Synchronous detection method and system for small molecules and proteins
By using carboxyl fluorescent microspheres labeled with antibodies and microsphere array biochips combined with deep learning models in saliva samples, highly sensitive simultaneous detection of small molecules and proteins was achieved, solving the problem of matrix effect in saliva and improving detection efficiency and sensitivity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SUZHOU CHUXINDAKANG BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-02-11
- Publication Date
- 2026-05-15
AI Technical Summary
Traditional methods cannot achieve simultaneous high-sensitivity detection of small molecules and proteins. In particular, the matrix effect in saliva samples leads to reduced detection sensitivity and repeatability. Existing technologies cannot meet the needs of small molecule and protein detection.
Small molecule and protein detection antibodies labeled with carboxyl fluorescent microspheres were used to prepare a microsphere array biochip. By combining competitive reaction and sandwich immunoassay, a deep learning model was used to identify fluorescence images, the saliva buffer system was optimized, and parallel channels were designed for simultaneous detection.
It enables simultaneous detection of small molecules and proteins with high sensitivity (fg/ml level), solves the problem of matrix interference in saliva, improves detection throughput and sensitivity, and meets the needs of clinical diagnosis.
Smart Images
Figure CN122042975A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of microfluidics, and in particular to a method and system for the simultaneous detection of small molecules and proteins. Background Technology
[0002] Currently, rapid clinical and field testing often requires the simultaneous monitoring of small molecule and protein biomarkers. However, traditional small molecule detection relies on competitive immunoassays, which suffer from low sensitivity and narrow dynamic range; protein detection primarily uses sandwich immunoassays, which are incompatible with the needs of small molecule detection, resulting in the inability to simultaneously acquire information on two key biomarkers. The mucins, enzymes, electrolytes, and non-specific binding substances in saliva can cause severe matrix effects, significantly reducing detection sensitivity and repeatability. Existing commercial kits are mostly designed for serum / plasma, and their performance drops drastically when used directly with saliva. Traditional ELISA detection limits for small molecules are typically in the ng / mL range, making it difficult to meet the detection needs of low concentrations of target substances (such as cortisol) in saliva. Although single-molecule technologies such as Simoa can improve sensitivity to fg / mL, they do not effectively integrate competitive and sandwich immunoassays and do not solve the matrix interference problem in saliva samples, limiting their application in point-of-care testing (POCT) scenarios.
[0003] Therefore, existing technologies still need to be improved and developed. Summary of the Invention
[0004] Based on this, this application provides a method and system for the simultaneous detection of small molecules and proteins, which can achieve simultaneous quantitative detection of small molecules and proteins with high sensitivity and high specificity.
[0005] To achieve the above objectives, embodiments of this application provide a method for the simultaneous detection of small molecules and proteins, comprising: Carboxyl-labeled fluorescent microspheres were used to prepare small molecule detection antibodies and protein detection antibodies, respectively. A mixed detection antibody was obtained by mixing a small molecule detection antibody labeled with carboxyl fluorescent microspheres with a protein detection antibody labeled with carboxyl fluorescent microspheres. Microsphere-labeled antibodies were prepared to capture small molecule antigens and proteins, respectively. Fabrication of a microsphere array biochip with two detection zones; The microsphere-labeled capture small molecule antigens and protein antibodies are spotted onto the two detection areas of the microsphere array biochip using an automated spotting machine or a pipette, respectively. The test sample and the mixed detection antibody were mixed in different proportions and incubated at 25℃ to 37℃ for 1 min to 10 min to carry out a competitive reaction, resulting in sample mixtures with different proportions. 10 μL to 50 μL of sample mixtures in different proportions were slowly added to the two detection zones of the microsphere array biochip. Slowly add 50 μL to 200 μL of cleaning solution to the two detection zones of the microsphere array biochip; Two detection areas of the microsphere array biochip were scanned and imaged separately. The fluorescence images obtained from the scanned images were then input into a deep learning model to identify all fluorescence spot information in the fluorescence images. The concentrations of small molecules and proteins in the sample to be tested were obtained through the Poisson distribution principle or by establishing a standard curve.
[0006] Preferably, the preparation method of the carboxyl fluorescent microsphere-labeled small molecule detection antibody or protein detection antibody includes the following steps: Take 20 μL of fluorescent microsphere solution into a centrifuge tube, add 180 μL of coupling solution, and disperse by sonication; Add 5 to 20 μL of freshly prepared 10 mg / mL NHS solution and 1 to 10 μL of 10 mg / mL EDC solution, and stir at room temperature for 15 to 30 minutes. After activation, centrifuge to remove the supernatant, and then sonicate to redisperse. Add 0.002 mg to 0.005 mg of small molecule antibody or protein antibody to the activated fluorescent microsphere solution and react at room temperature for 1 to 3 hours. Add 20 μL of blocking solution and incubate for 1 to 3 hours; After sealing, centrifuge to remove the supernatant, wash once with preservation solution, add preservation solution, add 100ul of preservation solution to redisperse, and store at 4℃.
[0007] Preferably, the method for preparing the microsphere-labeled antibody for capturing small molecule antigens or proteins includes the following steps: Take 100 μL of microsphere solution into a centrifuge tube, centrifuge to separate the supernatant, add 200 μL of coupling solution, and sonicate to disperse; Add 10 to 50 μL of freshly prepared 10 mg / mL NHS solution and 5 to 20 μL of 10 mg / mL EDC solution, and stir at room temperature for 15 to 30 minutes. After activation, the supernatant is removed by centrifugation and then redispersed by ultrasonication. Add 0.01 mg to 0.05 mg of the small molecule antigen or protein antibody to be coupled to the activated microsphere solution and react at room temperature for 1 to 3 hours. Add 20 μL of blocking solution and incubate for 1 to 3 hours; After sealing, centrifuge to remove the supernatant, wash once with preservation solution, add 200 μL of preservation solution for redispersibility, and store at 4°C.
[0008] Preferably, the step of spotting the microsphere-labeled small molecule antigens and protein antibodies onto the two detection areas of the microsphere array biochip using an automated spotting machine or a pipette includes the following steps: Using an automated spotting machine, 0.5 μL to 2 μL of microsphere-labeled capture small molecule antigens and protein antibodies are extracted and spotted onto the two detection areas of the microsphere array biochip in a five-row, five-column array with a spraying volume of 0.02 μL to 0.08 μL / drop. Alternatively, 0.5 μL to 5 μL of microsphere-labeled small molecule antigens or protein antibodies can be pipetted onto two detection areas of the microsphere array biochip, allowing the microspheres to disperse evenly and fall into the micro-trap structure.
[0009] Preferably, the pipette takes 0.5 μL to 5 μL of microsphere-labeled small molecule antigen and protein antibody and drops it onto two detection areas of the microsphere array biochip, so that the microspheres are uniformly dispersed and fall into the micro-trap structure. The process further includes the following steps: After the microspheres have settled for 5 minutes, gently absorb the excess microsphere dilution solution with a clean non-woven cloth, and dry the cleaning solution in an oven at 60°C.
[0010] Preferably, the method further includes the following step: preparing a reaction solution; Add 6.057g of Tris base to a 1L volumetric flask and dissolve it in about 800mL of distilled water; Add 5.021g Triton X-100, 11.688g NaCl, 5.042g BSA, 10.021g sucrose, 5.101g hydroxypropyl-β-cyclodextrin, and 0.511g PC-300 in sequence, and stir with a magnetic stirrer or stirring rod until completely dissolved; Add distilled water to a final volume of 1L; The pH of the solution was adjusted to 8.0 using HCl.
[0011] Preferably, the method for preparing the microsphere array biochip includes the following steps: Polymer materials are processed by injection molding, embossing, molding, and hot pressing, while inorganic materials are processed by photolithography, etching, and deposition. The processed chips are surface treated by physical adsorption, hydrophilic reagent spraying, coating or chemical vapor deposition to obtain microchannel contact angles of 5 to 70 degrees.
[0012] Preferably, the microsphere array biochip includes 2-16 parallel channels, each channel including two physically separated detection areas, the two detection areas being used to detect small molecules and proteins respectively.
[0013] Preferably, the sample to be tested is mixed with the mixed detection antibody in a ratio of 1:1, 3:1, or 6:1.
[0014] This application also provides a system for the simultaneous detection of small molecules and proteins, for implementing the above-described method for the simultaneous detection of small molecules and proteins, comprising: a microsphere array biochip; After spotting, the microsphere array biochip is bonded to the cover plate by ultrasonic welding or double-sided adhesive.
[0015] The method for simultaneous detection of small molecules and proteins provided by this invention has the following advantages and beneficial effects: On the single-molecule detection platform, competitive immunoassay for small molecules and sandwich immunoassay for proteins are physically separated, using the same reagent labeling method and reaction system to simultaneously carry out immunoreactions for small molecules and proteins, thus solving the compatibility problem between the two types of analysis modes.
[0016] By increasing the reaction ratio between fluorescent microspheres and saliva samples, the saliva samples are diluted to reduce the impact of non-specific adsorption of reagents by components such as mucin, amylase, IgA, and bacterial DNA in saliva. Optimize the dedicated buffer system for saliva samples by adding non-ionic detergents, inert proteins, and solubilizers to formulate a microenvironment buffer solution suitable for the simultaneous detection of small and large molecules. Microspheres labeled with small molecules and proteins are pre-embedded manually or automatically. Through the secondary flow effect, microgrooves of appropriate size capture individual microspheres. The microspheres are pre-placed in arrayed microgrooves, which enables the detection sensitivity to reach the fg / ml level. This solves the problem of low analyte content in saliva (concentration is only 1 / 10 to 1 / 3 of that in blood). By designing 2-16 parallel channels, 2-16 samples can be detected simultaneously, increasing the detection throughput. Attached Figure Description
[0017] Figure 1 This is a schematic flowchart of an embodiment of the method for simultaneous detection of small molecules and proteins according to this application.
[0018] Figure 2 To obtain the IL-6 and COR dual detection curve using the simultaneous detection method for small molecules and proteins proposed in this application. Detailed Implementation
[0019] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings, which illustrate preferred embodiments of the application. However, this application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of this application will be thorough and complete.
[0020] It should be noted that when a component is considered to be "connected" to another component, it can be directly connected to and integrated with the other component, or there may be an intervening component present. The term "mounted" and similar expressions used in this document are for illustrative purposes only.
[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.
[0022] This application provides a dual immunoassay integration method based on single-molecule technology, which enables the simultaneous detection of small molecules (such as hormones, drugs, and narcotic metabolites) and proteins (such as cytokines and tumor markers); it effectively improves detection sensitivity (small molecules at the pg / ml level and proteins at the fg / ml level) and dynamic range (above 3 logs), meeting the precision requirements of clinical diagnosis.
[0023] like Figure 1 As shown, a method for the simultaneous detection of small molecules and proteins is provided, comprising: S100: Prepare small molecule detection antibodies and protein detection antibodies labeled with carboxyl fluorescent microspheres, respectively; S200. Mix the small molecule detection antibody labeled with carboxyl fluorescent microspheres with the protein detection antibody labeled with carboxyl fluorescent microspheres to obtain a mixed detection antibody; S300: Prepare microsphere-labeled small molecule antigen and protein antibodies respectively; S400, Fabrication of a microsphere array biochip with two detection zones; S500 uses an automatic spotter or pipette to spot the microsphere-labeled capture small molecule antigens and protein antibodies onto the two detection areas of the microsphere array biochip, respectively. S600. Mix the sample to be tested with the mixed detection antibody in different proportions and incubate at 25℃ to 37℃ for 1 min to 10 min to carry out a competitive reaction to obtain sample mixtures in different proportions. S700: Slowly add 10ul to 50ul of sample mixtures in different proportions to the two detection areas of the microsphere array biochip. S800: Slowly add 50 μL to 200 μL of cleaning solution to the two detection areas of the microsphere array biochip. The S900 performs scanning imaging on two detection areas of the microsphere array biochip, and inputs the fluorescence images obtained from the scanning imaging into a deep learning model to identify all fluorescence spot information in the fluorescence image. The concentration of small molecules and proteins in the sample to be tested is obtained through the Poisson distribution principle or by establishing a standard curve.
[0024] Example 1: Taking the dual detection of the large molecule protein interleukin-6 (IL-6) and the small molecule cortisol (COR) as an example: I. Preparation of the reaction solution: 1.1 Weighing: Tris base: 6.057g, Triton X-100: 5.021g, NaCl: 11.688g, Bovine serum albumin (BSA): 5.042g, Sucrose: 10.021g, Hydroxypropyl-β-cyclodextrin: 5.101g, PC-300: 0.511g 1.2 Dissolving steps: (1) Add the weighed Tris base to a 1L volumetric flask and dissolve it in about 800 mL of distilled water.
[0025] (2) Then add Triton X-100, NaCl, BSA, sucrose, hydroxypropyl-β-cyclodextrin and PC-300 in sequence, and stir with a magnetic stirrer or stirring rod until completely dissolved.
[0026] (3) Finally, add distilled water to 1L.
[0027] (4) Adjust pH: Use HCl to adjust the pH of the solution to 8.0.
[0028] The prepared buffer system is a Tris-HCl buffer system (pH 8.0), which balances protein stability and small molecule antigen solubility.
[0029] BSA is used to block non-specific sites, sucrose is used to improve system stability, and hydroxypropyl-β-cyclodextrin is used to improve the solubility of key components in the reagent, such as antibodies, small molecule antigens, and fat-soluble vitamins, hormones, and small molecules detected in the sample, thereby enhancing detection performance. Triton X-100 reduces saliva viscosity and breaks down the mucus structure, allowing for full antigen release. PC-300 inhibits salivary microorganisms and prevents sample / reagent deterioration. NaCl maintains ionic strength.
[0030] This solution is suitable for IL-6 + COR simultaneous detection reaction / dilution system, especially for saliva detection, where the saliva sample to be tested is stored in this reaction solution.
[0031] II. Reagent Preparation: 2.1 Preparation of carboxyl-labeled fluorescent microsphere antibodies for IL-6 and COR detection (1) Sonicate the fluorescent microsphere solution for 1 min to disperse it evenly; (2) Take 20 μL of nanosphere suspension into a centrifuge tube, add 180 μL of coupling solution, and disperse by ultrasonication; (3) Add 10 μl of freshly prepared NHS (concentration of 10 mg / ml) and 5 μl of EDC (concentration of 10 mg / ml), and stir at room temperature for 30 min; (4) After activation, centrifuge the suspension at 15000 r / min for 10 min, remove the supernatant, and then sonicate to redisperse; (5) Add 0.005 mg of antibody (IL6 or COR antibody) to the activated quantum dot solution and react at room temperature for 3 h; (6) Add 20 μL of blocking solution and block for 3 h; (7) After the reaction is complete, centrifuge to remove the supernatant, wash once with the preservation solution, add the reaction solution to redisperse, and store at 4°C.
[0032] 2.2 Preparation of microsphere-labeled capture COR-BSA hapten / IL-6 primary antibody The small molecule antigen conjugate is bound via a two-step method that uses EDC and NHS to activate and capture the carboxyl groups of the microspheres.
[0033] (1) Take 100 μL of microspheres into a centrifuge tube, centrifuge at 15000 r / min for 10 min, remove the supernatant, add 200 μL of coupling solution, and disperse by sonication.
[0034] (2) Add 20 μl of freshly prepared NHS (concentration of 10 mg / ml) and 100 μl of EDC (concentration of 10 mg / ml), and stir at room temperature for 30 min; (3) After activation, at 15000 r / min for 10 min, remove the supernatant and re-disperse by sonication; (4) Add 0.01-0.05 mg of the COR-BSA hapten / IL-6 primary antibody to be coupled to the activated microsphere solution and react at room temperature for 3 h; (5) Add 20 μL of blocking solution and block for 3 h; (6) After the reaction is complete, centrifuge to separate the supernatant, wash once with the preservation solution, add the reaction solution to redisperse, and store at 4°C.
[0035] The above-described method for preparing microsphere-labeled capture COR-BSA hapten / IL-6 primary antibody requires no antibody pre-modification, has high coupling efficiency and stable binding, and is suitable for long-term storage.
[0036] Of course, microsphere-labeled capture COR-BSA hapten / IL-6 primary antibody can also be prepared via indirect coupling (streptavidin-biotin method): based on the specific high affinity binding of streptavidin (SA) to biotin (Kd≈10-15mol / L), SA is first covalently coupled to the surface of microspheres, and then specifically binds to the pre-labeled biotin hapten / antibody to achieve indirect antibody immobilization. This method allows for flexible antibody replacement, is highly versatile, and theoretically, one SA can link to four biotin haptens / antibodies, thereby enhancing the detection signal.
[0037] III. Fabrication of Microsphere Array Biochips 3.1 Microsphere array biochips can be manufactured using PDMS through injection molding, embossing, molding, hot pressing, etc. Each channel of the chip is designed with two physically separated detection areas, each pre-embedded with either COR-BSA or IL-6 microspheres (see S500 for details; microsphere-labeled small molecule antigens and protein antibodies are spotted onto the two detection areas of the microsphere array biochip using an automated spotting machine or pipette). IL-6 microspheres can be pre-embedded in the upper row and COR-BSA microspheres in the lower row. The test results are the fluorescence values of IL-6 and COR from different samples. Each channel has inlet and outlet ports, and 2-16 independent channels can be designed side-by-side.
[0038] 3.2 Before using the chip, a suitable surface treatment method can be selected. Chemical vapor deposition (APTES) can be used to obtain a microchannel contact angle of 60 degrees. After modification, the surface reduces non-specific protein adsorption and background noise. Depending on the surface treatment, surfaces with different hydrophilicities can be obtained, and passive or self-driven sample introduction can be selected.
[0039] IV. Sample Counting: Automated Spotting: Using an automated spotting machine, 0.5 μL of COR-BSA and IL-6 microspheres (obtained via the microsphere-labeled capture COR-BSA hapten / IL-6 primary antibody preparation in section 2.2) were separately spotted onto different detection areas of the chip in a 5x5 dot matrix at a spray rate of 0.02 μL / drop. The cleaning solution was dried at 60°C to obtain a COR-BSA / IL-6 dual-detection chip with a monodisperse array of microspheres. This embodiment uses an automated spotting method.
[0040] Of course, manual spotting can also be used. The specific steps for manual spotting are as follows: Take 0.5~5ul of COR-BSA and IL-6 microspheres (obtained by preparing microsphere-labeled capture COR-BSA hapten / IL-6 primary antibody through 2.2) with a pipette and drop them onto the detection area of the chip, so that the microspheres are evenly dispersed and fall into the micro-trap structure. After the microspheres settle for 5 minutes, gently absorb the excess microsphere dilution solution with a clean non-woven cloth, and dry the cleaning solution in an oven at 60°C, thus forming a microsphere array in the detection area of the chip.
[0041] After the sample is applied, the chip is bonded to the cover plate by ultrasonic welding, double-sided adhesive, etc., to form a microfluidic chip with detection reagents.
[0042] V. Simultaneous detection in dual modes: protein sandwich double antibody and small molecule competitive reaction. 5.1 Seven mixed antigen samples of COR and IL-6 were prepared using artificial saliva matrix. Table 1 shows the concentrations of COR and IL-6 in different mixed samples.
[0043] 5.2 Mixed detection antibodies: 5ug / ml fluorescent microspheres COR and 5ug / ml fluorescent microspheres IL-6 were mixed at a volume ratio of 1:1.
[0044] 5.3 Test Procedure: (1) Using an automated pipette, 20 μL of the 7 samples prepared in 5.1 and the mixed detection antibody prepared in 5.2 were respectively aspirated, mixed in a ratio of 3:1, and incubated at 37°C for 5 min to carry out the immune reaction; (2) After the reaction is complete, slowly add 20 μL of different sample mixtures to the two detection areas of the microsphere array biochip (5 μL / min) and incubate for 2 min; (3) Take 100ul of cleaning solution and slowly add it into the microsphere array biochip (50ul / min) to remove quantum dots and other impurities that did not participate in the competitive reaction.
[0045] The entire testing time is between 8 and 26 minutes, with the fastest sample loading time being 8 minutes.
[0046] (4) Detection After the above reaction is completed, a fluorescence scanner is used to excite the microspheres with a 365 / 15nm laser source. Bright spot signals are collected through a 610 / 20nm receiving filter. Laser autofocus is used to rapidly scan and image each region. A pre-trained deep learning model is used to identify bright spots in the image, completing the detection of one region in as little as 3 seconds. The number of microspheres in each detection region remains constant within a certain range. This stability makes the distribution of single-molecule events on the microspheres more uniform. The dark-field detection signal can be directly analyzed using the Poisson distribution law, eliminating the need for an additional bright-field microsphere identification step, simplifying the microsphere identification process and improving detection efficiency. Therefore, for low-abundance samples, the number of luminescent microspheres is counted using the Poisson distribution probability formula, while for high-concentration samples, the fluorescence brightness is tested. Finally, two different standard curves are established (see...). Figure 2 The concentrations of the two indicators to be measured were obtained.
[0047] VI. Test Results: Table 2 shows the number of bright spots for COR and IL-6 in different mixed samples.
[0048] 6.2 According to Figure 2 The following performance indicators can be obtained:
[0049] 6.3 Conclusion: This study achieves simultaneous dual-mode (competitive assay and double-antibody sandwich) detection of small molecules and proteins on a single-molecule platform, saving sample volume (<20 μL), time (<20 minutes), and cost. A dedicated saliva buffer system significantly suppresses matrix effects, improving detection sensitivity and stability. The detection LOD for COR is 0.074 ng / mL, and for IL-6 it is 0.085 pg / mL, with sensitivity reaching the fg~pg / mL level, meeting the requirements for trace biomarker detection in saliva. It is suitable for various applications such as drug screening, stress monitoring, inflammation assessment, and early tumor screening, and has the potential for POCT translation. By increasing the reaction ratio between fluorescent microspheres and saliva samples, the influence of complex components such as mucin and amylase in saliva samples is reduced, eliminating the need for saliva sample pretreatment and simplifying the detection process.
[0050] This embodiment utilizes single-molecule detection technology (such as single-molecule immune array technology, Simoa) to achieve highly sensitive and specific simultaneous quantitative detection of small molecules (such as hormones, drugs, drug metabolites, etc.) and large protein molecules (such as cytokines, tumor markers, etc.) coexisting in saliva samples, and effectively eliminates interference from the saliva matrix by optimizing the reagent system.
[0051] In summary, the present application provides a method and system for the simultaneous detection of small molecules and proteins. On a single-molecule detection platform, competitive immunoassay for small molecules and sandwich immunoassay for proteins are physically partitioned and use the same set of reagent labeling methods and reaction systems to simultaneously perform immunoreactions of small molecules and proteins, thus solving the compatibility problem between the two analytical modes.
[0052] By increasing the reaction ratio between fluorescent microspheres and saliva samples, the saliva samples are diluted, reducing the impact of non-specific adsorption of reagents by components such as mucin, amylase, IgA, and bacterial DNA in saliva. A dedicated buffer system for saliva samples is optimized by adding non-ionic detergents, inert proteins, and solubilizers to create a microenvironment buffer solution suitable for the simultaneous detection of small and large molecules. Microspheres labeled with small molecules and proteins are pre-embedded manually or automatically, and individual microspheres are captured by appropriately sized microgrooves through the secondary flow effect. These microspheres are pre-placed in arrayed microgrooves, achieving detection sensitivity at the fg / ml level, thus solving the problem of low analyte concentration in saliva (only 1 / 10 to 1 / 3 of that in blood). By designing 2-16 parallel channels, 2-16 samples can be detected simultaneously, increasing throughput.
[0053] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0054] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.
Claims
1. A method for the simultaneous detection of small molecules and proteins, characterized in that, Includes the following steps: Carboxyl-labeled fluorescent microspheres were used to prepare small molecule detection antibodies and protein detection antibodies, respectively. A mixed detection antibody was obtained by mixing a small molecule detection antibody labeled with carboxyl fluorescent microspheres with a protein detection antibody labeled with carboxyl fluorescent microspheres. Microsphere-labeled antibodies were prepared to capture small molecule antigens and proteins, respectively. Fabrication of a microsphere array biochip with two detection zones; The microsphere-labeled capture small molecule antigens and protein antibodies are spotted onto the two detection areas of the microsphere array biochip using an automated spotting machine or a pipette, respectively. The test sample and the mixed detection antibody were mixed in different proportions and incubated at 25℃ to 37℃ for 1 min to 10 min to carry out a competitive reaction, resulting in sample mixtures with different proportions. 10 μL to 50 μL of sample mixtures in different proportions were slowly added to the two detection zones of the microsphere array biochip. Slowly add 50 μL to 200 μL of cleaning solution to the two detection zones of the microsphere array biochip; Two detection areas of the microsphere array biochip were scanned and imaged separately. The fluorescence images obtained from the scanned images were then input into a deep learning model to identify all fluorescence spot information in the fluorescence images. The concentrations of small molecules and proteins in the sample to be tested were obtained through the Poisson distribution principle or by establishing a standard curve.
2. The method for simultaneous detection of small molecules and proteins according to claim 1, characterized in that, The preparation method of the carboxyl fluorescent microsphere-labeled small molecule detection antibody or protein detection antibody includes the following steps: Take 20 μL of fluorescent microsphere solution into a centrifuge tube, add 180 μL of coupling solution, and disperse by sonication; Add 5 to 20 μL of freshly prepared 10 mg / mL NHS solution and 1 to 10 μL of 10 mg / mL EDC solution, and stir at room temperature for 15 to 30 minutes. After activation, centrifuge to remove the supernatant, and then sonicate to redisperse. Add 0.002 mg to 0.005 mg of small molecule antibody or protein antibody to the activated fluorescent microsphere solution and react at room temperature for 1 to 3 hours. Add 20 μL of blocking solution and incubate for 1 to 3 hours; After sealing, centrifuge to remove the supernatant, wash once with preservation solution, add preservation solution, add 100ul of preservation solution to redisperse, and store at 4℃.
3. The method for simultaneous detection of small molecules and proteins according to claim 1, characterized in that, The method for preparing the microsphere-labeled antibody for capturing small molecule antigens or proteins includes the following steps: Take 100 μL of microsphere solution into a centrifuge tube, centrifuge to separate the supernatant, add 200 μL of coupling solution, and sonicate to disperse; Add 10 to 50 μL of freshly prepared 10 mg / mL NHS solution and 5 to 20 μL of 10 mg / mL EDC solution, and stir at room temperature for 15 to 30 minutes. After activation, the supernatant is removed by centrifugation and then redispersed by ultrasonication. Add 0.01 mg to 0.05 mg of the small molecule antigen or protein antibody to be coupled to the activated microsphere solution and react at room temperature for 1 to 3 hours. Add 20 μL of blocking solution and incubate for 1 to 3 hours; After sealing, centrifuge to remove the supernatant, wash once with preservation solution, add 200 μL of preservation solution for redispersibility, and store at 4°C.
4. The method for simultaneous detection of small molecules and proteins according to claim 1, characterized in that, The process of spotting microsphere-labeled capture small molecule antigens and protein antibodies onto two detection areas of the microsphere array biochip using an automated spotting machine or a pipette includes the following steps: Using an automated spotting machine, 0.5 μL to 2 μL of microsphere-labeled capture small molecule antigens and protein antibodies are extracted and spotted onto the two detection areas of the microsphere array biochip in a five-row, five-column array with a spraying volume of 0.02 μL to 0.08 μL / drop. Alternatively, 0.5 μL to 5 μL of microsphere-labeled small molecule antigens or protein antibodies can be pipetted onto two detection areas of the microsphere array biochip, allowing the microspheres to disperse evenly and fall into the micro-trap structure.
5. The method for simultaneous detection of small molecules and proteins according to claim 4, characterized in that, The pipette takes 0.5 μL to 5 μL of microsphere-labeled small molecule antigens and protein antibodies and drops them onto two detection areas of the microsphere array biochip. After the microspheres are evenly dispersed and fall into the micro-trap structure, the following steps are also included: After the microspheres have settled for 5 minutes, gently absorb the excess microsphere dilution solution with a clean non-woven cloth, and dry the cleaning solution in an oven at 60°C.
6. The method for simultaneous detection of small molecules and proteins according to claim 1, characterized in that, It also includes the following steps: preparing the reaction solution; Add 6.057g of Tris base to a 1L volumetric flask and dissolve it in about 800mL of distilled water; Add 5.021g Triton X-100, 11.688g NaCl, 5.042g BSA, 10.021g sucrose, 5.101g hydroxypropyl-β-cyclodextrin, and 0.511g PC-300 in sequence, and stir with a magnetic stirrer or stirring rod until completely dissolved; Add distilled water to a final volume of 1L; The pH of the solution was adjusted to 8.0 using HCl.
7. The method for simultaneous detection of small molecules and proteins according to claim 1, characterized in that, The fabrication method of the microsphere array biochip includes the following steps: Polymer materials are processed by injection molding, embossing, molding, and hot pressing, while inorganic materials are processed by photolithography, etching, and deposition. The processed chips are surface treated by physical adsorption, hydrophilic reagent spraying, coating or chemical vapor deposition to obtain microchannel contact angles of 5 to 70 degrees.
8. The method for simultaneous detection of small molecules and proteins according to claim 7, characterized in that, The microsphere array biochip includes 2-16 parallel channels, each channel including two physically separated detection areas, which are used to detect small molecules and proteins, respectively.
9. The method for simultaneous detection of small molecules and proteins according to claim 1, characterized in that, The test sample and the mixed detection antibody are mixed in a ratio of 1:1, 3:1, or 6:
1.
10. A system for simultaneous detection of small molecules and proteins, used to implement the method for simultaneous detection of small molecules and proteins according to any one of claims 1-9, characterized in that, include: Microsphere array biochip; After spotting, the microsphere array biochip is bonded to the cover plate by ultrasonic welding or double-sided adhesive.