Electrochemical immunosensor for detecting adiponectin and quantitative detection method thereof

Abstract

The invention discloses a preparation method of an electrochemical immunosensor for detecting adiponectin and a method for quantitatively detecting the adiponectin by adopting the electrochemical immunosensor, provides a new strategy for fixing an adiponectin antibody by a one-step method, and constructs the electrochemical immunosensor based on a gold sputtering silver wire screen printing electrode (Au / AgSPE). The rapid, high-sensitivity and stable detection of the adiponectin is realized, so that the conversion of the adiponectin detection technology to instant detection application is promoted. Only a portable electrochemical workstation is needed in the detection process, and large biochemical analysis equipment is not needed. And the screen printing electrode and the sputtering technology support large-scale production, and cost reduction and popularization are facilitated.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, and in particular to an electrochemical immunosensor for detecting adiponectin and a quantitative detection method thereof. Background Technology

[0002] Adiponectin (APN), an important adipose-derived factor secreted by adipose tissue, plays a crucial role in regulating physiological processes such as lipid metabolism, energy balance, insulin sensitivity, and inflammatory responses. Its serum levels are 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 disease prediction, treatment efficacy assessment, and clinical decision-making.

[0003] Currently, the most commonly used methods for adiponectin detection are enzyme-linked immunosorbent assay (ELISA), turbidimetric immunoassay (LTIA), and chemiluminescent enzyme immunoassay (CLEIA). Radioimmunoassay (RIA) can also be used to measure adiponectin. ELISA has a long processing time (2-3 hours), requires large sample volumes (100-200 μL), and involves complex procedures (centrifugation, incubation, blocking, washing, etc.), requiring specialized instruments (ELISA readers) and technical personnel. While turbidimetric immunoassay significantly reduces processing time (10 minutes), it still relies on large sample volumes, specialized instruments (fully automated biochemical analyzers), and offline blood centrifugation. Chemiluminescent enzyme immunoassay also requires a fully automated biochemical analyzer and involves cumbersome steps such as centrifugation, incubation, and washing, with a processing time of 2-3 hours. Radioimmunoassay has been gradually phased out due to issues such as poor reagent stability and environmental pollution. These tests often require collecting a certain number of samples for centralized testing, resulting in long sample turnaround times, increased testing costs and waiting times for test subjects, and low testing efficiency. Currently, the market lacks inexpensive, convenient, rapid, and accurate adiponectin point-of-care testing technologies, significantly limiting the widespread application of adiponectin testing. Therefore, developing new adiponectin detection methods, especially low-cost, highly sensitive, and rapid methods for detecting trace amounts of samples, presents greater technical challenges and broader application prospects.

[0004] Electrochemical immunosensors are a class of immunosensors that combine highly specific antigen-antibody reactions with electrochemical analysis techniques, using antigens or antibodies as biorecognition elements. The performance of these sensors largely depends on the biofunctionalization modification strategy of the molecular recognition interface. Current research typically uses reagents such as EDC / NHS, 6-MH / CNBr, 3-GOPS, or Mix&Go™ to immobilize antibodies on various electrode substrates. However, these immobilization methods generally share a common drawback: they usually require two or more steps to achieve effective antibody immobilization. This complex modification process not only introduces batch-to-batch variability but also increases manufacturing costs and time, ultimately limiting the reproducibility, stability, and detection efficiency of the sensor.

[0005] Therefore, the core technological bottleneck currently facing the field of adiponectin detection lies in how to develop an efficient, stable, and simple antibody immobilization method to overcome the complexity, inconsistency, and low efficiency caused by multi-step modification, thereby constructing a rapid, low-cost, and highly sensitive adiponectin immunosensor that meets clinical requirements. Summary of the Invention

[0006] This invention aims to address the shortcomings of existing technologies by proposing a novel one-step antibody immobilization strategy to construct an electrochemical immunosensor based on a gold sputtered silver wire mesh printed electrode (Au / AgSPE), thereby achieving rapid, highly sensitive, and stable detection of adiponectin and promoting the transformation of adiponectin detection technology towards point-of-care testing applications.

[0007] A method for preparing an electrochemical immunosensor for detecting adiponectin includes the following steps: (1) Place the silver wire mesh printed electrode in a vacuum ion sputtering instrument and sputter a layer of nanoscale gold film on the surface of the silver working electrode; (2) Thiolized adiponectin monoclonal antibody was drop-coated onto the surface of the gold film working electrode, placed in a constant temperature and humidity cabinet, left to stand, rinsed the electrode surface with deionized water, and dried with nitrogen to obtain a screen-printed electrode modified with adiponectin monoclonal antibody. (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.

[0008] The method for preparing an electrochemical immunosensor for detecting adiponectin according to the present invention, wherein in step (1), the sputtering time of the vacuum ion sputtering instrument is 80-100 s and the sputtering current is 20-30 mA.

[0009] The method for preparing an electrochemical immunosensor for detecting adiponectin according to the present invention, wherein in step (2), the concentration of thiolated adiponectin monoclonal antibody is 50 μg / ml and the amount used is 15 μL.

[0010] The method for preparing an electrochemical immunosensor for detecting adiponectin according to the present invention, wherein in step (3), the concentration of bovine serum albumin is 1% and the amount used is 20 μL.

[0011] The method for preparing an electrochemical immunosensor for detecting adiponectin according to the present invention, wherein the temperature of the constant temperature and humidity chamber in steps (2) and (3) is 37 ℃ and the humidity is 80%; the standing time is 45-60 min.

[0012] The product obtained by the preparation method of the electrochemical immunosensor for detecting adiponectin described in this invention.

[0013] The electrochemical immunosensor for detecting adiponectin of the present invention provides a method for quantitative detection of adiponectin in solution, comprising the following steps: 20 μL of the adiponectin solution to be tested was dropped onto the electrochemical immunosensor and incubated at room temperature for 15 min. Subsequently, the electrode 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 solution containing 0.1 M KCl was dropped onto the sensor surface. [Fe(CN The solution was tested using differential pulse voltammetry, and the adiponectin content in the solution was calculated based on the results using a standard curve.

[0014] The quantitative detection method for adiponectin in solution according to the present invention includes the following steps for preparing the standard curve: (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 seven electrochemical immunosensors obtained in step (A) to the electrochemical workstation for testing, and add 20 μL of 100 mM solution containing 0.1 M KCl. [Fe(CN The solution was analyzed using differential pulse voltammetry, and the peak current showed a linear relationship with the logarithm of the adiponectin solution concentration, thus obtaining a standard curve.

[0015] The quantitative detection method for adiponectin in solution according to the present invention, wherein the parameters of the differential pulse voltammetry detection during the detection of the test solution and the standard solution 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.

[0016] The present invention discloses a method for quantitative detection of adiponectin in solution, wherein the preparation method of the test sample solution is as follows: a biological sample is diluted 10 times with a 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.

[0017] The electrochemical immunosensor for detecting adiponectin in this invention differs from existing technologies in that: The electrochemical immunosensor for detecting adiponectin of this invention is not only highly efficient and stable, but also has a simple method for immobilizing biorecognition elements to overcome the complexity, inconsistency, and low efficiency caused by multi-step modification, thereby constructing a high-sensitivity adiponectin immunosensor that is fast, low-cost, and meets clinical requirements.

[0018] This invention proposes a highly efficient antibody immobilization method: using thiolized antibodies, the terminal thiol groups (-S) of which directly covalently bind to gold nanoparticles on the surface of a gold-sputtered silver screen-printed electrode, achieving one-step antibody immobilization on the electrode. The entire sensor fabrication time is only 90 minutes. This overcomes the shortcomings of traditional methods (such as EDC / NHS) which require multiple reaction steps, significantly simplifies the modification process, reduces batch-to-batch variability, and improves preparation efficiency and consistency.

[0019] This invention develops a novel electrode substrate: a gold-modified silver screen-printed electrode (Au / AgSPE) is designed and fabricated as a sensing substrate. This substrate combines the excellent conductivity of silver with the superior biocompatibility and stability of gold, overcoming the interference of polymer binders in traditional gold screen-printed electrodes and significantly improving the reproducibility of the electrode.

[0020] This invention develops a rapid adiponectin detection method: the developed adiponectin immunosensor specifically binds to adiponectin and completes the detection within 15 minutes, reducing the total analysis time by 11 times compared to the traditional ELISA method (from 160 minutes to 15 minutes). The detection limit is 0.097 mg / L, meeting the sensitivity requirements for quantitative adiponectin detection in clinical settings. The method is simple to operate and rapid, providing a powerful new tool for rapid on-site screening and dynamic monitoring of adiponectin.

[0021] The electrochemical immunosensor for detecting adiponectin and its detection method of the present invention will be further described below with reference to the accompanying drawings. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the fabrication process of the electrochemical immunosensor for detecting adiponectin in this invention; Figure 2 This is a schematic diagram illustrating the principle of the electrochemical immunosensor for detecting adiponectin in this invention. Figure 3 This is a graph showing the comparison of the response values ​​of Au / AgSPEs and AuSPEs to 10 mg / L adiponectin in this invention; Figure 4 The electrochemical voltammetric curves and standard curves of adiponectin at different concentrations are shown in this invention.

[0023] Figure 5 This is an electrochemical voltammetric curve of adiponectin in plasma samples from actual sample testing. Detailed Implementation

[0024] I. Fabrication method of electrochemical immunosensor: This invention uses a silver wire-printed electrode as a substrate, modifies the surface of the silver working electrode using ion sputtering technology, and then sequentially performs antibody modification and electrode sealing, such as... Figure 1 As shown. The specific production process is as follows: Step 1: Place the silver screen-printed electrode (Changsha Sanjun Technology Co., Ltd.; Model: D-SPE; Working electrode: Ag, diameter 3mm; Counter electrode: Carbon; Reference electrode: Silver) in a vacuum ion sputtering instrument (Cressington 108Auto) to sputter a nanoscale gold film on the surface of the silver working electrode. The sputtering time is 80-100 s and the sputtering current is 20-30 mA.

[0025] Step 2: 15 μL of 50 μg / ml thiolated adiponectin monoclonal antibody (Wuhan Yunclone Technology Co., Ltd., catalog number: IS005) was drop-coated onto the surface of the gold film working electrode and placed in a constant temperature and humidity chamber (Stokes ICTHI-150) at 37 ℃ and 80% humidity for 45-60 min. The electrode surface was rinsed with deionized water and dried with nitrogen gas to obtain the adiponectin monoclonal antibody-modified screen-printed electrode.

[0026] 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.

[0027] II. Construction of Standard Curve

[0028] The specific steps for creating a standard curve include the following: (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 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.

[0029] III. Actual Sample Testing

[0030] 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 The solution was detected using differential pulse voltammetry 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 detecting the plasma sample in this embodiment are as follows: Figure 5 As shown, the adiponectin content in the sample was calculated to be 20.1 mg / L based on the standard curve.

[0031] IV. Detection Principle of the Electrochemical Immunosensor of the Present Invention

[0032] The working principle of an adiponectin monoclonal antibody-modified electrochemical immunosensor, such as... Figure 2 As shown.

[0033] 20 μL of adiponectin solution was drop-coated onto the prepared immunosensor and incubated at room temperature for 15 min. The electrode was then rinsed with deionized water to remove non-specifically adsorbed adiponectin and dried under a nitrogen stream. 20 μL of a 100 mM solution containing 0.1 M KCl was then drop-coated onto the sensor surface. [Fe(CN The solution was analyzed using differential pulse voltammetry (DPV). The parameters were 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. When an APN is not present, [Fe(CN)6] 4 It can freely reach the electrode surface and produces a distinct oxidation peak near 0.23 V. However, when APN is present, its specific binding to the antibody increases the steric hindrance of the electrode surface, hindering the absorption of [Fe(CN)6]. 4 The electron transfer process of the probe leads to a significant reduction in the electrochemical signal.

[0034] V. Comparison with results from gold wire mesh printed electrodes

[0035] For comparison, all immunosensor fabrication and detection steps were performed on gold screen-printed electrodes (AuSPEs) using the same process for performance evaluation. Figure 3 As shown, gold-modified silver screen-printed electrodes (Au / AgSPEs) were fabricated in 100 mM [Fe(CN)2] solution containing 0.1 M KCl. After 10 repeated measurements in solution, it showed significantly enhanced reproducibility with a relative standard deviation (RSD) of 6.3%, which is 4.2 times better than that of traditional gold screen printed electrodes (RSD = 26.3%).

[0036] The electrochemical immunosensor for detecting adiponectin of the present invention has the following advantages: 1. Significantly improves testing efficiency and meets the needs of rapid diagnosis: It achieves a rapid process of "completing the test in 15 minutes", and the total analysis time is reduced by 11 times compared with the traditional ELISA (160 minutes). The whole process is fast and easy to operate, providing a high-performance testing solution for clinical testing and primary healthcare institutions.

[0037] 2. Simplify the preparation process and improve reproducibility and stability: By directly covalently binding thiolized antibodies to gold nanoparticles, the traditional multi-step coupling process (such as EDC / NHS) is replaced, avoiding complex modification steps and reducing batch-to-batch differences.

[0038] 3. Achieve high sensitivity and low detection limit: The detection limit is 0.097 mg / L, which meets the sensitivity requirements for clinical quantitative detection of adiponectin and can be used for accurate detection of low concentration samples.

[0039] 4. Reduced costs and equipment dependence: The detection process requires only a portable electrochemical workstation, eliminating the need for large-scale biochemical analysis equipment. Screen-printed electrodes and sputtering processes support large-scale production, which helps reduce costs and facilitates wider adoption.

[0040] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

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 layer of nanoscale gold film on the surface of the silver working electrode; (2) Thiolized adiponectin monoclonal antibody was drop-coated onto the surface of the gold film working electrode, placed in a constant temperature and humidity cabinet, left to stand, rinsed the electrode surface with deionized water, and dried with nitrogen to obtain a screen-printed electrode modified with adiponectin monoclonal antibody. (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: In step (1), the sputtering time of the vacuum ion sputtering instrument is 80-100 s and the sputtering current is 20-30 mA.

3. The method for preparing the electrochemical immunosensor for detecting adiponectin according to claim 2, characterized in that: In step (2), the concentration of thiolated adiponectin monoclonal antibody is 50 μg / ml, and the amount used is 15 μL.

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

5. The method for preparing the electrochemical immunosensor for detecting adiponectin according to claim 4, characterized in that: The temperature of the constant temperature and humidity cabinet mentioned in steps (2) and (3) is 37 ℃ and the humidity is 80%; the standing time is 45-60 min.

6. The product prepared by the method for preparing the electrochemical immunosensor for detecting adiponectin according to any one of claims 1 to 5.

7. A method for quantitative detection of adiponectin in solution using the electrochemical immunosensor for detecting adiponectin as described in claim 6, 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; then, the sensor was connected to an electrochemical workstation, and 20 μL of 100 mM solution containing 0.1 M KCl was added. [Fe(CN The solution was tested using differential pulse voltammetry, and the adiponectin content in the solution was calculated based on the results using a standard curve.

8. The method for quantitative detection of adiponectin in solution according to claim 7, 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 seven electrochemical immunosensors obtained in step (A) to the electrochemical workstation for testing, and add 20 μL of 100 mM solution containing 0.1 M KCl. [Fe(CN The solution was analyzed using differential pulse voltammetry, and the peak current showed a linear relationship with the logarithm of the adiponectin solution concentration, thus obtaining a standard curve.

9. The method for quantitative detection of adiponectin in solution according to claim 8, characterized in that: The parameters for differential pulse voltammetry detection during the testing of the test solution and standard solution 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.

10. The method for quantitative detection of adiponectin in solution according to claim 7, 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.

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