Protein detection method, detection device and application

Abstract

The invention provides a protein detection method which comprises the following steps: fixing a probe type pattern coding chip coupled with a specific capture antibody in a detection device, and simultaneously, respectively sealing SAPE and a detection antibody in the detection device; during detection, the detection antibody and the SAPE are dissolved respectively, and then detection and analysis are carried out through the probe type graph coding chip based on an ELISA principle, so that a protein quantitative analysis result can be obtained. According to the technical scheme provided by the invention, a Labin-Tip technology is adopted, so that the processes of sampling a sample, washing, hybridizing a detection antibody, washing, combining a fluorescence labeled streptavidin with a biotin labeled detection antibody and the like can be completed in the Labin-Tip device; the kit has the advantages of being rapid and convenient to complete, high in sensitivity, good in specificity, small in sample dosage and wide in detection range for multiple detection.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a protein detection method and detection device and application thereof. Background Art

[0002] Multiplex immunoassays have been widely used in clinical diagnosis, therapeutics, drug discovery, and clinical proteomics research. As the number of protein biomarkers for 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, easy false positives, time-consuming and labor-intensive, the new suspension array has a wide linear range, is easy to operate, and can perform efficient multiplex detection.

[0003] Microsphere suspension chip is a new biochip technology platform of Luminex xMAP technology. As the earliest biochip technology certified by the U.S. Food and Drug Administration (FDA) for clinical diagnosis, Luminex xMAP technology has become one of the most widely used multiplex detection technologies. It can achieve qualitative and quantitative purposes by jointly detecting microsphere coding and reporter fluorescence through red and green laser beams. It is a new generation of high-throughput molecular detection technology platform after gene chips and protein chips.

[0004] However, Luminex's xMAP technology also has some shortcomings. For example, due to the large and complex contents in serum, when detecting a certain antibody, the detection background signal is too high, which reduces the signal-to-noise ratio and the credibility of the experimental results. And although Luminex has made great efforts in recent decades to optimize microspheres and detection platforms, continuously improve detection accuracy, improve the performance of air compressors, increase detection weights and detection throughput, it is inevitable that it has brought higher technical complexity and high costs.

[0005] Graphics are another popular code system for suspension arrays, which use a set of visually distinguishable patterns, such as embedded barcodes or physical shapes, to identify different analyte particles. Similar to the xMAP system, which is particle-based, graphic suspension arrays are pseudo-homogeneous assays with near-solution diffusion dynamics, resulting in higher mixing efficiency. Importantly, graphic-coded particles have unique features that overcome the shortcomings of color-coded beads, such as better particle shape / size consistency, digital vs. analog decoding processes, and greater flexibility in choosing materials with different chemical, mechanical, and / or optical properties for custom microparticles.

[0006] To date, most proposed graphic suspension arrays focus on multiplexed detection of nucleic acids, and less work has been done for immunoassays of protein analytes, partly due to complex analytical development issues such as analyte fragility, reagent reproducibility, nonspecific binding, etc. A review of existing publications in this field shows that only a few works have proposed feasible methods for LOD (limit of detection) of protein determination at 1 pg / mL level or higher; and in the use of suspension arrays for protein quantitative analysis based on the ELISA principle, since the suspension chip is not fixed in the array mode, the washing process requires long natural sedimentation or multiple centrifugation during the reaction stage before multiplexed detection, which inevitably leads to a significant increase in the overall reaction time, time-consuming and labor-intensive, and has the disadvantages of poor repeatability, easy occurrence of false positives, time-consuming and labor-intensive, etc. Summary of the invention

[0007] In view of the technical problems of the existing human serum protein analysis methods such as poor technical repeatability, easy occurrence of false positives, time-consuming and labor-intensive, the present invention 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, short detection time, etc.

[0008] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: a protein detection method, comprising fixing a probe-type graphic coding chip coupled with a specific capture antibody in a detection device, and at the same time, sealing SAPE (R-phycoerythrin labeled streptavidin) and a detection antibody in the detection device respectively; during detection, the detection antibody and SAPE are dissolved respectively, or the detection antibody and SAPE solution are pre-configured 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 coding chip.

[0009] Preferably, 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 the SAPE solution are directly configured and placed in a pipetting workstation; the sample to be tested is placed in the pipetting workstation, and the volume of the solvent entering the storage tube is controlled, and the detection antibody and the SAPE are dissolved in sequence and then enter the detection tube; or the detection antibody and the SAPE solution are directly configured and directly transferred into the detection tube to achieve quantitative detection and analysis of the protein.

[0010] Preferably, the probe-type graphic coding chip comprises a graphic coding chip subjected to surface modification and then coupled with probe molecules on the graphic coding chip to obtain a probe-type graphic coding chip.

[0011] Preferably, the probe molecule is a specific capture antibody.

[0012] Preferably, the graphic coding chip is a coding suspension chip based on silica particles.

[0013] Preferably, the method for preparing the probe-type pattern coding chip comprises the following steps:

[0014] (1) dispersing a silicon dioxide-based coded suspension chip in an ethanol solution of aminosilane (APDMS) to react, so that the coded suspension chip is surface-modified with amino groups to obtain the amino-modified coded suspension chip;

[0015] (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;

[0016] (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.

[0017] Preferably, 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 containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0018] Preferably, the coupling reaction is carried out at 0-4°C for 6h-12h.

[0019] Preferably, the probe molecule solution is a NaAc-HAc buffer of the probe molecule.

[0020] Preferably, 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 enter the detection device respectively and in turn, and quantitative detection and analysis of the protein is achieved through the probe-type graphic coding chip.

[0021] Preferably, the detection device at least comprises a detection tube and a storage tube, and the probe-type graphic encoding chip is built into the detection tube.

[0022] Preferably, the detection antibody and SAPE are built into the inner wall of the storage tube, and after being dissolved by blowing and beating at a pipetting workstation, they enter the detection tube respectively to react with the graphic coding chip; or, the detection antibody and SAPE are dissolved and directly configured into a solution, and then blown and beated by a pipette gun or a pipetting workstation, and then contact and react with the graphic coding chip in the detection tube in turn to achieve quantitative detection and analysis of the protein.

[0023] Specifically, the protein detection method based on Lab-in-Tip technology includes the following steps:

[0024] S1. The probe-type pattern coding chip coupled with a specific capture antibody is fixed to the inner wall of the detection tube in the detection device by natural sedimentation;

[0025] S2. preparing a detection antibody solution and / or SAPE solution, or placing the detection antibody and / or SAPE inside the detection device;

[0026] S3. Add the protein sample solution to be tested to the sample well plate, and place the well plate into the pipetting workstation;

[0027] S4. Connecting the detection device to the pipetting workstation or pipetting gun;

[0028] S5. Place the well plate with the phosphate buffer solution in the pipetting workstation, dissolve the detection antibody built into the detection device to form a solution, or place the configured detection antibody solution in the well plate, and use a pipette gun or a pipetting workstation to blow the solution so that the detection antibody solution reacts with the graphic coding sheet; after completion, wash the well plate;

[0029] 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;

[0030] 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.

[0031] In order to achieve another object, the present invention also provides a detection device based on Lab-in-Tip technology, which is applied to the above-mentioned protein detection method for qualitative or quantitative detection of protein.

[0032] Preferably, the detection device comprises a detection tube and a storage tube that are detachably connected.

[0033] The storage tube has a pipette tip structure based on the Lab-in-Tip technology.

[0034] Preferably, 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.

[0035] Preferably, the probe-type graphic encoding chip is fixed in the detection device.

[0036] Preferably, the detection antibody and / or SAPE can enter the detection tube to react with the probe-type graphic encoding chip.

[0037] Preferably, 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 the solution flowing through.

[0038] Preferably, the positions where the detection antibody and SAPE are freeze-dried are different, 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.

[0039] Preferably, the detection tube is equipped with a probe-type graphic coding chip.

[0040] 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 graphic encoding chip.

[0041] As another preferred embodiment, the detection antibody and SAPE are pre-configured into solutions, respectively, and then transferred to the detection tube to react with the specific capture antibody coupled to the surface of the probe-type pattern encoding chip.

[0042] Preferably, the storage portion includes a first storage area and a second storage area.

[0043] Preferably, the first storage area is arranged 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.

[0044] Preferably, 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.

[0045] Preferably, the storage tube and the detection tube are connected via a connecting piece or can be directly connected without a connecting component.

[0046] Preferably, the direct connection setting includes connecting the storage tube and the detection tube by means of threads, snaps, 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 the present invention.

[0047] Preferably, 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 of the connector is connected to the detection tube.

[0048] Preferably, 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.

[0049] Preferably, the detection tube comprises any one of a capillary tube, a plastic tube, a quartz tube, and a glass tube, and the surface-modified probe-type graphic encoding chip can be fixed on the inner wall of the pipette tip tube by natural sedimentation.

[0050] Preferably, the inner wall of the pipette is sequentially immobilized with a biotinylated detection antibody mixture and SAPE.

[0051] The protein detection method using the above-mentioned Lab-in-Tip technology can be applied to protein quantitative analysis based on the ELISA principle. In particular, the probe-type graphic coding chip used in the Lab-in-Tip technology provides a 128-fold coding space, that is, 128-fold detection can be completed in a single pipette tip, and a sensitivity of less than 1pg / ml can be achieved within a reaction time of 1h.

[0052] The technical effects of the technical solution of the present invention are as follows:

[0053] 1. By adopting the technical solution of the present invention, the Lab-in-Tip technology used can complete the processes of sampling, washing, hybridization of detection antibodies, washing, and combining fluorescently labeled streptavidin with biotin-labeled detection antibodies in the pipette tip of this reaction, which has the advantages of completing multiple detections quickly, conveniently, with high sensitivity, good specificity, small sample usage, and a wide detection range.

[0054] 2. By adopting the technical solution of the present invention, 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, greatly shorten the time of the washing step, and can perform fast, convenient, high-throughput, repeatable, highly sensitive, and wide linear range multiple detection, and can achieve a sensitivity of less than 1 pg / mL in a reaction time of just 1 hour.

[0055] 3. By adopting the technical solution of the present invention, a detection device based on the 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

[0056] Figure 1 This is a schematic diagram of the structure of the Lab-in-Tip device provided in Example 1 of the present invention.

[0057] Figure 2 This is a diagram of a graphics coding chip provided in Example 1 of the present invention.

[0058] Figure 3 This is a performance standard curve diagram of IL-8 in Example 1 of the present invention within a reaction time of 1 h.

[0059] Figure 4a-4m They 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 invention within a reaction time of 1 hour.

[0060] Figure 5 This is a performance standard curve diagram of the stability test of IL-8 in Example 1 of the present invention after being stored at 4° C. for 3 months.

[0061] Figure 6a-6d They are performance standard curves of Example 4 of the present invention for performing quadruple detection in a total reaction volume of only 10 μL.

[0062] Figure 7a-7c They are respectively performance standard curves of triple detection within a reaction time of 15 min in Example 5 of the present invention. DETAILED DESCRIPTION

[0063] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the protection scope of the present invention.

[0064] The disclosures of all patent and non-patent literature cited herein are incorporated herein by reference in their entirety.

[0065] As used in the 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 need not be limited to only those elements, but may include other elements that are not explicitly listed or that are 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.

[0066] In addition, "a" or "an" is used to describe the elements and components described herein. This is done only for convenience and to provide a general sense of the scope of the invention. This description should be understood to include one or at least one, one or at least one, and the singular also includes the plural, unless it is obvious that it is intended otherwise.

[0067] 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 the present 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. If a conflict occurs, this specification and the definitions included therein shall prevail. In addition, materials, methods and examples are illustrative only, and not restrictive.

[0068] A protein detection method is a protein detection method based on Lab-in-Tip technology. Lab-in-Tip technology is a technical improvement based on the pipette tip of the prior art. The conical structure of the pipette tip is used to seal the detection antibody and SAPE at different positions on the inner wall of the pipette tip. The volume of the solvent sucked by the pipette tip is controlled by the pipette gun or the pipetting workstation to dissolve the detection antibody and SAPE step by step into the detection component fixed with the probe-type graphic encoding chip to achieve protein detection. The method is simple and fast, and the detection result is accurate. The device used is simple and low-cost. Only conventional pipette tips in the laboratory are needed to assemble and disassemble the detection device.

[0069] Furthermore, to realize the above-mentioned protein detection method, the present invention also provides a detection device, which performs quantitative detection of protein on the basis of Lab-in-Tip technology by simply modifying the pipette tip. The detection device is not only simple in structure and easy to assemble, but also only requires conventional laboratory equipment, such as pipette tips, silicone tubes, and capillaries to assemble the device. Moreover, the protein detection process is simple and the results are accurate. In particular, the detection time can be greatly shortened.

[0070] 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.

[0071] As a preferred embodiment, the direct connection setting includes connecting the storage tube and the detection tube by means of threads, snaps, 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 the present invention.

[0072] As a preferred embodiment, the storage tube and the detection tube are connected via a connector or directly.

[0073] Preferably, 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.

[0074] Preferably, the connecting piece is one of a latex tube, a rubber tube, a thermoplastic tube or a UV hose, which can seal and connect the storage tube and the detection tube.

[0075] Preferably, the detection tube comprises any one of a capillary tube, a plastic tube, a quartz tube, and a glass tube, and the surface-modified probe-type graphic encoding chip can be fixed on the inner wall of the pipette tip tube by a natural sedimentation method.

[0076] Preferably, the storage tube has a pipette tip structure; more preferably, the storage tube is a conical structure, including a tip and a tail; the tip is connected to the detection tube; the tail can be connected to a pipette gun or a pipette workstation. The pipette workstation used in the embodiment of the present invention is an Eppendorf pipette workstation.

[0077] Preferably, 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 the flow of the solution.

[0078] 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.

[0079] 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.

[0080] As another preferred embodiment, the detection antibody and SAPE are dissolved and directly configured into a solution, which is then blown by a pipette gun or a pipetting workstation to sequentially contact and react with the graphic coding chip in the detection tube to achieve quantitative detection and analysis of the protein.

[0081] Specifically, the steps of protein detection in combination with the above detection device include:

[0082] S1. Fixing the probe-type pattern coding chip coupled with a specific capture antibody on the inner wall of the detection tube by natural sedimentation;

[0083] S2..preparing a detection antibody solution and / or a SAPE solution, or placing the detection antibody and / or SAPE in the detection device;

[0084] S3. Add the protein sample solution to be tested to the sample well plate, and place the well plate into the pipetting workstation;

[0085] S4. Connect the Lab-in-Tip device to the pipetting workstation, so that the Lab-in-Tip device is pipetted on the well plate; after completion, clean it;

[0086] S5: Place the well plate with phosphate buffer in the pipetting workstation, and blow the Lab-in-Tip device on the well plate again to dissolve the sealed detection antibody first, so that it reacts with the graphic coding chip; after completion, wash it, or place the configured detection antibody solution in the well plate, so that the detection antibody solution directly reacts with the graphic coding chip;

[0087] S6. Repeat the previous step, and place the configured SAPE solution into the detection device, or dissolve the sealed SAPE again to react with the graphic coding chip; and then wash;

[0088] S7. Remove the detection unit for image data collection, 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.

[0089] As one of the preferred embodiments, the probe-type graphic coding chip comprises a graphic coding chip subjected to surface modification and then coupled with a probe molecule on the graphic coding chip to obtain a probe-type graphic coding chip; the probe molecule is a specific capture antibody; more preferably, the graphic coding chip is a coding suspension chip based on silica particles, which can provide 128-fold coding space.

[0090] As one of the preferred embodiments, the method for preparing the probe-type pattern coding chip comprises the following steps:

[0091] (1) dispersing a silicon dioxide-based coded suspension chip in an ethanol solution of aminosilane (APDMS) to react, so that the coded suspension chip is surface-modified with amino groups to obtain the amino-modified coded suspension chip;

[0092] (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;

[0093] (3) Activating the coding chip after surface modification in step (2), coupling it with the probe molecule solution, and coupling the probe molecule to the surface of the coding chip, thereby obtaining a probe-type graphic coding chip.

[0094] 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.

[0095] The technical scheme of the present invention, its implementation process and principle, etc. will be further explained and illustrated by specific embodiments as follows. It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. The described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention. Unless otherwise specified, the reagents and raw materials used in the following examples are commercially available, and the test methods in which the specific conditions are not specified are usually carried out under conventional conditions or according to the conditions recommended by each manufacturer. That is, unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the art. These technologies have been fully described in the existing literature.

[0096] Example 1

[0097] This embodiment provides a protein detection device based on Lab-in-Tip technology. For specific structure, see Figure 1 .

[0098] like Figure 1 As shown, the protein detection device comprises a detachable silicone tube 1, a capillary 2 and a pipette tip 3. The capillary 2 and the pipette tip 3 are respectively connected to the two ends of the silicone tube 1, and the sizes of the connecting parts of the silicone tube 1, the capillary 2 and the pipette tip 3 are matched to ensure that the entire connected protein detection device will not leak.

[0099] The inner wall of the capillary 2 is fixed with a probe-type pattern coding chip 4 coupled with different capture antibodies.

[0100] Preferably, 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.

[0101] The second storage area 6 is arranged at the rear end, that is, the area between the port close to the pipetting workstation and the middle 1 / 2 of the pipetting tip 3 .

[0102] In this embodiment, the first storage area 5 is immobilized with biotinylated detection antibody, and the second storage area 6 is immobilized with SAPE (fluorescently labeled streptavidin).

[0103] Preferably, the fixing method may be to directly fix the detection antibody and SAPE in the pipette tip 3 respectively by freeze-drying.

[0104] The freeze drying method includes: after the detection antibody is placed in the first storage area 5 and the SAPE is placed in the second storage area 6, the pipette tip 3 is placed in a -80°C refrigerator for 20 minutes. After the detection antibody and SAPE are in a solid state, the pipette tip 3 is placed in a freeze dryer for freeze drying for 1.5 hours.

[0105] Furthermore, the preparation method of the probe type pattern coding chip 4 includes:

[0106] (1) Provide a silicon dioxide-based coded suspension chip and select 2×10 5 The particle-coded suspension chip was dispersed in 1000 μL of 5% aminosilane (APDMS) ethanol solution (prepared with 95% ethanol), fully reacted for 30 minutes and then washed.

[0107] (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.

[0108] (3) Use 0.1 mol / L MES buffer (pH = 4.7) to prepare an activation solution containing 130 mmol / L EDC and 326 mmol / L NHS; at room temperature, allow the chip suspension and the activation solution to react for about 30 minutes and then wash, and then mix and react with the probe molecule solution (solvent is 0.1 mol / L NaAc-HAc buffer) at 4°C for 6 hours to 12 hours to obtain a probe-type image encoding chip, wherein the probe in this embodiment is a capture antibody.

[0109] See also Figure 2 , is the probe-type image coding chip prepared in this embodiment. The size of the chip is 14×25 μm, and it has 128-fold coding space, which can provide 128-fold detection for samples.

[0110] The probe-type image encoding chip is embedded in the inner wall of the capillary 2 by natural sedimentation method, and the silicone tube 1 is sequentially connected with the capillary 2 and the pipette tip 3 to assemble, thus obtaining a Lab-in-Tip detection device.

[0111] The protein detection is performed based on the above Lab-in-Tip detection device. The specific detection method includes the following steps:

[0112] a. After the pattern coding chip is precisely manufactured and released by photolithography, it is modified by carboxyl groups, and different types of specific capture antibodies are coated on 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;

[0113] b. Fixing different encoding chips coupled with different types of capture antibodies on the inner wall of capillary 2 by natural sedimentation;

[0114] c. After preparing the required concentration of biotinylated detection antibody mixture and SAPE, seal them at the front and rear ends of the pipette tip 3; the initial concentration of the detection antibody 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;

[0115] d. Connect the silicone tube 1, the square capillary 2, and the pipette tip 3 in sequence;

[0116] e. Add 50 μL of sample solution to each well of the 96-well plate, and place the 96-well plate in the liquid handling workstation;

[0117] f. Insert the Lab-in-Tip-based detection device into the machine tip in the liquid handling workstation, start the instrument, and allow the Lab-in-Tip-based detection device to pipette itself in the sample well for 30 minutes;

[0118] g. After the above pipetting is completed, in the pipetting workstation, the Lab-in-Tip-based detection device will automatically transfer to the wash tank and pipette up and down 3 times for cleaning;

[0119] h. Prepare another new 96-well plate, and dispense 70 μL of 1× phosphate buffer into each well, so that the Lab-in-Tip-based detection device is blown in the well for 20 minutes by itself, and the volume of phosphate buffer saline entering the pipette tip does not exceed 1 / 2 of the pipette tip, which can both blow and dissolve the detection antibody pre-sealed on the inner wall of the pipette tip 3, and will not affect the SAPE sealed in the second storage part 6 by the phosphate buffer. The final concentration of the antibody obtained after dissolution is 1 μg / mL, so that 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;

[0120] i. After the above-mentioned blowing and beating is completed, the Lab-in-Tip-based detection device will automatically transfer to the washing tank again and be blown up and down 3 times for cleaning;

[0121] j. Prepare a new 96-well plate, distribute 120 μL of phosphate buffer into each well, and allow the Lab-in-Tip device to blow and dissolve the pre-sealed SAPE in the well for 10 minutes. The volume of phosphate buffer entering the pipette tip exceeds the position of the second storage part 6 to ensure that the SAPE can be completely dissolved. The final concentration of the SAPE obtained after dissolution is 0.5 μg / mL, so that it reacts with the specific capture antibody on the surface of the multi-encoded chip on the inner wall of the capillary 2;

[0122] k. After the above pipetting is completed, the Lab-in-Tip-based detection device will automatically transfer to the washing tank of the pipetting workstation again and pipette up and down 5 times for cleaning;

[0123] 1. Remove the capillary 2 and directly use it for image data acquisition, and perform qualitative analysis or quantitative analysis to obtain the measurement results.

[0124] In this example, the sample liquid uses human cytokine IL-8 as a model protein analyte and is dissolved in a buffer solution; the initial concentration is 10 ng / mL, and after 5-fold dilution, the seventh concentration is 0.64 pg / mL.

[0125] After analysis by 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, and the number of various soluble components in the analysis sample is determined by the different fluorescence intensities on the different coded chips.

[0126] See also Figure 3 , is a standard curve of the detection performance of human cytokine IL-8 (purchased from R&D) within a reaction time of 1h, and the bottom dotted line is blank (blank, protein content is 0), that is, the detection line without analyte. Analysis shows that the technical solution of the present invention makes the sensitivity of protein quantitative analysis as high as pg / mL level (when the protein concentration is around 1pg / mL, the chip signal value is still clearly distinguished). The fluorescence intensity of the sample is matched with the standard curve to quantify the target protein.

[0127] Example 2

[0128] The difference between this embodiment and embodiment 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).

[0129] Thirteen protein analytes were mixed, and thirteen detection antibodies were also pre-mixed and sealed in a Lab-in-Tip pipette tip. The specific operation steps were the same as those in Example 1, and this example completed the above-mentioned thirteen-fold detection within a reaction time of 1 hour.

[0130] Test results reference Figure 4a , 4b , 4c, 4d, 4e, 4f, 4g, 4h, 4i, 4j, 4k, 4l, and 4m. It can be seen from the standard curve that the sensitivity of protein quantitative analysis is as high as pg / mL level, and when the protein concentration is around 1pg / mL, the chip signal values ​​are still clearly distinguished.

[0131] Example 3

[0132] This embodiment is the same as embodiment 1, except that the assembled Lab-in-Tip detection device of the embodiment is placed in a refrigerator at 4° C. for 3 months before detection.

[0133] Test results see Figure 5 The sensitivity of protein quantitative analysis can still be maintained at the pg / mL level. Specifically, the storage signal value fluctuates within a 20% error range and does not decrease significantly. At the same time, the detection sensitivity of IL-1β detected in the method can reach 1 pg / mL.

[0134] Example 4

[0135] The difference between this embodiment and embodiment 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 the quadruple detection is completed with a sample volume of 10 μL.

[0136] The four protein analytes were mixed, and the four detection antibodies were also pre-mixed and sealed in a Lab-in-Tip pipette tip. Through the Lab-in-Tip technology, the 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).

[0137] Test results reference Figure 6a , 6b , 6c, 6d. It can be seen from the standard curve that the sensitivity of protein quantitative analysis is as high as pg / mL level. When the protein concentration is around 1pg / mL, the chip signal values ​​are still clearly distinguished.

[0138] Example 5

[0139] The difference between this embodiment and embodiment 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.

[0140] The three protein analytes were mixed, and the three detection antibodies were also pre-mixed and sealed in a Lab-in-Tip pipette tip. The triple detection was completed in a reaction time of only 15 minutes (the reaction time in Example 1 was 1 hour) using the Lab-in-Tip technology.

[0141] Test results reference Figure 7a , 7b , 7c. As can be seen from the standard curve, the sensitivity of protein quantitative analysis is as high as pg / mL level (when the protein concentration is around 1pg / mL, the chip signal value is still clearly distinguished)

[0142] It can be seen from the test results of Examples 1-5 that the protein concentration can be detected at the 1pg / mL level, the protein is around 1pg / ml (concentration 1pg / ml), the signal value is also clearly distinguished, and the signal value at this point is higher than the background signal value, and multiple detections can be completed. At best, the Lab-in-Tip detection device provided by the present invention can achieve 128-fold detection.

[0143] 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.

[0144] In Example 5, the triple detection was completed in 15 minutes, indicating that the method of the present invention can achieve rapid detection.

[0145] By using the Lab-in-Tip detection device provided by the present invention, the graphic coding chip can be fixed on 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 improved by only an commonly used biological pipette tip structure in the prior art, that is, it can complete the protein quantitative analysis based on the ELISA principle in a relatively short time; and it can achieve a sensitivity of 1 pg / mL within a reaction time of 15 minutes.

[0146] 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.

[0147] Obviously, the present invention 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, and can perform fast, convenient, high-throughput, low-sample-amount, good repeatability, high sensitivity, and wide linear range multiple detection.

[0148] The above are only preferred embodiments of the present invention, which do not limit the protection scope of the present invention. For those skilled in the art, the present invention may have various modifications and changes. Within the spirit and principle of the present invention, changes, modifications, replacements, integrations and parameter changes to these embodiments by conventional substitutions or by being able to achieve the same functions without departing from the principles and spirit of the present invention all fall within the protection scope of the present invention.

Claims

1. A protein detection method, comprising fixing a probe-type pattern coding chip coupled with a specific capture antibody in a detection device, and at the same time, respectively placing SAPE and a detection antibody in the detection device, or pre-preparing a SAPE solution and a detection antibody solution; 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 as claimed in claim 1, It is 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; And / or, 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 (APDMS) to react, 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.

3. The protein detection method according to claim 2, It is 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; And / or, the coupling reaction comprises being carried out at 0 to 4° C. for a reaction time of 6 h to 12 h; And / or, the probe molecule solution is a NaAc-HAc buffer of the probe molecule.

4. The protein detection method according to any one of claims 1 to 3, It is 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 at 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.

5. The protein detection method according to any one of claims 1 to 3, It is 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.

6. A detection device, used for qualitative or quantitative detection of protein in the protein detection method according to any one of claims 1 to 5; the detection device comprises a detection tube and a storage tube that are detachably connected; The storage tube has a pipette tip structure based on Lab-in-Tip technology; And / or, the storage tube is a conical structure, including a tip portion and a tail portion; the tip portion is connected to the detection tube; the tail portion 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.

7. The detection device according to claim 6, It is 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 tapered 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.

8. The detection device according to claim 7, It is 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; 9. The detection device according to any one of claims 6 to 8, It is 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 hose, and can seal and connect the storage tube and the detection tube; And / or, 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 encoding chip can be fixed on the inner wall of the storage tube by a natural sedimentation method.

10. Use of the protein detection method according to any one of claims 1 to 6 in protein quantitative analysis.

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