Electrochemical immunosensor for detecting adiponectin as well as preparation method and application of electrochemical immunosensor

Abstract

The invention belongs to the technical field of electrochemical analysis and detection, and particularly relates to an electrochemical immunosensor for detecting adiponectin as well as a preparation method and application of the electrochemical immunosensor. The preparation method comprises the following steps: (1) connecting a screen-printed electrode SPCE to an electrochemical workstation by using a 0.01 M PBS (Phosphate Buffer Solution) with the pH value of 7.4, performing-0.2 V-1. 2V cyclic voltammetry scanning, and activating for 10 circles under 50mV / S; and (2) by taking 0.01-1M H2SO4 as a base solution, connecting the screen-printed electrode SPCE to an electrochemical workstation, carrying out electro-polymerization on poly-o-phenylenediamine in a 50-100mM o-phenylenediamine and NH2-UiO-66 solution to form a poly-o-phenylenediamine and NH2-UiO-66 composite modified electrode, washing with deionized water, and drying at room temperature. (3) soaking the modified electrode in a glutaraldehyde solution for 10-70 minutes, cleaning with deionized water, drying at room temperature, adding 10-100 [mu] L of ADPN antibody solution and 100-600 [mu] g / mL of ADPN antibody solution, and incubating; and (4) washing with ionized water, drying at room temperature, preventing non-specific binding sites at 37 DEG C for 0.5-1.5 hours by using 3-10 microliters of 0.8-1.2 wt% casein solution, and washing the screen-printed electrode again to obtain the electrochemical immunosensor.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical analysis and detection technology, and more specifically, relates to an electrochemical immunosensor for detecting adiponectin, its preparation method, and its application. Background Technology

[0002] Adiponectin, a core metabolic regulator secreted by adipose tissue, has become a research hotspot in life sciences and medicine in recent years due to its crucial role in the body's metabolic network and its cross-system regulatory effects. With the continuous rise in the incidence of metabolic diseases (such as type 2 diabetes and obesity), cardiovascular diseases, cancer, and neurodegenerative diseases, elucidating the mechanism of action of adiponectin and establishing accurate and efficient detection methods are of significant scientific and clinical value for early diagnosis, risk assessment, and targeted intervention. Currently, the main detection methods for adiponectin include enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and traditional electrochemical sensor methods, but all have significant technical bottlenecks: ELISA is cumbersome to operate, has a long detection cycle (requiring 4-6 hours), a detection limit of only 0.1 μg / mL, and cannot achieve real-time detection; HPLC has high equipment costs and complex sample pretreatment, making it unsuitable for primary healthcare settings; traditional electrochemical sensors often use single conductive polymers or NH2-UiO-66s materials for modification, resulting in low electron transfer rates (e.g., the electron transfer rate of a single PoPD-modified electrode). Problems such as low antibody immobilization (≤50 μg / cm²) and weak anti-interference ability (selectivity coefficient for interfering substances in blood ≤50) lead to insufficient detection sensitivity and specificity, making it difficult to meet the needs of accurate detection of adiponectin in complex samples. Therefore, developing an adiponectin detection sensor that combines high sensitivity, high specificity, and convenience has become an urgent technical problem to be solved in this field.

[0003] The close link between adiponectin and metabolism is primarily reflected in the fact that adiponectin, as a key biomarker of metabolic state, directly reflects the degree of metabolic disorder. Accurate detection of adiponectin levels is a core element in elucidating metabolic regulation mechanisms, assessing metabolic disease risk, and monitoring intervention effectiveness. From a metabolic regulation perspective, adiponectin regulates glucose and lipid metabolism through the AdipoR1 / 2-AMPK / PPARα signaling axis. Its serum levels are closely related to insulin resistance, glycemic control, and lipid profiles (such as triglycerides). For example, serum adiponectin levels are generally lower in patients with type 2 diabetes mellitus (T2DM), and the degree of decrease is negatively correlated with the homeostasis model of insulin resistance (HOMA-IR). High-sensitivity detection of adiponectin using electrochemical sensors can detect metabolic abnormalities early, providing a basis for early warning of metabolic diseases such as diabetes. Simultaneously, in metabolic intervention studies (such as drug therapy and lifestyle interventions), real-time monitoring of adiponectin level changes can directly reflect the metabolic regulatory effects of intervention measures, contributing to the optimization of precise intervention programs. Furthermore, the combined detection of adiponectin with other metabolic markers (such as glucose, triglycerides, and direct bilirubin) can further improve the diagnostic efficacy of metabolic disease complications (such as macrovascular disease). The multi-analyte simultaneous detection capability of electrochemical sensors provides technical support for achieving this goal, and it is expected that an integrated detection device combining adiponectin with multiple metabolic markers can be developed, providing an efficient tool for the comprehensive assessment of metabolic diseases.

[0004] Conductive polymers (such as polyaniline, polypyrrole, and poly(o-phenylenediamine)) have attracted much attention as core materials for electrochemical sensors due to their unique redox activity and processability. Among them, the amino groups on the surface of poly(o-phenylenediamine) (PoPD) films... The amino groups (-NH-) and imino groups can be chemically modified to introduce specific recognition groups, providing a material basis for the accurate detection of adiponectin. For example, the dopamine sensor constructed by the team at Beijing Normal University through thiophosphorylation modification of PoPD aptamers retained more than 90% of the signal response after 1 hour of ExoI enzyme treatment. This type of modification strategy can be transferred to the construction of adiponectin aptamer sensors. By modifying the surface of PoPD membranes with adiponectin-specific aptamers, the specific binding reaction between the aptamers and adiponectin triggers changes in electrical signals, achieving highly specific and sensitive detection of adiponectin. This provides an innovative technical path for adiponectin metabolism-related research and clinical detection. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide an electrochemical immunosensor of conductive polymer and NH2-UiO-66 composite material, its preparation method, and its application in ADPN detection. This invention provides a new method for detecting adiponectin by providing a new method for antibody immobilization, preparing poly(o-phenylenediamine) PoPD and using it as a sensing platform, immobilizing ADPN antibodies with glutaraldehyde, and using an electrochemical immunosensor for ADPN analysis.

[0006] The core innovation of this invention lies in the construction of a PoPD and NH2-UiO-66 composite modification layer. NH2-UiO-66 (Zr-based) possesses an ultra-high specific surface area and abundant amino active sites, significantly increasing antibody immobilization. Poly(o-phenylenediamine) (PoPD) exhibits excellent electron transport properties, accelerating electron transfer on the electrode surface and enhancing detection sensitivity. The composite film formed by electropolymerization achieves synergistic optimization of "high antibody loading" and "high electron transport efficiency," overcoming the performance bottleneck of traditional single-material modified electrodes. Furthermore, this invention innovatively uses casein as a blocking agent, improving the non-specific binding blocking rate by more than 30% compared to traditional BSA blocking agents, further enhancing detection specificity. The poly(o-phenylenediamine) modification of the screen-printed electrode is performed using electropolymerization of o-phenylenediamine. Electropolymerization of o-phenylenediamine is carried out in a sulfuric acid solution using cyclic voltammetry. After electrode modification, the electrode is thoroughly cleaned and immersed in glutaraldehyde. After incubation with ADPN antibody, casein incubation is used to prevent non-specific binding, also serving to block the sensor.

[0007] The inventors discovered that casein provides a better blocking effect than the commonly used bovine serum albumin (BSA). In this invention, the screen-printed electrode includes a carbon working electrode, a carbon counter electrode, and an Ag / AgCl reference electrode. The immunosensor is prepared by sequentially performing electrode surface activation, electropolymerization, antibody immobilization-Ab incubation, and blocking with a blocking solution-casein to prevent non-specific binding on the screen-printed electrode.

[0008] A method for preparing an electrochemical immunosensor for detecting adiponectin (ADPN) includes the following steps: (1) Activation of screen-printed carbon electrode: The screen-printed electrode SPCE was connected to the electrochemical workstation with 0.01M pH7.4 PBS as the base solution and activated for 10 cycles at 50mV / S by cyclic voltammetry scan of -0.2V-1.2V. (2) Electropolymerization of o-phenylenediamine and NH2-UiO-66: Using 0.01-1M H2SO4 as the base solution, the screen-printed electrode SPCE was connected to the electrochemical workstation. In a 50-100 mM o-phenylenediamine and NH2-UiO-66 solution, o-phenylenediamine was electropolymerized to form a PoPD and NH2-UiO-66 composite modified electrode. After rinsing with deionized water, it was dried at room temperature. (3) Immobilize ADPN antibody: Immerse the modified electrode in glutaraldehyde solution for 10-70 min, wash with deionized water and dry at room temperature, then add 10-100 μL of ADPN antibody solution with a concentration of 100-600 μg / mL and incubate. (4) Casein blocking: After washing with deionized water, dry at room temperature, use 3-10 μL of 0.8-1.2 wt% casein solution at 37℃ to block non-specific binding sites for 0.5-1.5 h, and rinse the screen-printed electrode again to obtain the electrochemical immunosensor.

[0009] In a further optimization of this technical solution, step (2) employs cyclic voltammetric electropolymerization with a voltage of -0.2 to 1.2V, a scan rate of 40 to 60 mV / s, and a cycle count of 25 to 35 times.

[0010] This technical solution is further optimized by using a cyclic voltammetry method with a voltage of -0.2 to 1.2 V, a scan rate of 50 mV / s, 28 to 30 cycles, and a 50 mM solution of o-phenylenediamine and NH2-UiO-66.

[0011] In a further optimization of this technical solution, the mass ratio of NH2-UiO-66 to poly(o-phenylene diamine) is 1:5-1:10.

[0012] In a further optimization of this technical solution, step (3) involves immersing the modified electrode in a 2.5% glutaraldehyde solution for 1-6 hours, followed by rinsing with deionized water.

[0013] In a further optimization of this technical solution, step (4) involves blocking non-specific binding sites with 8 μL of 1 wt% casein solution at 37°C for 1-1.5 h.

[0014] An electrochemical immunosensor for detecting adiponectin was prepared using the above-described technical solution.

[0015] An electrochemical immunosensor method for detecting adiponectin in solution includes the following steps: A series of adiponectin-specific antigen solutions of standard concentrations are prepared, and 6-10 μL of each solution is dropped onto the surface of the electrochemical immunosensor. A square wave voltammetry measurement at a frequency of 15 Hz is performed in 0.01 M PBS at pH 7.4 under a potential scan of -1 to 0.2 V. A standard curve is established with the concentration of adiponectin-specific antigen as the abscissa and the electrochemical current intensity as the ordinate, yielding a linear equation. The sample solution is then detected and calculated based on the standard curve or the linear equation.

[0016] In a further optimization of this technical solution, the sample solution is urine, blood, serum, plasma, or sweat.

[0017] Further optimization of this technical solution resulted in a detection limit of 0.02 μg / mL for adiponectin, a linear range of 0.5-35 μg / mL, and a relative standard deviation of ≤2.0%.

[0018] Unlike existing technologies, the above technical solution has the following beneficial effects: This invention proposes a novel strategy for detecting adiponectin using an electrochemical immunosensing platform based on a conductive polymer and an NH2-UiO-66 composite material. The strategy involves immobilizing antibodies with poly(o-phenylenediamine) and NH2-UiO-66 and glutaraldehyde, using poly(o-phenylenediamine) and NH2-UiO-66 as the electrosignal medium and antibody carrier and sensing platform for adiponectin detection. Simultaneously, this invention constructs an electrochemical immunosensor for adiponectin (ADPN) analysis by preparing poly(o-phenylenediamine) and directly using it as the electrochemical immunosensing platform. The electrochemical immunosensor for ADPN detection is constructed based on a screen-printed electrode (SPCE), which is flexible and can be integrated into wearable devices as a flexible biosensor. In this invention, the electrochemical current intensity difference shows a linear relationship with the adiponectin antigen concentration between 1-35 μg / mL, with the linear equation being y = 0.3318x + 4.8673 (R²). 2 =0.9948), with a linear correlation coefficient of 0.9948 and a detection limit of 0.02 μg / mL. The poly(o-phenylenediamine) and NH2-UiO-66 / glutaraldehyde screen-printed electrode sensor of this invention exhibits a wide linear range and a low detection limit for adiponectin, while also demonstrating good anti-interference performance, thus providing a new method for further detection of adiponectin. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the preparation process of the electrochemical immunosensing platform of the present invention and its application in the detection of adiponectin. Figure 2 This describes the carbon particle morphology on the surface of the working electrode of the bare wire mesh electrode of the present invention. Figure 3 This is the surface morphology of the working electrode after electropolymerization modification on the screen electrode of the present invention; Figure 4 This is the surface morphology of the working electrode after electropolymerization / immersion in glutaraldehyde on the wire mesh electrode of the present invention; Figure 5 This is the surface morphology of the working electrode after electropolymerization / immersion in glutaraldehyde / antibody incubation on the screen electrode of the present invention; Figure 6 This is the surface morphology of the working electrode after electropolymerization / immersion in glutaraldehyde / antibody incubation / casein blocking on the screen electrode of the present invention; Figure 7 The EIS plots for this invention show that the measurement frequency range of the EIS for bare SPCE, PoPD and NH2-UiO-66 / SPCE, GA / PoPD and NH2-UiO-66 / SPCE, Ab / GA / PoPD and NH2-UiO-66 / SPCE, and CN / Ab / GA / PoPD and NH2-UiO-66 / SPCE is 0.02 Hz-100 kHz, and the amplitude is 5 mV / s. Figure 8 The CV measurement scanning range of this invention is -1.2-0.2V, the scanning rate is 150mV / s, and the samples are bare SPCE, PoPD and NH2-UiO-66 / SPCE, GA / PoPD and NH2-UiO-66 / SPCE, Ab / GA / PoPD and NH2-UiO-66 / SPCE, and CN / Ab / GA / PoPD and NH2-UiO-66 / SPCE. Figure 9 The 2.5% glutaraldehyde soaking time of this invention has been optimized; Figure 10 To optimize the incubation concentration of the ADPN antibody in this invention; Figure 11 The incubation time for the ADPN antibody of this invention was optimized; Figure 12 The incubation time for the ADPN antigen in this invention was optimized; Figure 13 The SWV detection results of the antigen standard solutions of the present invention represent the detection results of the immunosensor at concentrations of 0.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 35 pg / mL, respectively. Figure 14 The responses of different interfering agents to SWV in this invention were measured using 10 μg / mL BSA, 10 μg / mL IL-6, 10 μg / mL HSA, IgG, 0.01M PBS, and 20 μg / mL ADPN. Figure 15 This is to ensure the batch repeatability of the sensor in this invention. Detailed Implementation

[0020] To explain in detail the technical content, structural features, objectives, and effects of the technical solution, the following description is provided in conjunction with specific embodiments and accompanying drawings.

[0021] This invention is based on the specific binding of antigen and antibody. A novel immunosensor is designed by modifying a screen-printed electrode with poly(o-phenylenediamine) and NH2-UiO-66 to obtain a modified electrode, then immobilizing an ADPN antibody on the electrode surface using glutaraldehyde crosslinking, and combining this with signal amplification technology. Specifically, poly(o-phenylenediamine) and NH2-UiO-66 are electropolymerized onto a screen-printed electrode, followed by chemical crosslinking of the antibody with glutaraldehyde. The fabrication process of the biosensor was characterized using scanning electron microscopy, Fourier transform infrared spectroscopy, and cyclic voltammetry. The fabricated biosensor was detected using square wave impulse voltammetry (SWV).

[0022] The preparation method of the electrochemical immunosensor for detecting adiponectin includes the following steps: 1. Activation of screen-printed carbon electrode: The screen-printed carbon electrode was immersed in 0.01M pH7.4 PBS solution containing 0.1M KCl. The electrode was activated for 10 cycles at 50mV / S by cyclic voltammetry scans from -0.2V to 1.2V. 2. Electropolymerization of o-phenylenediamine and NH2-UiO-66: Using 0.01M PBS as the substrate, the screen-printed electrode SPCE was connected to the electrochemical workstation. Electropolymerization of o-phenylenediamine (PoPD) was performed in 50-100 mM o-phenylenediamine and NH2-UiO-66 solutions using cyclic voltammetry at a voltage of -0.2-1.2 V, a scan rate of 40-60 mV / s, and 25-35 cycles. 3. Immobilize ADPN antibody: After electropolymerizing o-phenylenediamine, the screen-printed electrode is cleaned with deionized water, soaked in glutaraldehyde, dried at room temperature, and then 10 μL of ADPN antibody solution is added to the surface of the modified electrode and incubated. 4. Casein blocking: Use 3-8 μL of 0.8-1.2 wt% casein (CS) solution to block non-specific binding sites at 37°C for 0.5-1.5 hours, and rinse the electrode again to obtain the electrochemical immunosensor.

[0023] In the electrochemical immunosensor described above, the voltage for cyclic voltammetry in step 2 is -0.2 to 1.2 V, the scan rate is 50 mV / s, and the cycle duration is 28 to 30 cycles. The optimal concentrations of o-phenylenediamine and NH₂-UiO₆₆ solution are 50 mM. In step 4, the modified electrode is immersed in a 2.5% glutaraldehyde solution for 1 to 6 hours, followed by rinsing with deionized water. Then, it is blocked with 8 μL of casein solution (1 wt%) at 37°C to inhibit non-specific binding sites for 1 to 1.5 hours. The optimal immersion time in the glutaraldehyde solution is 1 hour. Furthermore, under these optimal conditions, SWV detection can be performed after incubation with ADPN sample solution.

[0024] An electrochemical immunosensor method for detecting adiponectin in solution includes the following steps: A series of adiponectin-specific antigen solutions of standard concentrations are prepared, and 6-10 μL of each solution is dropped onto the surface of the electrochemical immunosensor. A square-wave voltammetry measurement at a frequency of 15 Hz is performed in 0.01M PBS at pH 7.4 under a potential scan of -1 to 0.2 V. A standard curve is established with the concentration of adiponectin-specific antigen as the abscissa and the electrochemical current intensity as the ordinate, yielding a linear equation. The sample solution is then detected and calculated based on the standard curve or the linear equation.

[0025] Sample solutions can be urine, blood, serum, plasma, or sweat. The detection limit for adiponectin is 0.02 μg / mL, the linear range is 0.5–35 μg / mL, and the relative standard deviation is ≤2.0%.

[0026] The working principle of this invention is as follows: As shown in Figure 1, based on the electropolymer and NH2-UiO-66s composite material and the specific binding of antigen and antibody, and by modifying the screen-printed electrode with poly(o-phenylenediamine) to obtain a poly(o-phenylenediamine) modified electrode, the ADPN antibody is cross-linked with glutaraldehyde and immobilized on the electrode surface, thereby realizing the detection of ADPN. PoPD not only improves the electron transfer rate, but also provides a platform for antibody immobilization.

[0027] (1) SEM characterization The surface morphology of the prepared electrode was characterized using scanning electron microscopy (SEM). Figure 2 In the process, a large number of carbon particles are distributed on the surface of the bare SPCE electrode. Figure 3 In the process of polymerization, poly(o-phenylenediamine) and NH2-UiO-66 are uniformly coated on the surface of SPCE. Figure 4-6 In addition to glutaraldehyde, adiponectin antibody, and casein, the surface roughness of the electrode decreased significantly.

[0028] (2) Electrochemical characterization – EIS characterization The interfacial properties of the modified electrode were analyzed using EIS. Charge transfer resistance R CT The value reflects [Fe(CN6)] 3- / 4- The electron transfer kinetics of redox probes can be measured by... Nyquist The diameter of the high-frequency semicircle in the diagram is used to determine this. For example... Figure 7 As shown, EIS analysis revealed that the bare SPCE value significantly decreased after POPD and NH2-UiO-66 modification. However, due to the presence of glutaraldehyde, which inhibits electron transfer, the resistance increased when glutaraldehyde was modified on the electrode surface. Subsequently, after antibody modification, a gradual increase in resistance was observed. The decrease in current and the accompanying increase in current can be explained by the negatively charged phosphate backbone and [Fe(CN6)]. 3- / 4- The electrostatic repulsion generated between them, and the non-conductive properties of the proteins present on the electrode surface.

[0029] (3) Electrochemical characterization – CV characterization like Figure 8 The stepwise fabrication process of the immunosensor, characterized by CV assays performed in 0.01 M PBS buffer, is shown. The peak current of the redox peak of the bare SPCE significantly increased after modification with POPD and NH2-UiO-66. However, the immobilization of glutaraldehyde on the electrode surface led to a decrease in the peak current, indicating an increase in electron impedance due to the presence of glutaraldehyde. Immobilization with adiponectin antibody further reduced the peak current. Subsequently, blocking with casein resulted in a further decrease in the peak current due to the presence of casein, collectively demonstrating the successful fabrication of the immunosensor.

[0030] (4) Optimization of test conditions To explore optimal analytical performance, relevant experimental parameters were systematically optimized. The soaking time in 2.5% glutaraldehyde was preliminarily optimized to improve electrocatalytic activity and reduce the cost of developing immunosensors. Figure 9 In the assay, the immunosensor exhibited the best performance after soaking in 2.5% glutaraldehyde for 1 hour, after which the current began to decrease. Therefore, the optimal soaking time in 2.5% glutaraldehyde is 1 hour. The incubation concentration of adiponectin antibody was optimized. Figure 10 As shown, the current difference reached its maximum at a concentration of 300 μg / mL for adiponectin antibody. Therefore, 300 μg / mL of adiponectin antibody was used in the experiment. The incubation time of the antibody also has a significant impact on the performance of the immunosensor. Figure 11 As shown, when the antibody incubation time increased from 10 min to 110 min, the peak current response was highest at 70 min. After that, the current response no longer increased progressively. Therefore, the optimal incubation time for the antibody was chosen to be 70 min. Generally, the incubation time of the antigen affects the performance of the entire reaction system. Figure 12The results showed that extending the incubation time from 5 minutes to 40 minutes resulted in a rapid increase in the current response. However, after 30 minutes, a slight decrease in the current value of the immunosensor was observed. Therefore, the optimal immunoreaction time was selected as 30 minutes.

[0031] (5) Drawing working curves A series of standard concentrations of adiponectin antigen solutions were prepared. Under optimal experimental conditions, the changes in electrochemical current intensity of these standard concentrations of ADPN-specific antigen were measured. In other words, the electrochemical immunosensors with different concentrations of ADPN-specific antigen were recorded. Figure 13 As shown, this figure is a square-wave voltammogram of the electrochemical immunosensor for different specific antigens. The working curves are plotted with the concentration of the ADPN-specific antigen on the x-axis and the change in electrochemical current intensity on the y-axis, as shown below. Figure 13 As shown, the peak current difference exhibits a linear relationship with the ADPN antigen concentration between 0.5 and 35 μg / mL, and the linear equation of the working curve is y = 0.3318x + 4.8673 (R²). 2 =0.9948), the linear correlation coefficient was 0.9948, and the detection limit was 0.02 μg / mL.

[0032] (6) Anti-interference performance study: like Figure 14 As shown, in order to study the response of biosensors to common proteins in blood samples, BSA, IL-6, IgG, HSA, and PBS were selected as interfering substances. Figure 14 As shown, the selectivity of the immunoassay is acceptable, and nonspecific adsorption is negligible.

[0033] (7) Inter-batch reproducibility study: To investigate the reproducibility of the immunosensors, a series of electrochemical adiponectin immunosensors were prepared using the same method to detect samples with an antigen concentration of 10 μg / mL. For example... Figure 15 The relative standard deviation (RSD) calculated using the response current value is 1.69%, confirming that the immunosensor manufacturing process of this study has high reproducibility.

[0034] (9) Accuracy analysis of real samples To verify the accuracy of the electrochemical immunosensor prepared in this invention in the detection of actual samples, real sample tests were conducted, and the results are shown in Table 1. The table uses the Iprocon detection value as a reference standard, and the sensor's detection results were examined through parallel experiments (parallel 1, parallel 2, and parallel 3). The data show that the values ​​measured by the sensor of this invention are in high agreement with the reference values, and the parallel experimental results also show good repeatability, indicating that this sensor can be used for the accurate detection of adiponectin (ADPN) in actual samples.

[0035] Table 1. Accuracy Analysis of Real Samples This invention innovatively employs an electropolymerization process to form a uniform composite film of PoPD and NH2-UiO-66 on the surface of a screen-printed electrode (SPCE). Leveraging the synergistic effect of the high electron transport rate of PoPD and the high specific surface area of ​​NH2-UiO-66, ADPN antibodies are immobilized via covalent cross-linking with glutaraldehyde, and non-specific binding sites are blocked with casein, thus constructing a highly efficient immune recognition system. Compared with existing adiponectin detection sensors, the composite modified electrode of this invention overcomes the technical bottlenecks of insufficient specific surface area, low antibody immobilization, and low electron transport efficiency of traditional conductive polymer modified electrodes. It exhibits a wide detection linear range of 0.5-35 μg / mL and a detection limit as low as 0.02 μg / mL. The relative standard deviation (RSD) is only 1.69%, and the selectivity coefficients for interfering substances such as BSA, IL-6, IgG, and HSA in blood are all greater than 100. The sensor of this invention has the potential for flexible wearable integration, is easy to operate and low in cost, and can realize rapid and accurate detection of ADPN in complex samples such as urine, blood and serum, providing an innovative technical means for the early diagnosis of metabolic diseases, cardiovascular diseases and other diseases, while providing a general strategy for the construction of immune sensing platforms for other biomarkers.

[0036] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Unless otherwise specified, an element defined by the phrase "comprising..." or "including..." does not exclude the presence of additional elements in the process, method, article, or terminal device that includes said element. Additionally, in this document, "greater than," "less than," "exceeding," etc., are understood to exclude the stated number; "above," "below," "within," etc., are understood to include the stated number.

[0037] Although the above embodiments have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the above descriptions are merely embodiments of the present invention and do not limit the scope of patent protection of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A method for preparing an electrochemical immunosensor for detecting adiponectin, characterized by, Includes the following steps: (1) Activation of screen-printed carbon electrode: The screen-printed electrode SPCE was connected to the electrochemical workstation with 0.01M pH7.4 PBS as the base solution and activated for 10 cycles at 50mV / S by cyclic voltammetry scan of -0.2V-1.2V. (2) Electropolymerization of o-phenylenediamine and NH2-UiO-66: Using 0.01-1M H2SO4 as the base solution, the screen-printed electrode SPCE was connected to the electrochemical workstation. In a mixed solution containing 50-100 mM o-phenylenediamine and NH2-UiO-66, o-phenylenediamine was electropolymerized to form a composite modified electrode of o-phenylenediamine and NH2-UiO-66. After rinsing with deionized water, it was dried at room temperature. (3) Immobilize ADPN antibody: Immerse the modified electrode in glutaraldehyde solution for 10-70 min, wash with deionized water and dry at room temperature, then add 10-100 μL of ADPN antibody solution with a concentration of 100-600 μg / mL and incubate. (4) Casein blocking: After antibody incubation, wash with deionized water, dry at room temperature, and block non-specific binding sites with 3-10 μL of 0.8-1.2 wt% casein solution at 37°C for 0.5-1.5 h. Rinse the screen-printed electrode again to obtain the electrochemical immunosensor.

2. The preparation method of the electrochemical immunosensor for detecting adiponectin according to claim 1, wherein, In step (2), cyclic voltammetric electropolymerization is used with a voltage of -0.2 to 1.2V, a scan rate of 40 to 60 mV / s, and 25 to 35 cycles.

3. The method for preparing the electrochemical immunosensor for detecting adiponectin as described in claim 2, characterized in that, The cyclic voltammetry was performed at a voltage of -0.2 to 1.2 V, a scan rate of 50 mV / s, for 28 to 30 cycles, using 50 mM o-phenylenediamine and NH2-UiO-66 solution.

4. The method for preparing the electrochemical immunosensor for detecting adiponectin as described in claim 1, characterized in that, The mass ratio of NH2-UiO-66 to poly(o-phenylene diamine) is 1:5 to 1:

10.

5. The method for preparing the electrochemical immunosensor for detecting adiponectin as described in claim 1, characterized in that, In step (3), the modified electrode is immersed in 2.5% glutaraldehyde solution for 1-6 hours and then rinsed with deionized water.

6. The method for preparing the electrochemical immunosensor for detecting adiponectin as described in claim 1, characterized in that, In step (4), the non-specific binding sites are blocked by using 8 μL of 1 wt% casein solution at 37°C for 1-1.5 h.

7. An electrochemical immunosensor for detecting adiponectin is prepared using the preparation method described in any one of claims 1-6.

8. A method for detecting adiponectin in solution using the electrochemical immunosensor for detecting adiponectin as described in claim 7, characterized in that: The procedure includes the following steps: preparing a series of adiponectin-specific antigen solutions of standard concentrations, and applying 6-10 μL of each solution to the surface of an electrochemical immunosensor. Under a potential scan of -1 to 0.2 V, square wave voltammetry at a frequency of 15 Hz is performed in 0.01 M PBS at pH 7.

4. A standard curve is established with the concentration of adiponectin-specific antigen as the abscissa and the electrochemical current intensity as the ordinate, yielding a linear equation. The sample solution is then detected and calculated based on the standard curve or linear equation.

9. The method for detecting adiponectin as described in claim 8, characterized in that: The sample solution is urine, blood, serum, plasma, or sweat.

10. The method for detecting adiponectin as described in claim 8, characterized in that: The detection limit for adiponectin is 0.02 μg / mL, the linear range is 0.5-35 μg / mL, and the relative standard deviation is ≤2.0%.

Images