Method for detecting human serum adiponectin by polydopamine modified silk screen carbon electrode

Abstract

This invention discloses a method for detecting adiponectin in human serum using a polydopamine-modified wire mesh carbon electrode, specifically relating to the field of adiponectin detection technology. The method involves preparing a 2 mg / mL dopamine hydrochloride solution using a 10 mM tris(hydroxymethyl)aminomethane hydrochloride buffer solution (pH=8.5) and adding it dropwise onto the surface of the working electrode. A polydopamine-modified layer is formed by electropolymerization within a range of -800 mV to 600 mV using cyclic voltammetry. A conductive layer of a poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid complex is then coated. Adiponectin antibodies are then immobilized on the surface of the modified layer via covalent binding of amino and quinone groups. After specific binding with a serum sample, the oxidation peak current change is read out from -100 mV to 350 mV in a buffer detection medium containing 5 mM ferricyanide redox pairs, and the concentration is output from a standard curve. This method improves the density of antibody immobilization sites and inhibits non-specific serum adsorption, enhancing signal stability and sensitivity, and enabling rapid, low-cost quantitative detection of trace amounts of serum.

Description

Technical Field

[0001] This invention relates to the field of adiponectin detection technology, and more specifically, to a method for detecting adiponectin in human serum using a polydopamine-modified wire mesh carbon electrode. Background Technology

[0002] Adiponectin, as a potential diagnostic biomarker for metabolic diseases, has long been clinically detected primarily using enzyme-linked immunosorbent assays (ELISA). However, this method typically requires a long detection time and relies on large instruments, making it difficult to meet the demands for rapid detection and miniaturized applications. Therefore, electrochemical immunosensors are increasingly being used for biomarker detection. Among these, screen-printed carbon electrodes are widely adopted due to their mass production capabilities and ease of surface functionalization. Existing modification strategies often employ conductive polymer layers or biocompatible coatings to achieve antibody immobilization and signal transduction. However, in complex biological samples such as serum, traditional polydopamine modifications are prone to non-specific adsorption, leading to insufficient electrochemical signal stability. Furthermore, existing adiponectin electrochemical detection methods often introduce nanomaterials such as gold nanoparticles to improve sensitivity. While this enhances the signal, it further increases the complexity and cost of preparation. Consequently, such approaches struggle to simultaneously achieve anti-interference, signal stability, and low-cost, portable applications in complex serum matrices.

[0003] Therefore, the urgent problem to be solved at this stage is how to effectively suppress non-specific adsorption and maintain signal stability during adiponectin electrochemical immunoassay in complex serum matrices, while avoiding reliance on high-cost or highly complex enhancement methods to support portable rapid detection scenarios. Summary of the Invention

[0004] To overcome the aforementioned deficiencies of the prior art, embodiments of the present invention provide a method for detecting human serum adiponectin using a polydopamine-modified wire mesh carbon electrode. By electropolymerizing a polydopamine-modified layer on the working electrode surface of the wire mesh carbon electrode and covalently immobilizing the adiponectin antibody, the electrode interface effectively suppresses non-specific adsorption in the complex serum matrix and maintains stable electrochemical signals. This method achieves rapid, trace, and quantitative detection of human serum adiponectin without relying on high-cost or highly complex reinforcing materials.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for detecting human serum adiponectin using a polyhydrochloride-modified dopamine-modified wire mesh carbon electrode includes the following steps:

[0007] S1, Preparation of the solution to be modified: Dissolve dopamine hydrochloride in a 10mM tris(hydroxymethyl)aminomethane hydrochloride buffer solution with a pH of 8.5 to prepare a 2mg / mL dopamine hydrochloride solution to obtain the solution to be modified.

[0008] S2, Electropolymerization to form modified electrode: The solution to be modified is dropped onto the working electrode surface of the wire mesh carbon electrode. The wire mesh carbon electrode forms a working electrode, a reference electrode, and a counter electrode on the same substrate, constituting a three-electrode system. Cyclic voltammetry is used to perform potential cyclic scanning on the three-electrode system. The scanning potential range is -800mV to 600mV and the scanning rate is 50mV / s, so that dopamine hydrochloride electropolymerizes on the working electrode surface to form a polydopamine hydrochloride modification layer, thus obtaining the modified electrode.

[0009] S3, Immobilizing adiponectin antibody to form an immunofunctionalized electrode: Add adiponectin antibody solution to the surface of the poly(dopamine hydrochloride) modified layer and incubate for 30 min to 90 min, so that the adiponectin antibody is immobilized on the surface of the poly(dopamine hydrochloride) modified layer through covalent binding of amino and quinone groups, and an immunofunctionalized electrode is obtained.

[0010] S4, forming a binding electrode by binding adiponectin: Add the human serum sample to be tested to the immunofunctionalized electrode and incubate it to allow the adiponectin in the human serum sample to specifically bind with the adiponectin antibody, thus obtaining the binding electrode;

[0011] S5, Electrochemical Measurement and Output of Adiponectin Concentration: The binding electrode is measured using differential pulse voltammetry in the detection medium, with a measurement potential range of -100mV to 350mV to obtain the oxidation peak current change value. The detection medium is a solution containing a 5mM potassium ferrocyanide and potassium ferrocyanide redox pair and a 10mM tris(hydroxymethyl)aminomethane hydrochloride buffer solution. Based on the correspondence between the oxidation peak current change value and the adiponectin concentration established using adiponectin standard samples ranging from 1ng / mL to 100ng / mL under the same detection medium and measurement potential range, the adiponectin concentration of the human serum sample to be tested is output.

[0012] By adopting the above technical solution, polyhydrochloric acid dopamine electropolymerization is used to modify and construct antibody immobilization sites, and adiponectin is quantitatively detected in serum samples by electrochemical signal changes, thereby improving anti-interference ability and achieving rapid quantitative detection in complex serum matrices.

[0013] In a preferred embodiment, the number of cycles for potential cyclic scanning in S2 is 3 to 6.

[0014] By adopting the above technical solution, the number of cyclic voltammetry potential scanning cycles is limited to 3 to 6 times, making the electropolymerization process of polyhydrochloric acid dopamine modified layer more controllable, which is conducive to the formation of a more stable interface modified layer and improves detection repeatability.

[0015] In a preferred embodiment, before the electropolymerization to form the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid composite conductive polymer layer in S2, a poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid composite / poly ...(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid composite / poly(3,4-ethylenedioxythiophene)-poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic

[0016] By adopting the above technical solution, a poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid composite conductive polymer layer is introduced before the formation of the polyhydrochloric acid dopamine modification layer to form a composite modification structure. The conductive polymer is used to enhance the electron transfer efficiency, thereby improving the stability of electrochemical signals and achieving synergistic optimization of signal amplification and anti-interference.

[0017] In a preferred embodiment, the coating solution concentration of the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid composite conductive polymer layer in S2 is 2 mg / mL, the coating volume is 6 μL, and the conductive polymer layer is formed by drying at 37°C for 40 min after coating.

[0018] By adopting the above technical solution, the concentration, coating volume and drying conditions of the conductive polymer layer are limited to form a uniform conductive layer, thereby reducing the difference at the electrode interface and improving the consistency and detection stability of the composite modified structure.

[0019] In a preferred embodiment, the polydopamine hydrochloride modified layer formed by electropolymerization in S2 has a thickness of 50 nm.

[0020] By adopting the above technical solution, the thickness of the polyhydrochloric acid dopamine modification layer is limited to about 50 nm, so that the thickness of the interface modification layer is within a controllable range, which is conducive to obtaining a uniform and stable modification interface and supporting subsequent antibody immobilization and stable detection.

[0021] In a preferred embodiment, the concentration of adiponectin antibody in the adiponectin antibody solution in S3 is 50 μg / mL.

[0022] By adopting the above technical solution, the concentration of adiponectin antibody is limited to 50 μg / mL, making the antibody fixation conditions more controllable, thereby improving the reproducibility of antibody fixation and supporting the quantitative detection of adiponectin.

[0023] In a preferred embodiment, the incubation time of the adiponectin antibody solution in S3 is 30 min to 90 min.

[0024] By adopting the above technical solution, the fixation time of adiponectin antibody is limited to 30 min to 90 min, which makes the covalent fixation reaction of amino and quinone groups more complete, thereby improving the stability of the immunofunctionalized electrode and enhancing the reliability of detection.

[0025] In a preferred embodiment, the incubation temperature of the adiponectin antibody solution in S3 is room temperature or 37°C.

[0026] By adopting the above technical solution, the incubation temperature of adiponectin antibody is limited to room temperature or 37°C and precisely controlled, thereby maintaining the stability of biomolecular interactions and improving the stability of the detection process.

[0027] In a preferred embodiment, the binding electrode is rinsed before the differential pulse voltammetry measurement in step S5. The rinsing process includes rinsing the working electrode surface of the binding electrode with tris(hydroxymethyl)aminomethane hydrochloride buffer to remove unbound human serum components before performing the differential pulse voltammetry measurement.

[0028] By employing the above technical solution, the binding electrode is rinsed with buffer solution before differential pulse voltammetry measurement to remove unbound serum components, thereby reducing background interference from the complex serum matrix and improving the accuracy of electrochemical readout.

[0029] In a preferred embodiment, before outputting the adiponectin concentration of the human serum sample to be tested in step S5, background subtraction processing is performed on the current response signal obtained by differential pulse voltammetry. The background subtraction processing includes: using the measurement signal of the immunofunctionalized electrode without the human serum sample to be tested in the detection medium as the background signal, and subtracting the measurement signal of the binding electrode in the detection medium from the background signal to obtain the oxidation peak current change value.

[0030] By adopting the above technical solution, the oxidation peak current change value is obtained by background subtraction of the current response signal acquired by differential pulse voltammetry, thereby reducing the influence of background interference on signal readout and improving the accuracy of concentration output in complex serum matrix.

[0031] The technical effects and advantages of this invention are as follows:

[0032] A 2 mg / mL dopamine hydrochloride solution was prepared using a 10 mM tris(hydroxymethyl)aminomethane hydrochloride buffer solution at pH 8.5. A polydopamine hydrochloride modified layer was formed by electropolymerization using cyclic voltammetry within a potential range of -800 mV to 600 mV. Adiponectin antibody was then immobilized on the surface of this modified layer via amino-quinone covalent bonding. Human serum samples were then added to achieve specific binding. Differential pulse voltammetry was then used in a detection medium containing a 5 mM ferricyanide / ferrocyanide redox pair within a potential range of -100 mV to 350 mV. The oxidation peak current change is read out within a potential range of mV, and the adiponectin concentration is output according to the standard curve. The polyhydrochloric acid dopamine modification layer can form a uniform polymer film with a thickness of about 50nm and provide a higher surface amino density, thereby providing more covalent fixation sites for adiponectin antibodies and enhancing their anti-fouling and anti-interference capabilities. Combined with ferricyanide redox pair-mediated differential pulse voltammetry readout, rapid quantitative detection of trace serum samples can be achieved, balancing detection sensitivity, signal stability and clinical applicability without relying on expensive nanomaterials for enhancement. Attached Figure Description

[0033] Figure 1 This is a flowchart of the detection method of the present invention;

[0034] Figure 2 This is a schematic diagram of the detection system of the present invention;

[0035] Figure 3 This is a schematic flowchart of the two electrode modification methods of the present invention;

[0036] Figure 4 This is a comparison chart of the fluctuations in five detections of serum samples with the same concentration of adiponectin under different electrode modification methods according to the present invention;

[0037] Figure 5 This is a comparison chart of signal retention rates after 30 days of storage at 4°C for different electrode modification methods according to the present invention.

[0038] Figure 6 This is a comparison chart showing the adsorption capacity of whole serum proteins by different electrode modification methods according to the present invention. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Figure 2 middle Working electrode modification module: Demonstrates the electrode modification process, which is the core innovation of this invention.

[0041] DA Solution Preparation: Preparation process of dopamine hydrochloride solution;

[0042] PDA HCl Electropolymerization: The electropolymerization process of poly(dopamine hydrochloride);

[0043] PEDOT PSS Coating: The PEDOT PSS pre-coating step in Method 2;

[0044] Immune layer: Antibody fixation functional module

[0045] Antibody Immobilization: The process of immobilizing adiponectin antibodies;

[0046] Electrochemical Cell: Three-Electrode Detection System

[0047] Reference Electrode;

[0048] Counter Electrode: Counter electrode;

[0049] Electrolyte: A Tris buffer electrolyte containing iron ions;

[0050] Signal transduction: Electrochemical signal processing module

[0051] Potentiostat: Signal acquisition for electrochemical workstations;

[0052] Figure 2 The working electrode modification module demonstrates an integrated process of two innovative modification methods (PDA-HCl direct modification and PEDOTPSS-assisted modification), which achieves dynamic parameter adjustment through electrochemical feedback optimization (Potentiostat→PDA HCl Electropolymerization), a key difference from traditional static modification methods.

[0053] Figure 3 middle

[0054] Method 1: PDA HCl Modification: Direct Electropolymerization Modification Process

[0055] Prepare DA Solution: Prepare 2 mg / ml dopamine hydrochloride Tris buffer solution;

[0056] CV Electropolymerization: Cyclic voltammetry-driven polymerization;

[0057] Method 2: PEDOTPSS-assisted composite modification process

[0058] Coat PEDOT PSS: Spin-coated PEDOT PSS conductive layer;

[0059] Fixture Electropolymerization;

[0060] Common steps: Standardized testing process after modification

[0061] Antibody Immobilization: The antibody immobilization step;

[0062] Serum Detection: The stage of serum sample detection;

[0063] Figure 3 The differences between the two innovative modification methods were clearly compared, especially the combination of PEDOTPSS pre-coating and fixture-assisted electropolymerization in Method 2, which achieves more uniform polymer film growth through physical constraint (fixture), which is the key to improving detection reproducibility.

[0064] I. Overview

[0065] A method for detecting adiponectin in human serum using a poly(dopamine hydrochloride) modified carbon mesh electrode includes two specific implementation schemes. Method one involves adding dopamine hydrochloride solution to the electrode surface and performing electropolymerization modification, followed by immobilization of adiponectin antibodies, ultimately achieving serum sample detection. Method two adds a poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid complex coating step before electrode modification and optimizes the antibody immobilization time. Both methods employ cyclic voltammetry and differential pulse voltammetry for signal detection. The differential pulse voltammetry detection potential range is -100mV to 350mV, and the detection medium is a tris(hydroxymethyl)aminomethane buffer system containing ferricyanide redox pairs. This invention is simple to operate, low in cost, and suitable for rapid quantitative detection of serum adiponectin in clinical settings.

[0066] II. Detection System and Workflow

[0067] See Figure 2 , 3 The detection system consists of an electrode module, a signal processing module, and a detection environment control module. The electrode module uses a screen-printed carbon electrode (SPCE) as its core carrier. The SPCE is connected to the electrochemical workstation via a standard three-electrode system (working electrode, reference electrode, and counter electrode) to form an electrochemical detection circuit. The surface of the working electrode is functionalized with a polydopamine hydrochloride or a poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid composite / polydopamine hydrochloride composite layer to form a biocompatible working electrode interface.

[0068] The signal processing module integrates both cyclic voltammetry (CV) and differential pulse voltammetry (DPV) detection modes, acquiring current response signals through a preset potential scan range. The system incorporates a tris(hydroxymethyl)aminomethane buffer containing a ferricyanide redox pair as an electron mediator, converting the specific binding event of adiponectin to the antibody into a quantifiable electrical signal through a redox reaction. The detection environment control module precisely regulates the incubation temperature (room temperature or isothermal 37°C) and incubation time (15–90 min) to maintain the stability of biomolecular interactions. The system workflow consists of three stages: constructing a polymer-modified layer to form antibody immobilization sites; antigen-antibody binding to capture adiponectin in serum; and quantifying the target analyte using changes in electrochemical signals. The signal processing algorithm automatically removes background interference to ensure detection accuracy in complex serum matrices.

[0069] III. Detailed Introduction of Technical Principles

[0070] 1. Electrode modification process

[0071] Methods for constructing polydopamine hydrochloride (PDA-HCl) modified layers are provided, offering two modification pathways: Method 1 and Method 2.

[0072] 1.1 Method 1: Direct electropolymerization modification of polydopamine hydrochloride

[0073] (1) Preparation of dopamine hydrochloride solution

[0074] A 2 mg / mL dopamine hydrochloride (DA) solution was used, with 10 mM tris(hydroxymethyl)aminomethane hydrochloride buffer (Tris-HCl) at pH 8.5.

[0075] (2) Mechanism of dopamine oxidative self-polymerization

[0076] This alkaline environment promotes the oxidative self-polymerization of dopamine through the following reaction:

[0077] Equation (1):

[0078] Wherein: DA represents dopamine hydrochloride monomer; PDA-HCl is the electropolymerization product; Dissolved oxygen; As a byproduct;

[0079] (3) Electropolymerization film formation parameters

[0080] During the modification process, cyclic voltammetry (CV) was used to scan within the potential range of -800mV to 600mV at a scan rate of 50mV / s.

[0081] (4) Film formation results and interfacial site indices

[0082] The above parameter settings can ensure the formation of a uniform polymer film with a thickness of about 50 nm. The surface amino density, as measured by X-ray photoelectron spectroscopy (XPS), can reach 8.2 ± 0.3 amino / nm², which is significantly higher than the 5.1 ± 0.4 amino / nm² of traditional polydopamine modification.

[0083] 1.2. Method 2: Composite modification assisted by pre-coating of poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid composite

[0084] (1) Parameters of conductive polymer pre-coating

[0085] Method 2 introduces a pre-coating step using poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid composite (PEDOT:PSS), with a solution concentration of 2 mg / mL and a drop volume of 6 μL; the solution is then dried at 37 °C for 40 min to form a conductive layer.

[0086] (2) Results of improved interface resistance

[0087] The formed conductive layer can reduce the electrode interface resistance from 1.2±0.2kΩ of the original screen-printed carbon electrode to 0.4±0.1kΩ;

[0088] (3) Optimization of subsequent polyhydrochloric acid dopamine electropolymerization cycle number

[0089] The subsequent PDA-HCl electropolymerization cycle count was optimized to 3–6 times;

[0090] (4) Results and calculations of the improvement in electrochemical active area

[0091] At this point, the electrochemically active area of ​​the modified layer can reach 0.28 ± 0.03 cm², calculated by the Randles–Sevcik equation, which is about 40% higher than that of the unmodified electrode.

[0092] To facilitate the reproduction of experimental calculations, the Randles–Sevcik equation is written as:

[0093] Equation (2):

[0094] in, denoted as peak current (A), n as electron transfer number, A as electrochemical active area (cm²), D as diffusion coefficient (cm² / s), C as electroactive substance concentration (mol / cm³), and v as scan rate (V / s).

[0095] 2. Antibody fixation mechanism

[0096] Adiponectin antibody (ADP-Ab) is immobilized on the surface of PDA-HCl via amino-quinone covalent binding. The reaction process can be described as follows:

[0097] Equation (3)

[0098] in, Quinone refers to the quinone group site on the surface of polydopamine hydrochloride; The amino group represents the amino site on the adiponectin antibody molecule; The base is a covalently fixed product;

[0099] Immobilization efficiency was monitored in real time using a quartz crystal microbalance (QCM). The results showed that at an antibody concentration of 50 μg / mL, the antibody loading for Method 1 (30 min immobilization) was 3.2 ± 0.2 ng / mm², while that for Method 2 (90 min immobilization) reached 4.8 ± 0.3 ng / mm². Antibody orientation was verified by antigen-binding capacity testing, showing that the PEDOT:PSS layer in Method 2 increased the exposure rate of effective antibody binding sites to 78 ± 5% (compared to 62 ± 7% in Method 1).

[0100]

[0101] Table 1 Antibody Loading Capacity (QCM)

[0102]

[0103] Table 2 Comparison of Antibody Effective Binding Site Exposure Rate

[0104] 3. Serum detection kinetics

[0105] The binding of adiponectin (ADP) in the serum sample (4 μL) to the immobilized antibody followed the Langmuir adsorption model:

[0106] Equation (4)

[0107] Where: θ is the surface coverage; K is the binding constant, which is fitted as follows: ADP represents the concentration of adiponectin.

[0108] 4. Relationship between differential pulse voltammetry readout and quantification

[0109] Differential pulse voltammetry (DPV) detection uses a solution containing 5 mM A 10 mM tris(hydroxymethyl)aminomethane buffer solution was used as the detection medium, and measurements were taken within a potential range of -100 mV to 350 mV. Oxidation peak current variation ( The relationship between adiponectin concentration and concentration (1–100 ng / mL) is linear:

[0110] Equation (5)

[0111] in: The value of the oxidation peak current change is (μA); ADP is the adiponectin concentration (ng / mL); 0.18 is the linear fitting slope; 2.1 is the linear fitting intercept. The goodness of fit is denoted as .

[0112] Method 2 achieved a limit of detection (LOD) as low as 0.3 ng / mL (signal-to-noise ratio S / N=3), an improvement over Method 1 (0.8 ng / mL). For ease of reference, the linear range and LOD are summarized in Table 3:

[0113]

[0114] Table 3 Comparison of Quantitative Performance

[0115] 5. Interference Suppression Strategy

[0116] See Figure 4 – Figure 6 The PDA-HCl modified layer inhibits serum interference through the following mechanism:

[0117] (1) Electrostatic repulsion: Under pH 7.4 conditions, PDA-HCl is positively charged (Zeta potential +12.3mV), which can repel positively charged interfering proteins in serum (such as albumin). );

[0118] (2) Steric hindrance: The pore size distribution of the polymer layer was determined by the BJH method, showing that 85% of the pores were less than 5 nm, which could block the non-specific adsorption of macromolecules.

[0119] (3) Ferricyanide-mediated signal amplification: Electron transfer rate constant on PEDOT:PSS modified electrode ( The velocity reached 0.021 cm / s, which is 3 times higher than that of the bare electrode, enhancing the recognition of specific signals.

[0120]

[0121] Table 4 Comparison of Interface Properties

[0122] IV. Validation of Detection Reproducibility and Clinical Comparison

[0123] 1. Detection reproducibility verification

[0124] The relative standard deviation (RSD) for detecting 20 ng / mL adiponectin samples using the same batch of electrodes (n=5) was 4.7%, and the inter-batch RSD was 6.3%.

[0125] 2. Accelerate stability verification

[0126] Accelerated stability testing (stored at 4℃ for 30 days) showed a signal attenuation rate of less than 8%, indicating that the modified layer has long-term stability (corresponding to...). Figure 5 (Signal retention rate proof diagram).

[0127] 3. Verification of clinical relevance with enzyme-linked immunosorbent assay (ELISA)

[0128] When clinical samples (n=32) were tested in comparison with enzyme-linked immunosorbent assay (ELISA), the correlation coefficient between the two methods was R=0.961 (slope=1.03), confirming the clinical applicability of the present invention.

[0129]

[0130] Table 5 Summary of reproducibility, stability and clinical relevance indicators

[0131] Example 1

[0132] Portable testing card integration solution

[0133] Miniaturization of the three-electrode system: The working electrode, reference electrode and counter electrode are arranged in a concentric circle structure, the electrode spacing is reduced to 1mm, and the overall size is 10mm×30mm.

[0134] Dry reagent pre-storage: The electrode surface is pre-coated with a lyophilized PEDOT:PSS / PDA-HCl composite layer, which is activated by adding 100μL of tris(hydroxymethyl)aminomethane buffer before use;

[0135] Sample loading optimization: The capillary channel was designed to enable automatic siphon loading of 4μL of serum, avoiding errors from manual sample addition; the test card can be stored at room temperature for 6 months.

[0136] Example 2

[0137] Multi-index joint detection array electrode

[0138] 4×4 electrode array: Sixteen independent working electrodes were fabricated on a 4cm² substrate, eight of which were directly modified with PDA-HCl (Method 1), and the other eight were modified with PEDOT:PSS / PDA-HCl composite (Method 2).

[0139] Differential antibody immobilization: odd-numbered electrodes immobilize adiponectin antibodies, while even-numbered electrodes immobilize antibodies against other metabolic markers (such as leptin and resistin), achieving multi-indicator differentiation through spatial coding;

[0140] Microfluidic integration: A PDMS microchannel layer is integrated above the array to enable automatic sample distribution to each detection unit after a single injection.

[0141] Example 3

[0142] Wearable Continuous Monitoring System

[0143] Flexible electrode substrate: Screen-printed carbon electrodes made of polyimide substrate, with a bending radius of up to 5mm;

[0144] Subcutaneous microexudate collection: Using a microneedle array, a small amount (<1μL) of sample is extracted from the subcutaneous interstitial fluid, and the sample is automatically injected once per hour;

[0145] Wireless signal transmission: The integrated Bluetooth module sends DPV data to the mobile terminal in real time for long-term adiponectin level tracking.

[0146] Example 4

[0147] Automated batch testing platform

[0148] 96-well plate electrode: The SPCE array is embedded in the bottom of a standard 96-well plate, with each well forming an independent detection unit;

[0149] Robotic arm-assisted finishing: Through program control, precise control of PEDOT:PSS spraying (accuracy ±0.5μL) and electropolymerization parameters (voltage ±5mV) can be achieved;

[0150] Multi-channel parallel detection: The integrated 8-channel electrochemical workstation can complete the detection of a whole plate of samples within 30 minutes; the platform can process more than 500 serum samples per day.

[0151] Example 5

[0152] Environmentally Adaptability Enhancement Design

[0153] Temperature compensation electrode: A thermosensitive polymer (LCST=32℃) is incorporated into the PDA-HCl modified layer. When the ambient temperature exceeds 35℃, it automatically shrinks and densifies to prevent the modified layer from swelling.

[0154] Anti-evaporation structure: A 5μm thick breathable and hydrophobic membrane (PTFE material) is covered on the electrode surface to reduce solvent evaporation during detection in arid regions;

[0155] Vibration-resistant fixing: Spring contact electrode connection is used to ensure stable contact even in vibrating environments such as mobile testing vehicles.

[0156] Working principle:

[0157] This invention utilizes cell-driven electropolymerization of dopamine hydrochloride on the surface of a screen-printed carbon electrode to form a polydopamine hydrochloride modified layer, leveraging its high amino density to provide covalent fixation sites for antibodies. Method two further introduces a conductive polymer pre-coating to reduce interfacial resistance and enhance the electrochemically active area and electron transfer capability. The binding of adiponectin to immobilized antibodies in serum conforms to the Langmuir adsorption model; in the presence of... In the tris(hydroxymethyl)aminomethane buffer detection medium, the oxidation peak current change was read out in the range of -100mV to 350mV by DPV, and the adiponectin concentration was output according to the linear relationship of Equation (5) and the fitting parameters. Interference suppression is achieved through the synergistic effect of electrostatic repulsion, steric hindrance and enhanced electron transfer, as shown in Table 4 and Figure 4 – Figure 6 The evidence provided is as shown.

[0158] Taking a common clinical example of borderline low-value adiponectin (5 μg / mL), traditional ELISA testing requires more than 5 hours, using 50 μL of serum sample, with results fluctuating between 4.8 and 5.2 μg / mL. Using Method 1 of this invention, the entire process can be completed in just 90 minutes, reducing the serum volume to 4 μL, and the results remain stable within the range of 4.95–5.05 μg / mL. The electrochemical signal is acquired via DPV; the concentration value is directly output after comparing the oxidation peak current change value ΔIp with the standard curve, eliminating the need for complex data processing. This example demonstrates improvements in detection speed, sample consumption, and result accuracy, and is suitable for clinical emergency or large-scale screening scenarios.

[0159] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting human serum adiponectin using a polyhydrochloride-modified dopamine-modified wire mesh carbon electrode, characterized in that, Includes the following steps: S1, Preparation of the solution to be modified: Dissolve dopamine hydrochloride in a 10mM tris(hydroxymethyl)aminomethane hydrochloride buffer solution with a pH of 8.5 to prepare a 2mg / mL dopamine hydrochloride solution to obtain the solution to be modified. S2, Electropolymerization to form modified electrode: The solution to be modified is dropped onto the working electrode surface of the wire mesh carbon electrode. The wire mesh carbon electrode forms a working electrode, a reference electrode, and a counter electrode on the same substrate, constituting a three-electrode system. Cyclic voltammetry is used to perform potential cyclic scanning on the three-electrode system. The scanning potential range is -800mV to 600mV and the scanning rate is 50mV / s, so that dopamine hydrochloride electropolymerizes on the working electrode surface to form a polydopamine hydrochloride modification layer, thus obtaining the modified electrode. S3, Immobilizing adiponectin antibody to form an immunofunctionalized electrode: Add adiponectin antibody solution to the surface of the poly(dopamine hydrochloride) modified layer and incubate for 30 min to 90 min, so that the adiponectin antibody is immobilized on the surface of the poly(dopamine hydrochloride) modified layer through covalent binding of amino and quinone groups, and an immunofunctionalized electrode is obtained. S4, forming a binding electrode by binding adiponectin: Add the human serum sample to be tested to the immunofunctionalized electrode and incubate it to allow the adiponectin in the human serum sample to specifically bind with the adiponectin antibody, thus obtaining the binding electrode; S5, Electrochemical Measurement and Output of Adiponectin Concentration: The binding electrode is measured using differential pulse voltammetry in the detection medium, with a measurement potential range of -100mV to 350mV to obtain the oxidation peak current change value. The detection medium is a solution containing a 5mM potassium ferrocyanide and potassium ferrocyanide redox pair and a 10mM tris(hydroxymethyl)aminomethane hydrochloride buffer solution. Based on the correspondence between the oxidation peak current change value and the adiponectin concentration established using adiponectin standard samples ranging from 1ng / mL to 100ng / mL under the same detection medium and measurement potential range, the adiponectin concentration of the human serum sample to be tested is output.

2. The detection method according to claim 1, characterized in that, The number of cycles for potential cyclic scanning in S2 is 3 to 6.

3. The detection method according to claim 1, characterized in that, Before the electropolymerization of the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid composite conductive polymer layer is formed on the working electrode surface of the screen carbon electrode in S2, so as to form a poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid composite / poly ...(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid composite / poly(3,4-ethylenedioxythiophene)-poly(3,4-ethylenedioxythiophene 4. The detection method according to claim 3, characterized in that, The coating solution concentration of the conductive polymer layer of poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid composite in S2 is 2 mg / mL, the coating volume is 6 μL, and the conductive polymer layer is formed by drying at 37°C for 40 min after coating.

5. The method according to claim 1 or 3, characterized in that, The polyhydrochloric acid dopamine-modified layer formed by electropolymerization in S2 has a thickness of 50 nm.

6. The detection method according to claim 1, characterized in that, The concentration of adiponectin antibody in the S3 solution is 50 μg / mL.

7. The detection method according to claim 1, characterized in that, The incubation time for the adiponectin antibody solution in S3 is 30 min to 90 min.

8. The detection method according to claim 1, characterized in that, The incubation temperature of the adiponectin antibody solution in S3 is room temperature or 37°C.

9. The detection method according to claim 1, characterized in that, Before performing differential pulse voltammetry measurement in S5, the binding electrode is rinsed. The rinsing process includes rinsing the working electrode surface of the binding electrode with tris(hydroxymethyl)aminomethane hydrochloride buffer to remove unbound human serum components before performing differential pulse voltammetry measurement.

10. The method according to claim 1, characterized in that, Before outputting the adiponectin concentration of the human serum sample in step S5, background subtraction processing is performed on the current response signal obtained by differential pulse voltammetry. The background subtraction processing includes: using the measurement signal of the immunofunctionalized electrode without the human serum sample as the background signal, and subtracting the measurement signal of the binding electrode in the detection medium from the background signal to obtain the oxidation peak current change value.

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