An immunoassay method based on tyramide signal amplification technology and blood glucose meter

Abstract

The application provides an immunodetection method based on tyramide signal amplification technology and a blood glucose meter, and belongs to the technical field of molecular diagnosis and detection. The detection method is as follows: first, a tyramide-specific enzyme conjugate is prepared through amide condensation reaction and click chemistry reaction, and horseradish peroxidase or a material with peroxidase-like activity is introduced as a catalyst through specific combination of an antigen and an antibody; then, the tyramide-specific enzyme conjugate is deposited based on tyramide signal amplification technology; finally, the concentration of a target analyte is detected by using a blood glucose meter. The tyramide signal amplification technology is combined with the portable blood glucose meter, the sensitivity of the immunoreaction is significantly improved, and high-sensitivity detection of micro trace proteins and other immunoreaction-based substances can be realized. The method is simple to operate and low in cost, and does not need expensive and precise instruments and professional technicians, and is especially suitable for resource-poor and laboratory-free areas.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostic detection technology, and more specifically to an immunoassay method based on tyramine signal amplification technology and a blood glucose meter. Background Technology

[0002] In the early stages of disease, the levels of protein biomarkers are low. To achieve highly sensitive detection of trace proteins, it is often necessary to develop highly sensitive immunoassay methods. Combining signal amplification techniques is an effective way to improve sensitivity. Among common amplification strategies, nucleic acid amplification reactions, while highly sensitive, are prone to contamination and costly, while multi-enzyme cascade reactions require complex method design. Tyramine signal amplification (TSA) is a simple and highly sensitive method that has been widely used in immunoassays. Tyramine can be coupled with different signal reporter molecules, leading to the development of immunoassay methods based on tyramine signal amplification, such as tyramine-coupled fluorophore signal amplification for detecting fluorescence signals, tyramine-coupled electroactive molecule signal amplification for detecting electrochemical signals, and tyramine-coupled protease signal amplification for detecting chemiluminescence and electrochemiluminescence signals. Because enzymes have high catalytic activity, increasing the enzyme content in the system can catalyze more substrates to react, and tyramine-coupled protease further improves the sensitivity of TSA detection. However, the above immunoassay techniques usually require specialized precision instruments and technical personnel to perform the tests in a laboratory, which limits their practical application (especially in resource-scarce areas with poor experimental conditions).

[0003] To overcome the aforementioned shortcomings, researchers applied portable personal glucose meters (PGMs) to immunoassay. PGMs are a commonly used tool for point-of-care diagnostics; they are simple to operate, providing easy readings in just a few seconds, and are not limited by laboratory conditions. By coupling invertase with a detection antibody, and following an immune reaction, the glucose obtained from the hydrolysis of sucrose by the invertase bound to the antibody serves as the detection signal. The portable glucose meter then reads the signal, enabling quantitative analysis of the target analyte. Xiang Yu et al. directly coupled detection antibodies in the immune reaction with sucrose invertase, realizing immunoassay detection based on portable PGM signal readout (Portable and quantitative detection of protein biomarkers and small molecular toxins using antibodies and ubiquitous personal glucose meters. "Analytical Chemistry 84.9(2012):4174); Leonard, Elissa K. et al. fused a detection antibody with two sucrose invertase molecules to prepare a fusion protein with both immunoaffinity and catalytic activity. They achieved quantitative detection of the target analyte by constructing an immunoassay platform combining the fusion protein with a portable blood glucose meter (Antibody-Invertase Fusion Protein Enables Quantitative Detection of SARS-CoV-2 Antibodies Using Widely Available Glucometers. "Journal of the American Chemical Society 144.25(2022):11226-11237.). However, the above methods can only couple or fuse a target protein with a limited number of specific enzyme molecules, resulting in low sensitivity and complex synthesis of signal reporter molecules. Summary of the Invention

[0004] The purpose of this invention is to provide an immunoassay method based on tyramine signal amplification technology and a blood glucose meter, so as to solve the problems of low sensitivity and complex operation of existing immunoassay methods.

[0005] This invention is implemented as follows: This invention provides an immunoassay method based on tyramine signal amplification technology and a blood glucose meter: First, a tyramine-specific enzyme conjugate (Tyr-specific enzyme) is prepared through amide condensation and click chemistry reactions, and horseradish peroxidase (HRP) or a material with peroxidase-like activity is introduced as a catalyst through antigen-antibody specific binding; then, the Tyr-specific enzyme conjugate is deposited based on tyramine signal amplification technology; finally, the concentration of the target analyte is detected using a blood glucose meter.

[0006] Specifically, the preparation steps include the following: (1) Preparation of Tyr-specific enzyme conjugate a. The specific enzyme is reacted with azide-polyethylene glycol-active ester (N3-PEG). 2000 -NHS) is coupled via an amide condensation reaction to obtain an azide-specific enzyme conjugate (N3-specific enzyme conjugate); the specific enzyme is an enzyme capable of hydrolyzing to produce glucose; b. Tyramine and dibenzocyclooctene-active ester (DBCO-NHS) are coupled via an amide condensation reaction to obtain tyramine-dibenzocyclooctene (Tyr-DBCO). c. N3-specific enzyme and Tyr-DBCO form a Tyr-specific enzyme conjugate through a click chemical reaction between DBCO and N3.

[0007] (2) Construct an immune system for the target analyte sample and introduce catalyst A, wherein catalyst A is HRP or a material with peroxidase-like activity.

[0008] (3) Deposition of Tyr-specific enzyme conjugates based on tyramine signal amplification technology Add the mixed solution of Tyr-specific enzyme conjugate and H2O2 obtained in step (1) to the immune system of the target analyte sample obtained in step (2). Under the catalysis of catalyst A, Tyr-specific enzyme conjugate is deposited in large quantities on catalyst A or its neighboring protein through the tyramine signal amplification reaction, forming a system with a large amount of Tyr-specific enzyme conjugate deposited.

[0009] In the presence of hydrogen peroxide, HRP modified on the detection antibody or materials with peroxidase-like activity catalyze tyramine to become a short-lived active intermediate. This intermediate covalently binds to amino acid residues of neighboring proteins of the HRP or peroxidase-like active material, resulting in the deposition of a large amount of Tyr-specific enzyme conjugate, thereby enhancing the detection signal.

[0010] (4) Use a blood glucose meter to detect the concentration of the target analyte. Add the substrate of the specific enzyme to the system of the large amount of Tyr-specific enzyme conjugate obtained in step (3), and hydrolyze the substrate into glucose under the catalysis of the specific enzyme; after the reaction is completed, use a blood glucose meter to determine the glucose content and record the blood glucose meter reading; based on the correspondence between the blood glucose meter reading and the concentration of the target analyte, the concentration of the target analyte sample to be tested can be indirectly obtained through the blood glucose meter reading.

[0011] In step (3), the specific enzyme in the Tyr-specific enzyme conjugate that is deposited in large quantities is an enzyme capable of hydrolyzing glucose. Therefore, under hydrolysis conditions, it can catalyze the corresponding substrate to glucose, which can then be detected by the blood glucose meter. The amount of specific enzyme introduced varies depending on the concentration of the target analyte sample, resulting in different glucose concentrations generated within the same time period. Therefore, we can indirectly obtain the concentration of the target antigen through the blood glucose meter reading.

[0012] The correlation between blood glucose meter readings and target analyte concentrations can be obtained using the following two methods: First, a standard curve is constructed between the concentration of the target analyte standard solution and the blood glucose meter reading; second, a point-to-point relationship between the target analyte concentration and the blood glucose meter reading is established through detection at multiple points.

[0013] Preferably, the specific enzyme is invertase, amylase, glucosidase, or lactase.

[0014] Preferably, in step (2), the immune system is a sandwich immune system, a direct immune system, an indirect immune system, or a competitive immune system.

[0015] Preferably, in step (2), the material with peroxidase-like activity is a G-quadruplex oligonucleotide with peroxidase-like activity or a nanomaterial with peroxidase-like activity.

[0016] Preferably, in step (4), the blood glucose meter is a portable blood glucose meter.

[0017] Preferably, in step (4), the hydrolysis conditions are 25-55℃ and low-speed oscillation for 1-2.5 h.

[0018] The detection method described in this invention has universal applicability. The target analyte is a protein biomarker, antibiotic, toxin molecule, cytokine, cell or microorganism that can be detected by immunoassay, such as carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), prostate-specific antigen (PSA) or SARS-CoV-2.

[0019] Furthermore, the present invention is also applicable to other catalytic reaction systems that can produce glucose.

[0020] The beneficial effects of this invention are as follows: This invention discloses an immunoassay method based on tyramine signal amplification technology and a blood glucose meter. It innovatively prepares a Tyr-specific enzyme conjugate through amide condensation and click chemistry reactions. This conjugate can be deposited in large quantities based on tyramine signal amplification technology, thereby amplifying the detection signal. This effectively solves the deficiency of previous methods where a target protein could only be coupled or fused with a limited number of specific enzyme molecules, greatly improving the sensitivity of immunoassay. Furthermore, the specific enzyme in the heavily deposited Tyr-specific enzyme conjugate can catalyze the hydrolysis of its substrate to glucose, allowing the blood glucose meter to directly read the signal. This invention pioneeringly combines tyramine signal amplification technology with a blood glucose meter, solving the problem of complex synthesis of signal reporter molecules and overcoming the limitations of specialized precision instruments and operating techniques. It can be performed without expensive and sophisticated instruments or specialized technicians, making it particularly suitable for resource-scarce areas and regions without laboratory facilities.

[0021] The detection method described in this invention combines the high-efficiency signal amplification capability of tyramine signal amplification technology with the simplicity of blood glucose meter detection, greatly expanding the application of tyramine signal amplification technology and significantly improving the sensitivity of blood glucose meter-based immunoassays. It enables highly sensitive detection of trace proteins and other substances based on immunoreaction. Compared with methods using invertase-labeled antibodies or fusion proteins, the method of this invention has advantages such as high sensitivity, good universality, simple operation, and low cost, and has promising development prospects. Attached Figure Description

[0022] Figure 1 The diagram shows the preparation of Tyr-inv (a) and the principle of the method for immunoassay using a blood glucose meter combined with tyramine signal amplification technology (b).

[0023] Figure 2 Figures (a and b) show the feasibility verification of this method. Figure (a) shows the feasibility verification for CEA detection: Column 1, Ab1 + Ab2-HRP + Tyr-inv; Column 2, Ab1 + CEA + Ab2-HRP + Inv; Column 3, Ab1 + CEA + Ab2-HRP + Tyr-inv. The CEA concentration is 100 pg / mL. Figure (b) shows the feasibility verification for AFP detection: Column 1, Ab1 + Ab2-HRP + Tyr-inv; Column 2, Ab1 + AFP + Ab2-HRP + Inv; Column 3, Ab1 + AFP + Ab2-HRP + Tyr-inv. The AFP concentration is 100 pg / mL. Error bars represent the standard deviation of three parallel experiments.

[0024] Figure 3These are standard curves showing the change in blood glucose meter signal value with target protein concentration. Figure (a) shows the standard working curve for CEA detection, and Figure (b) shows the standard working curve for AFP detection. The error bars represent the standard deviation of three parallel experiments. Detailed Implementation

[0025] The present invention will be further illustrated below with reference to the embodiments (using CEA and AFP as research objects, based on the reaction system of tyramine-sucrose invertase conjugate). The following embodiments are for illustration only and do not limit the scope of protection of the present invention in any way.

[0026] The processes and methods not described in detail in the following embodiments are conventional methods known in the art. All reagents used in the embodiments are analytically pure or chemically pure, and are commercially available or prepared by methods well known to those skilled in the art. The following embodiments achieve the objectives of the present invention.

[0027] Example 1 A method to improve the sensitivity of immune detection in portable blood glucose meters, the detection principle is as follows: Figure 1 As shown, the specific detection method is as follows: 1. Preparation of tyramine-sucrose invertase (Tyr-inv) conjugate.

[0028] like Figure 1 As shown in (a), N3-invertase and Tyr-DBCO were first prepared based on an amide condensation reaction. Then, Tyr-invertase was prepared based on a click chemistry reaction between N3 and DBCO.

[0029] Preparation of Tyr-DBCO: DBCO-NHS and N3-PEG were dissolved in DMSO. 2000 -NHS was prepared into stock solutions of 4 mg / mL and 5 mg / mL. Tyramine (Tyr) and invertase (Inv) were dissolved in deionized sterile water to prepare stock solutions of 1 mg / mL and 5 mg / mL, respectively. In a 1.5 mL centrifuge tube, 712 µL of 10 mM PBS buffer (pH=7.4), 82 µL of 1 mg / mL tyramine solution, and 6 µL of 4 mg / mL DBCO-NHS stock solution were added. After thorough mixing, the mixture was shaken at 4°C for 2 h to obtain the Tyr-DBCO conjugate.

[0030] Preparation of N3-invertase: Add 250 µL of 10 mM phosphate buffer, 540 µL of 5 mg / mL sucrose invertase stock solution, and 10 µL of 5 mg / mL N3-PEG to a centrifuge tube. 2000N3-inv was prepared by reacting -NHS stock solution with shaking at 4°C for 2 h. After the reaction was complete, N3-inv was purified by ultrafiltration using a 100 KD tube. The centrifuge parameters for purification were 4°C, 6000 rpm, and 10 min. Unreacted N3-PEG was removed by centrifugation. 2000 -NHS, retaining the reaction solution trapped in the ultrafiltration tube, this solution is the purified N3-inv solution.

[0031] Preparation of Tyr-inv: The purified N3-inv solution was mixed with the Tyr-DBCO reaction solution and reacted in a shaker at 4°C and 800 rpm for 4 h. After the reaction, the mixture was centrifuged at 4°C and 6000 rpm for 10 min using a 100 KD ultrafiltration tube. Finally, the product was washed twice with 10 mM phosphate buffer (pH=7.4), the coupling product was collected, resuspended in phosphate buffer, and stored at 4°C for later use.

[0032] 2. This method selects CEA and AFP as models for research, and uses CEA as an example to illustrate the construction of the sandwich immune system.

[0033] The principle of the method is as follows Figure 1 As shown in (b), 100 µL of dilution buffer for the capture antibody (5 µg / mL) was first added to each well of a 96-well plate, and incubated overnight at 4°C. After the reaction, the plate was washed three times with 200 µL of PBS solution containing 0.1% Tween-20 (PBS-T). Then, 200 µL of PBS buffer containing 2% BSA was added to each well, and the plate was blocked at room temperature for 1.5 h. The blocking buffer was then discarded. Next, 100 µL of a certain concentration of CEA solution was added to each well, and the plate was incubated at 37°C for 2 h. After the reaction, the reaction solution was discarded, and the plate was washed twice with PBS-T (200 µL / well) to remove unbound antigen. Finally, 100 µL of 5 µg / mL HRP-modified detection antibody was added to each well, and the plate was reacted at room temperature for 2 h. The reaction solution was then discarded, and the plate was washed three times with PBS-T to form a CEA sandwich immunoassay system.

[0034] 3. Deposition of Tyr-inv conjugates based on tyramine signal amplification technology.

[0035] Add 100 µL of a mixed solution of Tyr-inv and H2O2 to each well and react at room temperature for 35 min. The final concentration of H2O2 was 10 mM and the final concentration of Tyr-inv was 5 µM. After the reaction was complete, discard the reaction solution and wash three times with PBS-T buffer to obtain the system with deposited Tyr-inv conjugate.

[0036] 4. Use a blood glucose meter to detect the concentration of the target antigen. Add 100 µL of 0.25 M sucrose solution diluted with acetate-sodium acetate buffer (pH=5.0) to the system containing the deposited Tyr-inv conjugate obtained in step 3. Seal the microplate and incubate at 55°C with low-speed shaking for 2.5 h. After the reaction is complete, measure the glucose content using a Roche Excellence blood glucose meter and record the data.

[0037] The experimental procedure for AFP is the same as the one described above.

[0038] Example 2 To verify the feasibility of the method of the present invention, different reaction systems were designed for comparison in this embodiment. Experiments were conducted on CEA and AFP to demonstrate that the method of the present invention has universal applicability.

[0039] First, three reaction systems were designed to verify the feasibility of the method for detecting CEA in this invention. System 1 was a blank solution, System 2 was a system with unmodified invertase added, and System 3 was a system with Tyr-inv conjugate added.

[0040] like Figure 2 As shown in (a), column 1 is the blank solution with a low signal; column 2 is the system with unmodified invertase added, and its response signal is not much different from the blank, also low; column 3 is the system with Tyr-inv conjugate added, which produces a strong response signal. The above results indicate the successful coupling of Tyr-inv, and also indicate that the experimental results of system 3 are not due to non-specific adsorption, but rather a strong signal generated by tyramine deposition based on the TSA reaction.

[0041] Comparing the results of systems 1 and 3, only a very weak signal was generated when the target analyte was absent (blank). When the target antigen was present, a clear glucose signal could be detected, and the signals of the blank and the sample were clearly distinguishable, indicating that the detection method of the present invention is feasible.

[0042] Based on the principle of similarity, this embodiment also designed three reaction systems to explore the feasibility of the method for detecting AFP in this invention. The results are as follows: Figure 2 As shown in (b).

[0043] By comparing different reaction systems, the feasibility of this method was verified, and it was also shown that the immunoassay technology platform constructed by this invention has universal applicability.

[0044] Example 3 Under optimal reaction conditions, the linear range for detecting CEA and AFP using the method of this invention was determined. When the CEA concentration was in the range of 100 fg / mL to 10 ng / mL, the relative signal value ΔGMS of the blood glucose meter in this immune system showed a linear relationship with the logarithm of the CEA concentration, where ΔGMS is the difference in PGM signal values ​​between the sample group and the blank group. Figure 3 As shown in (a), its linear equation is ΔGMS = 3.105 + 2.021 logC CEA (pg / mL), linear correlation coefficient R 2 =0.9950, the linear range of this method for detecting CEA is up to 5 orders of magnitude.

[0045] This method was further applied to the detection of AFP, from Figure 3 (b) It can be seen that when the AFP concentration is in the range of 100 fg / mL to 10 ng / mL, the logarithm of the PGM signal and the AFP concentration show a good linear relationship, and the linear equation is ΔGMS = 3.988 + 1.978 logC AFP (pg / mL), linear correlation coefficient R 2 =0.9941.

[0046] The above results demonstrate that the immunoassay method of the present invention has good analytical capabilities and versatility.

[0047] Example 4 To verify the practicality of this method, the CEA content of three serum samples from the Affiliated Hospital of Hebei University was detected using the detection method described in Example 1.

[0048] The measured values ​​of the detection method of this invention were compared with the signal values ​​(reference values) measured by the Roche Cobase 601 electrochemiluminescence fully automated immunoassay system, and the results are shown in Table 1.

[0049] Table 1. Comparison of measured values ​​and reference values ​​using the detection method of this invention (CEA)

[0050] The detection method of this invention performs spiked recovery determination on actual samples. Different concentrations of CEA standard solution are added to a certain amount of serum sample, and the recovery rate is calculated to be in the range of 97.9%-109.3% (Table 2).

[0051] The definition of spiked recovery rate is as follows: when a certain amount of standard substance is added to a subsample of the same sample for determination, the result of the determination is subtracted from the result of the standard substance for determination, and the recovery rate is calculated.

[0052] Table 2. Detection of CEA recovery rate in actual samples

[0053] Similarly, three actual AFP samples from the hospital were tested and compared, and the results are shown in Table 3.

[0054] Table 3 Comparison of measured values ​​and reference values ​​using the detection method of this invention (AFP)

[0055] Meanwhile, a spiked recovery experiment was conducted on the serum samples. The results are shown in Table 4, with recovery rates ranging from 93.6% to 104.5%.

[0056] Table 4. Recovery rate of AFP in actual samples

[0057] The above results demonstrate that the detection method of the present invention has good accuracy and practicality, and can successfully detect serum samples.

Claims

1. An immunoassay method based on tyramine signal amplification technology and a blood glucose meter, characterized in that, Includes the following steps: (1) Preparation of tyramine-sucrose invertase conjugate a. Sucrose invertase is coupled with azide-polyethylene glycol-active ester via an amide condensation reaction to obtain an azide-sucrose invertase conjugate; b. Tyramine and dibenzocyclooctene-active ester are coupled via amide condensation to yield tyramine-dibenzocyclooctene; c. Tyramine-sucrose invertase conjugate and tyramine-dibenzocyclooctene form tyramine-sucrose invertase conjugate through a click chemical reaction between dibenzocyclooctene and the azide group; (2) Construct an immune system for the target analyte sample and introduce catalyst A, wherein catalyst A is horseradish peroxidase or a material with peroxidase-like activity; (3) Deposition of tyramine-sucrose invertase conjugate based on tyramine signal amplification technology Add the tyramine-sucrose invertase conjugate obtained in step (1) and H2O2 mixed solution to the immune system of the target analyte sample obtained in step (2). Under the catalysis of catalyst A, a large amount of tyramine-sucrose invertase conjugate is deposited on catalyst A or its adjacent protein through the tyramine signal amplification reaction, forming a system with a large amount of tyramine-sucrose invertase conjugate deposited. (4) Use a blood glucose meter to detect the concentration of the target analyte. Add sucrose to the system of a large amount of deposited tyramine-sucrose invertase conjugate obtained in step (3), and hydrolyze the sucrose into glucose under the catalysis of sucrose invertase; After the reaction is complete, a blood glucose meter is used to measure the glucose content and record the blood glucose meter reading. Based on the correspondence between the blood glucose meter reading and the concentration of the target analyte, the concentration of the target analyte sample is indirectly obtained through the blood glucose meter reading.

2. The method according to claim 1, characterized in that, In step (2), the target analyte is a protein marker, antibiotic, toxin molecule, cytokine, cell or microorganism that can be detected by immunoassay.

3. The method according to claim 1, characterized in that, In step (2), the immune system is a sandwich immune system, a direct immune system, an indirect immune system, or a competitive immune system.

4. The method according to claim 1, characterized in that, In step (4), the blood glucose meter is a portable blood glucose meter.

5. The method according to claim 1, characterized in that, In step (4), the hydrolysis conditions are 25-55℃ and low-speed oscillation for 1-2.5 h.

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