Single-molecule protein detection method based on microarray chip and chemiluminescence immunoassay technology

Abstract

The invention relates to the technical field of detection, and relates to a single-molecule protein detection method based on a microarray chip and a chemiluminescence immunoassay technology. An antigen-antibody complex reaction solution is randomly segmented into tens of thousands of microarray holes with the single-hole volume at the pL level by utilizing a microarray chip containing 0.5-200 thousands of micropores with the diameter of 5-30 [mu] m. Each micropore is used as an independent reaction unit for detection and comprises one or zero antigen-antibody compounds, a chromogenic substrate or exciting light is added to collect signal data in all the micropores, and the number of all the antigen-antibody compounds in a reaction system is directly and quantitatively detected according to a Poisson distribution calculation formula. And calculating the concentration of the antigen captured in the microarray chip. The method disclosed by the invention has the advantages of small background signal interference, high sensitivity, specificity, accuracy and precision, simplicity and rapidness in operation, low cost and obvious advantages in the technical field of single molecule detection, and the lower limit of detection reaches fg / ml.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and in particular to a method for detecting single-molecule proteins based on microarray chips and chemiluminescence immunoassay. Background Technology

[0002] Currently, the mainstream chemiluminescence immunoassay (CLIA) techniques mainly include direct chemiluminescence, enzyme-catalyzed chemiluminescence, electrochemiluminescence, and photo-induced chemiluminescence. Direct chemiluminescence immunoassay, represented by acridine esters, is a catalyst-free technique that directly emits light under alkaline conditions with an oxidant (such as hydrogen peroxide). It has extremely high luminescence efficiency, reaching maximum luminescence intensity approximately 0.4 seconds after the addition of the luminescent substrate.

[0003] Enzyme-catalyzed chemiluminescent immunoassay is a detection method that combines enzymatic reactions with chemiluminescence technology. Commonly used enzymes include horseradish peroxidase (HRP) and alkaline phosphatase (ALP). Luminol and its derivatives are common enzyme-catalyzed luminescent substrates for HRP. Under alkaline conditions, HRP catalyzes the redox process of an oxidant (such as hydrogen peroxide) and luminol, which emits light at approximately 425 nm. AMPPD is the most common enzyme-catalyzed reflective substrate for ALP. Under alkaline conditions, ALP catalyzes the removal of one phosphate group from AMPPD, forming the unstable intermediate AMPD. The cleavage of AMPD releases light at approximately 470 nm, which can last for tens of minutes.

[0004] Photochemiluminescence is a homogeneous immunoassay technique. This process utilizes photosensitive microspheres with a diameter of approximately 200 nm and luminescent microspheres, where antigen-antibody specific binding occurs through direct or indirect coupling, resulting in a distance of less than 200 nm between the two microspheres. When the photosensitive microspheres are irradiated with excitation light at a wavelength around 680 nm, the released singlet oxygen reacts with the luminescent microspheres, emitting light at a wavelength of approximately 610 nm. If the distance between the two microspheres is greater than 200 nm, the singlet oxygen, due to its short half-life, cannot react with the luminescent microspheres, and therefore no luminescence signal is generated.

[0005] With continuous development in scientific research and applications, the detection capabilities of the aforementioned chemiluminescence methods have approached their theoretical limits. However, the percentage of known proteins in the human body that can be reliably detected is still only a small fraction. Existing chemiluminescence immunoassay methods struggle to achieve quantitative detection of single-molecule proteins. Current chemiluminescence detection instruments rely on the overall light signal intensity in the solution and standards to calculate the concentration of the target protein in the sample, which, in principle, cannot achieve single-molecule protein detection. Therefore, some proteins with low expression abundance cannot be detected by chemiluminescence immunoassay, posing significant challenges to the development of new biomarkers, disease progression monitoring, and treatment prognosis monitoring.

[0006] SimoA technology, represented by Quanterix, currently exhibits excellent sensitivity, with a detection limit reaching fg / ml, demonstrating significant application potential in the detection of biomarkers for tumors and neurological diseases. However, the complexity of SimoA technology and the high cost of instruments and consumables limit its application and promotion in the clinical diagnostic market, hindering its wider adoption.

[0007] Existing technologies have also reported single-molecule protein detection chips based on electrochemiluminescence of ultramicroelectrode arrays, achieving highly sensitive qualitative detection of trace proteins using special chips containing tilted micro-trap arrays. However, the tilted micro-trap chips used in this technology are complex to prepare, the reaction process is complex and unstable, and achieving trace protein detection requires a high number and density of electrode arrays (greater than 40,000), which has generally been less than 1,000 previously. Therefore, there is an urgent need for a method for detecting single-molecule proteins that is easy to operate, highly sensitive, highly specific, rapid, accurate, and low-cost, requiring no expensive instruments, equipment, or consumables. Summary of the Invention

[0008] This invention provides a single-molecule protein detection method based on microarray chip and chemiluminescence immunoassay technology to overcome the above-mentioned shortcomings of the prior art.

[0009] Specifically, the present invention provides the following technical solutions.

[0010] In a first aspect, the present invention provides a single-molecule protein detection method based on microarray chip and chemiluminescence immunoassay technology. An antigen-antibody complex suitable for chemiluminescence immunoassay is loaded into a microarray chip with 50,000 to 200,000 micropores with a diameter of 5 to 30 μm on its surface. The chemiluminescence detection system identifies and focuses on the micropores, collects signal data within the micropores, and calculates and analyzes the concentration of the antigen within the microarray chip.

[0011] The chemiluminescence immunoassay detection method includes direct chemiluminescence immunoassay, enzyme-catalyzed chemiluminescence immunoassay, electrochemiluminescence immunoassay, and photo-induced chemiluminescence immunoassay.

[0012] Preferably, the chemiluminescence immunoassay method of the present invention is a direct chemiluminescence immunoassay method, an enzyme-catalyzed chemiluminescence immunoassay method, or a photo-induced chemiluminescence immunoassay method.

[0013] The antigen-antibody complex is a double-antibody sandwich complex or an antigen-antibody complex. The antigen-antibody complex refers to a complex formed by the reaction of a capture antibody (directly coupled to magnetic beads or coupled to magnetic beads via the streptavidin system) and a detection antibody (an antibody coupled to a chemiluminescent label or enzyme) with the target protein (antigen).

[0014] The microarray chip structure with 50,000-200,000 micropores with a diameter of 5-30 μm described in this invention is derived from the microfluidic chip described in Chinese Patent CN117899955A, with adaptive improvements made to the structure. Developed by Zhenzhun Biotechnology (Shanghai) Co., Ltd., this chip, combined with chemiluminescence immunoassay, achieves efficient, sensitive, specific, accurate, and rapid quantitative detection of single-molecule proteins. The product catalog number is CM0204.

[0015] The microarray chip with 50,000 to 200,000 micro-vias with a diameter of 5 to 30 μm on the surface as described in this invention includes a chip shell, a micro-via chip, and a substrate layer;

[0016] The microporous chip is mounted on top of the base plate layer with a gap to act as a microchannel layer. The chip housing and the base plate layer seal and cover the microporous chip with a gap.

[0017] The chip housing is provided with a sample dispensing port, which is suitable for injecting the reaction system to be tested into the microfluidic layer through the sample dispensing port, so that the reaction system to be tested passes through the micro-perforations on the microporous chip; the reaction system to be tested is drawn into the micro-perforations by the siphon effect and the hydrophilic effect of the hydrophilic groups in the micro-perforations; a luminescent substrate is injected into the microporous chip through the substrate port;

[0018] The microfluidic channel is disposed at the bottom of the micro-perforation; suitable for the reaction system to be tested to pass through the bottom of the micro-perforation;

[0019] A substrate aperture is provided on the opposite side of the sample loading aperture on the chip housing, which is suitable for injecting luminescent substrates (AMPPD, luminol, and hydrogen peroxide under alkaline conditions, etc.) into the microporous chip through the substrate aperture.

[0020] Preferably, the microporous layer has 200,000 micropores with a diameter of 10 μm.

[0021] Specifically, this invention provides a method for detecting single-molecule proteins based on direct chemiluminescence immunoassay, comprising the following steps:

[0022] (1) The antigen-antibody complexes connected with chemiluminescent labels are loaded into the microarray chip through the sample wells; each antigen-antibody complex contains a single protein to be tested.

[0023] (2) The microarray chip is identified by the chemiluminescence detection system, and the negative signal / background signal after the antigen-antibody complex transferred through the micropores is fixed and focused, such as... Figure 2 As shown;

[0024] (3) The substrate of the chemiluminescent label is added through the substrate wells, allowing the substrate to bind to the antigen-antibody complexes in all the chip wells; simultaneously, a chemiluminescent detection system is used to receive the positive signals generated within the micro-wells, such as... Figure 3 As shown; after signal collection is completed, the chemiluminescence system automatically performs threshold division, calibration and verification, and analyzes and counts the number of valid wells, negative wells and positive wells, removes invalid wells, and calculates the concentration of the single protein to be detected.

[0025] In the above-mentioned single-molecule protein detection method based on direct chemiluminescence immunoassay, the chemiluminescent label includes, but is not limited to, acrid esters, luminol and its derivatives, fluorescein and its derivatives, biphenyl derivatives, and metal complexes.

[0026] Specifically, this invention provides a method for detecting single-molecule proteins based on enzyme-catalyzed chemiluminescence immunoassay, comprising the following steps:

[0027] (1) The antigen-antibody complexes linked with labeled enzymes are loaded into the microarray chip through the sample wells; each antigen-antibody complex contains a single protein to be tested;

[0028] (2) The microarray chip is identified by the chemiluminescence detection system, and the negative signal / background signal after the antigen-antibody complex transferred by the micro-hole is fixed and focused;

[0029] (3) Add the catalytic substrate through the substrate wells so that the substrate binds to the antigen-antibody complex in all the chip wells. After incubation and catalysis for 5-15 minutes, the chemiluminescence detection system receives the positive signal generated in the micro-wells, automatically performs threshold division, calibration and verification, and analyzes and counts the number of valid wells, negative wells and positive wells, removes the number of invalid wells, and calculates the concentration of the single molecule protein to be detected.

[0030] In the above-mentioned single-molecule protein detection method based on enzyme-catalyzed chemiluminescent immunoassay, the labeling enzymes in step (1) include, but are not limited to, horseradish peroxidase (HRP) and alkaline phosphatase (ALP).

[0031] Specifically, this invention provides a method for detecting single-molecule proteins based on photo-induced chemiluminescence immunoassay, comprising the following steps:

[0032] (1) The antigen-antibody complex is loaded into the microarray chip through the sample well; the antigen-antibody complex is an antigen-antibody complex coupled or indirectly coupled with photosensitive microspheres and luminescent microspheres;

[0033] (2) The microarray chip is irradiated with excitation light of a specific wavelength emitted by the chemiluminescence detection system, and the emission light of different wavelengths is collected at the same time to complete the detection of single protein concentration.

[0034] Preferably, in step (2) above, the specific wavelength is 680nm and the different wavelengths are 610nm.

[0035] The single-molecule protein detection methods based on direct chemiluminescence immunoassay, enzyme-catalyzed chemiluminescence immunoassay, and photo-induced chemiluminescence immunoassay mentioned above all employ chemiluminescence detection methods that are well-known and commonly used in the field. The chemiluminescence detection system of this invention was developed by AccuONE Biotechnology (Shanghai) Co., Ltd., with catalog number IN0501 and model number AccuONE-CL100B.

[0036] Secondly, the present invention provides a single-molecule protein detection system, comprising a microarray chip with 50,000 to 200,000 micropores with a diameter of 5 to 30 μm on its surface, a chemiluminescence detection system, and a formulation for preparing antigen-antibody complexes.

[0037] The microarray chip includes a chip housing, a microporous chip, and a substrate layer;

[0038] The microporous chip is mounted on top of the base plate layer with a gap to act as a microchannel layer. The chip housing and the base plate layer seal and cover the microporous chip with a gap.

[0039] The chip housing is provided with a sample loading port and a substrate port, which are disposed on opposite sides of the chip housing. The test reaction system is injected into the microfluidic layer through the sample loading port, and the test reaction system is drawn into the micro-perforations on the microporous chip through the micro-perforations by the siphon effect and the hydrophilic effect of the hydrophilic groups in the micro-perforations. A luminescent substrate is injected into the microporous chip through the substrate port.

[0040] The microfluidic channel is located at the bottom of the micro-hole, and is suitable for the reaction system to be tested to pass through the bottom of the micro-hole.

[0041] Preferably, the microarray chip with 200,000 micro-holes with a diameter of 10 μm and the chemiluminescence detection system were both developed by Zhenzhun Biotechnology (Shanghai) Co., Ltd.

[0042] Preferably, in the single-molecule protein detection system, the preparations used to prepare antigen-antibody complexes include streptavidin-coated magnetic beads, biotin-coupled reagent kits, and one, two, or more antibodies against the same or different antigens.

[0043] Thirdly, the present invention provides any of the following applications of a microarray chip:

[0044] (1) The antigen-antibody complex is loaded onto the microarray chip, and the quantitative detection of single-molecule proteins is achieved by direct chemiluminescence immunoassay.

[0045] (2) The antigen-antibody complex is loaded onto the microarray chip, and the quantitative detection of single-molecule proteins is achieved by enzyme-catalyzed chemiluminescence immunoassay.

[0046] (3) The antigen-antibody complex is loaded onto the microarray chip, and the quantitative detection of single-molecule proteins is achieved by photo-induced chemiluminescence immunoassay.

[0047] The microarray chip includes a chip housing, a microporous chip, and a substrate layer;

[0048] The microporous chip is installed inside the chip housing, and the microporous layer has 50,000 to 200,000 micropores with a diameter of 5 to 30 μm. The chip housing is provided with microfluidic channels.

[0049] The chip housing is provided with a sample application port; it is suitable for injecting the test reaction system (antigen-antibody complex) into the microfluidic layer through the sample application port, so that the test reaction system passes through the micropores on the microporous chip, and the test reaction system is drawn into the micropores by the siphon effect and the hydrophilic effect of the hydrophilic groups in the micropores;

[0050] The microfluidic channel is disposed between the microporous chip and the substrate layer; it is suitable for the reaction system to be tested to pass through the bottom of the micropore;

[0051] A substrate aperture is provided on the opposite side of the sample loading aperture on the chip housing, which is suitable for injecting luminescent substrates (AMPPD, luminol, and hydrogen peroxide under alkaline conditions, etc.) into the microporous chip through the substrate aperture.

[0052] The beneficial effects of this invention are as follows:

[0053] 1. Compared to traditional chemiluminescence immunoassay techniques, which can only perform qualitative analysis or cannot achieve quantitative detection of single-molecule proteins, this invention introduces a microarray chip containing 5-20w wells with a diameter of 5-30µm, enabling direct quantitative detection. Simultaneously, it reduces background signal interference from the original single-tube reaction system, increasing the relative abundance of antigen-antibody complexes and significantly improving detection sensitivity to the single-molecule level, with a detection limit reaching fg / ml. Compared to Quanterix's SimoA detection technology, the sensitivity of this invention can reach the detection limit of SimoA technology, or even lower.

[0054] 2. The chip micropore diameter of this invention is relatively large, which improves the chip yield and reduces the technical requirements of chip manufacturing process. The chip design of this invention is compatible with multiple chemiluminescence detection methods. It can be used not only for enzyme-catalyzed chemiluminescence technology, but also in combination with direct chemiluminescence technology and photo-induced chemiluminescence technology, thus expanding the application range. Even when using enzyme-catalyzed chemiluminescence technology, it can generate a stronger signal, thereby improving the detection sensitivity. In terms of detection efficiency, the method of this invention can significantly shorten the test time. For example, the detection time using acridinium ester combined with the microarray chip of this invention is only a dozen minutes. In terms of detection cost, the instrument and consumable costs of the method of this invention are lower. The price of a single test is only one-tenth of that of SimoA technology, which has obvious advantages in long-term promotion and popularization of single-molecule detection technology.

[0055] 3. While existing chemiluminescence detection methods have achieved automation, few products can detect single-molecule proteins. The method of this invention does not alter the mainstream chemiluminescence operation process or the steps involved in forming the antigen-antibody complex. It only introduces a microarray chip and a chemiluminescence detection system in the detection stage, opening up new applications for microarray chips. Attached Figure Description

[0056] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0057] Figure 1 This is a schematic diagram of the microarray chip structure of the present invention, wherein hole A represents a sample injection hole, hole B represents a substrate hole, and C represents a selected schematic diagram of the microarray chip.

[0058] Figure 2The screenshot shows the actual working negative / background signal when using the microarray chip described in this invention to perform single-molecule protein detection using the direct chemiluminescence immunoassay method;

[0059] Figure 3 This is a screenshot of a positive signal observed when using the microarray chip described in this invention to detect single-molecule proteins using the direct chemiluminescence immunoassay method. Detailed Implementation

[0060] To more clearly illustrate the technical solution and optimization steps of the present invention, the detection method of the present invention will be further described in detail below with reference to specific embodiments. Obviously, the described embodiments are merely for illustrative purposes and do not limit the application scope of the detection method of the present invention. Based on the embodiments of the present invention, any modifications, equivalent substitutions, improvements, etc., made by all other embodiments obtained by those skilled in the art without creative effort should be included within the protection scope of the present invention.

[0061] The antibodies, modifying groups, and modification methods involved in this invention are derived from the following sources:

[0062] Streptospirin-coated magnetic beads: Beyotime, product number: P2151;

[0063] P-tau 217 antibody 1: ThermoFisher, catalog number: 44-744;

[0064] P-tau 217 antibody 2: ThermoFisher, catalog number: PA5-37639;

[0065] Acridinium ester: Meilun Biotechnology, Product No.: MT0013;

[0066] Biotin Coupling Kit: Abcam, Catalog No.: ab201795;

[0067] Alkaline phosphatase coupling kit: Abcam, catalog number: ab102850;

[0068] Streptavidin-coated photosensitive microspheres: Weidu Bio, catalog number: 67600000;

[0069] Luminescent microspheres: Weidu Bio, catalog number: 67700001;

[0070] P-tau 217 Detection Kit: Quanterix, P-Tau 217 ( ALZpath)Assay.

[0071] P-tau 217 Chemiluminescence Assay Kit: Novizan, Catalog No.: M4701CB.

[0072] P-tau 217 was serially diluted to obtain working solutions of 1000 fg / ml, 750 fg / ml, 500 fg / ml, 250 fg / ml, 50 fg / ml and 5 fg / ml.

[0073] The microarray chip and chemiluminescence detection system were developed by Zhenzhun Biotechnology (Shanghai) Co., Ltd.

[0074] The microarray chip used in this embodiment of the invention has 200,000 microvias with a diameter of 10 μm on its surface. The microarray chip includes a chip shell, a microvia chip, and a substrate layer. The microvia chip is mounted above the substrate layer with a gap to act as a microfluidic channel layer. The chip shell and the substrate layer seal over the microvia chip with a gap. The chip shell has a sample loading port and a substrate port, which are disposed opposite to each other on the chip shell. The reaction system to be tested is injected into the microfluidic layer through the sample loading port, so that the reaction system to be tested passes through the microvias on the microvia chip. The reaction system to be tested is drawn into the microvias by a siphon effect and the hydrophilic effect of the hydrophilic groups in the microvias. A luminescent substrate is injected into the microvia chip through the substrate port. The microfluidic channel is disposed at the bottom of the microvia, suitable for the reaction system to be tested to pass through the bottom of the microvia.

[0075] Example 1: Detection of P-tau 217 gradient working solutions using acridine ester and basic oxidant

[0076] Biotin-labeled P-tau 217 antibody 1, streptomycin-coated magnetic beads, and acridil ester-labeled P-tau 217 antibody 2 were mixed with six gradients of P-tau 217 working solution (1000 fg / ml, 750 fg / ml, 500 fg / ml, 250 fg / ml, 50 fg / ml, and 5 fg / ml) and one negative control at room temperature to form a double antibody sandwich complex. After incubation, excess antibody was removed by magnetic adsorption and the magnetic bead complex was washed several times, followed by elution with elution buffer. Each concentration was repeated three times.

[0077] The eluted seven groups of double-antibody sandwich complexes of different concentrations were loaded onto a microarray chip with 200,000 micropores of 10 μm diameter on the surface using a pipette; the sample loading process took no more than 5 seconds; each antigen-antibody complex contained a single protein to be tested.

[0078] The microarray chip loaded with the dual-antibody sandwich composite is placed under a chemiluminescence detection system, which then identifies and focuses the micropores.

[0079] A mixture of sodium hydroxide and hydrogen peroxide was injected into the chip through the substrate well using the sampling needle of the chemiluminescence detection system. At the same time, the chemiluminescence detection system received and captured the optical signal generated in the chip well.

[0080] The substrate injection, microarray focusing, optical signal detection, and imaging were all automatically completed by the Zhenzhun Biochemiluminescence Detection System. Finally, the chemiluminescence detection system automatically analyzed and calculated the concentration of antigen captured in the microarray chip. The results are shown in Table 1.

[0081] Example 2: Detection of P-tau 217 gradient working solution using alkaline phosphatase-catalyzed AMPPD.

[0082] Biotin-labeled P-tau 217 antibody 1, streptomycin-coated magnetic beads, and alkaline phosphatase (ALP)-labeled P-tau 217 antibody 2 were mixed with six gradients of P-tau 217 working solution (1000 fg / ml, 750 fg / ml, 500 fg / ml, 250 fg / ml, 50 fg / ml, and 5 fg / ml) and one negative control at room temperature to form a double antibody sandwich complex. After incubation, excess antibody was removed by magnetic adsorption and the magnetic bead complex was washed several times, followed by elution with elution buffer. Each concentration was repeated three times.

[0083] Seven groups of double-antibody sandwich complexes of different concentrations were eluted and loaded onto a microarray chip with 200,000 micropores of 10 μm diameter on the surface using a pipette; the sample loading process took no more than 5 seconds; each antigen-antibody complex contained a single protein to be tested.

[0084] Sodium hydroxide solution and alkaline phosphatase chemiluminescent substrate AMPPD were added to the microarray chip loaded with the double antibody sandwich complex. After incubation at room temperature for 10 min, the chip was placed under a chemiluminescence detection system. The chemiluminescence detection system then identified and focused on the microwells, and captured the positive signals generated in all the wells.

[0085] Finally, the chemiluminescence detection system will automatically analyze and calculate the concentration of antigen captured within the microarray chip, as shown in Table 1.

[0086] Example 3: Detection of P-tau 217 gradient working solution using photo-induced chemiluminescence (PRC) technology

[0087] Biotin-labeled P-tau 217 antibody 1 and P-tau 217 antibody 2 labeled luminescent microspheres were mixed with 6 gradients of P-tau 217 working solutions and 1 negative control reagent and incubated at room temperature for 15 min to obtain reaction solution 1. Each concentration was repeated 3 times.

[0088] Add streptavidin-coated photosensitive microspheres to the above 7 reaction solutions 1 respectively, mix and incubate at room temperature for 15 min to obtain reaction solution 2;

[0089] Seven sets of reaction solutions 2 were loaded into a microarray chip with 200,000 micropores of 10 μm in diameter; the sample loading process took no more than 5 seconds; the reaction solution 2 was an antigen-antibody complex (double antibody sandwich complex) coupled with photosensitive microspheres and luminescent microspheres; the micropores were identified and focused using a chemiluminescence detection system, and 680 nm excitation light was emitted to the seven chips in sequence, and the 610 nm wavelength light signal emitted from the chip pores was collected and photographed through a filter;

[0090] Finally, the chemiluminescence detection system will automatically analyze and calculate the concentration of antigen captured within the microarray chip, as shown in Table 1.

[0091] Example 4: Detection of P-tau 217 gradient working fluid using SimoA technology

[0092] At Quanterix HD-X platform, based on P-Tau 217 ( The ALZpath Assay kit was used to detect six gradients of P-tau 217 working solutions (1000 fg / ml, 750 fg / ml, 500 fg / ml, 250 fg / ml, 50 fg / ml and 5 fg / ml) and one negative control reagent, as shown in Table 1. Each concentration was tested in triplicate.

[0093] Example 5: Detection of P-tau 217 gradient working solution using conventional chemiluminescence technology

[0094] The p-Tau 217 protein chemiluminescence assay kit from Novizuma was used to detect six gradients of p-tau 217 working solutions (1000 fg / ml, 750 fg / ml, 500 fg / ml, 250 fg / ml, 50 fg / ml and 5 fg / ml) and one negative control reagent. Each concentration was tested in triplicate. There were no significant differences between batches. The average value of the three replicates was taken, as shown in Table 1.

[0095] Table 1. Comparison of P-tau217 detection results between the present invention technology and existing technologies.

[0096]

[0097] Note: The concentration data in the table are the average of three parallel replicates.

[0098] The detection results of each embodiment (Table 1) show that: 1. The microarray chip proposed in this invention can be combined with traditional direct (corresponding to the method in Example 1), indirect (corresponding to the method in Example 2), and photo-induced chemiluminescence (corresponding to the method in Example 3) techniques for the detection of extremely low concentrations of single-molecule proteins or small molecules; 2. The accuracy and detection limit of the detection method of this invention are not significantly different from Quanterix's SimoA technology; 3. The detection method of this invention has greatly improved accuracy and sensitivity compared to traditional chemiluminescence detection techniques; 4. The detection method of this invention is low in cost, short in time, simple, and efficient.

[0099] Example 6: Comparison of detection of other proteins

[0100] To demonstrate that the detection scope of this invention is not limited to mental illnesses or even to the detection of P-tau 217, this embodiment shows detection data for other types of biomarkers such as infectious diseases and immune inflammation.

[0101] The single-molecule immunoassay technique based on enzyme-catalyzed chemiluminescence mentioned in this invention (Example 2) and other detection methods were used to conduct comparative tests on inflammatory markers (IL-6) and infectious disease markers (IL-7).

[0102] IL-6 antibody 1: ThermoFisher, catalog number: 14-7068-81;

[0103] IL-6 antibody 2: ThermoFisher, catalog number: M620;

[0104] IL-7 antibody 1: ThermoFisher, catalog number: PA5-46944;

[0105] IL-7 antibody 2: ThermoFisher, catalog number: 500-P57-100UG;

[0106] IL-6 Detection Kit: Quanterix IL-6Advantage Kit

[0107] IL-7 test kit: Quanterix IL-7 Advantage Kit

[0108] IL-6 ELISA kit: Shanghai Keabo, catalog number: CB10373-Hu

[0109] IL-7 ELISA kit: Shanghai Keabo, catalog number: CB10375-Hu

[0110] IL-6 and IL-7 were serially diluted to obtain working solutions of 10 pg / ml, 1 pg / ml, 100 fg / ml, and 50 fg / ml, respectively.

[0111] Using the single-molecule immunoassay method based on enzyme-catalyzed chemiluminescence mentioned in this invention (Example 2), Quanterix's SimoA technology and Shanghai Keaibo's ELISA detection kit, we conducted comparative tests on four gradient working solutions and negative controls for IL-6 and IL-7, respectively. Each concentration was repeated three times. There was no significant difference between batches. The average value of the three replicates was taken. The results are shown in Table 2.

[0112] Table 2. Comparison of IL-6 and IL-7 detection results between the present invention's technology and existing technologies.

[0113]

[0114] Note: The concentration data in the table are the average of three parallel replicates.

[0115] The test results (Table 2) show that: 1. The single-molecule protein detection method based on microarray chip and chemiluminescence immunoassay technology of the present invention can detect different trace amounts of protein; 2. The accuracy of the detection method of the present invention is not significantly different from that of Quanterix's SimoA technology; 3. The detection method of the present invention has greatly improved accuracy and sensitivity compared with the traditional chemiluminescence detection technology (ELISA).

[0116] The above are merely some preferred embodiments of the single-molecule protein detection method proposed in this invention, and this invention is not limited to the contents of these embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the concept of this invention, and any changes and modifications made are within the protection scope of this invention.

Claims

1. A method for detecting single-molecule proteins based on microarray chips and chemiluminescence immunoassay, characterized in that, An antigen-antibody complex suitable for chemiluminescence immunoassay is loaded into a microarray chip with 50,000 to 200,000 micropores with a diameter of 5-30 μm on its surface. The chemiluminescence detection system identifies and focuses on the micropores, collects the signal data within the micropores, and calculates and analyzes the concentration of antigen within the microarray chip.

2. The single-molecule protein detection method according to claim 1, characterized in that, The chemiluminescence immunoassay detection methods include direct chemiluminescence immunoassay, enzyme-catalyzed chemiluminescence immunoassay, electrochemiluminescence immunoassay, and photo-induced chemiluminescence immunoassay.

3. The method for detecting single-molecule proteins according to claim 2, characterized in that, The chemiluminescence immunoassay method is a direct chemiluminescence immunoassay method, an enzyme-catalyzed chemiluminescence immunoassay method, or a photo-induced chemiluminescence immunoassay method; the antigen-antibody complex is a double-antibody sandwich complex or an antigen-antibody complex.

4. The method for detecting single-molecule proteins according to any one of claims 1-3, characterized in that, The microarray chip with 50,000 to 200,000 micro-vias with a diameter of 5-30 μm on its surface includes a chip shell, a micro-via chip, and a base plate layer. The microporous chip is mounted on top of the base plate layer with a gap to act as a microchannel layer. The chip housing and the base plate layer seal and cover the microporous chip with a gap. The chip housing is provided with a sample loading port and a substrate port, which are arranged opposite to each other on the chip housing; the reaction system to be tested is injected into the microfluidic layer through the sample loading port, so that the reaction system to be tested passes through the micropores on the microporous chip; the reaction system to be tested is drawn into the micropores by the siphon effect and the hydrophilic effect of the hydrophilic groups in the micropores; A light-emitting substrate is injected into the microporous chip through the substrate aperture; The microfluidic channel is disposed at the bottom of the micro-perforation; suitable for the reaction system to be tested to pass through the bottom of the micro-perforation.

5. The method for detecting single-molecule proteins according to claim 4, characterized in that, The microporous layer has 200,000 micropores with a diameter of 10 μm.

6. The method for detecting single-molecule proteins according to claim 4, characterized in that, Includes the following steps: (1) The antigen-antibody complexes connected with chemiluminescent labels are loaded into the microarray chip through the sample wells; each antigen-antibody complex contains a single protein to be tested. (2) The microarray chip is identified by the chemiluminescence detection system, and the substrate of the chemiluminescence label is added through the substrate well, so that the substrate binds to the antigen-antibody complex in all the chip wells; (3) The positive signal generated in the micro-well is received by the chemiluminescence detection system; after the signal collection is completed, the chemiluminescence detection system performs threshold division, calibration and verification, and analyzes and counts the number of valid wells, the number of negative wells and the number of positive wells, removes the number of invalid wells, and calculates the specific concentration of protein in the sample to be tested.

7. The method for detecting single-molecule proteins according to claim 4, characterized in that, Includes the following steps: (1) The antigen-antibody complexes linked with labeled enzymes are loaded into the microarray chip through the sample wells; each antigen-antibody complex contains a single protein to be tested; (2) The microarray chip is identified by the chemiluminescence detection system, and the negative signal / background signal after the antigen-antibody complex transferred by the micro-hole is fixed and focused; (3) Add the catalytic substrate through the substrate well, so that the substrate binds to the antigen-antibody complex in the chip well. After incubation and catalysis for 5-15 min, the positive signal generated in the micro-well is received by the chemiluminescence detection system. Threshold division, calibration and testing are performed, and the number of effective wells, negative wells and positive wells are analyzed and counted. The number of invalid wells is removed, and the concentration of the single molecule protein to be detected is calculated.

8. The method for detecting single-molecule proteins according to claim 4, characterized in that, Includes the following steps: (1) The antigen-antibody complex is loaded into the microarray chip through the sample well; the antigen-antibody complex is an antigen-antibody complex of photosensitive microspheres and luminescent microspheres directly or indirectly coupled. (2) The microarray chip is irradiated with excitation light of a specific wavelength emitted by the chemiluminescence detection system, and the emission light of different wavelengths is collected at the same time to complete the detection of single protein concentration.

9. A single-molecule protein detection system, comprising a microarray chip with 50,000 to 200,000 micropores with a diameter of 5-30 μm on its surface, a chemiluminescence detection system, and a preparation for preparing antigen-antibody complexes; The microarray chip includes a chip housing, a microporous chip, and a substrate layer; The microporous chip is mounted on top of the base plate layer with a gap to act as a microchannel layer. The chip housing and the base plate layer seal and cover the microporous chip with a gap. The chip housing is provided with a sample loading port and a substrate port, which are disposed on opposite sides of the chip housing. The test reaction system is injected into the microfluidic layer through the sample loading port, and the test reaction system is drawn into the micro-perforations on the microporous chip through the micro-perforations by the siphon effect and the hydrophilic effect of the hydrophilic groups in the micro-perforations. A luminescent substrate is injected into the microporous chip through the substrate port. The microfluidic channel is located at the bottom of the micro-hole, and is suitable for the reaction system to be tested to pass through the bottom of the micro-hole.

10. Any of the following applications of a microarray chip: (1) The antigen-antibody complex is loaded onto the microarray chip, and the quantitative detection of single-molecule proteins is achieved by direct chemiluminescence immunoassay. (2) The antigen-antibody complex is loaded onto the microarray chip, and the quantitative detection of single-molecule proteins is achieved by enzyme-catalyzed chemiluminescence immunoassay. (3) The antigen-antibody complex is loaded onto the microarray chip, and the quantitative detection of single-molecule proteins is achieved by photo-induced chemiluminescence immunoassay. The microarray chip includes a chip housing, a microporous chip, and a substrate layer; The microporous chip is installed inside the chip housing, and the microporous layer has 50,000 to 200,000 micropores with a diameter of 5 to 30 μm. The chip housing is provided with microfluidic channels. The chip housing is also provided with a sample loading port; suitable for injecting the test reaction system into the microfluidic layer through the sample loading port, so that the test reaction system passes through the micro-perforations on the microporous chip; the test reaction system is drawn into the micro-perforations by the siphon effect and the hydrophilic effect of the hydrophilic groups in the micro-perforations; The microfluidic channel is disposed between the microporous chip and the substrate layer; it is suitable for the reaction system to be tested to pass through the bottom of the micropore; A substrate hole is provided on the opposite side of the sample loading hole on the chip housing, which is suitable for injecting a light-emitting substrate into the microporous chip through the substrate hole.

Images