Protein detection device

Abstract

This application discloses a protein detection method and detection device, as well as their applications. The protein detection method involves immobilizing a probe-type graphic-coded chip bound to a specific capture antibody on a detection device, simultaneously encapsulating an SAPE and a detection antibody in the detection device. During detection, the detection antibody and SAPE are dissolved, and then detected and analyzed using the probe-type graphic-coded chip based on the ELISA principle to obtain quantitative protein analysis results. By adopting the technical solution of this application, the adopted Lab-in-Tip technology can complete the steps of sampling, washing, detection antibody hybridization, washing, and binding of fluorescently labeled streptavidin and biotin-labeled detection antibody within the Lab-in-Tip device, offering multiple detection advantages, including speed, simplicity, high sensitivity, high specificity, small sample requirements, and a wide detection range.

Description

[Technical Field]

[0001] (Related Applications) This application claims priority to a Chinese patent application for invention, application number CN202311657451.8, filed on December 5, 2023, and entitled "Protein detection method and detection device and application thereof," the entire contents of which are incorporated herein by reference.

[0002] (Technical field) The present application relates to the technical field of biological detection, and in particular to protein detection methods and devices and their applications. [Background technology]

[0003] Multiplexed immunoassays are widely used in clinical diagnostics, therapeutics, drug discovery, and clinical proteomics research. The number of protein biomarkers used in clinical and pharmaceutical applications reaches hundreds, creating a demand for time-saving and cost-effective analytical strategies for multiplexed immunoassays. Liquid-phase microarray technology (suspension array technology, also known as suspension array technology) is a new, high-throughput biochip technology that combines flow cytometry, laser technology, and applied fluidics for protein and nucleic acid detection. Compared with traditional enzyme-linked immunosorbent assay (ELISA) technology, which has the disadvantages of low reproducibility, susceptibility to false positives, and time and labor consumption, new types of suspension arrays have a wide linear range, are easy to operate, and can perform highly efficient multiplexed assays.

[0004] The microsphere suspension microarray is a new biochip technology platform from Luminex xMAP Technology, which is one of the earliest FDA-approved biochip technologies for clinical diagnostics and one of the most widely used multiplexing technologies. It simultaneously detects the microsphere coding and reporter fluorescence with red and green laser light, enabling qualitative and quantitative analysis. It is a new-generation high-throughput molecular detection technology platform following GeneChip and ProteinChip.

[0005] However, Luminex's xMAP technology also has drawbacks. For example, due to the large and complex contents in serum, when detecting certain antibodies, the background signal can be too high, resulting in a low S / N ratio and reduced reliability of experimental results. Over the past few decades, Luminex has made great efforts to optimize its microspheres and detection platform, continuously improving detection accuracy, improving the performance of its air compressor, increasing the number of detection weights, and increasing detection throughput, but this inevitably results in higher technical complexity and higher costs.

[0006] Graphical coding is another popular coding system for suspension arrays, using a set of visually distinguishable patterns, such as barcodes and physical shapes, to identify different particles for analysis. Similar to particle-based xMAP systems, graphical suspension arrays are pseudo-homogeneous assays that approximate solution-diffusion kinetics, resulting in higher mixing efficiency. Importantly, graphically encoded particles offer unique features that overcome the drawbacks of color-coded beads, including better particle shape / size consistency, digital and analog decoding processes, and greater flexibility in selecting materials with different chemical, mechanical, and optical properties for custom particles.

[0007] To date, most proposed graphical suspension arrays have focused on multiplexed detection of nucleic acids, while little research has been done on immunoassays for protein analytes. This is partly due to complex analytical development issues, such as analyte fragility, reagent reproducibility, and nonspecific binding. Existing publications in this field reveal few methods for measuring LOD (limit of detection) for proteins at levels above 1 pg / mL. Furthermore, in the case of protein quantification methods based on the ELISA principle using suspension arrays, because the suspension chips are not immobilized in an array pattern, washing steps are required in the reaction stage before multiplexing. The washing steps in the reaction stage before multiplexing require prolonged natural sedimentation or multiple centrifugations, which inevitably significantly lengthen the overall reaction time, resulting in time-consuming and labor-intensive procedures, poor reproducibility, and a susceptibility to false positives. Summary of the Invention [Problem to be solved by the invention]

[0008] In response to existing techniques for performing human serum protein analysis, such as poor technical reproducibility, a high incidence of false positives, and time-consuming and labor-intensive technical problems, the present application provides a protein detection method and detection device based on Lab-in-Tip technology, and applications thereof, which have advantages such as good reproducibility, a simple detection device, and a short detection time. [Means for solving the problem]

[0009] To achieve the above objectives, the present application provides the following technical solution: a protein detection method includes the steps of fixing a probe-type graphic-coded chip bound to a specific capture antibody to a detection device, and simultaneously encapsulating SAPE (R-phycoerythrin-labeled streptavidin) and a detection antibody in the detection device, respectively; and during detection, after dissolving the detection antibody and SAPE, respectively, or pre-setting the detection antibody and SAPE solutions, respectively, detecting and analyzing the protein using the probe-type graphic-coded chip based on the ELISA principle to obtain quantitative analysis results.

[0010] In some embodiments, the detection device is based on Lab-in-Tip technology and specifically includes a detection tube and a storage tube, and the probe-type graphic-coded chip to which a specific capture antibody is bound is incorporated into the detection tube, and the SAPE and the detection antibody are incorporated into the inner wall of the storage tube; or the detection antibody and SAPE solution are directly placed in a pipetting workstation, and the sample to be detected is placed in the pipetting workstation, and the volume of solvent entering the storage tube is controlled to sequentially dissolve the detection antibody and the SAPE before they enter the detection tube, or the detection antibody and SAPE solution are directly transferred into the detection tube, thereby achieving quantitative detection and analysis of proteins.

[0011] In some embodiments, the probe-type graphic-coded chip comprises surface-modifying a graphic-coded chip and then binding a probe molecule onto the graphic-coded chip to obtain a probe-type graphic-coded chip.

[0012] In some embodiments, the probe molecule is a specific capture antibody.

[0013] In some embodiments, the graphic encoded chip is a silica particle-based encoded suspension chip.

[0014] In some embodiments, the method for preparing the probe-based graphic-encoded chip comprises the steps of: (1) dispersing and reacting a silica-based coded suspension chip in an ethanol solution of aminosilane (APDMS) to subject the coded suspension chip to amino surface modification, thereby obtaining an amino-modified coded suspension chip; (2) dispersing the amino-modified graphic-encoded chip obtained by the reaction in step (1) in a butanedioic anhydride solution and reacting with shaking at room temperature to perform surface modification, thereby obtaining the carboxyl-modified graphic-encoded chip; (3) The carboxyl group-modified graphic-coded chip obtained in step (2) is activated and then subjected to a binding reaction with a probe molecule solution, and the probe molecule is bound to the surface of the graphic-coded chip, thereby obtaining the probe-type graphic-coded chip.

[0015] Furthermore, in step (3), the activation reaction is carried out at room temperature for 20 to 40 min, and the activation solution is a morpholineethanesulfonic acid (MES) buffer solution containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0016] In some embodiments, the binding reaction is carried out at 0 to 4° C., and the reaction time is 6 to 12 hours (h).

[0017] In some embodiments, the probe molecule solution is a NaAc-HAc buffer solution of probe molecules.

[0018] In some embodiments, the detection device is based on Lab-in-Tip technology, in which the probe-type graphic-encoded chip bound to a specific capture antibody is built into the detection device, and a detection antibody solution and an SAPE solution are sequentially introduced into the detection device, thereby realizing quantitative detection and analysis of proteins using the probe-type graphic-encoded chip.

[0019] In some embodiments, the detection device comprises at least a detection tube and a storage tube, and the probe-type graphic-encoded chip is housed within the detection tube.

[0020] In some embodiments, the detection antibody and SAPE are embedded in the inner wall of the storage tube, and are dissolved by spraying using a pipetting workstation, and then enter the detection tube, respectively, to react with the graphic-coded tip. Alternatively, the detection antibody and SAPE are dissolved to prepare a solution directly, and then the solution is sequentially contacted with and reacted with the graphic-coded tip in the detection tube through the spraying action of a pipette gun or a pipetting workstation, thereby achieving quantitative detection and analysis of proteins.

[0021] Specifically, the protein detection method based on Lab-in-Tip technology includes the following steps: S1. Immobilizing a probe-type graphic-coded chip bound with a specific capture antibody on the inner wall of a detection tube in the detection device by natural precipitation; S2. Prepare a detection antibody solution and / or SAPE solution or incorporate a detection antibody and / or SAPE into the detection device; S3. Add the protein sample solution to be detected to the sample well plate, and place the well plate on the pipetting workstation; S4. Connecting the detection device to the pipetting workstation or pipette gun; S5. Place the well plate into which the phosphate buffer solution is to be dispensed into the pipetting workstation, dissolve the detection antibody contained in the detection device to form a solution, or place the detection antibody solution disposed in the well plate, and spray the solution using a pipette gun or the pipetting workstation to react the detection antibody solution with the graphic code chip, and then wash it after completion; S6. Repeat the previous operation, and allow the disposed SAPE solution to enter the detection device, or dissolve the SAPE contained in the detection device, react with the probe-type graphic-encoded chip, and then wash it after completion; S7. The detection tube is taken out for image data collection, and the probe-type graphic-coded chip in the detection tube is subjected to qualitative or quantitative analysis to obtain measurement results.

[0022] To achieve another object, the present application further provides a method for qualitatively or quantitatively detecting proteins by applying a detection device based on Lab-in-Tip technology to the above-mentioned protein detection method.

[0023] In some embodiments, the detection device comprises a detection tube and a storage tube that are removably connected.

[0024] Furthermore, the storage tube has a pipette tip structure based on Lab-in-Tip technology.

[0025] Furthermore, the storage tube is a conical structure and includes a tip and a tail, the tip being connectable to the detection tube and the tail being connectable to a pipette gun or a pipetting workstation.

[0026] Furthermore, the probe-type graphic-encoded chip is fixed in the detection device.

[0027] In some embodiments, the detection antibody and / or SAPE can enter the detection tube and react with the probe-type graphic-encoded chip.

[0028] In some embodiments, the detection antibody and SAPE are freeze-dried directly onto the inner wall of the storage tube by lyophilization, and the flow of solution therethrough dissolves the detection antibody and SAPE, respectively.

[0029] In some embodiments, the detection antibody and SAPE are freeze-dried at different locations, with the detection antibody being encapsulated on the surface of the inner wall at one end of the storage tube closer to the detection tube, and the SAPE being encapsulated at the tail end of the storage tube closer to the conical pipette tip.

[0030] Preferably, the detection tube contains a probe-type graphic-encoded chip.

[0031] In a preferred embodiment, the detection antibody and SAPE can be encapsulated on the inner wall surface of the storage tube by freeze-drying, and after dissolving, can enter the detection tube and react with the specific capture antibody bound to the surface of the probe-type graphic-encoded chip.

[0032] In another preferred embodiment, the detection antibody and SAPE are each prepared in advance in solution, then transferred to the detection tube and reacted with the specific capture antibody bound to the surface of the probe-type graphic-encoded chip.

[0033] Furthermore, the storage unit includes a first storage area and a second storage area.

[0034] Furthermore, the first storage region is located between the tip and half of the storage tube, and the detection antibody is encapsulated in the inner wall of the first storage region.

[0035] Furthermore, the second storage region is located between the tail end and 1 / 2 of the storage tube, and the SAPE is enclosed in the inner wall of the second storage region.

[0036] Furthermore, the storage tube and the detection tube are connected via a connecting member, or directly without any connecting member.

[0037] Furthermore, the direct connection can be achieved by connecting the storage tube and the detection tube using a connection method such as a screw member, a snap member, or an encapsulating member, or by adopting a prior art device that can encapsulate and connect the two, all of which are within the scope of protection of this application.

[0038] Furthermore, the storage tube and the detection tube are connected via a connecting member, one end of which is connected to the tip of the storage tube and the other end of which is connected to the detection tube.

[0039] Furthermore, the connecting member is any one of a latex tube, a rubber tube, a thermoplastic tube, and a UV adhesive tube, and is capable of sealing and connecting the storage tube and the detection tube.

[0040] Furthermore, the detection tube may include any one of a capillary tube, a plastic tube, a quartz tube, and a glass tube, and the probe-type graphic-encoded tip after surface modification may be fixed to the inner wall of the pipette tip tube by natural precipitation.

[0041] Furthermore, a biotinylated detection antibody mixture and SAPE are immobilized sequentially on the inner wall of the transfer tube.

[0042] The protein detection method using the above-mentioned Lab-in-Tip technology can be applied to quantitative protein analysis based on the ELISA principle. In particular, the probe-type graphic coding chip used in Lab-in-Tip technology provides a 128-plex coding space, meaning that 128-plex detection can be achieved with a single pipette tip, and a sensitivity of less than 1 pg / mL can be obtained within a reaction time of 1 hour. [Effects of the Invention]

[0043] The technical solution of this application has the following technical effects: 1. By adopting the technical solution of this application, the adopted Lab-in-Tip technology can complete the steps of sampling, washing, hybridization of detection antibodies, washing, binding of fluorescently labeled streptavidin and biotin-labeled detection antibodies, etc. within this one reactive pipette tip, which has multiple detection advantages such as speed, simplicity, high sensitivity, high specificity, small sample volume, and wide detection range.

[0044] 2. By adopting the technical solution of this application, the probe-type graphic coding chip used in the adopted Lab-in-Tip technology provides a 128-multiplex code space, that is, 128-multiplex detection can be completed with a single pipette tip, the amount of sample required in the reaction process can be greatly reduced, and the time for the washing process can be greatly shortened. This enables multiplex detection that is fast, simple, high-throughput, has good reproducibility, high sensitivity, and a wide linear range, and can achieve a sensitivity of less than 1 pg / mL within a short reaction time of 1 hour.

[0045] 3. By adopting the technical solution of the present application, a detection device based on Lab-in-Tip technology can be assembled using commonly used laboratory equipment, and the detection device can perform quantitative protein detection by simply modifying pipette tips. The detection device has a simple structure and is easy to assemble. The assembly components can be selected from commonly used laboratory equipment such as pipette tips, silica tubes, and capillary tubes. The protein detection process is simple and the results are accurate, and the detection time can be significantly reduced. [Brief explanation of the drawings]

[0046] [Figure 1] 1 is a schematic diagram showing the structure of the Lab-in-Tip device provided in Example 1 of the present application. [Figure 2] FIG. 1 shows a graphic-encoded chip provided in Example 1 of the present application. [Figure 3] FIG. 1 shows a performance standard curve for IL-8 within a 1-hour reaction time in Example 1 of the present application. [Figure 4a] FIG. 1 shows a performance standard curve for detection of IL-1β within a reaction time of 1 hour in Example 2 of the present application. [Figure 4b] FIG. 1 shows a performance standard curve for detection of IL-2 within a reaction time of 1 hour in Example 2 of the present application. [Figure 4c] FIG. 1 shows a performance standard curve for detection of IL-4 within a reaction time of 1 hour in Example 2 of the present application. [Figure 4d] FIG. 1 shows a performance standard curve for detection of IL-5 within a reaction time of 1 hour in Example 2 of the present application. [Figure 4e] FIG. 1 shows a performance standard curve for detection of IL-6 within a reaction time of 1 hour in Example 2 of the present application. [Figure 4f] FIG. 1 shows a performance standard curve for detection of IL-8 performed within a reaction time of 1 hour in Example 2 of the present application. [Figure 4g] FIG. 1 shows a performance standard curve for detection of IL-10 performed within a reaction time of 1 hour in Example 2 of the present application. [Figure 4h] FIG. 1 shows a performance standard curve for detection of IL-12 within a reaction time of 1 hour in Example 2 of the present application. [Figure 4i] FIG. 1 shows a performance standard curve for detection of IL-17 performed within a reaction time of 1 hour in Example 2 of the present application. [Figure 4j] FIG. 1 shows a performance standard curve for detection of TNF-α within a reaction time of 1 hour in Example 2 of the present application. [Figure 4k] FIG. 1 shows a performance standard curve for detection of IFN-α within a reaction time of 1 hour in Example 2 of the present application. [Figure 4l] FIG. 1 shows a performance standard curve for detection of IFN-γ within a reaction time of 1 hour in Example 2 of the present application. [Figure 4m] FIG. 1 shows a performance standard curve for detection of GM-CSF within a reaction time of 1 hour in Example 2 of the present application. [Figure 5] FIG. 1 shows a performance standard curve for detecting stability of IL-8 after storage at 4° C. for 3 months in Example 1 of the present application. [Figure 6a] FIG. 1 shows a performance standard curve for IL-1β detection in only a total reaction volume of 10 μL in Example 4 of the present application. [Figure 6b] FIG. 1 shows the performance standard curve for IL-4 detection in Example 4 of the present application with a total reaction volume of only 10 μL. [Figure 6c]FIG. 1 shows the performance standard curve for IL-6 detection in Example 4 of the present application with a total reaction volume of only 10 μL. [Figure 6d] FIG. 1 shows the performance standard curve for IL-8 detection in Example 4 of the present application with a total reaction volume of only 10 μL. [Figure 7a] FIG. 1 shows a performance standard curve for IL-4 detection within a reaction time of 15 minutes (min) in Example 5 of the present application. [Figure 7b] FIG. 1 shows a performance standard curve for IL-6 detection within a reaction time of 15 minutes (min) in Example 5 of the present application. [Figure 7c] FIG. 1 shows a performance standard curve for IL-8 detection within a reaction time of 15 minutes (min) in Example 5 of the present application. DETAILED DESCRIPTION OF THE INVENTION

[0047] In order to make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application are clearly and completely described, but obviously, the described embodiments are only some of the embodiments of the present application, not all of the embodiments, and based on the embodiments of the present application, other embodiments obtained by those skilled in the art without any creative work are all included in the protection scope of the present application.

[0048] The entire disclosures of all patent and non-patent literature cited in this application are hereby incorporated by reference.

[0049] As used herein, terms such as "comprise," "comprise," "contain," "cover," "have," "bearing," and any variations thereof are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that includes a list of elements need not be limited to those elements, but may include other elements not expressly listed or elements inherent in the process, method, article, or apparatus. Furthermore, unless expressly stated to the contrary, "or" means inclusive rather than exclusive. For example, condition A or condition B satisfies any of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present). "One or more" is intended to cover a non-exclusive inclusion. 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.

[0050] Additionally, the terms "one" and "one kind" are used to describe elements and features described herein. This is used merely for convenience and to provide a general sense of the scope of the application. Unless it is clear that a different meaning is specifically intended, "one" or "at least one," "one kind" or "at least one kind," and "singular" should be understood to include "plural."

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Methods and materials similar or equivalent to those described herein can be used in carrying out or testing the disclosed composition embodiments, and suitable methods and materials are described below.All publications, patent applications, patents, and other references mentioned herein are incorporated herein in their entirety by reference unless a specific paragraph is cited.In the event of any discrepancy, the present specification and the definitions contained herein shall prevail.In addition, the materials, methods, and examples are illustrative and not limiting.

[0052] The protein detection method is based on Lab-in-Tip technology, which is an improved version of the prior art pipette tip of a pipette gun. It utilizes the conical structure of the pipette tip to encapsulate the detection antibody and SAPE at different positions on the inner wall of the pipette tip. The volume of solvent adsorbed by the pipette tip is controlled by the pipette gun or pipetting workstation, allowing the detection antibody and SAPE to gradually dissolve and enter the detection element with the probe-type graphic-coded tip fixed thereto, thereby achieving protein detection. This method is simple, rapid, and produces accurate detection results. The device used is simple and inexpensive, and the detection device can be assembled and disassembled using only ordinary laboratory pipette tips.

[0053] Furthermore, to realize the above-mentioned protein detection method, the present application also provides a detection device, which is based on Lab-in-Tip technology and performs quantitative protein detection after simply modifying a pipette tip. The detection device has a simple structure and is easy to assemble. The assembly components can be selected from ordinary laboratory equipment such as pipette tips, silica tubes, and capillary tubes. The protein detection process is simple and the results are accurate. In particular, the detection time can be greatly shortened.

[0054] Specifically, the detection device includes a detection tube and a storage tube that are detachably connected, and the storage tube and the detection tube are connected via a connecting member or directly.

[0055] In a preferred embodiment, the direct connection may be achieved by connecting the storage tube and the detection tube using a connection method such as a screw member, a snap member, or an encapsulating member, or by adopting a prior art device that can encapsulate and connect the two, all of which are within the scope of protection of this application.

[0056] In a preferred embodiment, the storage tube and the detection tube are connected via a connecting member or directly.

[0057] Furthermore, one end of the connecting member is connected to the tip of the storage tube, and the other end is connected to the detection tube.

[0058] Furthermore, the connecting member may be any one of a latex tube, a rubber tube, a thermoplastic tube, and a UV adhesive tube, and may enclose and connect the storage tube and the detection tube.

[0059] Furthermore, the detection tube may include any one of a capillary tube, a plastic tube, a quartz tube, and a glass tube, and the probe-type graphic-encoded tip after surface modification may be fixed to the inner wall of the pipette tip tube by natural precipitation.

[0060] Furthermore, the storage tube has a pipette tip structure, more preferably, the storage tube has a conical structure and includes a tip and a tail, the tip can be connected to the detection tube, and the tail can be connected to a pipette gun or a pipetting workstation. The pipetting workstation employed in the examples of this application is an Eppendorf pipetting workstation.

[0061] Furthermore, the detection antibody and SAPE can be freeze-dried directly onto the inner wall surface of the storage tube by freeze-drying, and the detection antibody and SAPE can be dissolved by the flow of the solution through it.

[0062] In a preferred embodiment, the storage section includes a first storage region and a second storage region, the first storage region being located between the tip and half of the storage tube, and the detection antibody being encapsulated in the inner wall of the first storage region.

[0063] In a preferred embodiment, the second storage region is located between the tail end and half of the storage tube, and the SAPE is encapsulated in the inner wall of the second storage region.

[0064] In another preferred embodiment, the detection antibody and SAPE are dissolved and directly prepared into a solution, and then the solution is sequentially brought into contact with the graphic-coded tip in the detection tube through the spraying action of a pipette gun or a pipetting workstation, thereby realizing quantitative detection and analysis of proteins.

[0065] Specifically, protein detection combined with the above detection device includes the following steps: S1. Immobilizing a probe-type graphic-coded chip bound with a specific capture antibody on the inner wall of a detection tube by natural precipitation; S2. Prepare a detection antibody solution and / or SAPE solution or incorporate a detection antibody and / or SAPE into the detection device; S3. Add the protein sample solution to be detected to the sample well plate, and place the well plate on the pipetting workstation. S4. Connect the Lab-in-Tip device to the pipetting workstation and allow the Lab-in-Tip device to spray on the well plate, then rinse and rinse. S5. The well plate into which the phosphate buffer solution has been dispensed is placed on the pipetting workstation, and the Lab-in-Tip device sprays the well plate again to first dissolve the encapsulated detection antibody and react with the graphic-coded chip, and then washes it after completion; or the detection antibody solution placed on the well plate is placed on the well plate, and the detection antibody solution is directly reacted with the graphic-coded chip; S6. Repeat the previous operation, place the disposed SAPE solution into the detection device, or dissolve the encapsulated SAPE again, react with the graphic-encoded chip, and then wash after completion; S7. The detection unit is taken out for image data collection, and is imaged with an optical channel of a set wavelength, and the target substance in the liquid phase system therein is qualitatively or quantitatively analyzed to obtain a measurement result.

[0066] In one preferred embodiment, the probe-type graphic-coded chip comprises surface-modifying the graphic-coded chip and then binding a probe molecule onto the graphic-coded chip to obtain a probe-type graphic-coded chip, wherein the probe molecule is a specific capture antibody. More preferably, the graphic-coded chip is a silica particle-based coded suspension chip, which can provide a 128-fold coded space.

[0067] In one preferred embodiment, the method for preparing a probe-type graphic-encoded chip comprises the following steps: (1) dispersing and reacting a silica-based coded suspension chip in an ethanol solution of aminosilane (APDMS) to subject the coded suspension chip to amino surface modification, thereby obtaining an amino-modified coded suspension chip; (2) dispersing the amino-modified graphic-encoded chip obtained by the reaction in step (1) in a butanedioic anhydride solution and reacting with shaking at room temperature to perform surface modification, thereby obtaining the carboxyl-modified graphic-encoded chip; (3) The coded chip after surface modification in step (2) is activated and reacted with a probe molecule solution to bind the probe molecule to the surface of the coded chip, thereby obtaining a probe-type graphic coded chip.

[0068] Alternatively, the modification and detection of the above-mentioned probe-type graphic coding chip can be referred to the relevant technical solutions disclosed in Chinese Invention Patent CN114965397A.

[0069] The technical solutions, implementation processes, and principles of the present application will be further explained and illustrated through specific examples. The following detailed description is illustrative and is used to further explain the present application. Please note that the described examples are only some of the examples of the present application, and do not represent all of the examples. Other examples obtained by those skilled in the art based on the examples of the present application without creative effort are all within the scope of protection of the present application. Unless otherwise specified, all reagents and raw materials used in the following examples are commercially available, and test methods for which specific conditions are not specified generally follow conventional conditions or conditions recommended by each manufacturer. That is, unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present application use conventional techniques in the relevant technical field. These techniques are well described in existing literature.

[0070] Example 1 This embodiment provides a protein detection device based on Lab-in-Tip technology, the specific structure of which can be seen in FIG.

[0071] As shown in Figure 1, the protein detection device is composed of a detachable silica tube 1, a capillary tube 2, and a pipette tip 3. The capillary tube 2 and the pipette tip 3 are connected to both ends of the silica tube 1, and the sizes of the connections of the silica tube 1, the capillary tube 2, and the pipette tip 3 are matched so that no liquid leaks from the entire connected protein detection device.

[0072] A probe-type graphic-encoded chip 4 with different capture antibodies bound thereto is fixed to the inner wall of the capillary tube 2 .

[0073] Specifically, the pipette tip 3 has a conical structure, and its inner wall includes a first storage area 5 and a second storage area 6, and the first storage area 5 is located at one end of the connecting silica tube 1, i.e., in the area between the end close to the tip of the pipette tip 3 and the middle half of the pipette tip 3.

[0074] The second storage area 6 is located at the tail end, ie in the area between the end connected to the pipetting workstation and the middle half of the pipette tip 3 .

[0075] In this example, a biotinylated detection antibody is immobilized in the first conserved region 5 , and SAPE (fluorescently labeled streptavidin) is immobilized in the second conserved region 6 .

[0076] Specifically, the immobilization method may be a freeze-drying method, and the detection antibody and SAPE may be immobilized directly in the pipette tip 3, respectively.

[0077] The freeze-drying method includes the following: after placing the detection antibody in the first storage area 5 and the SAPE in the second storage area 6, the pipette tip 3 is placed in a refrigerator at -80°C to freeze for 20 minutes (min), and after the detection antibody and SAPE are in a solid state, the pipette tip 3 is placed in a freeze-dryer to freeze-dry for 1.5 hours.

[0078] Furthermore, the method for preparing the probe-type graphic-encoded chip 4 includes: (1) Prepare silica-based coded suspension chips and measure 2 x 10 5 Each coded suspension chip is selected and dispersed in 1000 μL of a 5% aminosilane (APDMS) ethanol solution (prepared with 95% ethanol), and allowed to react thoroughly for 30 minutes, followed by washing. (2) After discarding the supernatant, the suspended chips are dispersed in 1000 μL of 10% butanedioic anhydride solution, shaken overnight at room temperature, and then washed to obtain carboxylated chips. (3) Prepare an activation solution containing 130 mmol / L EDC and 326 mmol / L NHS in 0.1 mol / L MES buffer (pH 4.7). Mix the chip suspension with the activation solution at room temperature and react for about 30 minutes, then wash. Then mix with a probe molecule solution (solvent is 0.1 mol / L NaAc-HAc buffer) at 4°C and react for 6 to 12 hours to obtain a probe-type image-encoded chip. In this example, the probe is a capture antibody.

[0079] Referring to Figure 2, the probe-type image-encoded chip prepared in this example has a size of 14 x 25 μm, a 128-fold code space, and can provide 128-fold detection for the sample.

[0080] The probe-type image-encoded chip is embedded in the inner wall of the capillary tube 2 by natural sedimentation, and the silica tube 1, the capillary tube 2, and the pipette tip 3 are connected in sequence to assemble the chip, thereby obtaining a Lab-in-Tip detection device.

[0081] The protein is detected by the above-mentioned Lab-in-Tip based detection device, and the specific detection method includes the following steps: a. Graphically coded chips are precisely fabricated and released by photolithography, and then modified with carboxyl groups. Different specific capture antibodies are then encapsulated on different graphic coded chips to obtain different coded chips with different capture antibodies attached. In this example, the capture antibodies are named Purified anti-human IL-8 and Purified anti-human IL-1β. b) Different coded chips with different types of capture antibodies bound thereto are immobilized on the inner wall of the capillary tube 2 by spontaneous precipitation; c) After preparing a mixture of biotinylated detection antibodies and SAPE at desired concentrations, they are sealed in the front and rear ends of the pipette tip 3. The initial concentration of the detection antibodies is 35 μg / mL, the sampling volume is 2 μL, and the final concentration after dissolution is 1 μg / mL. The concentration of SAPE is 30 μg / mL, the sampling volume is 2 μL, and the final concentration after dissolution is 0.5 μg / mL. d. The silica tube 1 is connected to the square capillary tube 2 and the pipette tip 3 in sequence. 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. f. Inserting the Lab-in-Tip based detection device onto the mechanical pipette tip in the pipetting workstation and starting the device, the Lab-in-Tip based detection device sprays into the sample well for 30 minutes; g. After the spraying is completed, the Lab-in-Tip-based detection device is automatically transferred to the washing bath in the pipetting workstation and sprayed up and down three times for washing; h) Prepare another new 96-well plate, dispense 70 μL of 1× phosphate buffer solution into each well, and spray the Lab-in-Tip-based detection device into the wells for 20 minutes, ensuring that the volume of phosphate buffer solution in the pipette tip does not exceed half the length of the pipette tip, so that the detection antibody pre-encapsulated on the inner wall of the pipette tip 3 can be sprayed and dissolved, and the SAPE encapsulated in the second storage section 6 will not be affected by the phosphate buffer solution. The final concentration of the antibody obtained after dissolution is 1 μg / mL, which enters the capillary tube 2 and reacts with the specific capture antibody on the multi-coded chip surface on the inner wall of the capillary tube 2; After the spraying is completed, the Lab-in-Tip-based detection device is automatically transferred to the cleaning tank again and sprayed up and down three times for cleaning. j) Prepare another new 96-well plate, dispense 120 μL of phosphate buffer into each well, and allow the Lab-in-Tip device to spray itself into the well for 10 minutes to spray and dissolve the pre-encapsulated SAPE. The volume of phosphate buffer in the pipette tip exceeds the position of the second storage section 6, thereby completely dissolving the SAPE. The final concentration of SAPE obtained after dissolution is 0.5 μg / mL, which reacts with the specific capture antibody on the multi-coded chip surface on the inner wall of the capillary tube 2. k) After the spraying is completed, the Lab-in-Tip-based detection device is automatically transferred to the washing tank of the pipetting workstation again and sprayed up and down five times for cleaning; l. The capillary tube 2 is removed and used directly to collect image data, which is then processed for qualitative or quantitative analysis to obtain measurement results.

[0082] In this example, the sample solution uses human cytokine IL-8 as a model protein analyte, dissolved in a buffer solution, with an initial concentration of 10 ng / mL, and diluted 5 times to a final concentration of 0.64 pg / mL.

[0083] After analysis using the above detection method, specific capture antibodies are bound to specific coded chips in the detection tube to capture the target substance in the sample, and the amount of multiple soluble components in the sample is measured and analyzed based on the difference in the intensity of the fluorescence carried by the different coded chips.

[0084] Figure 3 shows the standard curve for the detection of human cytokine IL-8 (purchased from R&D) within one hour of reaction time. The dotted line at the bottom represents the blank (protein content: 0), i.e., the detection line for no analyte. The analysis shows that by adopting the technical solution of the present application, the sensitivity of protein quantification analysis is improved to the pg / mL level (when the protein concentration is around 1 pg / mL, the chip signal value is still clearly distinguishable). The target protein can be quantified by matching the fluorescence intensity of the sample to the standard curve.

[0085] Example 2 This example differs from Example 1 in the following respects: the samples to be detected were 13 types of human cytokines used as model protein analytes, namely, 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).

[0086] Thirteen types of protein analytes were mixed, and thirteen types of detection antibodies were also premixed 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 in this example, the above 13-plex detection was completed within a reaction time of 1 hour.

[0087] The detection results can be seen in Figures 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i, 4j, 4k, 4l, and 4m. As can be seen from the standard curve, the sensitivity of protein quantification analysis is improved to the pg / mL level, and when the protein concentration is around 1 pg / mL, the chip signal values ​​are still clearly distinguishable.

[0088] Example 3 This example is similar to Example 1 except for the following points. The assembled Lab-in-Tip detection device of this example is stored in a refrigerator at 4° C. for 3 months before detection.

[0089] The detection results can be seen in Figure 5, which shows that the quantitative analysis sensitivity of protein can still be maintained at the pg / mL level. Specifically, the stored 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 above method can reach 1 pg / mL.

[0090] Example 4 This example differs from Example 1 in the following respects: the samples to be detected were four human cytokines used as model protein analytes, namely IL-4, IL-6, IL-8, and IL-1β (all purchased from R&D), and quadruplicate detection was achieved with a sample volume of 10 μL.

[0091] Four types of protein analytes were mixed, and four types of detection antibodies were also premixed and sealed in a Lab-in-Tip pipette tip. Using Lab-in-Tip technology, quadruple detection was completed with only a 10 μL sample volume (the sample volume in Example 1 was 50 μL) within a reaction time of 1 hour.

[0092] The detection results can be seen in Figures 6a, 6b, 6c, and 6d. As can be seen from the standard curve, the sensitivity of protein quantitative analysis is improved to the pg / mL level, and when the protein concentration is around 1 pg / mL, the chip signal values ​​are still clearly distinguishable.

[0093] Example 5 This example differs from Example 1 in the following respects: the samples to be detected were three types of human cytokines used as model protein analytes, IL-4, IL-6, and IL-8 (all purchased from R&D), and triple detection was completed within a reaction time of 15 minutes.

[0094] Three types of protein analytes were mixed, and three types of detection antibodies were also premixed and sealed in a Lab-in-Tip pipette tip. Using the Lab-in-Tip technology, triple detection was completed within a reaction time of only 15 minutes (the reaction time in Example 1 was 1 hour).

[0095] The detection results can be seen in Figures 7a, 7b, and 7c. As can be seen from the standard curve, the sensitivity of protein quantification was improved to the pg / mL level (when the protein concentration was around 1 pg / mL, the chip signal values ​​were still clearly distinguishable).

[0096] As can be seen from the detection results of Examples 1 to 5, protein concentrations can be detected down to the 1 pg / mL level. When the protein concentration is around 1 pg / mL (concentration 1 pg / mL), the signal values ​​are clearly distinguishable. At this point, all signal values ​​are higher than the background signal value, making multiplex detection possible. At its highest, the Lab-in-Tip detection device provided in this application can achieve 128-plex detection.

[0097] Furthermore, in Example 4, highly sensitive (pg / mL level) detection and analysis was possible with only 10 μL of detection sample, and chip signal values ​​were clearly distinguishable.

[0098] In Example 5, triplex detection was completed in 15 minutes, demonstrating that rapid detection can be achieved using the method of the present application.

[0099] By adopting the Lab-in-Tip detection device provided in this application, a graphic-coded tip can be fixed to the inner wall of a capillary tube and assembled into the structure of a pipette tip commonly used in biology. At the same time, biotinylated detection antibodies and proteins such as SAPE can be pre-encapsulated inside the pipette tip. This Lab-in-Tip detection device, which improves on the prior art pipette tip structure commonly used in biology, can quickly achieve protein quantitative analysis based on the ELISA principle in a short time, and can achieve a sensitivity of 1 pg / mL within a 15-minute reaction time.

[0100] In particular, in actual detection, a detection device commonly used in laboratories can be selected and assembled, and highly sensitive detection can be achieved without the need for a special detection device.

[0101] Apparently, this application is based on Lab-in-Tip technology, and the graphic coding chip used provides a 128-plex code space, that is, 128-plex detection can be completed with a single pipette tip, the amount of sample required in the reaction process can be greatly reduced, the time for the washing process can be greatly shortened, and multiplex detection can be achieved quickly, simply, with high throughput, good reproducibility, high sensitivity, and a wide linear range.

[0102] The above are only preferred embodiments of the present application, and do not limit the scope of protection of the present application, and those skilled in the art can make various modifications and variations to the present application. As long as they do not deviate from the spirit and principles of the present application, any modifications, alterations, substitutions, integrations and parameter changes made to these embodiments that achieve normal substitutions or similar functions and do not deviate from the principles and spirit of the present application shall all be included in the scope of protection of the present application.

[0103] (Addendum) (Appendix 1) Fixing a probe-type graphic-coded chip bound with a specific capture antibody to a detection device, and simultaneously placing SAPE and detection antibody into said detection device respectively, or presetting a solution of SAPE and detection antibody; A protein detection method characterized by comprising the steps of: dissolving a detection antibody and an SAPE in the detection device, respectively, during detection; or transferring a predetermined SAPE solution and a detection antibody solution to the detection device; and detecting and analyzing the SAPE using the probe-type graphic-coded chip to obtain a quantitative analysis result of the protein.

[0104] (Appendix 2) The method for producing the probe-type graphic-coded chip includes surface-modifying a graphic-coded chip and then binding a probe molecule onto the graphic-coded chip to obtain the probe-type graphic-coded chip; and / or said probe molecule is a specific capture antibody; And / or the protein detection method described in Appendix 1, characterized in that the graphic-coded chip is a coded suspension chip based on silica particles.

[0105] (Appendix 3) The method for preparing the probe-type graphic-encoded chip includes: (1) dispersing and reacting silica-based coded suspension chips in an ethanol solution of aminosilane to perform amino surface modification on the coded suspension chips, thereby obtaining amino-modified coded suspension chips; (2) dispersing the amino-modified graphic-encoded chip obtained by the reaction in step (1) in a butanedioic anhydride solution and reacting with shaking at room temperature to perform surface modification, thereby obtaining a carboxyl-modified graphic-encoded chip; (3) subjecting the carboxyl group-modified graphic-coded chip obtained in step (2) to an activation reaction, subjecting it to a binding reaction with a probe molecule solution, and binding the probe molecule to the surface of the graphic-coded chip to obtain the probe-type graphic-coded chip.

[0106] (Appendix 4) The protein detection method according to claim 3, wherein in step (3), the activation reaction is carried out at room temperature for 20 to 40 minutes, and the activation solution is an MES buffer solution containing EDC and NHS.

[0107] (Appendix 5) In step (3), the binding reaction is carried out at 0 to 4°C and the reaction time is 6 to 12 hours; And / or the protein detection method described in Appendix 3, characterized in that the probe molecule solution is a NaAc-HAc buffer solution of the probe molecule.

[0108] (Appendix 6) The detection device is based on Lab-in-Tip technology, and the probe-type graphic-coded chip bound to a specific capture antibody is built into the detection device, and a detection antibody solution and an SAPE solution are sequentially introduced into the detection device, respectively, to realize quantitative detection and analysis of proteins by the probe-type graphic-coded chip. Preferably, the detection device includes at least a detection tube and a storage tube, and the probe-type graphic-coded chip is built into the detection tube; A protein detection method described in any one of Appendix 1 to 5, characterized in that the detection antibody and SAPE are embedded in the inner wall of the storage tube, and after being sprayed and dissolved by a pipetting workstation, they enter the detection tube respectively and react with the graphic-coded chip, or a predetermined detection antibody solution and SAPE solution directly enter the detection tube and react with the probe-type graphic-coded chip.

[0109] (Appendix 7) S1. Immobilizing a probe-type graphic-coded chip bound with a specific capture antibody in the detection device by spontaneous precipitation; S2. Preparing a detection antibody solution and / or SAPE solution or incorporating a detection antibody and / or SAPE into the detection device; S3. Adding a protein sample solution to be detected to a sample well plate and placing the well plate on the pipetting workstation; S4. Connecting the detection device to the pipetting workstation or pipette gun; S5. Place the well plate into which the phosphate buffer solution is dispensed on the pipetting workstation, dissolve the detection antibody contained in the detection device to form a solution, or place the detection antibody solution on the well plate, and spray the solution using a pipette gun or a pipetting workstation to react the detection antibody solution with the graphic code chip, and then wash the chip after completion; S6. Repeat the previous operation, disposing the disposed SAPE solution in the detection device or dissolving the SAPE contained in the detection device, reacting with the probe-type graphic-encoded chip, and then washing after completion; S7. A protein detection method described in any one of appendices 1 to 5, characterized in that it includes a step of removing the detection tube for image data collection and subjecting the probe-type graphic-coded chip in the detection tube to qualitative or quantitative analysis to obtain measurement results.

[0110] (Appendix 8) A detection device that is applied to qualitatively or quantitatively detect a protein in the protein detection method described in any one of Appendixes 1 to 7, characterized in that the detection device comprises a detection tube and a storage tube that are detachably connected.

[0111] (Appendix 9) The detection device described in Appendix 8, wherein the storage tube has a pipette tip structure based on Lab-in-Tip technology.

[0112] (Appendix 10) the storage tube is a conical structure and includes a tip and a tail, the tip being connected to the detection tube and the tail being connected to a pipette gun or a pipetting workstation; The probe-type graphic-encoded chip is fixed in the detection device; 9. The detection device according to claim 8, wherein the detection antibody and / or SAPE can enter the detection tube and react with the probe-type graphic-encoded chip.

[0113] (Appendix 11) The detection antibody and the SAPE are respectively sealed at different positions on the inner wall surface of the storage tube, and after dissolving, are introduced into the detection tube, respectively. Preferably, the detection antibody is sealed on the inner wall surface of one end of the storage tube close to the detection tube, and the SAPE is sealed at the tail end of a conical pipette tip close to the storage tube at the rear end. Alternatively, the detection device described in Appendix 10 is characterized in that the detection antibody and SAPE are dissolved to form a solution, and then the solution is sequentially contacted and reacted with the graphic-coded chip in the detection tube by the spraying action of a pipette gun or a pipetting workstation, thereby achieving quantitative detection and analysis of proteins.

[0114] (Appendix 12) The detection antibody and SAPE are stored in the storage tubes by freeze-drying, or solutions of the detection antibody and SAPE are added to the storage tubes, respectively, and dissolved therein, and then introduced into the detection tube to react with the specific capture antibody bound to the surface of the probe-type graphic-encoded chip; the storage unit includes a first storage area and a second storage area; the first storage region is located between the tip and 1 / 2 of the storage tube, and the detection antibody is encapsulated in the inner wall of the first storage region; 12. The detection device of claim 11, wherein the second storage area is located between the tail end and half of the storage tube, and the SAPE is enclosed within the inner wall of the second storage area.

[0115] (Appendix 13) the storage tube and the detection tube are connected by a connecting member or directly; one end of the connecting member is connected to the tip of the storage tube, and the other end is connected to the detection tube; 13. The detection device according to any one of appendices 8 to 12, wherein the connecting member is one of a latex tube, a rubber tube, a thermoplastic tube, or a UV-bonded tube, and is capable of sealing and connecting the storage tube and the detection tube.

[0116] (Appendix 14) The detection device described in Appendix 11, characterized in that the detection tube includes one of a capillary tube, a plastic tube, a quartz tube, and a glass tube, and the probe-type graphic-coded chip after surface modification can be fixed to the inner wall of the storage tube by natural precipitation.

[0117] (Appendix 15) Application of the protein detection method according to any one of claims 1 to 7 in protein quantitative analysis.

Claims

1. Fixing a probe-type graphic-coded chip bound with a specific capture antibody to a detection device, and simultaneously placing an SAPE and a detection antibody into said detection device, respectively, or presetting a SAPE solution and a detection antibody solution; A protein detection method characterized by comprising the steps of: dissolving a detection antibody and an SAPE in the detection device, respectively, during detection; or transferring a predetermined SAPE solution and a detection antibody solution to the detection device; and detecting and analyzing the detected antibody using the probe-type graphic-coded chip to obtain quantitative protein analysis results.

2. The method for producing the probe-type graphic-coded chip includes surface-modifying a graphic-coded chip and then binding a probe molecule onto the graphic-coded chip to obtain the probe-type graphic-coded chip; and / or said probe molecule is a specific capture antibody; And / or the graphic-encoded chip is a silica particle-based encoded suspension chip, The protein detection method according to claim 1, characterized in that:

3. The method for preparing the probe-type graphic-encoded chip includes: (1) dispersing and reacting silica-based coded suspension chips in an ethanol solution of aminosilane to perform amino surface modification on the coded suspension chips, thereby obtaining amino-modified coded suspension chips; (2) dispersing the amino-modified graphic-encoded chip obtained by the reaction in step (1) in a butanedioic anhydride solution and reacting it with shaking at room temperature to perform surface modification, thereby obtaining the carboxyl-modified graphic-encoded chip; (3) subjecting the carboxyl group-modified graphic-coded chip obtained in step (2) to an activation reaction, subjecting the carboxyl group-modified graphic-coded chip to a binding reaction with a probe molecule solution, and binding the probe molecule to the surface of the graphic-coded chip to obtain the probe-type graphic-coded chip.

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

5. In step (3), the binding reaction is carried out at 0 to 4°C and the reaction time is 6 to 12 hours; And / or the protein detection method according to claim 3, wherein the probe molecule solution is a NaAc-HAC buffer solution of the probe molecule.

6. The detection device is based on Lab-in-Tip technology, in which the probe-type graphic-encoded chip bound to a specific capture antibody is built into the detection device, and a detection antibody solution and an SAPE solution are sequentially introduced into the detection device, respectively, to realize quantitative detection and analysis of proteins by the probe-type graphic-encoded chip; Preferably, the detection device includes at least a detection tube and a storage tube, and the probe-type graphic-coded chip is built into the detection tube; 6. The protein detection method according to claim 1, wherein the detection antibody and SAPE are incorporated into the inner wall of the storage tube, and after being sprayed and dissolved by a pipetting workstation, enter the detection tube, respectively, to react with the graphic-coded chip, or a preset detection antibody solution and SAPE solution directly enter the detection tube to react with the probe-type graphic-coded chip.

7. S1. Immobilizing a probe-type graphic-coded chip bound with a specific capture antibody in the detection device by spontaneous precipitation; S2. Preparing a detection antibody solution and / or SAPE solution or incorporating a detection antibody and / or SAPE into the detection device; S3. Adding a protein sample solution to be detected to a sample well plate and placing the well plate on the pipetting workstation; S4. Connecting the detection device to the pipetting workstation or pipette gun; S5. Place the well plate into which the phosphate buffer solution is dispensed on the pipetting workstation, dissolve the detection antibody contained in the detection device to form a solution, or place the detection antibody solution on the well plate, and spray the solution using a pipette gun or a pipetting workstation to react the detection antibody solution with the graphic code chip, and then wash the chip after completion; S6. Repeat the previous operation, disposing the SAPE solution in the detection device or dissolving the SAPE contained in the detection device, reacting with the probe-type graphic-encoded chip, and then washing after completion; S7. The protein detection method according to any one of claims 1 to 5, further comprising the step of removing the detection tube for image data collection and subjecting the probe-type graphic-coded chip in the detection tube to qualitative or quantitative analysis to obtain a measurement result.

8. A detection device that is applied to qualitatively or quantitatively detect proteins in the protein detection method described in any one of claims 1 to 7, characterized in that the detection device comprises a detection tube and a storage tube that are detachably connected.

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

10. the storage tube is a conical structure and includes a tip and a tail, the tip being connected to the detection tube and the tail being connected to a pipette gun or a pipetting workstation; The probe-type graphic-encoded chip is fixed in the detection device; 9. The detection device according to claim 8, wherein the detection antibody and / or SAPE can enter the detection tube and react with the probe-type graphic-encoded chip.

11. The detection antibody and the SAPE are respectively sealed at different positions on the inner wall surface of the storage tube, and after dissolving, are respectively introduced into the detection tube. Preferably, the detection antibody is sealed on the inner wall surface of one end of the storage tube close to the detection tube, and the SAPE is sealed at the tail end of a conical pipette tip close to the storage tube at the rear end. Alternatively, the detection device according to claim 10, wherein the detection antibody and SAPE are dissolved to form a solution, and then the solution is sequentially brought into contact with and reacted with the graphic-coded chip in the detection tube by the spraying action of a pipette gun or a pipetting workstation, thereby realizing quantitative detection and analysis of proteins.

12. The detection antibody and SAPE are stored in the storage tubes by freeze-drying, or solutions of the detection antibody and SAPE are added to the storage tubes, respectively, and dissolved therein, and then introduced into the detection tube to react with the specific capture antibody bound to the surface of the probe-type graphic-encoded chip; the storage unit includes a first storage area and a second storage area; the first storage region is located between the tip and half of the storage tube, and the detection antibody is encapsulated in an inner wall of the first storage region; 12. The detection device of claim 11, wherein the second storage region is located between the tail end and half of the storage tube, and the SAPE is encapsulated in an inner wall of the second storage region.

13. the storage tube and the detection tube are connected by a connecting member or directly; one end of the connecting member is connected to the tip of the storage tube, and the other end is connected to the detection tube; 13. The detection device according to claim 8, wherein the connecting member is one of a latex tube, a rubber tube, a thermoplastic tube, and a UV adhesive tube, and is capable of sealing and connecting the storage tube and the detection tube.

14. 12. The detection device according to claim 11, wherein the detection tube comprises one of a capillary tube, a plastic tube, a quartz tube, and a glass tube, and the probe-type graphic-encoded chip after surface modification can be fixed to the inner wall of the storage tube by natural precipitation.

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

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