CN118393131A - Immunoassay method constructed by using syringe - Google Patents

Abstract

The invention discloses an immunoassay method constructed by using a syringe, wherein the syringe is used as a measuring and discharging tool of working solution, a construction site of an immunoassay system and a gas generating container related to the concentration of a target object. Fixing a capturing component on the inner cylinder wall of the injector, and constructing a sandwich or competitive immunoassay system of a target object on the inner cylinder wall by taking a probe-platinum nanoparticle or a catalase marked antibody or antigen as a detection component; the probe captured in the assay system catalyzes the decomposition of the H ₂O₂ substrate and generates oxygen in the syringe; connecting an injection port of the injector with a communicating pipe or a barometer or a hydraulic gauge to acquire a distance signal of discharged liquid in the communicating pipe or an air pressure or hydraulic signal in the injector; and establishing a quantitative relation between the concentration of the target object and the corresponding signal, and realizing immunoassay. The method has the advantages of simple operation, low use cost and low requirements on professional skills by means of the structural and functional advantages of the injector.

Description

An immunoassay method using a syringe

Technical Field

The invention relates to the technical field of immunoassay, and in particular to an immunoassay method constructed by using a syringe.

Background technique

Immunoassays are widely used in the fields of disease diagnosis, food safety and environmental monitoring. How to make the detection process simpler, reduce costs and professional skills, and make the method more suitable for ordinary people to use, has important practical significance. Since no analytical instrument is required, the immunoassay method based on the gas generation mechanism has attracted widespread research interest. This method usually uses materials such as platinum nanoparticles with certain biological or chemical catalytic activity as immunoassay probes, and uses the probe to catalyze substrates such as _H2O2 to produce gases such as _O2 , and then converts the volume of the gas _into a signal.

As a prior art, the general practice of the immunoassay method based on the gas generation mechanism is to construct an immunoassay system in a 96-well plate, a plastic centrifuge tube or a glass bottle modified with a functional group, or to transfer the constructed assay system to a special container, and use a sealed external barometer or a connecting device such as an open capillary to convert the concentration information of the antigen or antibody target into an air pressure signal or a liquid movement distance signal in the connecting device. This detection process has the following disadvantages: First, in the construction process of the immunoassay system, additional solution measurement and transfer tools such as pipettes are frequently used, or multiple liquid storage containers are involved, and the operation is cumbersome; second, due to the high requirements for the airtightness of the test container, these containers usually need to be sealed, such as additional sealing rings and sealing covers with capillary perforations, which increases the difficulty of operation and is prone to leakage in actual operation, thereby affecting the accuracy of the test results. In addition, there is another approach, which is to build an immunoassay system in a microfluidic chip, using a similar mechanism to generate gas in the sample pool of the chip, and then convert the gas volume signal into a dye movement distance signal driven by the air pressure in the microchannel. However, the structure of such chips is usually more complicated, and the preparation cost is also increased. Operations such as micro-volume addition and micro-channel connection require high professional skills and are not suitable for ordinary operators.

Summary of the invention

The technical problem to be solved by the present invention is to provide an immunoassay method constructed by using a syringe, which is based on a gas generation mechanism and has the characteristics of being easier to operate, lower manufacturing cost and lower requirements on professional skills.

The technical solution of the present invention is as follows:

An immunoassay method constructed using a syringe, wherein the immunoassay is performed according to the following steps:

Step 1: Using a syringe as a measuring and discharging tool for the working solution, first draw the antibody solution or antigen solution as the capture component into the barrel of the syringe, and fix the capture component on the inner barrel wall by physical adsorption method with the inner barrel wall as the base;

Step 2: Using the syringe to extract the cleaning liquid, and removing the residual components in the syringe by cleaning;

Step 3: Use the syringe to extract the protein blocking solution to achieve the blocking of the remaining sites on the inner wall of the syringe that are not bound to the capture component; then clean and remove the residual components in the syringe according to the second step;

Step 4: For constructing a sandwich immunoassay system, extract the target solution, and complete the specific recognition between the capture component and the target on the inner wall of the syringe; then clean and remove the residual components in the syringe according to the second step, and then carry out the fifth step, the sixth step and the seventh step in sequence;

For the construction of a competitive immunoassay system, a mixture of a target solution and a probe as a detection component, i.e., an antibody solution or an antigen solution labeled with platinum nanoparticles or catalase, is extracted, and competitive specific recognition between the capture component and the target and the detection component is completed on the inner wall of the syringe; then, the residual components in the syringe are removed by washing according to the second step, and then the sixth and seventh steps are performed in sequence;

Step 5: Extract the probe as the detection component - the antibody solution or antigen solution labeled with platinum nanoparticles or catalase, and construct a sandwich immunoassay system on the inner wall of the syringe; then clean and remove the residual components in the syringe according to the second step;

Step 6: extract H ₂ O ₂ substrate solution; connect the injection port of the syringe to the connecting tube or the air pressure gauge or the hydraulic pressure gauge to obtain the moving distance signal of the liquid discharged by the syringe in the connecting tube or the air pressure signal or the hydraulic pressure signal in the syringe;

Step 7: Establish a quantitative relationship between the target concentration and the corresponding signal, and realize immunoassay through this quantitative relationship.

Preferably, the specific determination steps are as follows:

Step A: Pull the piston rod of the syringe to quantitatively extract the antibody or antigen solution as the capture component, the extracted volume is less than half of the total volume of the barrel, and the capture component is fixed on the inner wall of the barrel; after placing it, push out the liquid;

Step B: extracting cleaning liquid so that the cleaning liquid covers the fixed position of the captured component on the inner cylinder wall in step A; brushing the aforementioned covered position by pulling and pushing the piston rod;

Step C: extracting bovine serum albumin blocking solution so that the solution covers the fixed position of the capture component on the inner tube wall in step A; after standing, push out the liquid and clean the syringe;

Step D: for constructing a sandwich immunoassay system, extract the antigen solution or antibody solution as the target component so that the liquid covers the fixed position of the capture component on the inner cylinder wall in step A; after placing, push out the liquid and clean the syringe; then perform steps E, F and G in sequence;

For constructing a competitive immunoassay system, extract a mixture of an antigen solution or an antibody solution as a target component and a probe-platinum nanoparticle-labeled or catalase-labeled antibody solution or a probe-platinum nanoparticle-labeled or catalase-labeled antigen solution as a detection component, so that the liquid covers the fixed position of the capture component on the inner cylinder wall in step A; push out the liquid after placing; then clean the syringe according to step B; then perform steps F and G in sequence;

Step E: extracting a probe-platinum nanoparticle-labeled or catalase-labeled antibody solution or a probe-platinum nanoparticle-labeled or catalase-labeled antigen solution as a detection component, so that the liquid covers the fixed position of the capture component on the inner tube wall in step A; after placement, push out the liquid and clean the syringe;

Step F: extracting a _{H2O2 substrate} solution with a concentration of 0.05% to 10%, so that the liquid covers the fixed position of the capture component on the inner tube wall in step A; connecting the liquid outlet of the syringe to a connecting tube, an air pressure gauge or a hydraulic pressure gauge; after reacting for 2 to 30 minutes, recording the moving distance signal of the liquid discharged by the syringe in the connecting tube, the air pressure signal or the hydraulic pressure signal in the syringe;

Step G: Establish a standard curve between the signal in step F and the target concentration in step D, and use the standard curve to achieve immunoassay of the target concentration.

Preferably, the concentration of the capture component in step A is 2-80 μg/mL; the syringe is placed in a 4-10 ^° C. environment for 12 hours or in a 30-38 ^° C. water bath for 2 hours.

Preferably, the cleaning solution in step B is a phosphate buffer or phosphate Tween buffer with a pH of 7.0 to 7.4; the piston rod is pulled and pushed 1 to 3 times to scrub the inner cylinder wall, and the cleaning solution is pushed out and the cleaning operation is repeated 1 to 3 times.

Preferably, the concentration of the bovine serum albumin blocking solution in step C is 0.5-10%; the syringe is placed in a 30-38 ^° C. water bath for 2 hours.

Preferably, after extracting the target component solution or the mixture of the target component solution and the detection component solution in step D, the syringe is placed in a 30-38 ^° C water bath for 10-30 minutes or at 25±5 ^° C room temperature for 0.25-1 hour.

Preferably, the detection component described in step E is an antibody solution labeled with platinum nanoparticles or an antigen solution labeled with platinum nanoparticles; the particle size of the platinum nanoparticles is 5 to 300 nm; the concentration of the platinum nanoparticles in the detection component is 0.1 to 0.5 mg/mL; after extracting the detection component solution, the syringe is placed in a 30 to 38 ^o C water bath for 15 to 30 minutes or at 25±5 ^o C room temperature for 0.25 to 1 hour.

Preferably, the concentration of H ₂ O ₂ in the H ₂ O ₂ substrate solution in step F is 0.05% to 1%.

Preferably, the placing in step A and step C refers to placing the syringe at 4-38 ^° C for 0.5-24 hours; the placing in step D and step E refers to placing the syringe at 4-38 ^° C for 0.1-4 hours.

Compared with the prior art, the present invention has the following beneficial effects:

First, in the process of constructing the immunoassay system of the present invention, a syringe can realize three functions: 1. as a tool for measuring and discharging the working solution; 2. as a construction site for the immunoassay system; 3. as a gas generation container associated with the concentration of the target substance. Syringes have been widely used in daily life, are simple to operate, and have low cost. With the above functions and advantages of syringes, it is possible to avoid the solution measuring and transfer tools such as pipettes or various liquid storage containers that are commonly used in the prior art, significantly simplifying the operation process, and is very suitable for people without relevant professional skills.

Second, the syringe itself is a quasi-sealed device that is easy to connect. When the injection port is connected to a signal conversion and reading device such as a connecting tube, a microchannel, a barometer or a hydraulic gauge, the airtightness of the measurement system can be fully guaranteed. The special sealing treatment of the measurement container in the prior art, such as the addition of a sealing gasket and a sealing cover with a capillary perforation, can be avoided to avoid leakage caused by operating difficulties, which is conducive to improving the accuracy of the detection.

Third, from the perspective of practical application, the technical solution of the present invention can be directly used in existing commercial syringes, especially commercial syringes with a capacity of 1 mL and a tube made of polypropylene (PP), and the connecting tube can directly use a commercial catheter. Therefore, the investment in later product development is relatively low. Combined with the above advantages, the technology of the present invention, as a quantitative on-site instant immunoassay technology, has greater practical feasibility.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a diagram of the syringe photo verification result of the sandwich immunoassay system for prostate-specific antigen (PSA) constructed in the syringe according to Example 1 of the present invention; wherein a: the capture component is none, the target component is PSA, and the detection component is mouse anti-human PSA monoclonal antibody labeled with platinum nanoparticles (Pt NPs); wherein b: the capture component is none, the target component is none, and the detection component is mouse anti-human PSA monoclonal antibody labeled with Pt NPs; wherein c: the capture component is mouse anti-human PSA monoclonal antibody, the target component is none, and the detection component is mouse anti-human PSA monoclonal antibody labeled with Pt NPs; wherein d: the capture component is mouse anti-human PSA monoclonal antibody, the target component is PSA, and the detection component is mouse anti-human PSA monoclonal antibody labeled with Pt NPs;

Fig. 2 is a diagram of the signal verification result of the moving distance of the liquid discharged from the syringe in the connecting tube of the sandwich immunoassay system for constructing PSA in the syringe according to Example 1 of the present invention; wherein a: the capture component is none, the target component is PSA, and the detection component is mouse anti-human PSA monoclonal antibody labeled with Pt NPs; wherein b: the capture component is none, the target component is none, and the detection component is mouse anti-human PSA monoclonal antibody labeled with Pt NPs; wherein c: the capture component is mouse anti-human PSA monoclonal antibody, the target component is none, and the detection component is mouse anti-human PSA monoclonal antibody labeled with Pt NPs; wherein d: the capture component is mouse anti-human PSA monoclonal antibody, the target component is PSA, and the detection component is mouse anti-human PSA monoclonal antibody labeled with Pt NPs;

FIG3 is a linear relationship curve for measuring different concentrations of PSA in Example 1 of the present invention;

FIG4 is a graph showing specific results of PSA determination in Example 1 of the present invention;

Fig. 5 is a diagram showing the verification results of a syringe photograph of a competitive immunoassay system for constructing urine microalbumin (mALB) in a syringe according to Example 2 of the present invention; a: the capture component is none, the target component is none, and the detection component is none; b: the capture component is none, the target component is none, and the detection component is mALB labeled with Pt NPs; c: the capture component is mouse anti-human mALB monoclonal antibody, the target component is none, and the detection component is mALB labeled with Pt NPs; d: the capture component is mouse anti-human mALB monoclonal antibody, the target component is mALB, and the detection component is mALB labeled with Pt NPs;

Fig. 6 is a diagram of the signal verification result of the moving distance of the liquid discharged from the syringe in the connecting tube of the competitive immunoassay system of mALB constructed in the syringe according to Example 2 of the present invention; wherein a: the capture component is none, the target component is none, and the detection component is none; wherein b: the capture component is none, the target component is none, and the detection component is mALB labeled with Pt NPs; wherein c: the capture component is mouse anti-human mALB monoclonal antibody, the target component is none, and the detection component is mALB labeled with Pt NPs; wherein d: the capture component is mouse anti-human mALB monoclonal antibody, the target component is mALB, and the detection component is mALB labeled with Pt NPs;

FIG7 is a linear relationship curve for mALB determination in the concentration range of 0.1 to 6.0 μg/mL in Example 2 of the present invention;

FIG8 is a linear relationship curve for mALB determination in the concentration range of 0.006 to 0.1 μg/mL according to Example 2 of the present invention;

FIG. 9 is a diagram showing the accuracy verification of mALB determination in actual urine samples according to the second embodiment of the present invention.

Detailed ways

The present invention is further described below in conjunction with specific measurement steps and technical effect experimental data. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

The determination steps of the following examples mainly include:

Step 1: Using a syringe as a measuring and discharging tool for the working solution, first draw the antibody solution or antigen solution as the capture component into the barrel of the syringe, and fix the capture component on the inner barrel wall by physical adsorption method with the inner barrel wall as the base;

Step 2: Using the syringe to extract the cleaning liquid, and removing the residual components in the syringe by cleaning;

Step 3: Use the syringe to extract the protein blocking solution to achieve the blocking of the remaining sites on the inner wall of the syringe that are not bound to the capture component; then clean and remove the residual components in the syringe according to the second step;

Step 4: For constructing a sandwich immunoassay system, extract the target solution, and complete the specific recognition between the capture component and the target on the inner wall of the syringe; then clean and remove the residual components in the syringe according to the second step, and then carry out the fifth step, the sixth step and the seventh step in sequence;

For the construction of a competitive immunoassay system, a mixture of a target solution and a probe as a detection component, i.e., an antibody solution or an antigen solution labeled with platinum nanoparticles or catalase, is extracted, and competitive specific recognition between the capture component and the target and the detection component is completed on the inner wall of the syringe; then, the residual components in the syringe are removed by washing according to the second step, and then the sixth and seventh steps are performed in sequence;

Step 5: Extract the probe as the detection component - the antibody solution or antigen solution labeled with platinum nanoparticles or catalase, and construct a sandwich immunoassay system on the inner wall of the syringe; then clean and remove the residual components in the syringe according to the second step;

Step 6: extract H ₂ O ₂ substrate solution; connect the injection port of the syringe to the connecting tube or the air pressure gauge or the hydraulic pressure gauge to obtain the moving distance signal of the liquid discharged by the syringe in the connecting tube or the air pressure signal or the hydraulic pressure signal in the syringe;

Step 7: Establish a quantitative relationship between the target concentration and the corresponding signal, and realize immunoassay through this quantitative relationship.

Embodiment 1

A prostate-specific antigen (PSA) immunoassay method constructed using a syringe, wherein the immunoassay system constructed in the syringe is a double antibody sandwich type. The specific assay steps are as follows:

Step A: Pull the piston rod of a commercially available disposable syringe with a capacity of 1 mL and a tube made of polypropylene (PP) to extract 0.3 mL of a mouse anti-human PSA monoclonal antibody solution with a concentration of 20 μg/mL as the capture component; place the syringe in a 4 ^o C refrigerator for 12 hours to push out the liquid; this step can fix the capture component to the inner wall surface of the syringe.

Step B: Draw 0.3 mL of 0.01 M phosphate-tween buffer (PBST) with a pH of 7.4 to allow the liquid to cover the fixed position of the captured component on the inner tube wall in step A; pull and push the piston rod three times in sequence to brush the aforementioned covered position and push out the cleaning liquid; repeat the operation three times; this step can remove the residual components in the syringe.

Step C: Draw 0.3 mL of 5% bovine serum albumin (BSA) solution to cover the fixed sites of the captured components on the inner tube wall in step A; place the syringe in a 37 ^o C water bath for 1 hour and push out the liquid; clean the syringe according to step B; this step can seal the remaining sites on the inner tube wall where the captured components are not adsorbed.

Step D: Draw 0.3 mL of PSA antigen solution as the target component so that the liquid covers the fixed position of the capture component on the inner tube wall in step A; place the syringe in a 37 ^o C water bath for 20 minutes and push out the liquid; clean the syringe according to step B; this step can complete the specific recognition of the capture component and the target on the inner tube wall.

Step E: Extract 0.3 mL of the detection component - mouse anti-human PSA monoclonal antibody solution labeled with platinum nanoparticles (Pt NPs), in which the concentration of Pt NPs is 0.25 mg/mL and the particle size of Pt NPs is 25 nm. Make the liquid cover the fixed part of the capture component on the inner cylinder wall in step A; place the syringe in a 37 ^o C water bath for 20 minutes and push out the liquid; clean the syringe according to step B; this step can complete the immune capture of the detection probe - Pt NPs on the inner cylinder wall.

The preparation steps of the Pt NPs-labeled mouse anti-human PSA monoclonal antibody solution are as follows:

(1) Preparation of Pt NPs: In an 80 ^° C water bath, an aqueous solution of ascorbic acid (AA) and an aqueous solution of _{chloroplatinic} acid ( _H2PtCl6 ) were mixed to a final concentration of 0.04 M AA and 0.1 mM _H2PtCl6 , respectively. After standing for 10 min, the mixture _was centrifuged at 15,000 rpm for 6 min and the supernatant was discarded. Following this operation, the precipitate was washed with water three times. The Pt NPs finally collected were ultrasonically dispersed in water at a concentration of 4 mg/mL.

(2) Labeling: Prepare a 1 mg/mL Pt NPs aqueous dispersion; adjust the pH of the dispersion to 8.5 with _{a K2CO3} aqueous solution; add a mouse anti-human PSA monoclonal antibody stock solution with a final concentration of 20 μg/mL; slowly shake at room temperature at 25±5 ^° C for 2 hours, add a BSA aqueous solution with a final concentration of 0.5%, and continue shaking for 0.5 hours; centrifuge at 8000 rpm for 5 minutes and discard the supernatant; wash the precipitate with water three times according to this operation.

(3) Storage: Vortex or ultrasonically disperse the precipitate obtained in the previous step in 0.01 M phosphate buffer (PBS) at pH 7.4. The dispersion contains 0.05% Triton X-100, 1.25% sucrose, and 0.5% BSA. The final concentration of Pt NPs is 0.25 mg/mL. Reserve.

Step F: Extract 0.3 mL of H ₂ O ₂ substrate reaction solution with a concentration of 0.15%, so that the liquid covers the fixed position of the capture component on the inner tube wall in step A; connect the injection port of the syringe with a transparent silicone tube with an inner diameter of 0.5 mm through a commercially available sharp needle, and the other end of the silicone tube is open; after 6 minutes of reaction, record the moving distance signal of the liquid in the silicone tube; this step can convert the target concentration in the immunoassay system in the syringe into the aforementioned distance signal. It should be noted that the gas in the syringe needs to be emptied in advance so that the discharged liquid can enter the tube or channel as soon as possible to improve the detection sensitivity.

Step G: Establish a standard curve between the moving distance signal in step F and the target PSA concentration in step D, establish a quantitative relationship between the two, and use the quantitative relationship to achieve quantitative immunoassay of PSA.

In the above process, the volume of each working solution is controlled by the scale lines on the syringe; during operation, it is necessary to ensure that the piston of the syringe does not contact the inner cylinder wall part where the captured component has been fixed (for example, before extracting the solution, the piston rod is pulled up to a certain empty distance in advance, and then the solution is extracted; when the solution is pushed out, the piston stops at the inner cylinder wall part where the captured component has been fixed to avoid touching the fixed captured component).

The immunoassay system was constructed with different components in the syringe, and the bubble generation and liquid movement distance control experiments were carried out for different systems. The control experiment results are shown in Figures 1 and 2. Only when the capture antibody, the target PSA with a concentration of 40 ng/mL and the detection antibody labeled with Pt NPs were used at the same time (Figures 1 and 2 (d), the bubble generation phenomenon can be observed in the syringe, proving that the Pt NPs probe was immunocaptured in the syringe. Correspondingly, the liquid injected by the syringe generated a distance signal of 85 mm in the silicone tube. There was no obvious bubble generation phenomenon in other control groups, and there was no obvious movement distance signal, all of which were less than 7.0 mm. This control experiment verified the successful construction of the sandwich immunoassay system of PSA in the syringe, and also proved that the concentration of the target PSA in the assay system can be converted into the movement distance signal of the liquid in the silicone tube.

As shown in Figure 3, within the concentration range of 2.0 to 50 ng/mL, the moving distance signal is linearly related to the PSA concentration (R ² =0.988), and a quantitative relationship between the two can be established. Calculated by 3 times the signal-to-noise ratio (S/N), the limit of detection (LOD) is 1.28 ng/mL, which is significantly lower than the clinical diagnostic threshold of prostate cancer - 4.0 ng/mL. As shown in Figure 4, compared with several other common proteins and other tumor markers commonly found in serum, all with a concentration of 200 ng/mL: human serum albumin (ALB) and immunoglobulin G (IgG), carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP), this method only produces a significant detection signal for the target PSA at a concentration of 40 ng/mL, proving that this method has a high specificity.

Embodiment 2

An immunoassay method for urine microalbumin (mALB) constructed using a syringe. The immunoassay system constructed in the syringe is a monoclonal antibody competitive type, which belongs to a competitive immunoassay method. The assay steps are as follows:

Step A: Pull the piston rod of a commercially available disposable syringe with a capacity of 1 mL and a tube made of polypropylene (PP) to extract 0.3 mL of the capture component - mouse anti-human mALB monoclonal antibody solution with a concentration of 20 μg/mL; place the syringe in a 4 ^o C refrigerator for 12 hours to push out the liquid; this step can fix the capture component to the inner wall surface of the syringe.

Step B: Draw 0.3 mL of 0.01 M phosphate-tween buffer (PBST) with a pH of 7.4 to allow the liquid to cover the fixed position of the captured component on the inner tube wall in step A; pull and push the piston rod three times in sequence to brush the aforementioned covered position and push out the cleaning liquid; repeat the operation three times; this step can remove the residual components in the syringe.

Step C: Draw 0.3 mL of 5% bovine serum albumin (BSA) solution to cover the fixed sites of the captured components on the inner tube wall in step A; place the syringe in a 37 ^o C water bath for 1 hour and push out the liquid; clean the syringe according to step B; this step can seal the remaining sites on the inner tube wall where the captured components are not adsorbed.

Step D: Draw 0.3 mL of a mixed solution of the target component (mALB antigen solution) and the detection component (mALB solution labeled with platinum nanoparticles (Pt NPs), where the final concentration of Pt NPs is 0.25 mg/mL, so that the liquid covers the fixed position of the capture component on the inner cylinder wall in step A; place the syringe in a 37 ^o C water bath for 30 minutes and push out the liquid; clean the syringe according to step B; this step can complete the specific recognition of the capture component and the target on the inner cylinder wall.

The preparation steps of the Pt NPs-labeled mALB solution are as follows:

(1) Preparation of Pt NPs: In an 80 ^° C water bath, an aqueous solution of ascorbic acid (AA) and an aqueous solution of _{chloroplatinic} acid ( _H2PtCl6 ) were mixed to a final concentration of 0.04 M AA and 0.1 mM _H2PtCl6 , respectively. After standing for 10 min, the mixture _was centrifuged at 15,000 rpm for 6 min and the supernatant was discarded. Following this operation, the precipitate was washed with water three times. The Pt NPs finally collected were ultrasonically dispersed in water at a concentration of 4 mg/mL.

(2) Labeling: Prepare a 1 mg/mL Pt NPs aqueous dispersion; adjust the pH of the dispersion to 8.5 with _{a K2CO3} aqueous solution; add a mALB stock solution with a final concentration of 20 μg/mL; slowly shake at room temperature (25±5 ^° C) for 2 hours, add a BSA aqueous solution with a final concentration of 0.5%, and continue shaking for 0.5 hours; centrifuge at 8000 rpm for 5 minutes and discard the supernatant; wash the precipitate with water three times according to this operation.

(3) Storage: Vortex or ultrasonically disperse the precipitate obtained in the previous step in 0.01 M phosphate buffer (PBS) at pH 7.4. The dispersion contains 0.05% Triton X-100, 1.25% sucrose, and 0.5% BSA. The final concentration of Pt NPs is 0.25 mg/mL. Reserve.

Step E: Extract 0.3 mL of H ₂ O ₂ substrate reaction solution with a concentration of 0.15%, so that the liquid covers the fixed position of the capture component on the inner tube wall in step A; connect the injection port of the syringe with a transparent silicone tube with an inner diameter of 0.5 mm through a commercially available sharp needle, and the other end of the silicone tube is open; after 6 minutes of reaction, record the moving distance signal of the liquid in the silicone tube; this step can convert the target concentration in the immunoassay system in the syringe into the aforementioned distance signal. It should be noted that the gas in the syringe needs to be emptied in advance so that the discharged liquid can enter the tube or channel as soon as possible to improve the detection sensitivity.

Step F: Establish a standard curve between the moving distance signal in step E and the target mALB concentration in step D, establish a quantitative relationship between the two, and use the quantitative relationship to achieve quantitative immunoassay of mALB.

In the above process, the volume of each working solution is controlled by the scale lines on the syringe; during operation, it is necessary to ensure that the piston of the syringe does not contact the inner cylinder wall part where the captured component has been fixed (for example, before extracting the solution, the piston rod is pulled up to a certain empty distance in advance, and then the solution is extracted; when the solution is pushed out, the piston stops at the inner cylinder wall part where the captured component has been fixed to avoid touching the fixed captured component).

The immunoassay system was constructed with different components in the syringe, and the bubble generation and liquid movement distance control experiments were carried out for different systems. The control experiment results are shown in Figures 5 and 6. Only when the capture antibody and Pt NPs-labeled mALB were used at the same time (Figures 5 and 6 (c-d), the bubble generation phenomenon can be observed in the syringe, and the injected liquid generated a movement distance signal of 148 to 220 mm in the silicone tube, proving that the Pt NPs probe was immunocaptured in the syringe. Among them, when the target mALB with a concentration of 0.5 μg/mL was present (Figures 5 and 6 (d), the bubble size in the syringe was significantly smaller than that in the absence of target mALB (Figures 5 and 6 (c), and the distance signal was also reduced accordingly. There was no obvious bubble generation phenomenon and movement distance signal in other control groups, all of which were less than 3.0 mm. This control experiment verified the successful construction of the competitive immunoassay system of mALB in the syringe, and also proved that the concentration of the target mALB in the assay system can be converted into the movement distance signal of the liquid in the silicone tube.

As shown in Figures 7 and 8, within the concentration range of 0.006-0.1 μg/mL and 0.1-6.0 μg/mL, the moving distance signal is linearly correlated with the mALB concentration (C _mALB ) and Lg C _mALB (R ² =0.990, 0.986), respectively, and a quantitative relationship between the two can be established. Calculated by 3 times the signal-to-noise ratio (S/N), the limit of detection (LOD) is 5.18 ng/mL, which is significantly lower than the health monitoring threshold of mALB - 20 μg/mL. As shown in Figure 9, the mALB concentration in 20 clinical urine samples was detected by this method and the conventional ELISA kit, and the urine samples were diluted 100 times. There is a good linear correlation between the two test results (R ² =0.983), and the slope of the linear equation is 1.001, indicating that there is a high consistency between the two test results.

Claims (9)

1. An immunoassay method using a syringe, characterized in that the immunoassay is performed according to the following steps: Step 1: Using a syringe as a measuring and discharging tool for the working solution, first draw the antibody solution or antigen solution as the capture component into the barrel of the syringe, and fix the capture component on the inner barrel wall by physical adsorption method with the inner barrel wall as the base; Step 2: Using the syringe to extract the cleaning liquid, and removing the residual components in the syringe by cleaning; Step 3: Use the syringe to extract the protein blocking solution to achieve the blocking of the remaining sites on the inner wall of the syringe that are not bound to the capture component; then clean and remove the residual components in the syringe according to the second step; Step 4: For constructing a sandwich immunoassay system, extract the target solution, and complete the specific recognition between the capture component and the target on the inner wall of the syringe; then clean and remove the residual components in the syringe according to the second step, and then carry out the fifth step, the sixth step and the seventh step in sequence; For the construction of a competitive immunoassay system, a mixture of a target solution and a probe as a detection component, i.e., an antibody solution or an antigen solution labeled with platinum nanoparticles or catalase, is extracted, and competitive specific recognition between the capture component and the target and the detection component is completed on the inner wall of the syringe; then, the residual components in the syringe are removed by washing according to the second step, and then the sixth and seventh steps are performed in sequence; Step 5: Extract the probe as the detection component - the antibody solution or antigen solution labeled with platinum nanoparticles or catalase, and construct a sandwich immunoassay system on the inner wall of the syringe; then clean and remove the residual components in the syringe according to the second step; Step 6: extract H ₂ O ₂ substrate solution; connect the injection port of the syringe to the connecting tube or the air pressure gauge or the hydraulic pressure gauge to obtain the moving distance signal of the liquid discharged by the syringe in the connecting tube or the air pressure signal or the hydraulic pressure signal in the syringe; Step 7: Establish a quantitative relationship between the target concentration and the corresponding signal, and realize immunoassay through this quantitative relationship. 2. The immunoassay method using a syringe as claimed in claim 1, wherein the specific assay steps are as follows: Step A: Pull the piston rod of the syringe to quantitatively extract the antibody or antigen solution as the capture component, the extracted volume is less than half of the total volume of the barrel, and the capture component is fixed on the inner wall of the barrel; after placing it, push out the liquid; Step B: extracting cleaning liquid so that the cleaning liquid covers the fixed position of the captured component on the inner cylinder wall in step A; brushing the aforementioned covered position by pulling and pushing the piston rod; Step C: extracting bovine serum albumin blocking solution so that the solution covers the fixed position of the capture component on the inner tube wall in step A; after standing, push out the liquid and clean the syringe; Step D: for constructing a sandwich immunoassay system, extract the antigen solution or antibody solution as the target component so that the liquid covers the fixed position of the capture component on the inner cylinder wall in step A; after placing, push out the liquid and clean the syringe; then perform steps E, F and G in sequence; For constructing a competitive immunoassay system, extract a mixture of an antigen solution or an antibody solution as a target component and a probe-platinum nanoparticle-labeled or catalase-labeled antibody solution or a probe-platinum nanoparticle-labeled or catalase-labeled antigen solution as a detection component, so that the liquid covers the fixed position of the capture component on the inner cylinder wall in step A; push out the liquid after placing; then clean the syringe according to step B; then perform steps F and G in sequence; Step E: extracting a probe-platinum nanoparticle-labeled or catalase-labeled antibody solution or a probe-platinum nanoparticle-labeled or catalase-labeled antigen solution as a detection component, so that the liquid covers the fixed position of the capture component on the inner tube wall in step A; after placement, push out the liquid and clean the syringe; Step F: extracting a _{H2O2 substrate} solution with a concentration of 0.05% to 10%, so that the liquid covers the fixed position of the capture component on the inner tube wall in step A; connecting the liquid outlet of the syringe to a connecting tube, an air pressure gauge or a hydraulic pressure gauge; after reacting for 2 to 30 minutes, recording the moving distance signal of the liquid discharged by the syringe in the connecting tube, the air pressure signal or the hydraulic pressure signal in the syringe; Step G: Establish a standard curve between the signal in step F and the target concentration in step D, and use the standard curve to achieve immunoassay of the target concentration. 3. The immunoassay method constructed using a syringe as claimed in claim 2, characterized in that: the concentration of the capture component in step A is 2-80 μg/mL; the syringe is placed in a 4-10 ^° C environment for 12 hours or in a 30-38 ^° C water bath for 2 hours. 4. The immunoassay method constructed by using a syringe as claimed in claim 2, characterized in that: the cleaning solution in step B is a phosphate buffer or a phosphate Tween buffer with a pH of 7.0 to 7.4; Pull and push the piston rod 1 to 3 times to clean the inner cylinder wall, push out the cleaning liquid and repeat the cleaning operation 1 to 3 times. 5. The immunoassay method using a syringe as claimed in claim 2, characterized in that: the concentration of the bovine serum albumin blocking solution in step C is 0.5-10%; and the syringe is placed in a 30-38 ^° C water bath for 2 hours. 6. The immunoassay method constructed by using a syringe as claimed in claim 2, characterized in that: after extracting the target component solution or the mixed solution of the target component solution and the detection component solution in step D, the syringe is placed in a 30-38 ^° C water bath for 10-30 minutes or at a room temperature of 25±5 ^° C for 0.25-1 hour. 7. The immunoassay method constructed using a syringe as claimed in claim 2, characterized in that: the detection component described in step E is an antibody solution labeled with platinum nanoparticles or an antigen solution labeled with platinum nanoparticles; the particle size of the platinum nanoparticles is 5 to 300 nm; the concentration of the platinum nanoparticles in the detection component is 0.1 to 0.5 mg/mL; after extracting the detection component solution, the syringe is placed in a 30 to 38 ^o C water bath for 15 to 30 minutes or at 25±5 ^o C room temperature for 0.25 to 1 hour. 8. The immunoassay method constructed by using a syringe as claimed in claim ₂ , characterized in that the concentration of _H2O2 in the _H2O2 substrate solution in step F is _0.05 % to 1%. 9. The immunoassay method constructed by using a syringe as claimed in claim 2, characterized in that: the placing in step A and step C refers to placing the syringe in an environment of 4 to 38 ^° C for 0.5 to 24 hours; the placing in step D and step E refers to placing the syringe in an environment of 4 to 38 ^° C for 0.1 to 4 hours.