Highly sensitive immunoassay methods and systems

Abstract

This application relates to a highly sensitive immunoassay method and system. A highly sensitive immunoassay method for a target protein is provided, comprising: capturing the target protein to obtain a solution containing immune complexes; spreading the solution containing immune complexes onto a detection carrier without the aid of a power source; immobilizing the immune complexes on at least one surface; and detecting and calculating the concentration of the immune complexes. The immunoassay method disclosed herein achieves highly sensitive detection of the target protein using a simple and low-cost consumable structure.

Description

[0001] Cross-references to related applications

[0002] This disclosure claims priority to Chinese Patent Application No. 2025102769187, filed on March 10, 2025, entitled "Highly Sensitive Immunoassay Method and System", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of immunoassay, specifically to a highly sensitive immunoassay method and system. Background Technology

[0004] Immunoassay is a chemical measurement method that utilizes the specific reaction between antigens and antibodies to identify analyte molecules. Reagents then cause the identified analyte molecules to generate signals for quantitative analysis. The analyte molecules can be either antigens or antibodies. Immunoassay was first invented by American scientists Berson and Yalow, for whom Yalow was awarded the Nobel Prize in Medicine in 1977. To date, after nearly 50 years of development, immunoassay has diversified into various analytical modes and types, with new methods constantly emerging.

[0005] Traditional immunoassays quantify samples by measuring the relationship between changes in the intensity of macroscopic signals in the sample and the concentration of the target molecule. These methods generally have low sensitivity, with detection limits typically around 10⁻⁶. -9 mol / L~10 -12 mol / L. While highly effective for diagnosing diseases with obvious symptoms, it is insufficient for early screening of serious illnesses, early diagnosis of neurological disorders, and early detection of cardiovascular diseases. Early diagnosis and treatment are the main means to improve patient survival rates. In the early stages of serious illnesses, the antigen concentration in the blood is around 10 mol / L. -15 At concentrations of mol / L or even lower, routine immunoassays cannot detect them, let alone measure them precisely. Due to the lack of effective early diagnostic methods, 80% of patients in my country are diagnosed at middle or late stages, missing the optimal treatment window. For example, the concentration of prostate-specific antigen (PSA) in post-prostate cancer patients is (0.1–100) × 10⁻⁶. -12 Such low concentrations (g / mL) are beyond the reach of traditional immunoassay techniques.

[0006] Single-molecule immunoassay based on microarrays can improve the sensitivity of immunoassays. This involves etching or casting thousands of micrometer-sized microwells on millimeter-scale chips, with each well typically around 40 fL in volume. By distributing magnetic beads containing captured immune complexes into individual microwells, the fluorescent dots are then counted using a high-resolution fluorescence microscope. A count of 10-1 can be achieved. - 18The detection sensitivity of mol / L meets the needs of most biomarker detection. However, compared with traditional immunoassay techniques, this technique is time-consuming, has complex consumable preparation processes, and both the detection instruments and consumables are relatively expensive, which to some extent limits its promotion and use.

[0007] Therefore, there is still a need to develop new, highly sensitive immunoassay methods. Summary of the Invention

[0008] [Technical problem to be solved]

[0009] The purpose of this invention is to solve the above-mentioned problems in the prior art and to provide an immunoassay method that is highly sensitive and simple.

[0010] [Technical Solution]

[0011] To achieve the above-mentioned technical effects, on the one hand, the present invention provides a highly sensitive immunoassay method for a target protein, comprising the following steps:

[0012] The target protein is captured, yielding a solution containing immune complexes;

[0013] The solution containing immune complexes is spread evenly onto the detection carrier without the aid of a power source;

[0014] Immunocomplexes are fixed on at least one surface;

[0015] The concentration of immune complexes was detected and calculated.

[0016] In this invention, the power unit is a component or device that provides continuous power (to drive the flow of liquid) in a liquid circuit. It can be a component or device that directly provides mechanical energy or provides mechanical energy output from an electric motor (or other prime mover); examples of directly providing mechanical energy include generating pressure on the solution through the gravity of a cover plate or generating pressure or thrust through manual transmission; preferably, a device that converts the mechanical energy output from an electric motor (or other prime mover) into hydraulic energy and provides pressurized oil to the hydraulic system is used. This device is the power element of the hydraulic system, and can be, for example, a common energy device used in hydraulic systems (such as pumps), air compression, or, for example, a magnet or a device that generates an electromagnetic field.

[0017] In some specific implementations, the power unit can actively provide continuous power.

[0018] In some specific implementations, the power unit can provide continuous external power.

[0019] In this article, "under the action of no power device" refers to the situation where no power device is used or there is no power device.

[0020] In a specific implementation, the solution containing the immune complex is spread onto the detection carrier by capillary action without the aid of a power source. The surface tension generated at the solid-liquid-gas three-phase contact line is the dominant, spontaneous, and continuous driving force, enabling the liquid to flow autonomously, directionally, and over long distances.

[0021] Orientation, for example, refers to the flow direction along the channels (cavities) formed by the surface, driven by capillary forces. Orientation is distinct from the circumferential direction of the droplet.

[0022] Preferably, the surface is a basically horizontal surface.

[0023] In one embodiment of the present invention, before spreading the solution containing the immune complex onto the detection carrier, the method further includes a step of loading the solution containing the immune complex onto the detection carrier.

[0024] Preferably, in this invention, the spreading step involves the solution containing the immune complex coming into contact with the solid wall of the detection carrier. Because the attraction between solution molecules is less than the adhesion between the solution and the solid wall, the solution will adhere to the solid wall and extend outward (away from the loading point) along the solid wall.

[0025] More preferably, in this invention, the spreading step is a step in which, when the solution containing the immune complex comes into contact with the solid wall of the detection carrier, the solution will adhere to the solid wall because the attraction between solution molecules is less than the adhesion between the solution and the solid wall, and will extend along one side (near the loading point) of the solid wall to the other side (away from the loading point).

[0026] In one embodiment of the present invention, the immune complex is an immune complex containing magnetic microparticles.

[0027] In one specific embodiment of the present invention, the magnetic particles are superparamagnetic particles.

[0028] In this invention, "magnetic microparticles", "magnetic particles", "magnetic granules", "magnetic beads", and "magnetic microspheres" can be used interchangeably.

[0029] In one embodiment of the present invention, the magnetic microparticles are selected from at least one of streptavidin magnetic microparticles, toluenesulfonyl magnetic microparticles, carboxyl magnetic microparticles, succinyl ester (NHS) magnetic beads, and amino magnetic microparticles.

[0030] In one embodiment of the present invention, the fixation on at least one surface is achieved by fixing the immune complex on at least one surface under the action of a magnetic field.

[0031] In this invention, the magnetic force refers to the force experienced by a magnetic medium after it has been magnetized by a constant magnetic field.

[0032] In a preferred embodiment of the present invention, the magnetic field force is a magnetic field force that is substantially uniformly distributed on the detection carrier.

[0033] In one embodiment of the present invention, the immune complex is an immune complex containing a fluorescent label.

[0034] In one embodiment of the present invention, the detection is the acquisition of fluorescence intensity.

[0035] In one specific embodiment of the present invention, the fluorescence intensity acquisition method is selected from at least one of imaging detection, radiance detection, and irradiance detection.

[0036] In one embodiment of the present invention, the detection carrier has a first surface and a second surface disposed opposite to each other, the first surface and the second surface being hydrophilic surfaces.

[0037] By using the first and second surfaces, the surface tension generated at the solid-liquid-gas three-phase contact line drives the liquid to flow over long distances. "Long distance" refers to the greater flow distance of the droplet compared to a detection carrier with a single detection surface (in which case the flow distance is determined by the contact angle of the detection surface and gravity, spreading along the perimeter).

[0038] In a specific embodiment, the first surface and / or the second surface are made of a light-transmitting material.

[0039] In one specific embodiment of the present invention, the light-transmitting material is a hydrophilic transparent material; for example, a hydrophilic transparent material with a water contact angle ≤85° and a light transmittance ≥50%; further, the light-transmitting material is selected from glass, acrylic sheet and other light-transmitting plastics.

[0040] In a preferred embodiment of the present invention, the light-transmitting material has a light transmittance of ≥70%.

[0041] In a preferred embodiment of the present invention, the light-transmitting material has a light transmittance of ≥85%.

[0042] In one specific embodiment of the present invention, the detection carrier is an open container with a distance of 0.01 to 0.4 mm between the first surface and the second surface.

[0043] In a preferred embodiment of the present invention, the spacing is 0.05 to 0.3 mm.

[0044] In one specific embodiment of the present invention, the method of capturing the target protein is to mix the capture protein, magnetic microparticles, test sample and marker to obtain a solution containing immune complex.

[0045] In one specific embodiment of the present invention, the method of capturing the target protein includes:

[0046] Mix the captured protein, magnetic microparticles, and the sample to be tested;

[0047] Then, a label is added to obtain a solution containing immune complexes.

[0048] In one specific embodiment of the present invention, the method of capturing the target protein includes:

[0049] Mix the test sample and the marker;

[0050] Then, capture proteins and magnetic microparticles are added to obtain a solution containing immune complexes.

[0051] In one specific embodiment of the present invention, the method of capturing the target protein is to mix magnetic microparticles coated with the capture protein, the test sample, and the label to obtain a solution containing immune complexes.

[0052] In one specific embodiment of the present invention, the method of capturing the target protein includes:

[0053] The magnetic microparticles coated with the captured protein and the sample to be tested were mixed.

[0054] Then, the marker is added and mixed to obtain a solution containing immune complexes.

[0055] In one specific embodiment of the present invention, the method of capturing the target protein includes:

[0056] Mix the markers and the test samples;

[0057] Then, magnetic microparticles coated with capture proteins are added and mixed to obtain a solution containing immune complexes.

[0058] On the other hand, the present invention provides a highly sensitive immunoassay method for a target protein, comprising the following steps:

[0059] The target protein is captured, yielding a solution containing immune complexes;

[0060] Without the aid of a power source, the solution containing immune complexes is spread evenly onto the detection carrier;

[0061] Under the influence of magnetic force, the immune complex is fixed on at least one surface;

[0062] The concentration of immune complexes was detected and calculated.

[0063] In one embodiment of the present invention, the immune complex is an immune complex containing magnetic microparticles.

[0064] In one specific embodiment of the present invention, the method of capturing the target protein is to mix the capture protein, magnetic microparticles, test sample and marker to obtain a solution containing immune complex.

[0065] In one specific embodiment of the present invention, the method of capturing the target protein includes:

[0066] Mix the captured protein, magnetic microparticles, and the sample to be tested;

[0067] Then, a label is added to obtain a solution containing immune complexes.

[0068] In one specific embodiment of the present invention, the method of capturing the target protein includes:

[0069] Mix the test sample and the marker;

[0070] Then, capture proteins and magnetic microparticles are added to obtain a solution containing immune complexes.

[0071] In one specific embodiment of the present invention, the method of capturing the target protein is to mix magnetic microparticles coated with the capture protein, the test sample, and the label to obtain a solution containing immune complexes.

[0072] In one specific embodiment of the present invention, the method of capturing the target protein includes:

[0073] The magnetic microparticles coated with the captured protein and the sample to be tested were mixed.

[0074] Then, the marker is added and mixed to obtain a solution containing immune complexes.

[0075] In one specific embodiment of the present invention, the method of capturing the target protein includes:

[0076] Mix the markers and the test samples;

[0077] Then, magnetic microparticles coated with capture proteins are added and mixed to obtain a solution containing immune complexes.

[0078] In one embodiment of the present invention, before spreading the solution containing the immune complex onto the detection carrier, the method further includes a step of loading the solution containing the immune complex onto the detection carrier.

[0079] In one embodiment of the present invention, the detection carrier has a first surface and a second surface disposed opposite to each other, the first surface and the second surface being hydrophilic surfaces.

[0080] In a specific embodiment, the first surface and / or the second surface are made of a light-transmitting material.

[0081] In one embodiment of the present invention, the light-transmitting material is a hydrophilic transparent material; for example, a hydrophilic transparent material with a water contact angle ≤85° and a light transmittance ≥50%; further, the light-transmitting material is selected from glass, acrylic sheet and other light-transmitting plastics.

[0082] In a preferred embodiment of the present invention, the light-transmitting material has a light transmittance of ≥70%.

[0083] In a preferred embodiment of the present invention, the light-transmitting material has a light transmittance of ≥85%.

[0084] In one specific embodiment of the present invention, the detection carrier is a carrier with openings at both ends.

[0085] In some embodiments, the distance between the first and second surfaces of the detection carrier is 0.01 to 0.4 mm.

[0086] In a preferred embodiment of the present invention, the spacing is 0.05 to 0.3 mm.

[0087] In a preferred embodiment of the present invention, the magnetic field force is a magnetic field force that is substantially uniformly distributed on the detection carrier.

[0088] In one embodiment of the present invention, the detection is the acquisition of fluorescence intensity.

[0089] In one specific embodiment of the present invention, the fluorescence intensity acquisition method is selected from at least one of imaging detection, radiance detection, and irradiance detection.

[0090] Furthermore, this invention provides a highly sensitive immunoassay method for a target protein, comprising the following steps:

[0091] The target protein is captured, yielding a solution containing immune complexes;

[0092] The solution containing immune complexes is loaded into the detection carrier;

[0093] Without the aid of a power source, the solution containing immune complexes is spread evenly onto the detection carrier;

[0094] Under the influence of magnetic force, the immune complex is fixed on at least one surface;

[0095] The concentration of immune complexes was detected and calculated.

[0096] In one embodiment of the present invention, the immune complex is an immune complex containing magnetic microparticles.

[0097] In one specific embodiment of the present invention, the method of capturing the target protein is to mix the capture protein, magnetic microparticles, test sample and marker to obtain a solution containing immune complex.

[0098] In one specific embodiment of the present invention, the method of capturing the target protein includes:

[0099] Mix the captured protein, magnetic microparticles, and the sample to be tested;

[0100] Then, a label is added to obtain a solution containing immune complexes.

[0101] In one specific embodiment of the present invention, the method of capturing the target protein includes:

[0102] Mix the test sample and the marker;

[0103] Then, capture proteins and magnetic microparticles are added to obtain a solution containing immune complexes.

[0104] In one specific embodiment of the present invention, the method of capturing the target protein is to mix magnetic microparticles coated with the capture protein, the test sample, and the label to obtain a solution containing immune complexes.

[0105] In one specific embodiment of the present invention, the method of capturing the target protein includes:

[0106] The magnetic microparticles coated with the captured protein and the sample to be tested were mixed.

[0107] Then, the marker is added and mixed to obtain a solution containing immune complexes.

[0108] In one specific embodiment of the present invention, the method of capturing the target protein includes:

[0109] Mix the markers and the test samples;

[0110] Then, magnetic microparticles coated with capture proteins are added and mixed to obtain a solution containing immune complexes.

[0111] In one embodiment of the present invention, the detection carrier has a first surface and a second surface disposed opposite to each other, the first surface and the second surface being hydrophilic surfaces.

[0112] In a specific embodiment, the first surface and / or the second surface are made of a light-transmitting material.

[0113] In one embodiment of the present invention, the light-transmitting material is a hydrophilic transparent material; for example, a hydrophilic transparent material with a water contact angle ≤85° and a light transmittance ≥50%; further, the light-transmitting material is selected from glass, acrylic sheet and other light-transmitting plastics.

[0114] In a preferred embodiment of the present invention, the light-transmitting material has a light transmittance of ≥70%.

[0115] In a preferred embodiment of the present invention, the light-transmitting material has a light transmittance of ≥85%.

[0116] In one specific embodiment of the present invention, the detection carrier is an open container with a distance of 0.01 to 0.4 mm between the first surface and the second surface.

[0117] In a preferred embodiment of the present invention, the spacing is 0.05 to 0.3 mm.

[0118] In a preferred embodiment of the present invention, the magnetic field force is a magnetic field force that is substantially uniformly distributed on the detection carrier.

[0119] In one embodiment of the present invention, the detection is the acquisition of fluorescence intensity.

[0120] In one specific embodiment of the present invention, the fluorescence intensity acquisition method is selected from at least one of imaging detection, radiance detection, and irradiance detection.

[0121] More specifically, the present invention provides an immunoassay method for highly sensitive target proteins, comprising the following steps:

[0122] The capture protein-coated magnetic microparticles, the test sample, and the label are mixed to obtain a solution containing immune complexes.

[0123] A solution containing immune complexes is loaded onto a detection carrier with a spacing of 0.01 mm to 0.4 mm between the first and second surfaces;

[0124] Without a power source, the detection carrier is filled with a solution containing immune complexes;

[0125] A magnet with a plane area more than 1 times that of the detection carrier is placed under the detection carrier to attract magnetic attraction.

[0126] Fluorescence intensity was collected, and the concentration of the target protein in the sample was calculated using analysis software.

[0127] In some embodiments, after the magnetic attraction step, the device further includes a step of demagnetizing and placing the detection carrier under the optical detection device for bottom focusing.

[0128] In a specific implementation, the marker is a detection protein that is directly or indirectly labeled with a signal.

[0129] In some embodiments, the capture protein or detection protein is selected from one of antibodies or fragments thereof, antigens, aptamers, and peptides. The detection protein is selected from one of antibodies or fragments thereof, Fab, antigens, aptamers, and peptides.

[0130] The antibody or its fragment is selected from, for example, antigen-binding fragments, Fab, Fab' fragments, F(ab')2 fragments, full-length polyclonal or monoclonal antibodies, antibody-like fragments, etc.

[0131] In a preferred embodiment of the present invention, the spacing is 0.05 to 0.3 mm.

[0132] In a preferred embodiment of the present invention, the magnetic attraction time is more than 3 seconds, and more preferably, the magnetic attraction time is 5 seconds to 1 minute.

[0133] In some embodiments, the specific steps for obtaining a solution containing immune complexes include:

[0134] Magnetic bead microparticles are covalently coupled with capture antibodies to obtain magnetic microparticles coated with capture antibodies;

[0135] The marker is covalently coupled to the detection antibody to obtain the marker-labeled detection antibody;

[0136] The capture antibody-coated magnetic microparticles, the sample to be tested, and the marker-labeled detection antibody are added to the reaction vessel and mixed to obtain a solution containing immune complexes.

[0137] In some embodiments, the specific steps for obtaining a solution containing immune complexes include:

[0138] Magnetic beads are covalently coupled with antigens to obtain antigen-coated magnetic particles;

[0139] The marker is covalently coupled to the detection antibody to obtain the marker-labeled detection antibody;

[0140] Antigen-coated magnetic microparticles, the sample to be tested, and marker-labeled detection antibodies are added to a reaction vessel and mixed to obtain a solution containing immune complexes.

[0141] In some embodiments, the specific steps for obtaining a solution containing immune complexes include:

[0142] Magnetic beads are covalently coupled with antigens to obtain antigen-coated magnetic particles;

[0143] The marker is covalently coupled to the antigen to obtain the marker-labeled antigen;

[0144] Antigen-coated magnetic microparticles, the sample to be tested, and a marker-labeled antigen are added to a reaction vessel and mixed to produce a solution containing immune complexes.

[0145] In some embodiments, the specific steps for obtaining a solution containing immune complexes include:

[0146] Magnetic bead microparticles are covalently coupled with capture antibodies to obtain magnetic microparticles coated with capture antibodies;

[0147] The marker is covalently coupled to the antigen to obtain the marker-labeled antigen;

[0148] The capture antibody-coated magnetic microparticles, the sample to be tested, and the marker-labeled antigen are added to the reaction vessel and mixed to obtain a solution containing immune complexes.

[0149] In some embodiments, the magnetic particles refer to magnetic particles with a particle size of 1.0 μm to 5.0 μm;

[0150] In some preferred embodiments, the magnetic particles refer to magnetic particles with a particle size of 1.0 μm to 3 μm; in some preferred embodiments, the magnetic particles refer to magnetic particles with a particle size of 1.0 μm to 1.5 μm; in one specific embodiment, the magnetic particles refer to magnetic particles with a particle size of 1.5 μm. The magnetic particles may optionally be carboxyl magnetic beads, streptavidin magnetic beads, toluenesulfonyl magnetic beads, etc.

[0151] In an exemplary embodiment, the signaling material is fluorescent microspheres. In some embodiments, the signaling material is fluorescent microspheres with a particle size of 100 nm to 300 nm; in some preferred embodiments, the signaling material is fluorescent microspheres with a particle size of 200 nm to 300 nm; in one specific embodiment, the signaling material is fluorescent microspheres with a particle size of 200 nm.

[0152] In one embodiment of the present invention, the detection carrier has a first surface and a second surface disposed opposite to each other, the first surface and / or the second surface being made of a light-transmitting material.

[0153] In one embodiment of the present invention, the light-transmitting material is a hydrophilic transparent material with a water contact angle ≤85° and a light transmittance ≥50%; further, the light-transmitting material is selected from glass, acrylic sheet and other light-transmitting plastics.

[0154] In a preferred embodiment of the present invention, the light-transmitting material has a light transmittance of ≥70%.

[0155] In a preferred embodiment of the present invention, the light-transmitting material has a light transmittance of ≥85%.

[0156] In some specific embodiments, the detection carrier is made of an acrylic bottom of 0.5 mm to 2 mm and an acrylic top of 0.5 mm to 2 mm.

[0157] In some specific embodiments, the acrylic cover plate is provided with an inlet and an outlet at both ends, respectively; the length from the inlet to the outlet is consistent with the length of the upper chamber of the detection carrier.

[0158] In some embodiments, the detection method includes a washing step before the immune complex is added to the detection carrier.

[0159] In some embodiments, the volume ratio of the capture protein-coated magnetic microparticles, the test sample, and the label is 2:2:(1-2).

[0160] In some embodiments, the mixing is carried out at 37°C for 10 to 40 minutes.

[0161] In some embodiments, the magnet is placed at a distance of 0.1 mm to 20 mm from the bottom surface of the detection carrier, or for example, attached to the bottom surface of the detection carrier; in some preferred embodiments, the magnet is placed at a distance of 10 mm from the bottom surface of the detection carrier. In some preferred embodiments, the magnet is in the shape of a cube or a cylinder.

[0162] In some embodiments, bottom focusing is performed using magnetic bead focusing in bright field or fluorescent focusing in dark field. In a preferred embodiment, bottom focusing is performed using magnetic bead focusing in bright field.

[0163] In some embodiments, the imaging detection refers to exposing the image to illumination light with a wavelength of 280nm to 800nm ​​for 10 to 800ms before imaging. In some preferred embodiments, the imaging exposure time is 400ms.

[0164] In some specific embodiments, the irradiation light may be one or more of the following wavelengths: 620nm to 760nm, 575nm to 600nm, 525nm to 575nm, 400nm to 490nm, and 280nm to 400nm.

[0165] In some embodiments, the illumination light is generated by at least one of a thermal radiation source, a gas discharge source, a solid-state source, an electroluminescent source, and a chemical source.

[0166] In some specific embodiments, the illumination light is generated by at least one of a mercury lamp, a light-emitting diode (LED), a laser diode, a gas laser, and a solid-state laser.

[0167] Compared with the prior art, the present invention has the following beneficial effects:

[0168] The unexpected discovery of this invention is that by first laying the beads in a self-spontaneous manner without a power source and then fixing the surface, the immune complexes captured by the magnetic beads can be spread out evenly, increasing the exposure probability of the fluorescence signal, thereby achieving highly sensitive detection of the target protein.

[0169] The immunoassay method of the present invention can also use disposable detection carriers, thus further avoiding the problem of cross-contamination caused by repeated use of detection carriers. The device using this immunoassay method can use a simple and low-cost consumable structure, avoiding the use of high-precision consumables and reducing batch-to-batch performance variations in reagents caused by consumables. Attached Figure Description

[0170] Figure 1 This is a schematic diagram of the structure of a detection mechanism according to the present invention;

[0171] Figure 2 This is a three-dimensional structural diagram of the detection chip of the present invention;

[0172] Figure 3 The ROC curve statistical results of plasma p-Tau217 detection in Example 12 are shown below (top: chemiluminescence group; bottom: high-sensitivity immunoassay group).

[0173] The following are the annotations in the attached diagram: 27 cover plate; 26 bottom plate; 28 sample inlet; 29 exhaust port. Detailed Implementation

[0174] The present invention will be further described and illustrated below with reference to embodiments thereof. Based on this disclosure, those skilled in the art should understand that many changes can be made to the specific embodiments disclosed without departing from the scope of the invention as defined below, while still obtaining the same or similar results. Therefore, the following embodiments are intended only to illustrate certain features of the invention and are not intended to illustrate the full scope of the invention.

[0175] A highly sensitive immunoassay method of the present invention includes the following steps:

[0176] First, a conventional immune reaction is performed, and the resulting immune complexes are dispersed in a resuspension. Preferably, the present invention uses magnetic microparticles with a particle size of 1.0 μm to 3.0 μm and fluorescent microspheres with a particle size of 100 nm to 300 nm.

[0177] Then, the immune complex is spread evenly on the detection chip by capillary action and captured by magnets in single or multiple layers.

[0178] The immune reaction includes a reaction method selected from the following: detection of target antigen by double antibody sandwich method, detection of target antibody by double antigen sandwich method, detection of target antibody by indirect method, detection of target antibody by capture method, and detection of target antigen by competitive method.

[0179] To ensure the uniform spreading of the immune complex, the present invention requires the detection chip to be designed as an ultra-thin plate consisting of a base plate and a cover plate with a spacing of 0.01 mm to 0.4 mm; both ends of the cover plate are provided with sample inlets and exhaust outlets; the base plate and cover plate of the detection chip are preferably made of organic transparent materials with a water contact angle ≤85° and a light transmittance ≥80%, such as acrylic sheets. When an aqueous liquid, such as an immune complex resuspension, is added to the detection chip, the liquid uniformly spreads throughout the chip cavity within 1 second under capillary action.

[0180] In some preferred embodiments, in order to ensure the precision of the detection method of the present invention, the cavity size of the detection chip is consistent with the volume of the added immune complex resuspension liquid.

[0181] After the immune complex spreads within the chamber of the detection chip via capillary action, the chip enters a magnetic field. The immune complex, suspended in the slits, is then drawn down by the magnetic force and settles to the bottom of the chip, achieving a flat distribution. Since the magnetic field lines of the magnet are denser closer to the edge in the axial magnetization direction, the size of the magnet and its relative position to the detection area also affect the distribution of magnetic particles. Experimental results show that the magnetic attraction effect is better when the area of ​​the magnet used is approximately the same as or larger than the detection area.

[0182] Finally, an optical module is used to capture signals from the detection chip. This invention prioritizes bottom focusing; the bottom focusing process includes magnetic bead focusing in bright field or fluorescence focusing in dark field.

[0183] Among them, focusing the magnetic bead in bright field refers to focusing the magnetic bead using an optical module under natural light or white light.

[0184] Fluorescence focusing in a dark field refers to focusing fluorescent microspheres using an optical module in a dark field after being illuminated by an excitation light source.

[0185] After imaging, the present invention obtains the concentration of the target protein by analyzing the fluorescence intensity.

[0186] In this paper, magnetic microparticles refer to particles with a diameter of 1.0 μm to 5.0 μm, such as 1.0 μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2.0 μm, 2.1 μm, 2.2 μm, 2.3 μm, 2.4 μm, 2.5 μm, 2.6 μm, 2.7 μm, and 2.8 μm. m, 2.9μm, 3.0μm, 3.1μm, 3.2μm, 3.3μm, 3.4μm, 3.5μm, 3.6μm, 3.7μm, 3.8μm, 3.9μm, 4 .0μm, 4.1μm, 4.2μm, 4.3μm, 4.4μm, 4.5μm, 4.6μm, 4.7μm, 4.8μm, 4.9μm or 5.0μm magnetic particles.

[0187] In this paper, the signaling material is a fluorescent microsphere with a particle size of 100nm to 300nm, such as 100nm, 120nm, 140nm, 160nm, 180nm, 200nm, 220nm, 240nm, 260nm, 280nm or 300nm.

[0188] In this paper, the distance between the first surface and the second surface is 0.01–0.40 mm, such as 0.01 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, 0.10 mm, 0.11 mm, 0.12 mm, 0.13 mm, 0.14 mm, 0.15 mm, 0.16 mm, 0.17 mm, 0.18 mm, etc. m, 0.19mm, 0.20mm, 0.21mm, 0.22mm, 0.23mm, 0.24mm, 0.25mm, 0.26mm, 0.27mm, 0.28mm, 0.29mm , 0.30mm, 0.31mm, 0.32mm, 0.33mm, 0.34mm, 0.35mm, 0.36mm, 0.37mm, 0.38mm, 0.39mm or 0.40mm.

[0189] In some embodiments, the method of this application does not include a cleaning step after the fixing step and before the detection step. In some embodiments, the method of this application does not include a cleaning step after the tiling step and before the detection step.

[0190] In a specific implementation, during the fixing step, the magnet and the detection carrier remain stationary relative to each other.

[0191] A testing mechanism of the present invention, such as Figures 1-2 As shown, it includes:

[0192] The chip loading module is used to carry the detection carrier and push the detection chip from the designated station to the magnetic suction station or the detection station via the track module.

[0193] A magnetic attraction device is installed below the magnetic attraction station and moves closer to or away from the magnetic attraction station as the magnetic flipping rod rotates.

[0194] An optical module is installed above the detection station to collect optical signals from the detection chip;

[0195] The control device includes a control chip loading module, a magnetic attraction device, and an optical module.

[0196] The detection mechanism of this invention places the sampled detection chip on the chip loading module and moves it to the designated station via the track module. Then, the track module continues to work, pushing the detection chip to the magnetic suction station. The magnetic suction device rotates via the magnet flipping rod and approaches the magnetic suction station for magnetic attraction. After the magnetic suction module is reset, the track module continues to push the detection chip to position it at the detection station. The optical module performs imaging detection. After the detection is completed, the track module can also move the detection chip away for unloading.

[0197] This invention provides an immunoassay analyzer, comprising the aforementioned detection mechanism and an immunoassay device, wherein the immunoassay device and the detection mechanism are connected by a corresponding track; wherein the immunoassay device includes...

[0198] A reagent compartment for storing reagents includes a reagent rack and a reagent cup for holding a reagent kit, and a turntable for holding the reagent kit. Below the turntable, there is a temperature control mechanism for providing the activation temperature of the reagent kit and a reagent cup mixing device for mixing the reagents.

[0199] The reaction chamber is used to provide the reaction tube temperature bath reaction conditions and to detect the reaction results; the reaction chamber includes a reaction tube support with a circular outer sawtooth structure, a reagent arm unit for adding reagents, a temperature control device for providing the reaction tube temperature bath environment, a cleaning station, and a waste liquid recovery unit at the outer diameter of the circular ring of the reaction tube support, and a reaction tube mixing device at the bottom of the reaction tube support.

[0200] Furthermore, the immunoassay analyzer of the present invention also includes a cleaning station for providing cleaning conditions; the cleaning station is equipped with a magnetic attraction device for magnetically attracting reactants.

[0201] Furthermore, the testing apparatus of the present invention can be used independently or in conjunction with existing immunoassay instruments.

[0202] A highly sensitive immunoassay method includes the following steps:

[0203] The captured protein-coated magnetic microparticles, the test sample, and the label were mixed in a reaction tube in the reaction chamber and reacted at 37°C for 10 to 40 minutes.

[0204] Then, after magnetic attraction using the magnetic attraction device in the cleaning station, the mixture is cleaned and 30 μL of resuspension buffer is added to obtain the immune complex.

[0205] Immunocomplexes are loaded onto the detection chip using a pipette or reagent delivery tubing.

[0206] The detection chip is placed on the chip loading module of the detection mechanism, and then sequentially undergoes magnetic attraction, movement under the optical module for exposure, and imaging detection by the optical module to obtain the concentration of the sample to be tested through analysis and calculation.

[0207] In some embodiments, the testing institution or immunoassay instrument of this application does not include a grating structure.

[0208] In the claims and the foregoing description, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," etc., should be understood as open-ended, meaning including but not limited to. Only the transitional phrases "consisting of" and "consisting substantially of" are closed or semi-closed transitional phrases, respectively.

[0209] In this article, magnetic beads, magnetic microparticles, magnetic particles, and magnetic microparticles are used interchangeably. They primarily refer to superparamagnetic magnetic particles used to capture target proteins.

[0210] The following sections provide additional information regarding the methods, materials, and parameters that can be used to practice the determinations described above.

[0211] Example 1: The following describes a non-limiting example of the preparation of 1.5 μm magnetic beads functionalized with protein capture antibodies.

[0212] Magnetic bead cleaning: Take 1.5μm carboxyl magnetic beads (purchased from Sichuan Ankerui New Material Technology Co., Ltd.) and sonicate them in a water bath for 2 min. Add 40μL of magnetic beads (4mg) to 960μL of 100mM 2-(N-morpholino)ethanesulfonic acid (MES, pH=6.0). After washing 3 times with 1mL of 100mM MES (pH=6.0), resuspend the magnetic beads in 200μL of 100mM MES (pH=6.0).

[0213] Preparation of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) solution: Prepare a mixture of 100 mg / mL EDC and 200 mg / mL NHS using 100 mM MES (pH = 6.0);

[0214] Magnetic bead activation: Add 200 μL of the prepared EDC and NHS mixture to the magnetic beads, mix at room temperature for 30 min, and remove the supernatant by magnetic aspiration. Wash twice with 400 μL of 100 mM MES (pH = 6.0), and then resuspend in 1 mL of 20 mM MES (pH = 6.0).

[0215] Antibody conjugation: Add 10 μg of capture antibody to the activated magnetic beads and mix at room temperature for 3 h;

[0216] Reaction termination: Remove all liquid with magnetic adsorption, add 800 μL of termination solution (1M Tris, pH 7.0) and 800 μL of 20mM MMES (pH = 7.0), and mix at room temperature for 1 h;

[0217] Washing: Wash 3 times with 1 mL of 100 mM Tris, pH 7.4, containing 10 g / L BSA buffer;

[0218] Resuspension: Resuspend in 10 mL buffer (100 mM Tris, pH 7.4, containing 10 g / L BSA) to obtain a final concentration of 0.4 mg / mL of capture antibody-coated magnetic beads (calculated as magnetic beads).

[0219] Example 2 describes a non-limiting example of the preparation of 1.0 μm magnetic beads functionalized with protein capture antibodies.

[0220] Magnetic bead cleaning: Take 1.0 μm carboxyl magnetic beads (purchased from Sichuan Ankerui New Material Technology Co., Ltd.), sonicate them in a water bath for 15 seconds, add 40 μL of magnetic beads (4 mg) to 960 μL of 100 mM MES (pH=6.0), add 1 mL of 100 mM MES (pH=6.0) to wash 3 times, and then resuspend the magnetic beads in 200 μL of 100 mM MES (pH=6.0).

[0221] Preparation of EDC / NHS solution: Prepare a mixture of 100 mg / mL EDC and 200 mg / mL NHS using 100 mM MES (pH = 6.0);

[0222] Magnetic bead activation: Add 200 μL of the prepared EDC and NHS mixture to the magnetic beads, mix at room temperature for 30 min, and remove the supernatant by magnetic aspiration. Wash twice with 400 μL of 100 mM MES (pH = 6.0), and then resuspend in 1 mL of 20 mM MES (pH = 6.0).

[0223] Antibody conjugation: Add 10 μg of capture antibody to the activated magnetic beads and mix at room temperature for 3 h;

[0224] Reaction termination: (First remove 800 μL of liquid with magnetic suction) Add 800 μL of termination solution (1M Tris pH 7.0) and mix at room temperature for 1 h;

[0225] Washing: Wash 3 times with 1 mL of 100 mM Tris, pH 7.4, containing 10 g / L BSA buffer;

[0226] Resuspension: Resuspend in 10 mL buffer (100 mM Tris, pH 7.4, containing 10 g / L BSA) to obtain a final concentration of 0.4 mg / mL of capture antibody-coated magnetic beads (calculated as magnetic beads).

[0227] Example 3 describes a non-limiting example of the preparation of 1.5 μm magnetic beads functionalized with protein capture antibodies.

[0228] Magnetic bead cleaning: Take 100 μL of streptavidin (SA) magnetic beads with a particle size of 1.5 μm (purchased from Sichuan Ankerui New Material Technology Co., Ltd.), wash 3 times with 100 mM Tris, pH=7.4, containing 10 g / L BSA diluted 5 times, and finally resuspend the magnetic beads in 500 μL.

[0229] Antibody-conjugated biotin N-hydroxysuccinimide ester (NHS-biotin): Add NHS-biotin rapidly to the antibody at a molar ratio of antibody to biotin of 1:20, vortex immediately to mix, and react at room temperature for 30 min; add 1% of 1M Tris of the total reaction solution volume, and react at room temperature for 10 min.

[0230] Antibody-conjugated magnetic beads: The antibody was added to the magnetic beads at a ratio of 10 μg antibody / mg magnetic beads, and the mixture was reacted by rolling at room temperature for 30 min.

[0231] Blocking: Add the appropriate volume of blocking agent (BSA) and react at room temperature for 30 minutes.

[0232] Washing: Wash 3 times with 1 mL of 100 mM Tris, pH 7.4, containing 10 g / L BSA buffer;

[0233] Resuspension: Resuspend in 100mM Tris, pH=7.4, with 10g / L BSA buffer to 0.4mg / ml, and store at 2-8℃.

[0234] Example 4: The following describes a non-limiting example of covalent coupling of fluorescent microspheres with a particle size of 200 nm to a detection antibody.

[0235] Cleaning of fluorescent microspheres: Take 25 μL of red fluorescent microspheres (1 wt%) with a particle size of 200 nm (purchased from Sichuan Ankerui New Material Technology Co., Ltd.) and add them to 250 μL of 20 mM MES (pH = 6.0);

[0236] Microsphere activation: Within 15 minutes before use, prepare 50 mg / mL EDC and 100 mg / mL NHS using 20 mM MES (pH = 6.0). Take 5 μL of each of the above EDC and NHS and quickly add them to the cleaned fluorescent microspheres. React in the dark for 20 minutes, then centrifuge at 18000 rcf for 10 minutes and remove the supernatant.

[0237] Washing: Resuspend in 300 μL of 20 mM MES (pH = 5.0), centrifuge at 18000 rcf for 10 min, remove supernatant; resuspend in 500 μL of labeling buffer (20 mM, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, HEPES, pH = 7.4);

[0238] Antibody conjugation: Take 0.2 mg of the detection antibody diluted with labeling buffer, add the above microspheres to the detection antibody, mix well, and react at room temperature in the dark for 2 hours;

[0239] Reaction termination: Add 800 μL of 50 mM glycine solution to terminate the reaction, and react at room temperature in the dark for 30 min;

[0240] Storage: Redissolve the fluorescent microspheres in borate buffer to a final concentration of 2 mg / mL, and store at 2-8°C.

[0241] Example 5: The following describes a non-limiting example of covalent coupling of fluorescent microspheres with a particle size of 300 nm to a detection antibody.

[0242] Cleaning of fluorescent microspheres: Take 25 μL of red fluorescent microspheres (1 wt%) with a particle size of 300 nm (purchased from Sichuan Ankerui New Material Technology Co., Ltd.) and add them to 250 μL of 20 mM MES;

[0243] Microsphere activation: Within 15 minutes before use, prepare 50 mg / mL EDC and 100 mg / mL NHS using 20 mM MES (pH = 6.0). Take 5 μL of each of the above EDC and NHS and quickly add them to the washed fluorescent microspheres. React in the dark for 20 minutes, then centrifuge at 18000 rcf for 15 minutes and remove the supernatant.

[0244] Washing: Resuspend in 300 μL of 20 mM MES, centrifuge at 18000 rcf for 15 min, remove supernatant; resuspend in 500 μL of labeling buffer (20 mM borate buffer, pH = 7.10);

[0245] Antibody conjugation: Take 0.15 mg of the detection antibody diluted with labeling buffer, add the above microspheres to the detection antibody and mix quickly. React at room temperature in the dark for 2 hours.

[0246] Reaction termination: Add 800 μL of 50 mM glycine solution to terminate the reaction, and react at room temperature in the dark for 30 min;

[0247] Storage: Reconstitute with borate buffer to a final concentration of 2 mg / mL, and store in a refrigerator at 2-8℃.

[0248] Example 6 describes a non-limiting example of capturing target proteins and forming immune complexes on magnetic beads.

[0249] Add 50 μL of test solution containing the target protein, 50 μL of magnetic beads coated with capture antibody, and 25 μL of fluorescent microsphere-detection antibody to a 96-well plate in sequence, and incubate at 37 °C with shaking for 20 min.

[0250] After incubation, place the 96-well plate on a plate washer with a magnetic plate for 40 seconds to remove the supernatant and excess fluorescent microspheres-detection antibodies. Add 200 μL of washing buffer to each well and vortex for 1 min to resuspend. Repeat the above washing steps 3 times to eliminate non-specific adsorption. Add 30 μL of resuspension buffer (100 mM Tris, pH 7.4, containing 10 g / L LBSA) to each well and vortex for 2 min to ensure that the immune complexes are evenly dispersed in the resuspension.

[0251] Example 7 describes a non-limiting example of fabricating a test chip.

[0252] A hydrophilic transparent acrylic sheet with a water contact angle ≤85° and a light transmittance ≥80% was selected for encapsulation. The encapsulation process involved a 1mm thick base plate 26 and a 0.8mm thick cover plate 27 with a spacing of 0.2mm and a capacity of approximately 20μL. The test chip, such as... Figure 2 As shown, both ends of the cover plate are provided with an inlet 28 and an exhaust port 29.

[0253] Example 8 describes a non-limiting example of the immune complex of the present invention being tiled on a capillary test chip.

[0254] 20 μL of the test solution was drawn from 30 μL of immune complex resuspension and added to the test chip. The test solution was rapidly and evenly spread by capillary action (1 s) until the chip chamber was completely filled.

[0255] Then attach a magnet (the same size as the detection chip) to the bottom of the detection chip, and let the magnet attract the bottom of the detection chip for 40 seconds; then remove the magnet.

[0256] The following experimental examples describe different methods for tiled on the test chip.

[0257] Test group 1 (n=3) 20 μL of the test solution (a solution of 1.5 μm carboxyl magnetic beads coated with IL-6 capture antibody) was added to the test chip. After the test solution rapidly and evenly spreads across the test chip via capillary action, it was magnetically attracted for 40 seconds. Bright-field imaging was performed using an optical detection device. Based on the equidistant sampling method, six positions were selected on each side of the central axis of the test chip for imaging, for a total of 12 imaging points. The number of magnetic beads was counted (1-6 are in the same column, 7-12 are corresponding parallel columns). The results are shown in the table below. The higher the imaging number, the farther the position is from the injection port.

[0258] Table 1

[0259]

[0260] After the 1.5μm magnetic beads coated with the capture antibody were magnetically laid out, the CV values ​​of the three parallel samples were all less than 5%, indicating that the 1.5μm magnetic beads coated with the capture antibody were evenly laid out in the test chip; the CV between parallel samples was less than 5%, indicating that the laying effect was stable.

[0261] Control group 2 (n=3) 20 μL of the test solution (a solution of 1.5 μm magnetic beads coated with capture antibody) was added to the test chip. After the test solution was rapidly and evenly spread on the test chip by capillary action, the bright field imaging detection was performed using the optical detection module. Based on the equidistant sampling method, six positions were selected on both sides of the central axis of the test chip for imaging. A total of 12 imaging operations were performed to calculate the number of magnetic beads. The results are shown in the table below.

[0262] Table 2

[0263]

[0264] Relying solely on capillary action for flattening, without the use of magnets, the magnetic beads will be layered within a 0.4mm deep chamber, and then slowly settle to the bottom of the test chip under gravity. Therefore, as the number of imaging sessions increases, the number of magnetic beads gradually increases, leading to a higher in-parallel CV value.

[0265] Control group 3 (n=3) The test chip is placed on a magnet, and 20 μL of the test solution (a solution of 1.5 μm magnetic beads coated with capture antibodies) is added to the test chip. After the test solution is rapidly and evenly spread across the test chip by capillary action, bright-field imaging detection is performed using an optical detection device. Based on the equidistant sampling method, 6 points are selected on the central axis of the test chip to calculate the number of magnetic beads.

[0266] Table 3

[0267]

[0268] The 1.5μm magnetic beads coated with the capture antibody were subjected to both capillary force and magnetic field during the laying process, which prevented them from being laid evenly in the test chip. The CV value of the number of beads in parallel was greater than 5%.

[0269] Control group 4 (n=3) The test chip was placed on a magnet, and 20 μL of the test solution (a solution of 1.5 μm magnetic beads coated with capture antibodies) was added to the test chip. After the test solution was rapidly and evenly spread on the test chip by capillary action, it was magnetically attracted for 40 seconds. Bright field imaging detection was performed using an optical detection device. Based on the equidistant sampling method, six positions were selected on both sides of the central axis of the test chip for imaging. A total of 12 imaging operations were performed to calculate the number of magnetic beads. The results are shown in the table below.

[0270] Table 4

[0271]

[0272] The 1.5μm magnetic beads coated with the capture antibody are subjected to both capillary force and magnetic field during the laying process. After the laying is completed, the magnetic attraction continues for 40s, which not only causes the CV within the parallel to be greater than 5%, but also causes the CV between parallels to be greater than 5%, thus affecting both the laying uniformity and laying stability.

[0273] Test group 5 (n=3) 20 μL of the test solution (a solution of 1.0 μm magnetic beads coated with capture antibody (purchased from Sichuan Ankerui New Material Technology Co., Ltd.)) was added to the test chip. After the test solution was rapidly and evenly spread across the test chip by capillary action, it was magnetically attracted for 40 seconds. Bright-field imaging detection was performed using an optical detection device. Based on the equidistant sampling method, six positions were selected on both sides of the central axis of the test chip for imaging. A total of 12 imaging operations were performed to calculate the number of magnetic beads. The results are shown in the table below.

[0274] Table 5

[0275]

[0276] After the 1.0 μm magnetic beads coated with capture antibodies were magnetically laid out, the CV value of the number of beads within each parallel was less than 5%, indicating that the 1.0 μm magnetic beads coated with capture antibodies were evenly laid out in the test chip; the CV value of the number of beads between parallels was less than 5%, indicating that the laying effect was stable.

[0277] Test group 6 (n=3)20 μL of the test solution (a solution of 3 μm magnetic beads coated with capture antibody (purchased from Sichuan Ankerui New Material Technology Co., Ltd.)) was added to the test chip. After the test solution was rapidly and evenly spread across the test chip by capillary action, it was magnetically attracted for 40 seconds. Bright-field imaging was performed using an optical detection device. Based on the equidistant sampling method, six positions were selected on both sides of the central axis of the test chip for imaging. A total of 12 imaging operations were performed to calculate the number of magnetic beads. The results are shown in the table below.

[0278] Table 6

[0279]

[0280] After the 3μm magnetic beads coated with the capture antibody were magnetically laid out, the CV values ​​of the number of beads within each parallel were all greater than 5%, indicating that the 3μm magnetic beads can still be uniformly laid out in the test chip, with an intra-parallel CV value of less than 10% and an inter-parallel CV value of less than 5%.

[0281] Example 9 The following non-limiting examples describe the detection of immune complexes on a test chip.

[0282] Magnetic beads with a diameter of 1.5 μm coated with anti-IL-6 capture antibody (purchased from Sichuan Ankerui New Material Technology Co., Ltd.) were prepared as described above. Anti-IL-6 detection antibody labeled with fluorescent microspheres and a particle size of 200 nm (purchased from Sichuan Ankerui New Material Technology Co., Ltd.) was also prepared as described above. These magnetic beads were incubated with IL-6 test solutions (0 pg / mL, 1 pg / mL, and 10 pg / mL) and the fluorescent microsphere-detection antibody to prepare immune complexes. The immune complexes were then added to the test chip, magnetically attracted, and fluorescence imaging was performed. Information was collected in 12 fixed areas of the test chip, and the results of the number of bright-field magnetic beads and the fluorescence intensity in the dark field were obtained. The distribution results are shown in the table below.

[0283] Table 7

[0284]

[0285] Table 8

[0286]

[0287] Table 9

[0288]

[0289] The signal-to-noise ratio (SNR) of fluorescence intensity was 264.88 for 1 and 0 pg / mL, and 2192.39 for 10 and 0 pg / mL. After magnetically scattering the immune complexes with 1.5 μm magnetic beads, bright-field imaging showed that the intra-parallel and inter-parallel CV values ​​of the immune complexes at IL-6 concentrations of 0 pg / mL, 1 pg / mL, and 10 pg / mL were all less than 5%, indicating that the immune complexes were evenly and stably scattered in the test chip. Fluorescence imaging results showed that the fluorescence intensity CV value was higher for immune complexes with low IL-6 concentrations, and decreased with increasing IL-6 concentration.

[0290] Example 10 The following non-limiting examples describe the distribution of immune complexes on a test chip.

[0291] Magnetic beads with a diameter of 1 μm coated with anti-IL-6 capture antibody (purchased from Sichuan Ankerui New Material Technology Co., Ltd.) were prepared as described above. Anti-IL-6 detection antibody labeled with fluorescent microspheres with a particle size of 200 nm (purchased from Sichuan Ankerui New Material Technology Co., Ltd.) was also prepared as described above. These magnetic beads were incubated with the IL-6 test solution (0 pg / mL, 1 pg / mL, and 10 pg / mL) and the fluorescent microsphere-detection antibody to prepare an immune complex. The immune complex was then added to the test chip, magnetically attracted, and bright-field and fluorescence imaging were performed. Information was collected in 12 fixed areas of the test chip, and the results of the number of bright-field magnetic beads and the fluorescence intensity in the dark field were obtained, as shown in the table below.

[0292] Table 10

[0293]

[0294] Table 11

[0295]

[0296] Table 12

[0297]

[0298] The signal-to-noise ratio (SNR) of fluorescence intensity was 66.35 for 1 and 0 pg / mL, and 643.42 for 10 and 0 pg / mL.

[0299] After magnetically scattering the immune complexes with 1μm magnetic beads, bright-field imaging showed that the intra-parallel and inter-parallel counts of immune complexes with IL-6 concentrations of 0 pg / mL, 1 pg / mL, and 10 pg / mL were all less than 5%, indicating that the immune complexes were evenly and stably scattered in the test chip. Fluorescence imaging revealed that the fluorescence intensity count of immune complexes with low IL-6 concentrations was higher, and the fluorescence intensity count decreased as the IL-6 concentration increased.

[0300] Example 11 The following non-limiting example describes the detection of IL-6.

[0301] Magnetic beads with a diameter of 1.5 μm coated with anti-IL-6 capture antibody (purchased from Sichuan Ankerui New Material Technology Co., Ltd.) were prepared as described above. Anti-IL-6 detection antibody labeled with fluorescent microspheres and a particle size of 200 nm (purchased from Sichuan Ankerui New Material Technology Co., Ltd.) was also prepared as described above. These magnetic beads were incubated together with the test solution containing different concentrations (0 pg / mL, 0.01 pg / mL, 0.1 pg / mL, 1 pg / mL, 10 pg / mL) of IL-6 and the fluorescent microsphere-detection antibody. After incubation, the 96-well plate was placed on a plate washer with a magnetic plate for 40 seconds to remove the supernatant and excess fluorescent microsphere-detection antibody. 200 μL of washing buffer was added to each well, and the plate was resuspended by shaking for 1 min. The washing steps were repeated three times to eliminate non-specific adsorption. Add 30 μL of resuspension buffer (100 mM Tris, pH 7.4, containing 10 g / L BSA) to each well and shake to resuspend for 2 min to ensure that the immune complexes are evenly dispersed in the resuspension.

[0302] Then, as described above, 20 μL of the resuspension was added to the test chip, magnetically attracted, and fluorescence imaging was performed. Based on the fluorescence intensity obtained from the imaging analysis, a standard curve was fitted using concentration as the x-axis and the average fluorescence intensity of all images at each concentration as the y-axis to analyze the sensitivity of the method. The four-parameter fitting results showed a good linear relationship between the signal value and the sample concentration, R... 2 =0.999998 is close to 1.

[0303] This example also includes tests on LOB and LOD.

[0304] Table 13

[0305]

[0306] By selecting concentrations between 1 and 5 times that of LOB (LOB = 6.7 fg / mL), samples of 12, 15, 18, 24, and 30 fg / mL were used for testing, and the results are shown in the table below.

[0307] Table 14

[0308]

[0309] In summary, the LOB detected by this invention is 6.7 fg / mL, and the LOD is updated to 14.6 fg / mL. The method of this invention exhibits extremely high sensitivity, representing a 100-fold improvement in sensitivity compared to the chemiluminescent IL-6 kit (LOD 1.4 pg / mL).

[0310] Example 12 The following non-limiting examples describe the detection of plasma p-Tau217.

[0311] SA magnetic beads with a diameter of 1.5 μm coated with anti-p-Tau217 capture antibody (purchased from Sichuan Ankerui New Material Technology Co., Ltd.) were prepared as described below. Anti-p-Tau217 detection antibody with a particle size of 200 nm labeled with fluorescent microspheres (purchased from Sichuan Ankerui New Material Technology Co., Ltd.) was prepared as described above.

[0312] Step A: Biotin-Ab2 labeling (antibody-labeled biotin ester)

[0313] 1. Antibody dialysis: The initial antibody concentration should not be less than 2 mg / mL. Dialyze with 0.02M PBS for 12 hours. Calculate the concentration using Nanodrop.

[0314] 2. Preparation of 2.5mM BNHS: 1mg BNHS + 868μL DMSO.

[0315] 3. Antibody-BNHS (Ab1:BNHS = 1:20): Add 1.33 μL of BNHS quickly to the antibody, vortex immediately to mix, and react at room temperature for 30 min.

[0316] 4. Termination: Add 1% of the total volume of the previous reaction solution with 1M Tris, mix well at room temperature and react for 10 min.

[0317] 5. Washing: Dialyze with 0.02M PBS for 12 hours.

[0318] 6. Storage: Store at 4℃. Calculate concentration using Nanodrop.

[0319] Step B: SA connected to Biotin-Ab2

[0320] 1. 200 μL SA (0.2 mg) was diluted to 0.5 mg / mL with boric acid reconstitution solution (pH 8.2);

[0321] 2. Replacement buffer: Centrifuge at 8500 rcf for 10 min, discard the supernatant, and resuspend in 400 μL of boric acid reconstitution solution (pH 8.2);

[0322] 3. Add Bio-Ab2: Take the appropriate mass of Bio-Ab2 and react at a ratio of 5 μg antibody / mg SA. Incubate at room temperature for 30 min.

[0323] 4. Blocking: Add 15 μg BSA and react at room temperature for 30 min.

[0324] 5. Washing: Centrifuge at 8500 rcf for 10 min, discard the supernatant, resuspend in 300 μL boric acid reconstitution solution (pH 8.2) by sonication for 2 min, and repeat the washing 1-2 times;

[0325] 6. Resuspension: Add boric acid reconstitution solution (pH 8.2) and mix well to a final concentration of 1 mg / mL;

[0326] These magnetic beads were incubated together with test solutions containing different concentrations of p-Tau217 and fluorescent microsphere-detection antibody. After incubation, the 96-well plate was placed on a plate washer with a magnetic plate for 40 seconds to remove the supernatant and excess fluorescent microsphere-detection antibody. 200 μL of washing buffer was added to each well, and the plate was vortexed for 1 min to resuspend. This washing step was repeated three times to eliminate non-specific adsorption. 30 μL of resuspension buffer was added to each well, and the plate was vortexed for 2 min to ensure uniform dispersion of the immune complexes in the resuspension.

[0327] Then, as described above, the resuspension was added to the test chip, and fluorescence imaging was performed after magnetic adsorption (i.e., the high-sensitivity immunoassay group). Simultaneously, the signal-to-noise ratio (SNR) of the chemiluminescence and high-sensitivity immunoassay groups was compared. A series of different concentrations of p-Tau217 (0, 0.5 pg / mL, 2.0 pg / mL, 10.0 pg / mL) were analyzed. The table shows the luminescence value, fluorescence intensity, and SNR corresponding to different concentrations of p-Tau217 calibrators. It can be seen that the SNR of the high-sensitivity immunoassay group is significantly higher than that of the chemiluminescence group (Table 15). Plasma samples from patients in a hospital (with clinical symptoms suspected to be AD) were used as positive samples, and plasma samples from healthy individuals were used as negative samples. Fourteen negative samples and nine positive samples were tested. ROC curve analysis was performed on the test results. Figure 3 The AUC value of chemiluminescence was 0.881 (P<0.001, 95% confidence interval 0.678-0.977), and the AUC value of high-sensitivity immunoassay was 0.933 (P<0.001, 95% confidence interval 0.946-0.995). The AUC value of high-sensitivity immunoassay was better than that of chemiluminescence, and both were able to effectively distinguish between healthy people and AD patients.

[0328] Table 15

[0329] p-Tau217 (pg / mL) High-sensitivity immunofluorescence intensity Chemiluminescent RLU 0 147011 1395 0.5 1409499 2886 2 5524155 7948 10 27620775 28001 S / N(0.5 / 0) 9.6 2.1 S / N(2 / 0) 37.6 5.7 S / N(10 / 0) 187.9 20.1

[0330] Example 13 The following non-limiting example describes the detection of serum Aβ1-42.

[0331] Magnetic beads with a diameter of 1.5 μm coated with anti-Aβ1-42 capture antibody (purchased from Sichuan Ankerui New Material Technology Co., Ltd.) were prepared as described above.

[0332] The following method was used to prepare fluorescent microsphere-labeled anti-Aβ1-42 detection antibody with a particle size of 200 nm (purchased from Sichuan Ankerui New Material Technology Co., Ltd.).

[0333] Step A: Biotin-Ab2 labeling (antibody-labeled biotin ester)

[0334] 1. Antibody dialysis: The initial antibody concentration should not be less than 2 mg / mL. Dialyze with 0.02M PBS for 12 hours. Calculate the concentration using Nanodrop.

[0335] 2. Preparation of 2.5mM BNHS: 1mg BNHS + 868μL DMSO.

[0336] 3. Antibody-BNHS (Ab1:BNHS = 1:20): Add 1.33 μL of BNHS quickly to the antibody, vortex immediately to mix, and react at room temperature for 30 min.

[0337] 4. Termination: Add 1% of the total volume of the previous reaction solution with 1M Tris, mix well at room temperature and react for 10 min.

[0338] 5. Washing: Dialyze with 0.02M PBS for 12 hours.

[0339] 6. Storage: Store at 4℃. Calculate concentration using Nanodrop.

[0340] Step B: SA connected to Biotin-Ab2

[0341] 1. 200 μL SA (0.2 mg) was diluted to 0.5 mg / mL with boric acid reconstitution solution (pH 8.2);

[0342] 2. Replacement buffer: Centrifuge at 8500 rcf for 10 min, discard the supernatant, and resuspend in 400 μL of boric acid reconstitution solution (pH 8.2);

[0343] 3. Add Bio-Ab2: Take the appropriate mass of Bio-Ab2 and react at a ratio of 5 μg antibody / mg SA. Incubate at room temperature for 30 min.

[0344] 4. Blocking: Add 15 μg BSA and react at room temperature for 30 min.

[0345] 5. Washing: Centrifuge at 8500 rcf for 10 min, discard the supernatant, resuspend in 300 μL boric acid reconstitution solution (pH 8.2) by sonication for 2 min, and repeat the washing 1-2 times;

[0346] 6. Resuspension: Add boric acid reconstitution solution (pH 8.2) and mix well to a final concentration of 1 mg / mL;

[0347] These magnetic beads were incubated together with test solutions containing different concentrations of Aβ1-42 and fluorescent microsphere-detection antibody. After incubation, the 96-well plate was placed on a plate washer with a magnetic plate for 40 seconds to remove the supernatant and excess fluorescent microsphere-detection antibody. 200 μL of washing buffer was added to each well, and the plate was vortexed for 1 min to resuspend. This washing step was repeated three times to eliminate non-specific adsorption. 30 μL of resuspension buffer was added to each well, and the plate was vortexed for 2 min to ensure uniform dispersion of the immune complexes in the resuspension.

[0348] Then, as described above, 20 μL of the resuspended solution was added to the test chip, magnetically attracted, and fluorescence imaging was performed. Based on the fluorescence intensity obtained from the imaging analysis, a standard curve was fitted with concentration as the x-axis and the average fluorescence intensity of all images at each concentration as the y-axis to analyze the sensitivity of the method. Compared with the chemiluminescent Aβ1-42 kit, this method exhibits higher sensitivity.

[0349] Although the present invention has been described herein with reference to illustrative embodiments, the above embodiments are merely preferred embodiments of the present invention, and the implementation of the present invention is not limited to the above embodiments. It should be understood that those skilled in the art can devise many other modifications and implementations, which will fall within the scope and spirit of the principles disclosed in this application.

Claims

1. A highly sensitive immunoassay method for a target protein, comprising: The target protein is captured, yielding a solution containing immune complexes; The solution containing the immune complex is spread evenly onto the detection carrier without the action of a power device; The immune complex is fixed on at least one surface; as well as The concentration of the immune complex was detected and calculated.

2. The immunoassay method according to claim 1, wherein, The process of spreading the solution containing immune complexes onto the detection carrier without the aid of a power source is achieved through the spontaneous spreading of the solution containing immune complexes.

3. The immunoassay method according to claim 1, wherein, The immune complex is an immune complex containing magnetic microparticles.

4. The immunoassay method according to claim 1, wherein, The fixation on at least one surface refers to fixing the immune complex on at least one surface under the action of a magnetic field.

5. The immunoassay method according to claim 1, wherein, The immune complex is an immune complex containing a fluorescent label.

6. The immunoassay method according to claim 1, wherein, The detection carrier has a first surface and a second surface arranged opposite to each other, and the first surface and the second surface are hydrophilic surfaces.

7. The immunoassay method according to claim 1, wherein, The detection carrier is an open container with a distance of 0.01 to 0.4 mm, preferably 0.05 to 0.3 mm, between the first and second surfaces.

8. The immunoassay method according to claim 3, wherein, The method for capturing the target protein involves mixing the capture protein, magnetic microparticles, the sample to be tested, and a label to obtain a solution containing an immune complex. The label is a detection protein that is directly or indirectly labeled with a signal.

9. The immunoassay method according to claim 8, wherein, The capture or detection protein is selected from antibodies or fragments thereof, aptamers, and peptides.

10. A highly sensitive immunoassay method for a target protein, comprising: The target protein is captured, yielding a solution containing immune complexes; Without the aid of a power source, the solution containing the immune complex is spread evenly onto the detection carrier; Under the action of a magnetic field, the immune complex is fixed on at least one surface; as well as The concentration of the immune complex was detected and calculated.

11. An immunoassay method for a highly sensitive target protein, comprising: The target protein is captured, yielding a solution containing immune complexes; The solution containing the immune complex is loaded into the detection carrier; Without the aid of a power source, the solution containing the immune complex is spread evenly onto the detection carrier; The immune complex is fixed on at least one surface under the action of a magnetic field. as well as The concentration of immune complexes was detected and calculated.

12. A highly sensitive immunoassay method for a target protein, comprising: The capture protein-coated magnetic microparticles, the test sample, and the label are mixed to obtain a solution containing immune complexes. The solution containing the immune complex is loaded onto a detection carrier with a spacing of 0.01 mm to 0.4 mm between the first and second surfaces; Without a power source, the solution containing the immune complex is filled into the detection carrier; A magnet with a plane area more than 1 times that of the detection carrier is placed under the detection carrier to attract magnetic attraction. as well as Fluorescence intensity was collected, and the concentration of the target protein in the sample was calculated using analysis software.

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