homogeneously with the sample. In yet other embodiments, the magnetically sensitive beads may be mixed less homogeneously with the sample, but one objective of the present invention is to optimize the position and concentration of the beads relative to the electrodes. Those skilled in the art will recognize that the magnetically sensitive beads of the present invention may be added to the biological sample before being introduced into the magnetic immunosensor device, for example, as an integral part of the blood collection device, or as a standard manual addition step. However, for the convenience of the user and to ensure a quality assay, the magnetically sensitive beads are preferably included in the immunosensor cartridge, for example, in one or more of the liquid storage capsules described above.

[0217] In some embodiments of the present invention, a sample, for example a whole blood sample, is collected and then modified by combining with a reagent including magnetically sensitive beads. In addition to the magnetically sensitive beads, the reagent may further include one or more of beads for reducing leukocyte interference, leukocyte killing reagents, buffers, salts, detergents, stabilizers, simple carbohydrates, complex carbohydrates, and various combinations thereof. The reagent may also include an enzyme-labeled antibody or antigen-binding portion thereof against the analyte (e.g., the labeled antibody or antigen-binding portion thereof described above). In some embodiments of the present invention, the additional components required for the assay are combined and included in a single reagent. In some embodiments of the present invention, at least two additional components required for the assay are combined and included in a single reagent, and other components required for the assay are included in separate reagents. In other embodiments of the present invention, the additional components required for the assay are individually included in separate reagents.

[0218] In some embodiments, magnetically sensitive beads are used to modify a biological sample, such as blood, in a first container or location, and then the sample is passed to a second container or location that contains a capture antibody and a signal antibody. In some embodiments, the magnetically sensitive beads are contained in a solution and mixed with the biological sample, and the resulting modified sample is introduced into a magnetic immunosensor cartridge. For example, a blood sample can be mixed with the magnetically sensitive beads to form a modified sample, which is then introduced into the device. In certain embodiments, a magnetic immunosensor device, such as a cartridge, includes a capsule that contains a liquid that contains magnetically sensitive beads, and the magnetically sensitive beads can be mixed with a biological sample in the device and then processed substantially as described herein to form an assay (e.g., a sandwich assay) for analyte detection.

[0219] Additionally, any immunoassay format known in the art may be modified to include the magnetically susceptible beads of the present invention, for example, by adding the beads in a sample pretreatment step. Pretreatment may be accomplished, for example, by incorporating the beads into a blood collection device, a separate container, or may occur in the immunoassay device itself by incorporating the beads as part of the assay method.

[0220] In some embodiments of the present invention, the beads are mobile, and thus can interact with the analyte. After binding to the analyte of interest, magnetic forces are used to concentrate the beads (either at the electrode or at a location spatially separated from the electrode) for washing purposes, and then at the electrode for measurement purposes. One advantage of using mobile beads according to the present invention is that their movement in the sample or fluid accelerates the binding reaction, making the capture step of the assay faster.

[0221] In some embodiments of the invention, additives may be included in the magnetic immunosensor device or used in conjunction with the assay. In some embodiments, an anticoagulant may be added. For example, heparin may be added to improve performance if the specimen was not collected in a heparinized tube or was not properly mixed in the heparinized tube. Any suitable amount of heparin may be added such that fresh, non-heparinized blood remains unclotted during the assay cycle of the cartridge, typically within the range of 2-20 minutes. In still other embodiments, one or more of Proclin, DEAE-Dextran, Tris buffer, and lactitol may be added as reagent stabilizers. In further embodiments, a surfactant such as polysorbate 20, also known as Tween® 20, may be added to reduce protein binding to plastic, which is a preferred material for the cartridge housing of the magnetic immunosensing device. The addition of a surfactant also facilitates uniform coating of the reagent on the plastic surface and minimizes crystallization of sugars (e.g., lactitol). In other embodiments of the invention, an antimicrobial or biocide (e.g., sodium azide) may be added to inhibit bacterial growth.

[0222] Computer Methods The methods described above can be performed in combination with one another, in many different orders, or multiple times, as required for a given diagnostic test. It is not necessary for one method to be completed before another method is performed.

[0223] The described method can be implemented by a diagnostic system using computer executable instructions. A computer program product or computer readable medium can include or store computer executable instructions. A computer program product or computer readable medium can include a hard disk drive, a flash memory, a read-only memory (ROM), a CD, a DVD, a cache, a random-access memory (RAM), and / or any other storage medium in which information is stored for any period of time (e.g., long term, permanent, short term, for temporary buffering, and / or for caching information). A computer program can include computer executable instructions. A computer readable medium can be a tangible or non-transitory computer readable medium. The term "computer readable" encompasses "machine readable."

[0224] Thus, also disclosed is a computer program comprising computer implementable instructions which, when executed by a diagnostic system, causes the diagnostic system to perform any of the methods described above. EXAMPLES

[0225] Example 1 - Magnet actuation used to spread beads over electrodes to increase signal response Human pooled plasma (Lit-heparin) supplemented with 625ng / ml of analyte (recombinant NT-pro-BNP) is measured using standard procedures. Magnetic particles (1μm epoxy beads) coated with capture antibody or its antigen-binding portion were mixed with plasma sample and sample diluent (25mM aqueous TBS) containing bovine serum albumin (5%), calcium chloride (20mM), magnesium chloride (20mM), Proclin 300 (0.05%) and Tween 20 (0.1%) and detection antibody labeled with horseradish peroxidase (HRP).

[0226] The reaction mixture was incubated in the tube for 2 min at room temperature, after which the magnetic particles were removed by a magnetic field and washed with an assay wash buffer containing a standard concentration of tween-20 in PBS (10 mM). The washed magnetic particles were driven at a specific flow rate (2 μl / sec) in the cartridge and localized on the electrode.

[0227] A magnetic field (0.4 Tesla) was applied on and off 12 times by using magnet actuation compared to the magnetic field applied without magnet actuation. A 3,3',5,5'-tetramethylbenzidine (TMB) solution was introduced into the electrode chamber. The assay response recorded for each sample (current after 60 seconds of chronoamperometry at -50 mV with IR compensation) is plotted to compare the signals obtained with and without actuation (Figure 1). As shown in Figure 1, the assay method provides a 1.5-fold signal increase using the optimized method.

[0228] Example 2 - Washing with and without magnetic actuation - Optimization of bead assay washing using magnetic actuation Analyte-free human plasma (EDTA) was measured in parallel using the standard procedure in a flow cell format, varying only the magnet actuation process.

[0229] Identical wash volumes (470 μl) were tested under standardized assay conditions. Plasma samples were mixed with sample diluent containing a detection antibody labeled with horseradish peroxidase (HRP) in a 1:1 ratio and magnetic particles coated with capture antibodies. The reaction mixture was incubated in the fluidic channel at 30° C. for 5 min, after which the magnetic particles were removed by a magnetic field and washed with assay wash buffer (only varying with or without the use of a magnet actuation process during washing).

[0230] The washed magnetic particles were resuspended and transported to a clean flow cell where the beads were pulled down onto a 2.5 mm electrode and the detection solution was introduced. Assay response currents recorded using chronoamperometry at -50 mV for 0 and 100 ng / L analyte samples using each wash process (varying magnet actuation) showed that using controlled mechanical magnet actuation during the wash process reduced nonspecific binding of the assay as defined by background measurements when no analyte was present (Figure 2). Figure 2 shows that the background signal is reduced by 4-fold by using the improved wash method.

[0231] Example 3 - Reduction of wash volume by magnet actuation process - Optimization of bead assay washing using magnet actuation Analyte-free human plasma (EDTA) and the same plasma supplemented with 100 ng / L analyte were measured in parallel using the standard procedure in the flow cell format, varying only the wash volume.

[0232] The same actuation method is used under standardized assay conditions. Plasma samples were mixed with sample diluent containing horseradish peroxidase (HRP)-labeled detection antibody in a 1:1 ratio and magnetic particles coated with capture antibody. The reaction mixture was incubated in the fluidic channel at 30° C. for 5 min, after which the magnetic particles were removed by a magnetic field and washed with assay wash buffer using sequential mechanical magnet actuation (varying only the total wash volumes used: 470 μl, 260 μl, 160 μl). The washed magnetic particles were resuspended and transported to a clean flow cell, where the beads were pulled down onto a 2.5 mm electrode and the detection solution was introduced.

[0233] Assay response currents recorded using chronoamperometry at -50 mV for 0 and 100 ng / L analyte samples using the same wash process (varying total wash volumes) showed that by using controlled mechanical magnet actuation during the wash process, not only could the assay have a lower background response, but the volume required to wash the assay could be further reduced from 470 μl to 160 μl (Figure 3). Figure 3 shows that the wash volume is reduced three-fold by using the improved wash method. This allows a smaller amount of wash solution to be stored on board the cartridge, thus reducing the required cartridge footprint. The smaller cartridge is more suitable for point-of-care testing devices, thereby meaning that this method is an improved method for use with such devices.

[0234] Example 4 - Signal Improvement Using Magnet Actuation - Signal to Noise Optimization Using Magnet Actuation Analyte-free human plasma (EDTA) was used in parallel with the same samples supplemented with 50 ng / L of analyte and measured in parallel using the standard procedure in the flow cell format, where only the magnet actuation process was changed before the measurement.

[0235] The same reagents were tested under standardized assay conditions. Plasma samples were mixed with sample diluent containing a detection antibody labeled with horseradish peroxidase (HRP) in a 1:1 ratio and magnetic particles coated with capture antibodies. The reaction mixture was incubated in the fluidic channel at 30°C for 5 minutes, after which the magnetic particles were removed by a magnetic field and washed with assay wash buffer.

[0236] The washed magnetic particles were resuspended and transported to a clean flow cell where the beads were pulled down onto a 2.5 mm electrode and detection solution was introduced prior to reading (comparing with and without magnet actuation). Assay response currents recorded using chronoamperometry at -50 mV for 0 and 50 ng / L analyte samples with and without magnet actuation showed that the use of controlled mechanical magnet actuation increased the specific signal and reduced nonspecific interactions reflected in improved assay performance (Figure 4).

[0237] Example 5 - Bead resuspension using an air-liquid interface - Optimization of bead resuspension using an air-liquid interface Analyte-free human plasma (EDTA) was supplemented with 100 ng / L analyte and measured in parallel using the standard procedure in a flow cell format, varying only the air-liquid interface used.

[0238] The same reagents were tested under standardized assay conditions. Plasma samples were mixed with sample diluent containing a detection antibody labeled with horseradish peroxidase (HRP) in a 1:1 ratio and magnetic particles coated with capture antibodies. The reaction mixture was incubated in the fluidic channel at 30°C for 5 minutes, after which the magnetic particles were removed by a magnetic field and washed with assay wash buffer. The washed magnetic particles were resuspended and transported to a clean flow cell with varying numbers of air-liquid interfaces used to transfer the beads.

[0239] If one interface was used during the resuspension and transfer of the magnetic beads, the flow cell was emptied using air flow and a single volume of wash buffer was used to resuspend the magnetic beads and transfer them to the sensor. If two interfaces were used during the resuspension and transfer of the magnetic beads, the flow cell was emptied using air flow and a first volume and a second volume of wash buffer were used to resuspend the magnetic beads and transfer them to the sensor. An air flow was used to generate an air-liquid interface between the first volume of wash buffer and the second volume of wash buffer.

[0240] The beads are lowered onto the 2.5 mm electrode and detection solution is introduced. Assay response currents recorded using chronoamperometry at -50 mV for 0 and 100 ng / L analyte samples with each interface replicate (varying interfaces) showed that increasing the number of interfaces increased the signal of the assay, reflected by an increased number / spreading of beads on the electrode surface (Figure 5).

[0241] Example 6 - Establishing the ability to measure precipitated TMB on magnetic beads using DPV Troponin-free serum was used as the matrix for the experiments and standard laboratory procedures were followed.

[0242] The capture antibody-coated magnetic particles were mixed with troponin-free serum and sample diluent (25 mM aqueous TBS) containing bovine serum albumin (5%), sodium chloride (400 mM), and detection antibody labeled with horseradish peroxidase (HRP). The reaction mixture was incubated in the well plate at 25° C. for 5 min, after which the magnetic particles were removed by a magnetic field and washed with assay wash buffer PBS-T 0.1%.

[0243] The washed magnetic particles were resuspended on the wells and a precipitating 3',3',5,5'-tetramethylbenzidine (TMB) solution was introduced and incubated for 1 min. The TMB was then removed by using a magnet to pull the magnetic particles to the bottom of the well plate. The beads were then resuspended, loaded into separate wells and pulled down onto the electrode surface. The assay response recorded for two concentrations, 0 ng / ml and 50 ng / ml, was recorded using differential pulse voltammetry (DPV) peak height signals and displays the signal-to-noise ratio (S / N) (Figure 7). These results confirmed the precipitation of the TMB substrate onto the magnetic beads.

[0244] According to the proposed method, the detection reaction can be stopped by precipitating TMB onto magnetic beads, using additional washing and optionally resuspension steps, which improve the signal-to-noise ratio by reducing the background without significantly affecting the detection signal.

[0245] Example 7 - Multiplex Assay The magnetic beads can be coated with antibodies against two or more different target analytes (e.g., NT-proBNP and troponin). The sample is mixed (or premixed) with a diluent and then incubated with a mixture of different second (i.e., detection) antibodies against each target analyte. For example, for the first analyte (e.g., NT-proBNP), the detection antibody can be labeled with alkaline phosphatase, and for the second analyte (e.g., troponin), the detection antibody can be labeled with HRP. This solution is then mixed with the magnetic particles and incubated at a certain temperature (as described herein) for a period of time (as described herein). The beads are then captured on the surface of the electrode using a magnet.

[0246] The beads with the analyte and the labeled secondary antibody are washed by resuspension and pulled down. After washing, two substrates are added sequentially to the beads: 1) a precipitating substrate for one of the first enzymes (e.g. an alkaline phosphatase substrate such as BCIP / NBT) is added and incubated for a period of time. This reacts and deposits a precipitate locally in the area of ​​the bead where the first analyte (e.g. NT-proBNP) was captured. The beads are then washed, thus removing the excess substrate and leaving only the precipitate formed on the beads. Subsequently, a suitable substrate for the second enzyme (e.g. an HRP substrate such as precipitating TMB) is added, which, like the first enzyme substrate, reacts and precipitates only in the area where the second analyte (e.g. troponin) was captured. The beads undergo one final wash and are then transferred to the detection electrode.

[0247] An electrochemical detection technique is then used (preferably differential pulse voltammetry, DPV), resulting in two characteristic peaks for each of the precipitating reagents, since they have distinct electrochemical profiles. The signal generated is proportional to the amount of each analyte. Similarly, beads can be coated with multiple precipitating electrochemical mediators with unique electrochemical profiles to create a more multiplexed option for parallel detection of different analytes.

[0248] The present invention is further described by the following numbered embodiments. 1) A method for measuring an analyte of interest in a biological sample, comprising: The biological sample is a magnetically susceptible bead conjugated to a first antibody or an antigen-binding portion thereof capable of binding to the analyte of interest; and A second antibody or antigen-binding portion thereof capable of binding to the analyte of interest conjugated to an enzyme. and combining with a composition comprising holding the magnetically susceptible beads on the electrodes using a magnetic field; contacting the magnetically susceptible beads with a substrate of the enzyme, where the enzyme substrate is converted by the enzyme into an electroactive molecule; and obtaining an electrochemical measurement using said electrode.

[0249] 2) A method for measuring one or more analytes of interest in a biological sample, comprising: The biological sample is magnetically sensitive beads conjugated to one or more antibodies or antigen-binding portions thereof, each antibody or antigen-binding portion thereof being capable of binding to one of the analytes of interest; and one or more second antibodies or antigen-binding portions thereof conjugated to an enzyme, each second antibody or antigen-binding portion thereof being capable of binding to one of the analytes of interest; and combining with a composition comprising holding the magnetically susceptible beads on the electrodes using a magnetic field; contacting the magnetically susceptible beads with one or more substrates of the enzyme, where the enzyme substrate is converted by the enzyme into an electroactive molecule; and obtaining an electrochemical measurement using said electrode.

[0250] 3) The method of embodiment 2, wherein the method is capable of detecting at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 different analytes of interest.

[0251] 4) The method of embodiment 2 or 3, wherein each magnetically susceptible bead is conjugated to a plurality of different antibodies or antigen-binding portions thereof, each antibody or antigen-binding portion thereof being capable of binding to one of the analytes of interest.

[0252] 5) The method of embodiment 2 or 3, wherein the magnetically susceptible beads comprise a plurality of different magnetically susceptible bead sets, each magnetically susceptible bead set being conjugated to a different antibody or antigen-binding portion thereof, each antibody or antigen-binding portion thereof being capable of binding to one of the analytes of interest.

[0253] 6) A method for measuring an analyte of interest in a biological sample according to any of the preceding embodiments, wherein the enzyme substrate is converted by the enzyme into a soluble electroactive molecule at the electrode.

[0254] 7) A method for measuring an analyte of interest in a biological sample according to any one of embodiments 1 to 5, wherein an enzyme substrate is converted by an enzyme into an electroactive molecule precipitated on a magnetically susceptible bead.

[0255] 8) A method for measuring an analyte of interest in a biological sample according to any of the preceding embodiments, further comprising incubating the combined biological sample and composition such that the one or more first antibodies and / or the one or more second antibodies bind to the analyte of interest.

[0256] 9) holding the magnetically susceptible beads in a fixed position using a magnetic field; Washing the magnetically sensitive beads 4. The method for measuring an analyte of interest in a biological sample of any preceding embodiment, further comprising:

[0257] 10) The method of embodiment 9, wherein the fixation location is located at an electrode. 11) The method of embodiment 9, wherein the fixed location is a location spatially separated from the electrode, and optionally, the spatially separated location is a blank or non-functional electrode.

[0258] 12) holding the magnetically susceptible beads in a first position using a magnetic field; washing the magnetically susceptible beads by modulating the magnetic field such that the magnetically susceptible beads are retained in a second position; 10. A method for measuring an analyte of interest in a biological sample according to any one of embodiments 1 to 9, further comprising:

[0259] 13) A method for measuring an analyte of interest in a biological sample according to any of the preceding embodiments, wherein the magnetic field is generated by a stationary magnet.

[0260] 14) A method for measuring an analyte of interest in a biological sample according to any of the preceding embodiments, wherein the magnetic field is generated by an electromagnet.

[0261] 15) A method for measuring an analyte of interest in a biological sample according to any one of embodiments 12 to 14, wherein the magnetic field is modulated by physically actuating a magnet.

[0262] 16) A method for measuring an analyte of interest in a biological sample according to any one of embodiments 12 to 14, wherein the magnetic field is modulated by controlling the current in an electromagnet.

[0263] 17) A method for measuring an analyte of interest in a biological sample according to any one of embodiments 9 to 15, wherein the magnetic field is located in a microfluidic device having a flow channel, and the magnetic field is modulated by actuating a magnet in a direction perpendicular to the direction of flow in the flow channel.

[0264] 18) A method for measuring an analyte of interest in a biological sample according to any one of embodiments 9 to 14 or 16, wherein the magnetic field is disposed in a microfluidic device having a flow channel, and the magnetic field is modulated by controlling a current in an electromagnet.

[0265] 19) A method for measuring an analyte of interest in a biological sample according to any one of embodiments 12 to 18, wherein the magnetically susceptible beads are moved between the first and second positions at least 20 times, for example at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times or at least 19 times.

[0266] 20) A method for measuring an analyte of interest in a biological sample according to any one of embodiments 12 to 18, wherein the magnetically susceptible beads are moved between the first and second positions 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times, preferably 12 times.

[0267] 21) A method for measuring an analyte of interest in a biological sample described in any one of embodiments 12 to 20, wherein the magnetic field is modulated such that the magnetically susceptible beads are periodically moved between a first position and a second position.

[0268] 22) The period during which the magnetically susceptible beads are in the first position and the second position is about 0.01 seconds to about 5 seconds, about 0.05 seconds to about 5 seconds, about 0.1 seconds to about 5 seconds, about 0.2 seconds to about 5 seconds, about 0.3 seconds to about 5 seconds, about 0.4 seconds to about 5 seconds, about 0.5 seconds to about 5 seconds, about 1 second to about 5 seconds, about 2 seconds to about 5 seconds, about 3 seconds to about 5 seconds, about 4 seconds to about 5 seconds, about 0.01 seconds to about 4 seconds, about 0.05 seconds to about 4 seconds, about 0.1 seconds to about 4 seconds, about 0.2 seconds to about 4 seconds, about 0.3 seconds to about 4 seconds, about 0.4 seconds to about 4 seconds, about 0.5 seconds to 4 seconds, 1 second to 4 seconds, 2 seconds to 4 seconds, 3 seconds to 4 seconds, 0.01 seconds to 3 seconds, 0.05 seconds to 3 seconds, 0.1 seconds to 3 seconds, 0.2 seconds to 3 seconds, 0.3 seconds to 3 seconds, 0.4 seconds to 3 seconds, 0.5 seconds to about 3 seconds, about 1 second to about 3 seconds, about 2 seconds to about 3 seconds, about 0.01 seconds to about 2 seconds, about 0.05 seconds to about 2 seconds, about 0.1 seconds to about 2 seconds, about 0.2 seconds to about 2 seconds, about 0.3 seconds to about 2 seconds, about 0.4 seconds to about 2 seconds, about 0.5 seconds to about 2 seconds, about 1 second to about 2 seconds, approximately 0.01 seconds to approximately 1 second, approximately 0.05 seconds to approximately 1 second, approximately 0.1 seconds to approximately 1 second, approximately 0.2 seconds to approximately 1 second, approximately 0.3 seconds to approximately 1 second, approximately 0.4 seconds to approximately 1 second, approximately 0.5 seconds to approximately 1 second, approximately 0.01 seconds to approximately 0.5 seconds, approximately 0.05 seconds to approximately 0.5 seconds , approximately 0.1 seconds to approximately 0.5 seconds, approximately 0.2 seconds to approximately 0.5 seconds, approximately 0.3 seconds to approximately 0.5 seconds, approximately 0.4 seconds to approximately 0.5 seconds, approximately 0.01 seconds to approximately 0.4 seconds, approximately 0.05 seconds to approximately 0.4 seconds, approximately 0.1 seconds to approximately 0.4 seconds, approximately 0.2 seconds to approximately 0.4 seconds, approximately 22. The method for measuring an analyte of interest in a biological sample according to embodiment 21, wherein the time is from 0.3 seconds to about 0.4 seconds, from about 0.01 seconds to about 0.3 seconds, from about 0.05 seconds to about 0.3 seconds, from about 0.1 seconds to about 0.3 seconds, from about 0.2 seconds to about 0.3 seconds, from about 0.01 seconds to about 0.2 seconds, from about 0.05 seconds to about 0.2 seconds, from about 0.1 seconds to about 0.2 seconds, from about 0.01 seconds to about 0.1 seconds, from about 0.05 seconds to about 0.1 seconds, from about 0.01 seconds to about 0.05 seconds, preferably from about 3 seconds to about 5 seconds, more preferably about 4 seconds.

[0269] 23) A method for measuring an analyte of interest in a biological sample according to any one of embodiments 9 to 22, wherein the magnetically susceptible beads are washed with a washing solution and / or air.

[0270] 24) A method for measuring an analyte of interest in a biological sample according to embodiment 23, wherein the magnetically susceptible beads are washed consecutively and separately using both a washing solution and air.

[0271] 25) A method for measuring an analyte of interest in a biological sample according to any one of embodiments 9 to 24, wherein the magnetically susceptible beads are washed alternately with washing solution and air at least two times, at least three times, at least four times or at least five times, preferably the magnetically susceptible beads are washed alternately with washing solution and air two times.

[0272] 26) A method for measuring an analyte of interest in a biological sample according to any of the preceding embodiments, wherein the biological sample is diluted prior to combining with the magnetically susceptible beads.

[0273] 27) A method for measuring an analyte of interest in a biological sample according to any of the preceding embodiments, wherein the electrode is a carbon ink electrode.

[0274] 28) A method for measuring an analyte of interest in a biological sample according to any of the preceding embodiments, wherein the analyte of interest is brain natriuretic peptide or N-terminal pro-BNP.

[0275] 29) A method for measuring an analyte of interest in a biological sample according to any one of embodiments 1 to 28, wherein the analyte of interest is cardiac troponin or cardiac troponin subunit I (cTnI).

[0276] 30) A method for measuring an analyte of interest in a biological sample according to any one of embodiments 2 to 27, wherein the one or more analytes of interest are selected from the list consisting of brain natriuretic peptide, N-terminal pro-BNP, cardiac troponin and cardiac troponin subunit I (cTnI).

[0277] 31) A method for measuring an analyte of interest in a biological sample according to any one of embodiments 2 to 27 or 30, wherein the one or more analytes of interest are N-terminal pro-BNP and cardiac troponin subunit I (cTnI).

[0278] 32) A method for measuring an analyte of interest in a biological sample according to any of the preceding embodiments, wherein the enzyme is horseradish peroxidase (HRP).

[0279] 33) A method for measuring an analyte of interest in a biological sample according to any one of embodiments 1 to 31, wherein the enzyme is alkaline phosphatase (ALP).

[0280] 34) A method for measuring an analyte of interest in a biological sample according to any one of embodiments 2 to 33, wherein at least one of the one or more second antibodies or antigen-binding portions thereof is conjugated to horseradish peroxidase (HRP) and at least one of the one or more second antibodies or antigen-binding portions thereof is conjugated to alkaline phosphatase (ALP).

[0281] 35) The method for measuring an analyte of interest in a biological sample according to any of the preceding embodiments, wherein the substrate for the enzyme is selected from the list consisting of 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS), o-phenylenediamine dihydrochloride (OPD), paranitrophenyl phosphate (PNPP) and BCIP / NBT (a combination of BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (nitroblue tetrazolium)).

[0282] 36) A method for measuring an analyte of interest in a biological sample according to embodiment 35, wherein the products of the enzymatic reaction are precipitated, and optionally the products are precipitated onto magnetically susceptible beads.

[0283] 37) A method for measuring an analyte of interest in a biological sample according to embodiment 35, wherein the substrate is 3,3',5,5'-tetramethylbenzidine (TMB) and the product of the enzymatic reaction is precipitated, optionally the product being precipitated onto magnetically susceptible beads.

[0284] 38) A method for measuring an analyte of interest in a biological sample according to embodiment 35, wherein the substrate is BCIP / NBT (a combination of BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (nitro blue tetrazolium)) and the products of the enzymatic reaction are precipitated, optionally the products being precipitated onto magnetically susceptible beads.

[0285] 39) A method for measuring an analyte of interest in a biological sample according to any of the preceding embodiments, wherein the electrochemical measurement is indicative of the concentration or amount of the analyte of interest.

[0286] 40) A method for measuring an analyte of interest in a biological sample according to embodiment 39, wherein the concentration or amount of the analyte of interest is determined by comparison with a reference solution.

[0287] 41) A method for measuring an analyte of interest in a biological sample according to any of the preceding embodiments, wherein the electrochemical measurement is an amperometric, voltametric, potentiometric, impedimetric, or electrochemical impedance spectroscopy measurement, preferably a chronoamperometric measurement.

[0288] 42) A method for measuring an analyte of interest in a biological sample according to any of the preceding embodiments, wherein the electrochemical measurement is differential pulse voltammetry (DPV).

[0289] 43) The method of any of the preceding embodiments, wherein the measuring method is a sandwich immunoassay.

[0290] 44) The method of any of the previous embodiments, wherein at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%) of the beads are retained on the surface of the electrode, measured by weight.

[0291] 45) A method for measuring an analyte of interest in a biological sample, comprising: The biological sample is a magnetically susceptible bead conjugated to a first antibody or an antigen-binding portion thereof capable of binding to the analyte of interest; and A second antibody or antigen-binding portion thereof capable of binding to the analyte of interest conjugated to an enzyme. and combining with a composition comprising holding the magnetically susceptible beads on the electrodes using a magnetic field; contacting the magnetically susceptible beads with a substrate of the enzyme, where the enzyme substrate is converted by the enzyme into a precipitated electroactive molecule; and obtaining an electrochemical measurement using said electrode.

[0292] 46) A method for measuring an analyte of interest in a biological sample, comprising: The biological sample is magnetically sensitive beads conjugated to one or more antibodies or antigen-binding portions thereof, each antibody or antigen-binding portion thereof being capable of binding to one of the analytes of interest; and one or more second antibodies or antigen-binding portions thereof conjugated to an enzyme, each second antibody or antigen-binding portion thereof being capable of binding to one of the analytes of interest; and combining with a composition comprising holding the magnetically susceptible beads on the electrodes using a magnetic field; contacting the magnetically susceptible beads with one or more substrates of the enzyme, where the enzyme substrate is converted by the enzyme into a precipitated electroactive molecule; and obtaining an electrochemical measurement using said electrode.

[0293] 47) The method of embodiment 46, wherein the method is capable of detecting at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 different analytes of interest.

[0294] 48) The method of any one of embodiments 45 or 46, wherein the electroactive molecule is precipitated on a magnetically susceptible bead.

[0295] 49) The method of any one of embodiments 45 to 47, wherein the one or more analytes of interest are N-terminal proBNP and cardiac troponin subunit I (cTnI).

[0296] 50) A method for measuring an analyte of interest in a biological sample according to any one of embodiments 45 to 48, wherein the enzyme is horseradish peroxidase (HRP).

[0297] 51) A method for measuring an analyte of interest in a biological sample according to any one of embodiments 45 to 48, wherein the enzyme is alkaline phosphatase (ALP).

[0298] 52) A method for measuring an analyte of interest in a biological sample according to any one of embodiments 46 to 49, wherein at least one of the one or more second antibodies or antigen-binding portions thereof is conjugated to horseradish peroxidase (HRP) and at least one of the one or more second antibodies or antigen-binding portions thereof is conjugated to alkaline phosphatase (ALP).

[0299] 53) The method for determining an analyte of interest in a biological sample according to any one of embodiments 45 to 51, wherein the substrate for the enzyme is selected from the list consisting of 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS), o-phenylenediamine dihydrochloride (OPD), paranitrophenyl phosphate (PNPP) and BCIP / NBT (BCIP (5-bromo-4-chloro-3-indolyl phosphate) in combination with NBT (nitroblue tetrazolium)).

[0300] 54) The method of any of the preceding embodiments, wherein the magnetically susceptible beads and biological sample are incubated at a temperature between 10°C and 50°C, optionally between 15°C and 45°C, further optionally between 20°C and 40°C, further optionally between 20°C and 30°C, further optionally between 25°C and 35°C, further optionally at 25°C, further optionally at 30°C, and further optionally at 40°C.

[0301] 55) A composition comprising a magnetically susceptible bead conjugated to a first antibody or an antigen-binding portion thereof capable of binding to an analyte of interest; a second antibody or antigen-binding portion thereof capable of binding to the analyte of interest conjugated to an enzyme; A composition comprising:

[0302] 56) A composition comprising magnetically sensitive beads conjugated to one or more antibodies or antigen-binding portions thereof, each antibody or antigen-binding portion thereof being capable of binding to an analyte of interest; one or more second antibodies or antigen-binding portions thereof conjugated to an enzyme, each of the second antibodies or antigen-binding portions thereof being capable of binding to one of the analytes of interest; A composition comprising:

[0303] 57) The composition of embodiment 56, wherein each magnetically susceptible bead is conjugated to a plurality of different antibodies or antigen-binding portions thereof, each antibody or antigen-binding portion thereof being capable of binding to one of the analytes of interest.

[0304] 58) The composition of embodiment 56, wherein the magnetically susceptible beads comprise a plurality of different magnetically susceptible bead sets, each magnetically susceptible bead set being conjugated to a different antibody or antigen-binding portion thereof, each antibody or antigen-binding portion thereof being capable of binding to one of the analytes of interest.

[0305] 59) A composition described in any one of embodiments 55 to 58, wherein the composition is capable of detecting at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 different analytes of interest.

[0306] 60) The composition of any one of embodiments 55 to 59, wherein the analyte of interest is brain natriuretic peptide or N-terminal pro-BNP.

[0307] 61) The composition of any one of embodiments 55 to 59, wherein the analyte of interest is cardiac troponin or cardiac troponin subunit I (cTnI).

[0308] 62) The composition of any one of embodiments 55 to 61, wherein the one or more analytes of interest are N-terminal proBNP and cardiac troponin subunit I (cTnI).

[0309] 63) The composition of any one of embodiments 55 to 62, wherein the enzyme is horseradish peroxidase (HRP) or alkaline phosphatase (ALP).

[0310] 64) The composition of any one of embodiments 55-63, wherein at least one of the one or more second antibodies or antigen-binding portions thereof is conjugated to horseradish peroxidase (HRP) and at least one of the one or more second antibodies or antigen-binding portions thereof is conjugated to alkaline phosphatase (ALP).

[0311] 65) The composition according to any one of embodiments 55 to 64, wherein the substrate of the enzyme is selected from the list consisting of 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS), o-phenylenediamine dihydrochloride (OPD), paranitrophenyl phosphate (PNPP) and BCIP / NBT (a combination of BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (nitroblue tetrazolium)).

[0312] 66) A kit for carrying out the method according to any one of embodiments 1 to 54, Magnetically sensitive beads; an immunoassay device comprising an electrode; a magnet positioned adjacent to the chip to hold the magnetically susceptible beads adjacent to the electrodes; A kit comprising:

[0313] 67) The kit of embodiment 66, further comprising a means for holding the magnetically susceptible beads in a separate location spatially separated from the electrodes.

[0314] 68) The kit of embodiment 67, wherein the means for holding the magnetically susceptible beads in a separate location spatially separated from the electrode is the same magnet used to hold the magnetically susceptible beads in close proximity to the electrode.

[0315] 69) The kit of embodiment 67, wherein the means for holding the magnetically susceptible beads at a separate location spatially separated from the electrode is a second magnet configured to hold the magnetically susceptible beads at a separate location spatially separated from the electrode.

[0316] 70) A kit described in any one of embodiments 66 to 69, wherein the magnet is a permanent magnet or an electromagnet.

[0317] 71) A method for measuring an analyte of interest in a biological sample, comprising: The biological sample is a magnetically susceptible bead conjugated to a first antibody or an antigen-binding portion thereof capable of binding to the analyte of interest; and A second antibody or antigen-binding portion thereof capable of binding to the analyte of interest conjugated to an enzyme. and combining with a composition comprising holding the magnetically susceptible beads on the electrodes using a magnetic field; contacting the magnetically susceptible beads with a substrate of the enzyme, where the enzyme substrate is converted by the enzyme into a precipitated electroactive molecule; obtaining electrochemical measurements using said electrodes, wherein a biological sample and a composition are combined at a location spatially separated from said electrodes, and optionally, the spatially separated location is a blank or non-functional electrode; A method comprising:

[0318] 72) A method for measuring an analyte of interest in a biological sample, comprising: The biological sample is magnetically sensitive beads conjugated to one or more antibodies or antigen-binding portions thereof, each antibody or antigen-binding portion thereof being capable of binding to one of the analytes of interest; and one or more second antibodies or antigen-binding portions thereof conjugated to an enzyme, each second antibody or antigen-binding portion thereof being capable of binding to one of the analytes of interest; and combining with a composition comprising holding the magnetically susceptible beads on the electrodes using a magnetic field; contacting the magnetically susceptible beads with one or more substrates of the enzyme, where the enzyme substrate is converted by the enzyme into a precipitated electroactive molecule; obtaining electrochemical measurements using said electrodes, wherein a biological sample and a composition are combined at a location spatially separated from said electrodes, and optionally, the spatially separated location is a blank or non-functional electrode; A method comprising:

[0319] 73) A method for measuring an analyte of interest in a biological sample according to any one of embodiments 71 or 72, wherein the magnetically susceptible beads are contacted with one or more substrates for the enzyme at a location spatially separated from the electrode.

[0320] 74) A method for measuring an analyte of interest in a biological sample described in any one of embodiments 71 to 73, wherein the magnetically susceptible beads are washed at a location spatially separated from the electrodes using a washing solution and / or air.

[0321] 75) A method for measuring an analyte of interest in a biological sample according to any one of embodiments 71 to 74, wherein the magnetically susceptible beads are washed consecutively and separately at a location spatially separated from the electrodes using both a washing solution and air.

[0322] 76) A method for measuring an analyte of interest in a biological sample according to any one of embodiments 71 to 75, wherein the magnetically susceptible beads are alternately washed at least two times, at least three times, at least four times or at least five times with a washing solution and air at a position spatially separated from the electrode, preferably the magnetically susceptible beads are alternately washed twice with a washing solution and air at a position spatially separated from the electrode.

[0323] 77) A method for measuring an analyte of interest in a biological sample described in any one of embodiments 71 to 76, wherein the biological sample and the composition are combined at a location spatially separated from the electrode, and then the magnetically susceptible beads are moved to the electrode to obtain an electrochemical measurement.

[0324] 78) A method for measuring an analyte of interest in a biological sample described in any one of embodiments 71 to 77, wherein magnetically susceptible beads are contacted with one or more substrates of an enzyme at a location spatially separated from the electrode, and the magnetically susceptible beads are then moved to the electrode and an electrochemical measurement is obtained.

[0325] 79) A method for measuring an analyte of interest in a biological sample described in any one of embodiments 71 to 78, wherein the magnetically sensitive beads are contacted with one or more substrates of the enzyme at a location spatially separated from the electrode, and then the magnetically sensitive beads are moved to the electrode and an electrochemical measurement is obtained.

[0326] 80) (i) the biological sample and the composition are combined at a location spatially separated from the electrode; and / or (ii) the magnetically susceptible beads are held at a spatial distance from the electrodes and washed with a washing solution and / or air; The magnetically susceptible beads are then moved to the electrodes to obtain electrochemical measurements, and optionally the spatially separated locations for combining the biological sample with the composition and the spatially separated locations for washing are the same. A method for measuring an analyte of interest in a biological sample described in any one of embodiments 71 to 79.

[0327] 81) (i) the biological sample and the composition are combined at a location spatially separated from the electrode; (ii) the magnetically susceptible beads are contacted with one or more substrates for the enzyme at a location spatially separated from said electrodes; and / or (iii) the magnetically susceptible beads are held at a spatial distance from the electrodes and washed with a washing solution and / or air; The magnetically susceptible beads are then moved to the electrodes to obtain electrochemical measurements, and optionally the spatially separated locations for combining the biological sample with the composition, for washing and / or for contacting the magnetically susceptible beads with one or more substrates of the enzyme are the same, a method for measuring an analyte of interest in a biological sample described in any one of embodiments 71 to 80.

Claims

1. 1. A method for measuring an analyte of interest in a biological sample, comprising: The biological sample is a magnetically susceptible bead conjugated to a first antibody or antigen-binding portion thereof capable of binding to the analyte of interest; and a second antibody or antigen-binding portion thereof capable of binding to the analyte of interest conjugated to an enzyme; and combining with a composition comprising holding the magnetically susceptible beads on an electrode using a magnetic field; contacting the magnetically susceptible beads with a substrate of the enzyme, wherein the enzyme substrate is converted by the enzyme into an electroactive molecule; obtaining electrochemical measurements using said electrodes; A method comprising:

2. 10. The method for determining an analyte of interest in a biological sample of claim 1, wherein the enzyme substrate is converted by the enzyme to a soluble electroactive molecule at the electrode.

3. 10. The method for determining an analyte of interest in a biological sample of claim 1, wherein the enzyme substrate is converted by the enzyme into an electroactive molecule that is precipitated onto the magnetically susceptible bead.

4. 10. The method for measuring an analyte of interest in a biological sample of claim 1, further comprising incubating the combined biological sample and composition so that the first antibody and / or the second antibody binds to the analyte of interest.

5. holding the magnetically susceptible beads in a fixed position using a magnetic field; washing the magnetically susceptible beads; 10. The method for determining an analyte of interest in a biological sample of claim 1, further comprising:

6. The fixed position is a) Located on the electrode; 10. The method of claim 5, wherein b) a location spatially separated from said electrode, optionally said spatially separated location is a blank or non-functional electrode.

7. holding the magnetically susceptible beads in a first position using a magnetic field; washing the magnetically susceptible beads by modulating the magnetic field such that the magnetically susceptible beads are held at a second position; 10. The method for determining an analyte of interest in a biological sample of claim 1, further comprising:

8. The magnetic field is a. A fixed magnet, or b) The method for determining an analyte of interest in a biological sample of claim 1, wherein the analyte is generated by an electromagnet.

9. 10. The method for determining an analyte of interest in a biological sample of claim 8, wherein the magnetic field is modulated by physically actuating the magnet.

10. 10. The method for measuring an analyte of interest in a biological sample of claim 8, wherein the magnetic field is located in a microfluidic device having a flow channel, and the magnetic field is modulated by actuating the magnet in a direction perpendicular to the direction of flow in the flow channel.

11. 8. The method for measuring an analyte of interest in a biological sample of claim 7, wherein the magnetically susceptible beads are moved between the first position and the second position at least 20 times, such as at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, or at least 19 times.

12. 6. The method for determining an analyte of interest in a biological sample of claim 5, wherein the magnetically susceptible beads are washed with a washing solution and / or air.

13. 13. The method for determining an analyte of interest in a biological sample of claim 12, wherein the magnetically susceptible beads are washed sequentially and separately using both a wash solution and air.

14. 6. The method for measuring an analyte of interest in a biological sample of claim 5, wherein the magnetically susceptible beads are washed alternately with a washing solution and air at least two times, at least three times, at least four times, or at least five times, preferably the magnetically susceptible beads are washed alternately with a washing solution and air twice.

15. 10. The method for determining an analyte of interest in a biological sample of claim 1, wherein the electrode is a carbon ink electrode.

16. 2. The method for measuring an analyte of interest in a biological sample of claim 1, wherein the analyte of interest is brain natriuretic peptide or N-terminal pro-BNP.

17. 2. The method for measuring an analyte of interest in a biological sample of claim 1, wherein the analyte of interest is cardiac troponin or cardiac troponin subunit I (cTnI).

18. 2. The method for determining an analyte of interest in a biological sample of claim 1, wherein the enzyme is horseradish peroxidase (HRP) or alkaline phosphatase (ALP).

19. 2. The method for determining an analyte of interest in a biological sample of claim 1, wherein the substrate for the enzyme is selected from the list consisting of 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS), o-phenylenediamine dihydrochloride (OPD), paranitrophenyl phosphate (PNPP), and BCIP / NBT (a combination of BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (nitroblue tetrazolium)).

20. 2. The method for measuring an analyte of interest in a biological sample according to claim 1, wherein the electrochemical measurement is an amperometric, voltammetric, potentiometric, impedimetric, or electrochemical impedance spectroscopy measurement, preferably a chronoamperometric measurement.

21. The method of claim 1, wherein the measuring method is a sandwich immunoassay.

22. 10. The method of claim 1, wherein at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%) of the beads are retained on the surface of the electrode, measured in weight percent.

23. A kit for carrying out the method according to any one of claims 1 to 22, comprising: magnetically susceptible beads; an immunoassay device comprising electrodes; a magnet positioned proximate to the tip to hold the magnetically susceptible beads proximate to the electrode; A kit comprising:

24. 24. The kit of claim 23, further comprising a means for holding the magnetically susceptible beads in a separate location spatially separated from the electrode.

25. 24. The kit of claim 23, wherein the magnet is a permanent magnet or an electromagnet.

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