An electrochemical immunosensor for detecting adiponectin and a quantitative detection method thereof

Abstract

The application discloses a preparation method of an electrochemical immunosensor for detecting adiponectin and a method for quantitatively detecting adiponectin by using the electrochemical immunosensor, proposes a new strategy for fixing adiponectin antibodies in one step, constructs an electrochemical immunosensor based on a gold sputtering silver screen-printed electrode (Au / AgSPE), and realizes rapid, high-sensitivity and stable detection of adiponectin, so as to promote the conversion of adiponectin detection technology to instant detection application. The detection process of the application only needs a portable electrochemical workstation, and does not need large biochemical analysis equipment. The screen-printed electrode and the sputtering process support large-scale production, and are favorable for reducing cost and popularization.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemistry, in particular to an electrochemical immunosensor for detecting adiponectin and a quantitative detection method thereof. BACKGROUND

[0002] Adiponectin (APN) is an important adipokine secreted by adipose tissue, which plays a key role in regulating lipid metabolism, energy balance, insulin sensitivity and inflammatory response. The serum level of adiponectin is significantly negatively correlated with the severity of metabolic diseases such as type 2 diabetes, obesity and atherosclerosis. Therefore, accurate detection of adiponectin is of great significance in early prediction of diseases, evaluation of therapeutic effect and clinical decision-making.

[0003] Currently, the most commonly used methods for detecting adiponectin are enzyme-linked immunosorbent assay (ELISA), immunoturbidimetry (LTIA) and chemiluminescent enzyme immunoassay (CLEIA). In addition, radioimmunoassay (RIA) can also be used to detect adiponectin. ELISA requires a long detection time (2-3 h), a large sample volume (100-200 μL), a complex detection procedure (centrifugation, incubation, blocking, washing, etc.), and a professional instrument (enzyme label instrument) and technical personnel. Although LTIA greatly shortens the detection time (10 min), it still requires a large sample volume, a professional instrument (full-automatic biochemical analyzer) and offline blood centrifugation. CLEIA also requires a full-automatic biochemical analyzer, centrifugation, incubation, washing and other complicated steps, and the detection time is 2-3 h. RIA has been gradually eliminated due to poor reagent stability and environmental pollution. These tests often require the collection of a certain batch of samples for centralized detection, which increases the detection cost and the waiting time of the subjects, and the detection efficiency is low. There is a lack of inexpensive, simple, rapid and accurate instant detection technology for adiponectin in the market, which greatly limits the popularization and application of adiponectin detection. Therefore, it is of great technical challenge and broad application prospect to develop new methods for detecting adiponectin, especially low-cost, high-sensitivity and rapid detection methods for micro-volume samples.

[0004] Electrochemical immunosensor is a kind of immunosensor which takes antigen or antibody as a biological recognition element, combines the highly specific reaction between antigen and antibody with electrochemical analysis technology. The performance of this kind of sensor depends largely on the biological functional modification strategy of the molecular recognition interface. Current research usually uses EDC / NHS, 6-MH / CNBr, 3-GOPS or Mix & GoTM reagents to fix the antibody on various electrode substrates. However, these immobilization methods have a common defect: they usually need two or more steps to complete the effective fixation of the antibody. The complex modification process not only introduces batch-to-batch variability, but also increases the manufacturing cost and time, ultimately restricting the reproducibility, stability and detection efficiency of the sensor.

[0005] Therefore, the core technical bottleneck in the current adiponectin detection field is: how to develop an efficient, stable and simple antibody immobilization method to overcome the complexity, inconsistency and inefficiency problems caused by multi-step modification, so as to build a fast, low-cost and high-sensitivity adiponectin immunosensor that meets the performance requirements of clinical applications. SUMMARY

[0006] The present application aims to overcome the defects of the prior art by proposing a new strategy for one-step antibody immobilization, constructing an electrochemical immunosensor based on gold sputtered silver screen-printed electrode (Au / AgSPE), and realizing rapid, high-sensitivity and stable detection of adiponectin, so as to promote the conversion of adiponectin detection technology to point-of-care testing applications.

[0007] A preparation method of an electrochemical immunosensor for detecting adiponectin, comprising the following steps:

[0008] (1) Place the silver screen-printed electrode in a vacuum ion sputtering instrument, and sputter a layer of nanoscale gold film on the surface of the silver working electrode;

[0009] (2) Drop the mercapto adiponectin monoclonal antibody on the surface of the gold film working electrode, place it in a constant temperature and humidity cabinet, stand still, rinse the electrode surface with deionized water, and dry it with nitrogen, to obtain a screen-printed electrode modified with adiponectin monoclonal antibody;

[0010] (3) Passivate the electrode surface, take the bovine serum albumin solution, drop it on the surface of the electrode, place it in a constant temperature and humidity cabinet, stand still, rinse the electrode surface with deionized water, and dry it with nitrogen, to obtain the final adiponectin immunosensor.

[0011] The preparation method of the electrochemical immunosensor for detecting adiponectin according to the present application, wherein the sputtering time of the vacuum ion sputtering instrument in step (1) is 80-100 s, and the sputtering current is 20-30 mA.

[0012] The preparation method of the electrochemical immunosensor for detecting adiponectin, wherein the concentration of the mercapto adiponectin monoclonal antibody in step (2) is 50 μg / ml, and the amount used is 15 μL.

[0013] The preparation method of the electrochemical immunosensor for detecting adiponectin, wherein the concentration of the bovine serum albumin in step (3) is 1%, and the amount used is 20 μL.

[0014] The preparation method of the electrochemical immunosensor for detecting adiponectin, wherein the temperature of the constant-temperature and constant-humidity cabinet in steps (2) and (3) is 37 ℃, the humidity is 80%, and the standing time is 45-60 min.

[0015] The product prepared by the preparation method of the electrochemical immunosensor for detecting adiponectin.

[0016] The method for quantitatively detecting adiponectin in a solution by using the electrochemical immunosensor for detecting adiponectin, comprising the following steps:

[0017] 20 μL of the adiponectin solution to be detected is drop-coated on the electrochemical immunosensor, and incubated at room temperature for 15 min; then, the electrode is washed with deionized water to remove the non-specifically adsorbed adiponectin, and dried under a nitrogen flow; the sensor is connected to an electrochemical workstation, 20 μL of 100 mM [Fe(CN ] solution containing 0.1 M KCl is drop-coated on the surface of the sensor, and the obtained result is calculated according to a standard curve to obtain the content of adiponectin in the solution.

[0018] The method for quantitatively detecting adiponectin in a solution, wherein the method for preparing the standard curve comprises the following steps:

[0019] (A) Preparation of standard working solution: adiponectin standard solutions with different concentrations are prepared, and the concentrations are 0, 0.5 mg / L, 1 mg / mL, 3 mg / L, 5 mg / L, 10 mg / L and 15 mg / L, a total of 7 concentrations; before determination, the adiponectin standard solutions with 7 concentrations are respectively drop-coated on the surfaces of 7 electrochemical immunosensors, and then incubated at room temperature for 15 min, and then washed with deionized water and dried with nitrogen.

[0020] (B) Drawing a standard curve: the 7 electrochemical immunosensors obtained in step (A) are respectively connected to an electrochemical workstation for testing, and 20 μL of 100 mM [Fe(CN The solution is detected by differential pulse voltammetry, and the peak current is linearly related to the logarithm of the concentration of the adiponectin solution.

[0021] The method for quantitatively detecting adiponectin in the solution, wherein the parameters for detecting the solution to be measured and the standard solution by the differential pulse voltammetry are as follows: a potential range of -0.1 to 0.5 V, an amplitude of 50 mV, a pulse width of 0.06 s, a pulse period of 0.5 s, and a sampling width of 0.02 s.

[0022] The method for quantitatively detecting adiponectin in the solution, wherein the preparation method of the sample solution to be measured is as follows: a biological sample is diluted 10 times by using a phosphate buffer solution to obtain the sample solution to be measured, wherein the biological sample is blood, plasma or serum, the concentration of the phosphate buffer solution is 0.01 M, and the pH value is 7.4.

[0023] The electrochemical immunosensor for detecting adiponectin is different from the prior art in that:

[0024] The electrochemical immunosensor for detecting adiponectin is not only efficient and stable but also has a simple biological recognition element fixing method, so as to overcome the complexity, inconsistency and low efficiency problems caused by multi-step modification, thereby constructing a high-sensitivity adiponectin immunosensor which is rapid, low in cost and meets the clinical requirements.

[0025] The application provides an efficient antibody fixing method: a thiolated antibody is used, the terminal thiol (-S) of the thiolated antibody is directly covalently combined with gold nanoparticles on the surface of a gold sputtering silver screen-printed electrode, one-step fixing of the antibody on the electrode is realized, and the overall preparation time of the sensor is only 90 minutes. The method overcomes the defects of multi-step reactions in traditional methods (such as EDC / NHS), significantly simplifies the modification process, reduces batch differences, and improves the preparation efficiency and consistency.

[0026] The application develops a new type of electrode substrate: a gold-modified silver screen-printed electrode (Au / AgSPE) is designed and prepared as a sensing substrate. The substrate combines the good conductivity of silver and the excellent biocompatibility and stability of gold, overcomes the interference of a polymer binder in a traditional gold screen-printed electrode, and significantly improves the reproducibility of the electrode.

[0027] The application develops a rapid adiponectin detection method: the developed adiponectin immunosensor can specifically combine with adiponectin in 15 minutes, the total analysis time is shortened by 11 times (from 160 minutes to 15 minutes) compared with a traditional ELISA method. The detection limit is 0.097 mg / L, which meets the sensitivity requirement of clinical quantitative detection of adiponectin. The operation is simple and rapid, and the method provides a powerful new tool for on-site rapid screening and dynamic monitoring of adiponectin.

[0028] The electrochemical immunosensor for detecting adiponectin and the detection method thereof will be further described below in combination with the drawings. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 A process diagram of the electrochemical immunosensor for detecting adiponectin in the present application is shown in the figure.

[0030] Figure 2 A principle diagram of the electrochemical immunosensor for detecting adiponectin in the present application is shown in the figure.

[0031] Figure 3 A comparison result diagram of the response values of Au / AgSPEs and AuSPEs to 10 mg / L adiponectin in the present application is shown in the figure.

[0032] Figure 4 Electrochemical voltammograms and standard curves of adiponectin with different concentrations in the present application are shown in the figures.

[0033] Figure 5 An electrochemical voltammogram of adiponectin in the plasma sample in the actual sample detection is shown in the figure. DETAILED DESCRIPTION

[0034] I. The preparation method of the electrochemical immunosensor

[0035] The silver screen-printed electrode is used as the substrate in the present application, the surface of the silver working electrode is modified by ion sputtering technology, and then the antibody modification and electrode sealing are sequentially performed, as shown in the figure. Figure 1 The specific preparation process is as follows:

[0036] Step 1: Place the silver screen-printed electrode (Changsha Sanjun Technology Co., Ltd.; Model: D-SPE; Working electrode: Ag, diameter 3 mm; Counter electrode: carbon; Reference electrode: silver) in a vacuum ion sputtering instrument (Cressington 108 Auto), sputter a layer of nanoscale gold film on the surface of the silver working electrode, and the sputtering time is 80-100 s and the sputtering current is 20-30 mA.

[0037] Step 2: Drop 15 μL of 50 μg / ml thiolated adiponectin monoclonal antibody (Wuhan Yunklon Technology Co., Ltd., Item No.: IS005) on the surface of the gold film working electrode, and place it in a constant temperature and humidity cabinet (Shiduoke ICTHI-150) at 37 ℃ and 80% humidity, and stand for 45-60 min. Rinse the electrode surface with deionized water and dry it with nitrogen to obtain a screen-printed electrode modified with adiponectin monoclonal antibody.

[0038] Step 3: Passivation of the electrode surface. Take 20 μL of a 1% bovine serum albumin (BSA) solution (Aladdin, catalog number: A104912), drop it onto the electrode surface, and place it in a constant temperature and humidity chamber at 37 ℃ and 80% humidity for 45-60 min. Rinse the electrode surface with deionized water and dry it with nitrogen gas. The final adiponectin immunosensor is obtained.

[0039] II. Construction of Standard Curve

[0040] The specific steps for creating a standard curve include the following:

[0041] (A) Preparation of standard working solutions: Adiponectin standard solutions of different concentrations were prepared, namely 0, 0.5 mg / L, 1 mg / mL, 3 mg / L, 5 mg / L, 10 mg / L and 15 mg / L, for a total of 7 concentrations; before the measurement, the 7 concentrations of adiponectin standard solutions were respectively dropped onto the surface of 7 electrochemical immunosensors, left to stand at room temperature for 15 min, then rinsed thoroughly with deionized water and dried with nitrogen gas;

[0042] (B) Plotting the standard curve: Connect the above 7 electrochemical immunosensors to the electrochemical workstation (Shanghai Chenhua, CHI760E), and add 20 μL of 100 mM KCl solution to each sensor. [Fe(CN The solution was scanned using differential pulse voltammetry (DPV) with a potential range of -0.1 to 0.5 V, an amplitude of 50 mV, a pulse width of 0.06 s, a pulse period of 0.5 s, and a sampling width of 0.02 s. The results were as follows: Figure 4 The differential pulse voltammetry curve shown in Figure A exhibits a linear relationship between the peak current and the logarithm of the adiponectin solution concentration. The standard curve is shown below. Figure 4 As shown in B, the linear equation for adiponectin concentrations ranging from 0.5 mg / L to 15 mg / L is i = -0.05118 log (C APN The correlation coefficient is 0.9989, with a value of 0.23237.

[0043] III. Actual Sample Testing

[0044] Biological samples (blood, plasma, serum) were diluted 10-fold with phosphate buffer (0.01 M, pH = 7.4). 20 μL of the diluted sample solution was dropped onto the electrochemical immunosensor and incubated at room temperature for 15 min. Subsequently, the sample was rinsed with deionized water and dried under a nitrogen atmosphere. The sensor was then connected to an electrochemical workstation, and 20 μL of 100 mM phosphate buffer containing 0.1 M KCl was dropped onto the sensor surface. [Fe(CN ] solution, using differential pulse voltammetry (DPV) with the following parameters: potential range -0.1 to 0.5 V, amplitude 50 mV, pulse width 0.06 s, pulse period 0.5 s, and sampling width 0.02 s. The results obtained after detection of the plasma sample in this example are shown in Figure 5 , and the content of adiponectin in the sample was calculated to be 20.1 mg / L according to the standard curve.

[0045] Fourth, the detection principle of the electrochemical immunosensor of the application

[0046] The working principle of the electrochemical immunosensor modified by the adiponectin monoclonal antibody is shown in Figure 2 .

[0047] On the prepared immunosensor, 20 μL of an adiponectin solution was dropped and incubated at room temperature for 15 min. Subsequently, the electrode was rinsed with deionized water to remove the non-specifically adsorbed adiponectin, and dried under a nitrogen stream. On the surface of the sensor, 20 μL of a 100 mM [Fe(CN [Fe(CN ] solution containing 0.1 M KCl was dropped, and differential pulse voltammetry (DPV) detection was performed. The parameters were as follows: potential range -0.1 to 0.5 V, amplitude 50 mV, pulse width 0.06 s, pulse period 0.5 s, and sampling width 0.02 s. When APN was not present, [Fe(CN)6] 4 could freely reach the electrode surface and produce a clear oxidation peak near 0.23 V. When APN was present, specific binding of APN to the antibody increased the steric hindrance of the electrode surface, hindering the electron transfer process of the probe, thereby causing a significant decrease in the electrochemical signal. 4

[0048] Fifth, comparison with the results of gold screen-printed electrodes

[0049] To make a comparison, all the immunosensor preparation and detection steps were performed on gold screen-printed electrodes (AuSPEs) in the same procedure for performance evaluation. As shown in Figure 3 , the gold-modified silver screen-printed electrodes (Au / AgSPEs) showed significantly enhanced reproducibility in 10 repeated measurements in a 100 mM [Fe(CN solution containing 0.1 M KCl, with a relative standard deviation (RSD) of 6.3%, which was 4.2 times higher than that of the traditional gold screen-printed electrodes (RSD = 26.3%).

[0050] ​The electrochemical immunosensor for detecting adiponectin has the following advantages:

[0051] 1. The detection efficiency is greatly improved to meet the requirement of rapid diagnosis: the rapid process of "15 minutes for completing detection" is realized, and the total analysis time is shortened by 11 times compared with the traditional ELISA (160 minutes). The whole process is rapid and the operation is simple, thereby providing a high-performance detection scheme for clinical examination and primary medical institutions.

[0052] 2. The preparation process is simplified, and the reproducibility and stability are improved: through the direct covalent combination of thiolated antibodies and gold nanoparticles, the traditional multi-step coupling process (such as EDC / NHS) is replaced, the complex modification steps are avoided, and the batch difference is reduced.

[0053] 3. High sensitivity and low detection limit are realized: the detection limit is 0.097 mg / L, which meets the sensitivity requirement of clinical quantitative detection of adiponectin, and can be used for accurate detection of low-concentration samples.

[0054] 4. The cost and equipment dependence are reduced: the detection process only needs a portable electrochemical workstation, without the need of large biochemical analysis equipment. The screen printing electrode and sputtering process support large-scale production, which is conducive to reducing the cost and promoting.

[0055] The above-described embodiments are only used to describe the preferred embodiments of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A method for preparing an electrochemical immunosensor for detecting adiponectin, characterized in that: Includes the following steps: (1) Place the silver wire mesh printed electrode in a vacuum ion sputtering instrument and sputter a nanoscale gold film on the surface of the silver working electrode; the sputtering time of the vacuum ion sputtering instrument is 80-100 s and the sputtering current is 20-30 mA. (2) The thiolized adiponectin monoclonal antibody was drop-coated onto the surface of the gold film working electrode, placed in a constant temperature and humidity cabinet, allowed to stand, rinsed the electrode surface with deionized water, and dried with nitrogen gas to obtain a screen-printed electrode modified with adiponectin monoclonal antibody; the concentration of the thiolized adiponectin monoclonal antibody was 50 μg / ml, and the amount used was 15 μL. (3) Passivate the electrode surface, take bovine serum albumin solution, drop it onto the electrode surface, place it in a constant temperature and humidity cabinet, let it stand, rinse the electrode surface with deionized water, and blow it dry with nitrogen to obtain the final adiponectin immunosensor.

2. The method for preparing the electrochemical immunosensor for detecting adiponectin according to claim 1, characterized in that: The temperature of the constant temperature and humidity cabinet mentioned in steps (2) and (3) is 37 ℃, the humidity is 80%, and the standing time is 45-60 min.

3. The method for preparing the electrochemical immunosensor for detecting adiponectin according to claim 2, characterized in that: In step (3), the concentration of bovine serum albumin is 1% and the amount used is 20 μL.

4. The product prepared by the method for preparing the electrochemical immunosensor for detecting adiponectin according to any one of claims 1 to 3.

5. A method for quantitative detection of adiponectin in solution using the product described in claim 4, characterized in that: Includes the following steps: 20 μL of the sample solution to be tested was dropped onto the electrochemical immunosensor and incubated at room temperature for 15 min. Then, it was rinsed with deionized water to remove non-specifically adsorbed adiponectin and dried under a nitrogen stream. The sensor was then connected to an electrochemical workstation, and 20 μL of a 100 mM K4[Fe(CN)6] solution containing 0.1 M KCl was added. Differential pulse voltammetry was used for detection, and the adiponectin content in the solution was calculated based on the results according to the standard curve. The parameters for the differential pulse voltammetry detection are set as follows: potential range -0.1 to 0.5 V, amplitude 50 mV, pulse width 0.06 s, pulse period 0.5 s, and sampling width 0.02 s.

6. The method for quantitative detection of adiponectin in solution according to claim 5, characterized in that: The method for creating the standard curve includes the following steps: (A) Preparation of standard working solutions: Adiponectin standard solutions of different concentrations were prepared, namely 0, 0.5 mg / L, 1 mg / mL, 3 mg / L, 5 mg / L, 10 mg / L and 15 mg / L, for a total of 7 concentrations; before the measurement, the 7 concentrations of adiponectin standard solutions were respectively dropped onto the surface of 7 electrochemical immunosensors, left to stand at room temperature for 15 min, then rinsed thoroughly with deionized water and dried with nitrogen gas; (B) Plotting the standard curve: Connect the 7 electrochemical immunosensors obtained in step (A) to the electrochemical workstation for testing. Add 20 μL of 100 mM K4[Fe(CN)6] solution containing 0.1 M KCl and detect using differential pulse voltammetry. The magnitude of the peak current is linearly related to the logarithm of the adiponectin solution concentration, thus obtaining the standard curve.

7. The method for quantitative detection of adiponectin in solution according to claim 5, characterized in that: The preparation method of the test sample solution is as follows: the biological sample is diluted 10 times with phosphate buffer solution to obtain the test sample solution, wherein the biological sample is blood, plasma or serum, and the concentration of the phosphate buffer solution is 0.01 M and the pH value is 7.4.

Images