Protein testing method, testing apparatus, and use

Abstract

Provided are a protein testing method, a testing apparatus, and a use. The protein testing method comprises: fixing in a testing apparatus probe-type graphic encoding chips (4) coupled to a specific capture antibody while separately sealing an SAPE and a detection antibody in the testing apparatus; and, during testing, separately dissolving the detection antibody and the SAPE, and then performing testing and analysis on the basis of the ELISA principle by means of the probe-type graphic encoding chips (4) to obtain results of protein quantitative analysis. According to the testing apparatus, Lab-in-Tip technology is used. Processes such as sampling, washing, hybridization and washing of detection antibodies, and binding of fluorescently-labeled streptavidin to biotin-labeled detection antibodies are completed within one testing apparatus, which has the advantages of completing multiple tests quickly and conveniently with high sensitivity, good specificity, small sample consumption, and a broad testing range.

Description

Protein detection method, detection device and application

[0001] This application claims priority to the Chinese invention patent application with application number CN202311657451.8, application date December 5, 2023, and titled “A protein detection method, detection device and application”. The above patent application is incorporated herein by reference in its entirety. Technical Field

[0002] The present application relates to the field of biological detection technology, and in particular to a protein detection method and detection device and application. Background Art

[0003] Multiplex immunoassays have been widely used in clinical diagnosis, therapeutics, drug discovery, and clinical proteomics research. As the number of protein biomarkers used in clinical and pharmaceutical applications reaches hundreds, this requires the use of time-saving, cost-effective and efficient analytical strategies - multiplex immunoassays. Liquid phase chip technology (suspension array technology, SAT), also known as suspension chip technology. It is a new, high-throughput biochip technology that combines flow cytometry, laser technology, and applied fluidics, and can be used for protein and nucleic acid detection. Compared with the traditional ELISA (enzyme-linked immunosorbent assay) technology, which has the disadvantages of poor reproducibility, prone to false positives, time-consuming and labor-intensive, the new suspension array has a wide linear range, is easy to operate, and can perform efficient multiple detection.

[0004] The microsphere suspension array is a new biochip platform based on Luminex xMAP technology. As the first biochip technology to be certified by the U.S. Food and Drug Administration (FDA) for clinical diagnostics, Luminex xMAP technology has become one of the most widely used multiplex detection technologies. Using red and green lasers to detect the microsphere's encoded and reporter fluorescence, it enables both qualitative and quantitative analysis. It represents a new generation of high-throughput molecular detection technology, following the success of gene chips and protein chips.

[0005] However, Luminex's xMAP technology also has some limitations. For example, due to the high and complex content of serum, the background signal when detecting a specific antibody is too high, resulting in a low signal-to-noise ratio and reduced confidence in the experimental results. Furthermore, despite Luminex's decades of continuous efforts to optimize microspheres and detection platforms, improving detection accuracy, enhancing air compressor performance, and increasing detection multiplexity and throughput, this inevitably leads to increased technical complexity and high costs.

[0006] Graphical coding is another popular coding system for suspension arrays, which uses a set of visually distinguishable patterns, such as embedded barcodes or physical shapes, to identify different analytical particles. Similar to the xMAP system, which is particle-based, graphical suspension arrays are pseudo-homogeneous assays with near-solution diffusion dynamics, resulting in higher mixing efficiency. Importantly, graphically coded particles offer unique features that overcome the shortcomings of color-coded beads, such as better particle shape / size consistency, digital versus analog decoding processes, and greater flexibility in selecting materials with varying chemical, mechanical, and / or optical properties for customized microparticles.

[0007] To date, most proposed graphical suspension arrays have focused on multiplexed detection of nucleic acids, with limited work on immunoassays for protein analytes, partly due to complex assay development issues such as analyte fragility, reagent reproducibility, and nonspecific binding. A review of existing publications in this field reveals that only a few studies have proposed feasible methods for protein assay LODs (limit of detection) at or above 1 pg / mL. Furthermore, in ELISA-based protein quantification using suspension arrays, since the suspension chips are not fixed in an array format, the washing process prior to multiplexed detection requires prolonged natural sedimentation or multiple centrifugation steps. This process inevitably results in significantly longer overall reaction times, is time-consuming and labor-intensive, and suffers from drawbacks such as poor reproducibility, prone to false positives, and is time-consuming and labor-intensive.

[0008] Summary of the Invention

[0009] In response to the technical problems of existing human serum protein analysis methods such as poor technical repeatability, prone to false positives, time-consuming and labor-intensive, this application provides a protein detection method and detection device based on Lab-in-Tip technology and its application, which has the advantages of good repeatability, simple detection device, and short detection time.

[0010] To achieve the above objectives, the present application provides the following technical solution: a protein detection method, comprising fixing a probe-type graphic encoding chip coupled with a specific capture antibody in a detection device, and at the same time, sealing SAPE (R-phycoerythrin-labeled streptavidin) and the detection antibody in the detection device respectively; during detection, the detection antibody and SAPE are dissolved separately, or the detection antibody and SAPE solution are pre-prepared respectively, and the results of protein quantitative analysis can be obtained by performing detection and analysis based on the ELISA principle through the probe-type graphic encoding chip.

[0011] In some embodiments, the detection device is based on Lab-in-Tip technology, specifically including a detection tube and a storage tube; the probe-type graphic encoding chip coupled with a specific capture antibody is built into the detection tube; the SAPE and the detection antibody are built into the inner wall of the storage tube; or the detection antibody and SAPE solution are directly configured and placed in a pipetting workstation; the sample to be tested is placed in the pipetting workstation, and by controlling the volume of the solvent entering the storage tube, the detection antibody and the SAPE are dissolved in sequence and then enter the detection tube; or the detection antibody and SAPE solution are directly configured and directly transferred into the detection tube to achieve quantitative detection and analysis of the protein.

[0012] In some embodiments, the probe-type pattern coding chip comprises modifying the surface of a pattern coding chip and then coupling probe molecules to the pattern coding chip to obtain a probe-type pattern coding chip.

[0013] In some embodiments, the probe molecule is a specific capture antibody.

[0014] In some embodiments, the graphic coding chip is a silica microparticle-based coding suspension chip.

[0015] In some embodiments, the method for preparing the probe-type pattern coding chip comprises the following steps:

[0016] (1) dispersing a silica-based coded suspension chip in an ethanol solution of aminosilane (APDMS) to react, thereby subjecting the coded suspension chip to amino surface modification to obtain the amino-modified coded suspension chip;

[0017] (2) dispersing the amino-modified graphic coding chip obtained after the reaction in step (1) in a succinic anhydride solution, and performing a surface modification by shaking the reaction at room temperature to further obtain the carboxyl-modified graphic coding chip;

[0018] (3) subjecting the carboxyl-modified graphic coding chip obtained in step (2) to an activation reaction, and subjecting it to a coupling reaction with a probe molecule solution, coupling the probe molecule to the surface of the graphic coding chip, thereby obtaining the probe-type graphic coding chip.

[0019] Furthermore, in step (3), the activation reaction is carried out at room temperature for 20 to 40 minutes; the activation solution is a morpholineethanesulfonic acid (MES) buffer solution containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0020] In some embodiments, the coupling reaction is carried out at 0-4°C for 6 hours to 12 hours.

[0021] In some embodiments, the probe molecule solution is a NaAc-HAc buffer of the probe molecule.

[0022] In some embodiments, the detection device is based on Lab-in-Tip technology, and the probe-type graphic coding chip coupled with a specific capture antibody is built into the detection device; the detection antibody solution and the SAPE solution enter the detection device respectively and sequentially, and quantitative detection and analysis of the protein is achieved through the probe-type graphic coding chip.

[0023] In some embodiments, the detection device comprises at least a detection tube and a storage tube, and the probe-type pattern coding chip is built into the detection tube.

[0024] In some embodiments, the detection antibody and SAPE are embedded in the inner wall of the storage tube, and after being dissolved by pipetting workstation, they enter the detection tube and react with the pattern coding chip respectively; or, the detection antibody and SAPE are dissolved and directly configured into a solution, and then, after being pipetted by a pipette gun or a pipetting workstation, they contact and react with the pattern coding chip in the detection tube in turn to achieve quantitative detection and analysis of the protein.

[0025] Specifically, the protein detection method based on Lab-in-Tip technology includes the following steps:

[0026] S1. The probe-type pattern coding chip coupled with a specific capture antibody is fixed to the inner wall of the detection tube within the detection device by natural sedimentation;

[0027] S2. preparing a detection antibody solution and / or SAPE solution, or incorporating a detection antibody and / or SAPE into the detection device;

[0028] S3. Add the test protein sample solution to the sample plate and place the plate into the pipetting workstation;

[0029] S4. Connecting the detection device to the pipetting workstation or pipette;

[0030] S5. Place the well plate containing the phosphate buffer solution in the pipetting workstation, dissolve the detection antibody in the detection device to form a solution, or place the prepared detection antibody solution in the well plate and pipette the solution through a pipette gun or a pipetting workstation to allow the detection antibody solution to react with the graphic coding sheet; then wash the well plate after completion.

[0031] S6. Repeat the previous step to add the SAPE solution to the detection device, or dissolve the SAPE built into the detection device to react with the probe-type pattern encoding chip; after completion, wash;

[0032] S7. The detection tube is taken out for image data acquisition, and the probe-type graphic coding chip in the detection tube is subjected to qualitative analysis or quantitative analysis to obtain a measurement result.

[0033] In order to achieve another purpose, the present application also provides a detection device based on Lab-in-Tip technology for use in the above-mentioned protein detection method to perform qualitative or quantitative detection of proteins.

[0034] In some embodiments, the detection device comprises a detection tube and a storage tube that are detachably connected.

[0035] Furthermore, the storage tube has a pipette tip structure based on Lab-in-Tip technology.

[0036] Furthermore, the storage tube is a conical structure, including a tip portion and a tail portion; the tip portion is connected to the detection tube; and the tail portion can be connected to a pipette gun or a pipetting workstation.

[0037] Furthermore, the probe-type graphic coding chip is fixed in the detection device.

[0038] In some embodiments, the detection antibody and / or SAPE can enter the detection tube and react with the probe-type pattern encoding chip.

[0039] In some embodiments, the detection antibody and SAPE are directly freeze-dried on the surface of the inner wall of the storage tube by freeze-drying, and the detection antibody and SAPE can be dissolved separately by flowing the solution.

[0040] In some embodiments, the detection antibody and SAPE are lyophilized at different locations, the detection antibody is sealed on the surface of the inner wall of one end of the storage tube close to the detection tube, and the SAPE is at the rear end close to the tail end of the tapered tip of the storage tube.

[0041] Preferably, the detection tube is equipped with a probe-type graphic coding chip.

[0042] As a preferred embodiment, the detection antibody and SAPE can be sealed on the inner wall surface of the storage tube by freeze-drying, and can enter the detection tube after being dissolved and react with the specific capture antibody coupled to the surface of the probe-type pattern encoding chip.

[0043] As another preferred embodiment, the detection antibody and SAPE are pre-configured into solutions, which are then transferred into the detection tube and react with the specific capture antibody coupled to the surface of the probe-type pattern encoding chip.

[0044] Furthermore, the storage portion includes a first storage area and a second storage area.

[0045] Furthermore, the first storage area is provided between the tip and 1 / 2 of the storage tube, and the detection antibody is sealed on the inner wall of the first storage area.

[0046] Furthermore, the second storage area is between the tail end and 1 / 2 of the storage tube, and the SAPE is sealed on the inner wall of the second storage area.

[0047] Furthermore, the storage tube and the detection tube are connected via a connector or can be directly connected without a connector.

[0048] Furthermore, the direct connection setting includes connecting the storage tube and the detection tube by means of threads, snap fasteners, seals, etc., or using devices in the prior art that can achieve sealing and connection between the two, all of which fall within the protection scope of this application.

[0049] Furthermore, the storage tube and the detection tube are connected via a connector, one end of the connector is connected to the tip of the storage tube, and the other end is connected to the detection tube.

[0050] Furthermore, the connecting piece is one of a latex tube, a rubber tube, a thermoplastic tube or a UV tube, and can seal and connect the storage tube and the detection tube.

[0051] Furthermore, the detection tube includes any one of a capillary tube, a plastic tube, a quartz tube, and a glass tube, and the surface-modified probe-type graphic coding chip can be fixed on the inner wall of the pipette tip tube by natural sedimentation.

[0052] Furthermore, the inner wall of the pipette is sequentially fixed with a biotinylated detection antibody mixture and SAPE.

[0053] The protein detection method using the aforementioned Lab-in-Tip technology can be applied to protein quantitative analysis based on the ELISA principle. In particular, the probe-type pattern coding chip used in the Lab-in-Tip technology provides a 128-fold coding space, which means that 128-fold detection can be completed in a single pipette tip, achieving a sensitivity of less than 1 pg / ml within a reaction time of 1 hour.

[0054] The technical effects of the technical solution of this application are:

[0055] 1. By adopting the technical solution of the present application, the Lab-in-Tip technology used can complete the processes of sample collection, washing, hybridization of detection antibodies, washing, and binding of fluorescent-labeled streptavidin and biotin-labeled detection antibodies within the pipette tip of this reaction, which has the advantages of completing multiple detections quickly, conveniently, with high sensitivity, good specificity, low sample consumption, and a wide detection range.

[0056] 2. By adopting the technical solution of the present application, the probe-type graphic coding chip using the Lab-in-Tip technology provides a 128-fold coding space, that is, 128-fold detection can be completed in a single pipette tip, which can greatly reduce the amount of sample required in the reaction process and greatly shorten the time of the washing step. It can perform fast, convenient, high-throughput, reproducible, highly sensitive, and wide-linear multiple detection, and can achieve a sensitivity of less than 1 pg / mL in a reaction time of just 1 hour.

[0057] 3. By adopting the technical solution of the present application, a detection device based on Lab-in-Tip technology can be assembled using conventional laboratory experimental equipment. The detection device can perform quantitative detection of protein by simply modifying the pipette tip. The detection device is not only simple in structure and easy to assemble, but also requires only conventional laboratory equipment such as pipette tips, silicone tubes, and capillaries to assemble. In addition, the protein detection process is simple and the results are accurate. In particular, the detection time can be greatly shortened. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIG1 is a schematic structural diagram of the Lab-in-Tip device provided in Example 1 of the present application.

[0059] Figure 2 is a diagram of the graphic coding chip provided in Example 1 of the present application.

[0060] FIG3 is a performance standard curve diagram of IL-8 within a 1-hour reaction time in Example 1 of the present application.

[0061] Figures 4a-4m are performance standard curves of the thirteen-fold detection of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, TNF-α, IFN-α, IFN-γ, and GM-CSF in Example 2 of the present application within a reaction time of 1 hour.

[0062] FIG5 is a performance standard curve diagram of the stability test of IL-8 after storage at 4° C. for 3 months in Example 1 of the present application.

[0063] Figures 6a-6d are performance standard curves of the quadruple assay performed in Example 4 of the present application in a total reaction volume of only 10 μL.

[0064] 7a-7c are performance standard curves of triple detection performed within a reaction time of 15 minutes in Example 5 of the present application. DETAILED DESCRIPTION

[0065] To make the purpose, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application are clearly and completely described. Obviously, the described embodiments are part of the embodiments of this application, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of this application without making creative efforts are within the scope of protection of this application.

[0066] The disclosures of all patent and non-patent literature cited in this application are incorporated herein by reference in their entirety.

[0067] As used in the present invention, the terms "comprises," "includes," "contains," "covers," "has," "with," or any other variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, article, or apparatus that includes a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to the process, method, article, or apparatus. In addition, unless expressly indicated to the contrary, "or" refers to an inclusive "or" rather than an exclusive "or." For example, conditions A or B satisfy any of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist). The phrase "one or more" is intended to cover non-exclusive inclusions. For example, one or more of A, B, and C means any of the following: A alone, B alone, C alone, a combination of A and B, a combination of B and C, a combination of A and C, or a combination of A, B, and C.

[0068] In addition, "a" or "an" is used to describe elements and components described herein. This is done merely for convenience and to provide a general sense of the scope of the application. This description should be understood to include one or at least one, a kind or at least one, and the singular also includes the plural unless it is obvious that it is intended otherwise.

[0069] Unless otherwise defined, the meaning of all technical and scientific terms used herein is the same as that generally understood by those of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed composition embodiments, suitable methods and materials are described below. Unless citing a specific paragraph, all publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, this specification and the definitions included therein shall prevail. In addition, materials, methods and examples are illustrative only and not restrictive.

[0070] A protein detection method based on Lab-in-Tip technology is described. This technology improves upon existing pipette tips. The conical structure of the pipette tip is utilized to seal the detection antibody and SAPE at different locations on the inner wall of the tip. The pipette tip is then controlled by the volume of solvent drawn up by the pipette tip via a pipette or a pipetting workstation to dissolve the detection antibody and SAPE in stages into a detection component affixed with a probe-type pattern encoding chip, enabling protein detection. This method is simple, rapid, and provides accurate detection results. The device employed is simple and low-cost, requiring only conventional laboratory pipette tips for assembly and disassembly.

[0071] Furthermore, to implement the above-mentioned protein detection method, the present application also provides a detection device, which, based on the Lab-in-Tip technology, performs quantitative protein detection by simply modifying the pipette tip. The detection device is not only simple in structure and easy to assemble, but also requires only conventional laboratory equipment such as pipette tips, silicone tubes, and capillaries to assemble. Moreover, the protein detection process is simple and the results are accurate. In particular, the detection time can be greatly shortened.

[0072] Specifically, the detection device includes a detection tube and a storage tube that are detachably connected; the storage tube and the detection tube are connected via a connector or are directly connected.

[0073] As a preferred embodiment, the direct connection setting includes connecting the storage tube and the detection tube by means of threads, snap fasteners, seals, etc., or using devices in the prior art that can achieve sealing and connection between the two, which all fall within the protection scope of this application.

[0074] As a preferred embodiment, the storage tube and the detection tube are connected via a connector or directly.

[0075] Furthermore, one end of the connecting piece is connected to the tip of the storage tube, and the other end is connected to the detection tube.

[0076] Furthermore, the connecting piece is one of a latex tube, a rubber tube, a thermoplastic tube or a UV tube, and can seal and connect the storage tube and the detection tube.

[0077] Furthermore, the detection tube includes any one of a capillary tube, a plastic tube, a quartz tube, and a glass tube, and can fix the surface-modified probe-type graphic coding chip on the inner wall of the pipette tip tube by natural sedimentation.

[0078] Furthermore, the storage tube has a pipette tip structure; more preferably, the storage tube is a tapered structure, including a tip and a tail; the tip is connected to the detection tube; and the tail can be connected to a pipette gun or a pipetting workstation. The pipetting workstation used in the embodiment of the present application is an Eppendorf pipetting workstation.

[0079] Furthermore, the detection antibody and SAPE are directly freeze-dried on the surface of the inner wall of the storage tube by freeze-drying, and the detection antibody and SAPE can be dissolved separately by flowing the solution.

[0080] As a preferred embodiment, the storage portion includes a first storage area and a second storage area. The first storage area is from the tip to 1 / 2 of the storage tube, and the detection antibody is sealed on the inner wall of the first storage area.

[0081] As a preferred embodiment, the second storage area is from the tail end to 1 / 2 of the storage tube, and the SAPE is sealed on the inner wall of the second storage area.

[0082] As another preferred embodiment, the detection antibody and SAPE are dissolved and directly prepared into a solution, which is then blown by a pipette gun or a pipetting workstation and sequentially contacted with the pattern coding chip in the detection tube to react, thereby achieving quantitative detection and analysis of the protein.

[0083] Specifically, the steps of protein detection in combination with the above detection device include:

[0084] S1. The probe-type pattern coding chip coupled with a specific capture antibody is fixed to the inner wall of the detection tube by natural sedimentation;

[0085] S2..preparing a detection antibody solution and / or a SAPE solution, or placing the detection antibody and / or SAPE into the detection device;

[0086] S3. Add the protein sample to be tested to the sample plate and place the plate into the pipetting workstation;

[0087] S4. Connect the Lab-in-Tip device to the pipetting workstation and pipette the Lab-in-Tip device into the well plate; then wash it after completion.

[0088] S5: Place the well plate with phosphate buffer solution in the pipetting workstation and pipette the Lab-in-Tip device on the well plate again to dissolve the sealed detection antibody and react it with the pattern coding chip; after the end, wash it, or place the prepared detection antibody solution in the well plate so that the detection antibody solution directly reacts with the pattern coding chip;

[0089] S6. Repeat the previous step to configure the SAPE solution into the detection device, or dissolve the sealed SAPE again to react with the graphic encoding chip; after completion, wash;

[0090] S7. Remove the detection unit for image data acquisition, and form an image in the optical channel of the set wavelength to perform qualitative or quantitative analysis on the target substance in the liquid phase system to obtain the measurement result.

[0091] As one of the preferred embodiments, the probe-type graphic coding chip includes surface-modifying a graphic coding chip and then coupling probe molecules to the graphic coding chip to obtain a probe-type graphic coding chip; the probe molecules are specific capture antibodies; more preferably, the graphic coding chip is a coding suspension chip based on silica particles, which can provide 128-fold coding space.

[0092] As one of the preferred embodiments, the method for preparing a probe-type pattern coding chip comprises the following steps:

[0093] (1) dispersing a silica-based coded suspension chip in an ethanol solution of aminosilane (APDMS) to react, thereby subjecting the coded suspension chip to amino surface modification to obtain the amino-modified coded suspension chip;

[0094] (2) dispersing the amino-modified graphic coding chip obtained after the reaction in step (1) in a succinic anhydride solution, and performing a surface modification by shaking the reaction at room temperature to further obtain the carboxyl-modified graphic coding chip;

[0095] (3) The coding chip after surface modification in step (2) is subjected to an activation reaction, and a coupling reaction is carried out with a probe molecule solution to couple the probe molecules to the surface of the coding chip, thereby obtaining a probe-type graphic coding chip.

[0096] Alternatively, the modification and detection of the above-mentioned probe-type pattern coding chip may also refer to the relevant technical solutions disclosed in Chinese invention patent CN114965397A.

[0097] The technical solutions, implementation processes and principles of the present application will be further explained below through specific examples. It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present application. The described embodiments are only a part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application. Unless otherwise stated, the reagents and raw materials used in the following examples are commercially available, and the test methods for which specific conditions are not specified are generally carried out under conventional conditions or according to the conditions recommended by the manufacturers. In other words, unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present application all adopt conventional techniques in the field of this technology. These technologies have been fully described in the existing literature.

[0098] Example 1

[0099] This embodiment provides a protein detection device based on Lab-in-Tip technology. The specific structure is shown in Figure 1.

[0100] As shown in Figure 1, the protein detection device includes a detachable silicone tube 1, a capillary tube 2, and a pipette tip 3. The capillary tube 2 and the pipette tip 3 are connected to the ends of the silicone tube 1. The dimensions of the connecting parts of the silicone tube 1, capillary tube 2, and pipette tip 3 are matched to ensure that the entire connected protein detection device will not leak.

[0101] The inner wall of the capillary 2 is fixed with a probe-type pattern coding chip 4 coupled with different capture antibodies.

[0102] Specifically, the pipette tip 3 is a conical structure, and its inner wall includes a first storage area 5 and a second storage area 6. The first storage area 5 is arranged at one end connected to the silicone tube 1, that is, the area between the end close to the tip of the pipette tip 3 and the middle 1 / 2 of the pipette tip 3.

[0103] The second storage area 6 is provided at the tail end, ie, the area between the port close to the pipetting workstation and the middle 1 / 2 of the pipetting tip 3 .

[0104] In this embodiment, the first storage area 5 is immobilized with a biotinylated detection antibody, and the second storage area 6 is immobilized with SAPE (fluorescently labeled streptavidin).

[0105] Specifically, the fixing method can be to directly fix the detection antibody and SAPE in the pipette tip 3 respectively by freeze-drying.

[0106] The freeze-drying method includes placing the detection antibody in the first storage area 5 and the SAPE in the second storage area 6, then freezing the pipette tip 3 in a -80°C freezer for 20 minutes. After the detection antibody and SAPE become solid, the pipette tip 3 is placed in a freeze dryer for freeze drying for 1.5 hours.

[0107] Furthermore, the preparation method of the probe-type pattern coding chip 4 includes:

[0108] (1) Provide a silicon dioxide-based coded suspension chip and select 2×10 5 The coded suspension chip was dispersed in 1000 μL of 5% aminosilane (APDMS) ethanol solution (prepared with 95% ethanol), and the solution was fully reacted for 30 minutes before washing.

[0109] (2) After discarding the supernatant, the suspended chip was dispersed in 1000 μL of 10% succinic anhydride solution, shaken at room temperature overnight, and then washed to obtain a carboxyl chip.

[0110] (3) An activation solution containing 130 mmol / L EDC and 326 mmol / L NHS was prepared using 0.1 mol / L MES buffer (pH = 4.7); the chip suspension and activation solution were mixed and reacted at room temperature for about 30 minutes and then washed. The mixture was then mixed and reacted with a probe molecule solution (solvent: 0.1 mol / L NaAc-HAc buffer) at 4°C for 6 to 12 hours to obtain a probe-type image encoding chip, wherein the probe in this embodiment is a capture antibody.

[0111] Refer to FIG2 , which shows the probe-type image coding chip prepared in this embodiment. The chip has a size of 14×25 μm and has a 128-fold coding space, which can provide 128-fold detection for samples.

[0112] The probe-type image encoding chip is embedded in the inner wall of the capillary 2 by the natural sedimentation method, and the silicone tube 1 is connected to the capillary 2 and the pipette tip 3 in sequence for assembly to obtain a Lab-in-Tip detection device.

[0113] Protein detection is performed based on the above-mentioned Lab-in-Tip detection device. The specific detection method includes the following steps:

[0114] a. After pattern-coded chips are precisely manufactured and released using a photolithography process and then modified with carboxyl groups, different types of specific capture antibodies are coated on the chips with different pattern codes to obtain different coding chips coupled with different types of capture antibodies. In this embodiment, the capture antibodies are named Purified anti-human IL-8 and Purified anti-human IL-1β.

[0115] b. Fixing different encoding chips coupled with different types of capture antibodies on the inner wall of capillary 2 by natural sedimentation;

[0116] c. Prepare a mixture of biotinylated detection antibodies and SAPE at the desired concentrations and seal them at the front and back ends of pipette tip 3; the initial concentration of the detection antibody is 35 μg / mL, the sample volume is 2 μL, and the final concentration after dissolution is 1 μg / mL; the concentration of SAPE is 30 μg / mL, the sample volume is 2 μL, and the final concentration after dissolution is 0.5 μg / mL;

[0117] d. Connect the silicone tube 1, the square capillary 2, and the pipette tip 3 in sequence;

[0118] e. Add 50 μL of sample solution to each well of a 96-well plate, and place the 96-well plate in a pipetting workstation;

[0119] f. Insert the Lab-in-Tip-based detection device into the pipette tip in the pipetting workstation, start the instrument, and allow the Lab-in-Tip-based detection device to pipette in the sample well for 30 minutes.

[0120] g. After the above pipetting is completed, the Lab-in-Tip-based detection device will automatically transfer to the wash tank in the pipetting workstation and pipette up and down three times for cleaning;

[0121] h. Prepare another new 96-well plate and dispense 70 μL of 1× phosphate buffer into each well. Allow the Lab-in-Tip-based detection device to pipette itself in the well for 20 minutes. The volume of phosphate buffered saline entering the pipette tip should not exceed 1 / 2 of the pipette tip. This allows the detection antibody pre-sealed on the inner wall of the pipette tip 3 to be dissolved by pipetting without affecting the SAPE sealed in the second storage portion 6 by the phosphate buffer. The final concentration of the antibody obtained after dissolution is 1 μg / mL. It enters the capillary 2 and reacts with the specific capture antibody on the surface of the multiple encoding chip on the inner wall of the capillary 2.

[0122] i. After the above pipetting is completed, the Lab-in-Tip-based detection device will automatically transfer to the wash tank again and pipette up and down three times for cleaning;

[0123] j. Prepare a new 96-well plate and dispense 120 μL of phosphate buffer into each well. Allow the Lab-in-Tip device to pipette itself in the well for 10 minutes to dissolve the pre-sealed SAPE. The volume of phosphate buffer entering the pipette tip exceeds the volume of the second reservoir 6 to ensure complete dissolution of the SAPE. The final concentration of SAPE after dissolution is 0.5 μg / mL, allowing it to react with the specific capture antibody on the surface of the multi-encoded chip on the inner wall of capillary 2.

[0124] k. After the above pipetting is completed, the Lab-in-Tip-based detection device will automatically transfer to the wash tank of the pipetting workstation again and pipette up and down 5 times for cleaning;

[0125] 1. Remove the capillary tube 2 and directly use it for image data acquisition, and perform qualitative or quantitative analysis to obtain the measurement results.

[0126] In this example, the sample liquid used human cytokine IL-8 as a model protein analyte and was dissolved in a buffer solution; the initial concentration was 10 ng / mL, and after 5-fold dilution, the seventh concentration was 0.64 pg / mL.

[0127] Through the above detection method, the specific coded chip in the detection tube is linked to the specific capture antibody to capture the analyte in the sample. The different fluorescence intensities on the different coded chips are used to determine the amount of various soluble components in the analysis sample.

[0128] Referring to Figure 3, a standard curve for the detection performance of the human cytokine IL-8 (purchased from R&D) within a 1-hour reaction time is shown. The bottom dashed line represents a blank (protein content of 0), i.e., a detection line without analyte. Analysis shows that the technical solution of this application enables protein quantification analysis with sensitivity as high as pg / mL (even at protein concentrations around 1 pg / mL, chip signal values ​​still clearly differentiate). The target protein is quantified by matching the sample's fluorescence intensity with the standard curve.

[0129] Example 2

[0130] The difference between this example and Example 1 is that the samples to be tested are thirteen human cytokines as model protein analytes, and the human cytokines are IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, TNF-α, IFN-α, IFN-γ, and GM-CSF (all purchased from R&D).

[0131] Thirteen protein analytes were mixed, and thirteen detection antibodies were also pre-mixed and sealed in a Lab-in-Tip pipette tip. Using Lab-in-Tip technology, the specific operating steps were the same as in Example 1, and this example completed the above-mentioned thirteen-plex detection within a reaction time of 1 hour.

[0132] The detection results are shown in Figures 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i, 4j, 4k, 4l, and 4m. As can be seen from the standard curves, the sensitivity of protein quantification is as high as pg / mL. At a protein concentration of around 1 pg / mL, the chip signal values ​​are still clearly distinguishable.

[0133] Example 3

[0134] This embodiment is the same as embodiment 1, except that the assembled Lab-in-Tip detection device of the embodiment is stored in a refrigerator at 4° C. for 3 months before testing.

[0135] The test results are shown in Figure 5. The sensitivity of protein quantitative analysis can still be maintained at the pg / mL level. Specifically, the storage signal value fluctuates within the 20% error range and does not decrease significantly. At the same time, the detection sensitivity of IL-1β detected by the method can reach 1 pg / mL.

[0136] Example 4

[0137] The difference between this example and Example 1 is that the samples to be tested are four human cytokines as model protein analytes, and the human cytokines are IL-4, IL-6, IL-8 and IL-1β (all purchased from R&D), and a sample volume of 10 μL is used to complete the quadruple detection.

[0138] The four protein analytes were mixed, and the four detection antibodies were also pre-mixed and sealed in a Lab-in-Tip pipette tip. Using Lab-in-Tip technology, quadruple detection was completed within a reaction time of 1 hour using only a 10uL sample volume (the sample volume in Example 1 was 50uL).

[0139] The test results are shown in Figures 6a, 6b, 6c, and 6d. As can be seen from the standard curves, the sensitivity of protein quantification is as high as the pg / mL level. When the protein concentration is around 1 pg / mL, the chip signal values ​​are still clearly distinguished.

[0140] Example 5

[0141] The difference between this example and Example 1 is that the samples to be tested are three human cytokines as model protein analytes, and the human cytokines are IL-4, IL-6, and IL-8 (all purchased from R&D), and the triple detection is completed within a reaction time of 15 minutes.

[0142] The three protein analytes were mixed, and the three detection antibodies were also pre-mixed and sealed in a Lab-in-Tip pipette tip. Using Lab-in-Tip technology, triple detection was completed in a reaction time of only 15 minutes (the reaction time in Example 1 was 1 hour).

[0143] The test results are shown in Figures 7a, 7b, and 7c. As can be seen from the standard curve, the sensitivity of protein quantification is as high as pg / mL (even at protein concentrations around 1 pg / mL, the chip signal values ​​are still clearly distinguishable).

[0144] It can be seen from the test results of Examples 1-5 that the protein concentration can be detected at the 1 pg / mL level, the protein is around 1 pg / ml (concentration 1 pg / ml), the signal value is also clearly distinguished, and the signal value at this point is higher than the background signal value, and multiple detection can be completed. At the highest, the Lab-in-Tip detection device provided in this application can achieve 128-plex detection.

[0145] Moreover, in Example 4, only 10 μL of the test sample is needed to complete high-sensitivity (pg / mL level) detection and analysis, and the chip signal values ​​can be clearly distinguished.

[0146] In Example 5, the triple detection was completed in 15 minutes, indicating that the method of the present application can achieve rapid detection.

[0147] Using the Lab-in-Tip detection device provided in this application, the graphic coding chip can be fixed to the inner wall of the capillary and assembled into the structure of a commonly used biological pipette tip; at the same time, biotinylated detection antibodies and proteins such as SAPE can be pre-sealed inside the pipette tip, thereby realizing a Lab-in-Tip detection device that is only improved by a commonly used biological pipette tip structure in the prior art, that is, it can complete protein quantitative analysis based on the ELISA principle in a relatively short time; and can achieve a sensitivity of 1 pg / mL within a reaction time of 15 minutes.

[0148] In particular, in actual testing, it is only necessary to select conventional laboratory testing equipment for assembly, and high-sensitivity testing can be achieved without the need for special testing devices.

[0149] Obviously, this application is based on Lab-in-Tip technology, and the graphic coding chip used provides 128-fold coding space, that is, 128-fold detection can be completed in a single pipette tip, which can greatly reduce the amount of sample required in the reaction process and greatly shorten the time of the washing step. It can perform fast, convenient, high-throughput, low-sample-volume, good repeatability, high sensitivity, and wide linear range multiple detection.

[0150] The above are merely preferred embodiments of the present application and are not intended to limit the scope of protection of the present application. Persons skilled in the art will readily appreciate that the present application may be modified and altered in various ways. Any changes, modifications, substitutions, integrations, or parameter changes to these embodiments, which fall within the spirit and principles of the present application and achieve the same functionality through conventional substitutions, without departing from the principles and spirit of the present application, fall within the scope of protection of the present application.

Claims

1. A protein detection method, characterized in that include: A probe-type pattern coding chip coupled with a specific capture antibody is fixed in a detection device, and at the same time, SAPE and a detection antibody are built into the detection device respectively, or a SAPE solution and a detection antibody solution are pre-configured; During detection, the detection antibody and SAPE are dissolved in the detection device respectively, or the pre-configured SAPE solution and the detection antibody solution are transferred to the detection device, and the results of protein quantitative analysis can be obtained by detection and analysis using the probe-type pattern encoding chip.

2. The protein detection method according to claim 1, characterized in that: The probe type pattern coding chip comprises: modifying the surface of the pattern coding chip, and coupling the probe molecule to the pattern coding chip to obtain the probe type pattern coding chip; and / or, the probe molecule is a specific capture antibody; And / or, the graphic coding chip is a coding suspension chip based on silica particles.

3. The protein detection method according to claim 2, characterized in that: The method for preparing the probe-type pattern coding chip comprises the following steps: (1) dispersing a silicon dioxide-based coded suspension chip in an ethanol solution of aminosilane for reaction, so that the coded suspension chip is surface-modified with amino groups to obtain the amino-modified coded suspension chip; (2) dispersing the amino-modified graphic coding chip obtained after the reaction in step (1) in a succinic anhydride solution, and performing a surface modification by shaking the reaction at room temperature, thereby obtaining a carboxyl-modified graphic coding chip; (3) subjecting the carboxyl-modified graphic coding chip obtained in step (2) to an activation reaction, and subjecting it to a coupling reaction with a probe molecule solution, coupling the probe molecule to the surface of the graphic coding chip, thereby obtaining the probe-type graphic coding chip.

4. The protein detection method according to claim 3, characterized in that: In step (3), the activation reaction is carried out at room temperature for 20 to 40 minutes; the activation solution is a MES buffer solution containing EDC and NHS.

5. The protein detection method according to claim 3, characterized in that: In step (3), the coupling reaction is carried out at 0 to 4° C. for 6 to 12 hours; And / or, the probe molecule solution is a NaAc-HAc buffer of the probe molecule.

6. The protein detection method according to any one of claims 1 to 5, characterized in that: The detection device is based on Lab-in-Tip technology, and the probe-type graphic coding chip coupled with specific capture antibodies is built into the detection device; The detection antibody solution and the SAPE solution are respectively and sequentially introduced into the detection device, and the quantitative detection and analysis of the protein is realized through the probe-type pattern coding chip; Preferably, the detection device comprises at least a detection tube and a storage tube, and the probe-type graphic encoding chip is built into the detection tube; The detection antibody and SAPE are built into the inner wall of the storage tube, and after being dissolved by blowing in the pipetting workstation, they enter the detection tube to react with the graphic coding chip respectively; or, the pre-configured detection antibody solution and SAPE solution directly enter the detection tube in sequence to react with the probe-type graphic coding chip.

7. The protein detection method according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1. The probe-type pattern coding chip coupled with a specific capture antibody is fixed in the detection device by natural sedimentation; S2. preparing a detection antibody solution and / or SAPE solution, or placing the detection antibody and / or SAPE inside the detection device; S3. Add the protein sample solution to be tested to the sample well plate, and place the well plate into the pipetting workstation; S4. Connecting the detection device to the pipetting workstation or pipetting gun; S5: placing the well plate for dispensing phosphate buffer into the pipetting workstation, dissolving the detection antibody built into the detection device to form a solution, or placing the configured detection antibody solution into the well plate, and blowing the solution with a pipette gun or a pipetting workstation to make the detection antibody solution react with the graphic coding sheet; after completion, washing is performed; S6. Repeat the previous step, and place the configured SAPE solution into the detection device, or dissolve the SAPE built into the detection device to react with the probe-type pattern encoding chip; after completion, wash; S7. The detection tube is taken out for image data acquisition, and the probe-type graphic coding chip in the detection tube is subjected to qualitative analysis or quantitative analysis to obtain the measurement result.

8. A detection device, used for qualitative or quantitative detection of protein in the protein detection method according to any one of claims 1 to 7; the detection device comprises a detection tube and a storage tube that are detachably connected.

9. The detection device according to claim 8, characterized in that: The storage tube has a pipette tip structure based on the Lab-in-Tip technology.

10. The detection device according to claim 8, characterized in that: The storage tube is a conical structure, including a tip and a tail; the tip is connected to the detection tube; the tail is connected to a pipette gun or a pipette workstation; The probe-type graphic encoding chip is fixed in the detection device; The detection antibody and / or SAPE can enter the detection tube to react with the probe-type graphic encoding chip.

11. The detection device according to claim 10, characterized in that: The detection antibody and SAPE are sealed at different positions on the inner wall surface of the storage tube, and enter the detection tube respectively after being dissolved; preferably, the detection antibody is sealed on the surface of the inner wall of one end of the storage tube close to the detection tube, and the SAPE is at the rear end close to the tail end of the conical pipette tip of the storage tube; Alternatively, the detection antibody and SAPE are dissolved into a solution and then blown by a pipette gun or a pipetting workstation to sequentially contact and react with the pattern coding chip in the detection tube to achieve quantitative detection and analysis of the protein.

12. The detection device according to claim 11, characterized in that: The detection antibody and SAPE can be stored in the storage tube by freeze-drying, or the detection antibody and SAPE solution can be added to the storage tube, dissolved, and then enter the detection tube to react with the specific capture antibody coupled to the surface of the probe-type pattern encoding chip; The storage portion includes a first storage area and a second storage area; The first storage area is between the tip and 1 / 2 of the storage tube, and the detection antibody is sealed on the inner wall of the first storage area; The second storage area is between the tail end and 1 / 2 of the storage tube, and the SAPE is sealed on the inner wall of the second storage area.

13. The detection device according to any one of claims 8 to 12, characterized in that: The storage tube and the detection tube are connected via a connector or directly connected; One end of the connecting piece is connected to the tip of the storage tube, and the other end is connected to the detection tube; The connecting piece is one of a latex tube, a rubber tube, a thermoplastic tube or a UV tube, and can seal and connect the storage tube and the detection tube.

14. The detection device according to claim 11, characterized in that: The detection tube includes any one of a capillary tube, a plastic tube, a quartz tube, and a glass tube, and can fix the surface-modified probe-type graphic encoding chip on the inner wall of the storage tube by a natural sedimentation method.

15. Use of the protein detection method according to any one of claims 1 to 7 in protein quantitative analysis.

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