阅读说明：本技术 一种电化学生物传感器及其制备方法和应用 (Electrochemical biosensor and preparation method and application thereof ) 是由 杜欣 张振国 张丛丛 于 2021-07-23 设计创作，主要内容包括：本发明涉及化学成分检测领域,为了解决现有瘦素的生物传感器存在的灵敏度、选择性、抗干扰性、重现性和稳定性都较差以及无法对不同BMI的人群进行检测的问题,本发明提出一种电化学生物传感器及其制备方法和应用,使用还原氧化石墨烯-金纳米复合材料(rGO-Au)修饰电极和生物纳米胶囊(ZZ-BNC)来垂直定位抗体检测瘦素。检测范围为0.001～1000pg/mL,检出限为0.00087pg/mL。与现有的传感器相比,本发明所述生物传感器具有更高的选择性、灵敏度和抗干扰能力。(The invention relates to the field of chemical component detection, and provides an electrochemical biosensor and a preparation method and application thereof, aiming at solving the problems that the existing leptin biosensor has poor sensitivity, selectivity, anti-interference performance, reproducibility and stability and cannot detect people with different BMIs. The detection range is 0.001-1000pg/mL, and the detection limit is 0.00087 pg/mL. Compared with the existing sensor, the biosensor has higher selectivity, sensitivity and anti-interference capability.)

1. An electrochemical biosensor, comprising: the graphene oxide-gold nano composite material is prepared by using a graphene oxide-gold nano composite material, a biological nano capsule, a leptin antibody and an electrode, wherein the graphene oxide-gold nano composite material, the biological nano capsule and the leptin antibody are modified on the surface of the electrode.

2. The electrochemical biosensor of claim 1, wherein the electrode is a glassy carbon electrode.

3. The electrochemical biosensor as claimed in claim 1, wherein the biological nanocapsule is formed by serially fusing hepatitis b virus envelope L protein and Fc-binding Z domain derived from protein a.

4. The method for preparing an electrochemical biosensor according to any one of claims 1 to 3, wherein the reduced graphene oxide-gold nanocomposite, the biological nanocapsule, and the leptin antibody are placed on the surface of the electrode, and dried, respectively.

5. The method for preparing an electrochemical biosensor according to claim 4, wherein the reduced graphene oxide-gold nanocomposite is placed on the surface of an electrode, dried, the biological nanocapsule is placed on the electrode for incubation, and the leptin antibody is placed on the electrode for incubation.

6. The method for preparing an electrochemical biosensor according to claim 4, wherein the reduced graphene oxide-gold nanocomposite is in a suspension with a concentration of 2-10 mg/mL.

7. The method for preparing an electrochemical biosensor according to claim 4, wherein the concentration of the bio-nanocapsule is 0.01-0.5 μ g/mL;

preferably, the leptin antibody concentration is 5-45 ng/mL.

8. Use of an electrochemical biosensor according to any one of claims 1 to 3 for the detection of leptin.

9. A leptin electrochemical biosensor comprising the electrochemical biosensor of any one of claims 1 to 3.

10. The leptin electrochemical biosensor of claim 9, wherein the leptin electrochemical biosensor is used to detect leptin levels in diet induced obesity symptoms.

Technical Field

The invention relates to the field of chemical component detection, in particular to an electrochemical biosensor and a preparation method and application thereof, and more particularly relates to an electrochemical biosensor for leptin detection and a preparation method and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

The rapidly rising rate of obesity has accelerated the outbreak of type-2 diabetes over the past decades, which has become one of the most serious public health burdens worldwide. Obesity not only causes cardiovascular disease, but also increases the incidence of hypertension and coronary heart disease. Leptin is a protein hormone secreted by adipose tissue, and can also be used as a marker of non-alcoholic fatty liver disease. Radioimmunoassay and enzyme-linked immunoassay are the major methods for the detection of leptin at present. However, these detection methods also have some limitations and limitations, such as expensive equipment and complicated procedures. Therefore, the development of a simple, efficient and sensitive method for detecting leptin, especially for detecting the actual sample effectively, has been the focus of attention.

Electrochemical immunosensors have gained wide attention in clinical disease diagnosis due to the advantages of specificity and affinity of antibody-antigen reaction, high sensitivity, low cost, high efficiency, portability and the like. Recently, it has been reported that, for example, Liu and the like synthesize cerium niobate/cerium oxide hollow nanospheres by a template-free hydrothermal technique to detect leptin. However, they did not test a population of different BMIs.

In addition, the inventor researches and discovers that the existing biosensor related to the leptin has low sensitivity, generally 0.001-0.042 mug/mL, and the selectivity, the anti-interference performance, the reproducibility and the stability of the biosensor of the leptin need to be improved or cannot be compatible with the performances, so that the use of the biosensor of the leptin is limited.

Disclosure of Invention

In order to improve the sensitivity, selectivity, anti-interference performance, reproducibility and stability of the conventional electrochemical biosensor for leptin, the invention provides the electrochemical biosensor and a preparation method and application thereof, and a reduced graphene oxide-gold nano composite material (rGO-Au) modified electrode and a biological nanocapsule (ZZ-BNC) are used for detecting leptin in a vertical positioning mode for the first time. The detection range is 0.001-1000pg/mL, and the detection limit is 0.00087 pg/mL. Compared with the existing sensor, the biosensor has higher selectivity, sensitivity and anti-interference capability. Meanwhile, the biosensor can detect leptin in a diet-induced obesity mouse model and is well matched with an enzyme-linked immunosorbent assay (ELISA) detection result. The results of the biosensor measurements vary among people with different Body Mass Indices (BMI). Therefore, the electrochemical biosensor based on the directional antibody immobilization is an effective platform for detecting leptin in practical samples and can be widely applied to the evaluation of obesity diseases.

Specifically, the invention is realized by the following technical scheme:

in a first aspect of the present invention, there is provided an electrochemical biosensor comprising: the graphene oxide-gold nano composite material is prepared by using a graphene oxide-gold nano composite material, a biological nano capsule, a leptin antibody and an electrode, wherein the graphene oxide-gold nano composite material, the biological nano capsule and the leptin antibody are modified on the surface of the electrode.

In a second aspect of the present invention, a method for preparing an electrochemical biosensor is provided, in which a reduced graphene oxide-gold nanocomposite, a biological nanocapsule, and a leptin antibody are placed on the surface of an electrode, and dried.

In a third aspect of the invention, an electrochemical biosensor is provided for detecting leptin.

In a fourth aspect of the invention, a leptin electrochemical biosensor is provided, which comprises an electrochemical biosensor.

One or more of the technical schemes have the following beneficial effects:

1) after ZZ-BNC is dripped on the surface of the rGO-Au/electrode, the peak current is reduced, which indicates that ZZ-BNC is successfully combined with Au nanoparticles. Upon addition of leptin antibody, the current further decreased, indicating that the ZZ terminus of ZZ-BNC successfully bound to the antibody, indicating that ZZ-BNC may act as a bridge.

2) The leptin biosensor constructed by one or more technical schemes of the invention shows a wide linear range from 0.001 to 1000pg/mL, and the detection limit is 0.00087 pg/mL. The linear regression equation was calculated as I ═ 3.94lg C (leptin) +38.15 (R)²0.999). The novel immunosensor is compared with the linear range, detection limit and sensitivity of the previous immunosensorCompared with the prior art, the leptin immunosensor prepared by the method has wider linear detection range and higher sensitivity.

3) The leptin electrochemical biosensor has good selectivity, interference resistance, reproducibility and stability. In a single component, the electrochemical biosensor has the highest sensitivity to leptin, and in a mixed system, as long as the system contains leptin, the electrochemical biosensor can still detect the existence of leptin and has the same sensitivity as the single leptin component, which shows that the electrochemical biosensor has stronger selectivity and anti-interference performance.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a flow chart of the preparation of the leptin immunosensor in example 1 of the present invention;

FIG. 2 is a graph of the morphology, structure and electrochemical properties of the nanocomposite material of example 2 of the invention. Transmission electron microscope images of graphene oxide (a), reduced graphene oxide (B), and reduced graphene oxide-gold (C). (D) Raman spectra images of graphene oxide, reduced graphene oxide, and reduced graphene oxide-gold. (E, F) atomic force microscopy images of rGO-Au-anti-leptin (E) and rGO-Au-ZZ-BNC-anti-leptin (F).

FIG. 3 is a graph showing the results of example 2 of the present invention, wherein (A) is represented by 10mM K₃[Fe(CN)₆]Recorded cyclic voltammograms indicating graphene oxide, reduced graphene oxide and reduced graphene oxide-gold produced by the electrode. Kinetic analysis of (B, C) rGO-Au/GCE showed that the reaction modifying the electrode was a diffusion controlled surface reaction. (B) And (3) performing kinetic analysis on the rGO-Au/GCE at a scanning rate of 10-200 mV/s. (C) Oxidation peak current (Ipa) and reduction peak current (Ipc) with square root of scan rate (v [ ])^{1 /2}) Is used. (D) At 10mM K₃[Fe(CN)₆]rGO-Au obtained in (1)Differential pulse voltage measurement of/GCE, rGO-Au-ZZ-BNC-anti-leptin/GCE and rGO-Au-ZZ-BNC-anti-leptin-BSA/GCE.

FIG. 4 shows the measurement of 10mM K in example 2 of the present invention₃[Fe(CN)₆]Linear range and performance of the immunosensor for leptin. (A) Differential pulse voltammetry is used for detecting leptin with different concentrations of 0.001-1000 pg/mL. (B) And (3) detecting a calibration curve of the leptin sensitivity by the immunosensor. (C-F) immunosensor measures the performance of leptin. The results obtained using this immunosensor have high sensitivity (C), interference immunity (D), stability (E) and reproducibility (F). Error bars represent mean ± standard deviation (n ═ 3).

FIG. 5 shows the detection of leptin in real samples according to example 2 of the present invention. (A) HE staining pattern of fat tissue of epididymis in NFD mice. (B) HE staining pattern of fat tissue of epididymis in high-fat mice. (C) Mice with different feeding patterns were tested for leptin by electrochemical analysis and ELISA. (D) Serum leptin levels in different BMI populations.

FIG. 6 shows the effect of different concentrations of leptin antibody (A) and different incubation times (B) on leptin immunosensors in example 2 of the present invention;

FIG. 7 is a graph showing the body weight change of a normal combination control group in example 2 of the present invention;

FIG. 8 is a graph of serum leptin levels in normal and obese humans, in accordance with example 2 of the present invention.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

Electrochemical immunosensors have gained wide attention in clinical disease diagnosis due to the advantages of specificity and affinity of antibody-antigen reaction, high sensitivity, low cost, high efficiency, portability and the like. Recently, it has been reported that, for example, Liu and the like synthesize cerium niobate/cerium oxide hollow nanospheres by a template-free hydrothermal technique to detect leptin. However, they did not test a population of different BMIs.

In addition, the inventor researches and discovers that the existing biosensor related to the leptin has low sensitivity, generally 0.001-0.042 mug/mL, and the selectivity, the anti-interference performance, the reproducibility and the stability of the biosensor of the leptin need to be improved or cannot be compatible with the performances, so that the use of the biosensor of the leptin is limited.

In order to solve the problems, the invention provides an electrochemical biosensor, and a preparation method and application thereof, wherein a reduced graphene oxide-gold nanocomposite (rGO-Au) modified electrode and a biological nanocapsule (ZZ-BNC) are used for vertically positioning an antibody to detect leptin. The detection range is 0.001-1000pg/mL, and the detection limit is 0.00087 pg/mL. Compared with the existing sensor, the biosensor has higher selectivity, sensitivity and anti-interference capability. Meanwhile, the biosensor can detect leptin in a diet-induced obesity mouse model and is well matched with an enzyme-linked immunosorbent assay (ELISA) detection result. The results of the biosensor measurements vary among people with different Body Mass Indices (BMI). Therefore, the electrochemical biosensor based on the directional antibody immobilization is an effective platform for detecting leptin in practical samples and can be widely applied to the evaluation of obesity diseases.

The directional immobilization of antibodies on the sensor chip surface is one of the important criteria for improving the sensitivity and specificity of immunosensors. In general, antibody attachment is primarily through chemical cross-linking with gold, or through antibody attachment to protein a or protein G. These immobilization methods typically use reagents-non-specific reactions with the amino groups of the protein, binding antibodies or other interacting molecules to the sensor surface, and thus Fv is not a targeting solvent.

ZZ-BNC is a substance which is formed by serially fusing hepatitis B virus envelope L protein and Fc-binding Z domain derived from protein A and is used for vertically fixing an antibody so as to enhance the binding capacity of the antigen and the antibody. Research shows that ZZ-BNC actually improves the sensitivity, antigen binding capacity and affinity of the immunosensor due to the special directional immobilization of antibodies. In addition, the nano material with excellent conductivity is selected, and the improvement of the vertical fixation of the antibody has important significance for improving the analysis performance of the sensor.

Specifically, the invention is realized by the following technical scheme:

in a first aspect of the present invention, there is provided an electrochemical biosensor comprising: the graphene oxide-gold nanoparticle composite material comprises a reduced graphene oxide-gold (rGO-Au) nanocomposite material, a biological nanocapsule, a leptin antibody and an electrode, wherein the reduced graphene oxide-gold nanocomposite material, the biological nanocapsule and the leptin antibody are modified on the surface of the electrode.

In some embodiments of the invention, the rGO-Au nano composite material is used for a modified electrode, and has the advantages of high stability, good biocompatibility, high conductivity and large specific surface area, and the advantages provide good conditions for biospecific recognition and electronic signal transduction. Therefore, it can be used as a modified material of a glassy carbon electrode. And then, constructing an immunosensor for rapidly detecting leptin in human serum by using ZZ-BNC as an electrode material and an antibody binding agent. And the serum leptin levels of mice with different dietary modes and different BMI populations are detected simultaneously, so that scientific basis is provided for further researching the pathogenesis of obesity.

In some embodiments, the electrode is a glassy carbon electrode, and the biological nanocapsule is formed by serially fusing hepatitis B virus envelope L protein and an Fc-binding Z domain derived from protein A.

In a second aspect of the present invention, a method for preparing an electrochemical biosensor is provided, in which a reduced graphene oxide-gold nanocomposite, a biological nanocapsule, and a leptin antibody are placed on the surface of an electrode, and dried.

When different materials are placed on the surface of the electrode, the former material is ensured to be dried and fixed, and the latter component is prevented from influencing the former component.

Experimental research shows that the biological nanocapsule can serve as an adhesive to connect the reduced graphene oxide-gold nano composite material and the leptin antibody, and is beneficial to stable existence of the three on an electrode, so that the electrochemical biosensor has good stability. Therefore, in some embodiments, the reduced graphene oxide-gold nanocomposite is placed on the surface of an electrode, dried, then the biological nanocapsule is placed on the electrode for incubation, and then the leptin antibody is placed on the electrode for incubation, so as to obtain the graphene/gold composite material.

In order to ensure uniform loading of functional ingredients on the electrode, in some embodiments, the reduced graphene oxide-gold nanocomposite is in suspension at a concentration of 2-10 mg/mL. Experimental research shows that when the concentration of the reduced graphene oxide-gold nanocomposite suspension is 5mg/mL, the loading effect on the surface of the electrode is the best.

In some embodiments, the biological nanocapsule concentration is 0.01-0.5 μ g/mL, preferably 0.1 μ g/mL; the concentration of the leptin antibody is 5-45ng/mL, and 25ng/mL is preferable.

The reduced graphene oxide-gold nano composite material is a suspension, the biological nanocapsule and a leptin antibody solvent, and the solvents are PBS.

The nano material improves the electrochemical performance, the biological nano capsule is used for fixing an antibody, and the antibody is used for capturing leptin.

And incubating the reduced graphene oxide-gold nano composite material and the electrode modified by the biological nano capsule with a leptin antibody for 30, 60, 90, 120 and 150min so as to optimize the incubation time of the immunosensor system. The incubation time of the leptin antibody greatly affects the reaction of the immunosensor. At shorter incubation times, the leptin antibody may not be effectively immobilized on the electrode surface. However, if the time is too long, the quality of protein adsorbed on the surface of the electrode may be too high, and the surface of the electrode may be insulated or even damaged. Therefore, 120min was finally selected as the optimal incubation time for the leptin antibody.

The rGO-Au nano composite material is used for modifying the electrode, has high stability, good biocompatibility, high conductivity and large specific surface area, and provides good conditions for biospecific recognition and electronic signal transduction. Therefore, it can be used as a modified material of a glassy carbon electrode. And then, constructing an immunosensor for rapidly detecting leptin in human serum by using ZZ-BNC as an electrode material and an antibody binding agent. And the serum leptin levels of mice with different dietary modes and different BMI populations are detected simultaneously, so that scientific basis is provided for further researching the pathogenesis of obesity.

The reduced graphene oxide-gold nanocomposite (rGO-Au) was slightly modified on the basis of the previous research methods to prepare a homogeneous rGO-Au nanocomposite. 20mg of graphene oxide was placed in 20mL of ultrapure water and ultrasonically dispersed for 2 hours. Then 100. mu.L of HAuCl was added at a concentration of 20mM₄The solution was stirred for 30min and then placed in a water bath at 95 ℃. Simultaneously adding 2mL of 50% hydrazine hydrate solution as a reducing agent, and reacting for 2h at 95 ℃. Then 15mg PVP was added and stirred for 1 h. The homogeneous solution was clarified by centrifugation at 14000rpm and the precipitate was washed at least three times with a mixture of ethanol and bi-distilled water. Finally, the volume was stabilized to 5mg/mL and stored in a 4 ℃ refrigerator. The reduced graphene oxide is prepared by a similar method without adding HAuCl₄And (3) solution.

In a third aspect of the invention, an electrochemical biosensor is provided for detecting leptin.

In a fourth aspect of the invention, a leptin electrochemical biosensor is provided, which comprises an electrochemical biosensor.

Since obesity has many causes, and the symptoms of obesity of different causes are different, the relief scheme is different.

The causes of obesity include, but are not limited to, genetic factors, dietary factors, activity factors, gender and occupation factors, age factors, mental factors, metabolic factors, endocrine factors, trace elements, sleep factors, and the like.

Experimental research shows that when the leptin electrochemical biosensor is used for detecting the content of leptin in diet-induced obesity, the detection limit is lower, the detection range is wide, and the selectivity and the stability are better.

The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.

Example 1

(1) Preparation of rGO-Au

20mg of graphene oxide was placed in 20mL of ultrapure water and ultrasonically dispersed for 2 hours. Then 100. mu.L of HAuCl was added at a concentration of 20mM₄The solution was stirred for 30min and then placed in a water bath at 95 ℃. Simultaneously adding 2mL of 50% hydrazine hydrate solution as a reducing agent, and reacting for 2h at 95 ℃. Then 15mg PVP was added and stirred for 1 h. The homogeneous solution was clarified by centrifugation at 14000rpm and the precipitate was washed at least three times with a mixture of ethanol and bi-distilled water. Finally, the volume was stabilized to 5mg/mL and stored in a 4 ℃ refrigerator. The reduced graphene oxide is prepared by a similar method without adding HAuCl₄And (3) solution.

(2) Electrode modification

The GCE surface was carefully polished with 0.3 and 0.05 μm diameter alumina powder to remove the oxide layer. Then the surface of the electrode is cleaned by ultrasonic wave by using double distilled water and ethanol to remove other physical absorption substances. The GCE was then immediately dried under nitrogen. Placing 10 μ L of rGO-Au suspension (5mg/mL) on the surface of GCE, naturally drying, dropping 10 μ L of 0.1 μ g/mL ZZ-BNC on the electrode, incubating for 60min, then incubating for 120min with 10 μ L of 25ng/mL leptin antibody (anti-leptin) to obtain rGO-Au-ZZ-BNC-anti-leptin/GCE, finally adding 10 μ L of 1% Bovine Serum Albumin (BSA), incubating for 30min, and cutting off non-specific sites to obtain rGO-Au-ZZ-BNC-anti-leptin-BSA/GCE (figure 1). After each step of modification, the electrodes were gently washed with Phosphate Buffered Saline (PBS).

(3) Characterization of ZZ-BNC

ZZ-BNC (0.1. mu.g/mL, 10. mu.L) was added dropwise to the coverslip and incubated at room temperature for 60 min. Then anti-leptin (25ng/mL 10. mu.L) was incubated for 120 min. After drying in air, observation was performed using an Atomic Force Microscope (AFM).

(4) Introduction to detection methods

By using cyclesThe electrode modification process was characterized by Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV). At 10mM potassium ferricyanide (K)₃[Fe(CN)₆]) Electrochemical detection was performed in solution. In addition, since DPV is the most sensitive detection method in voltammetry, it is also applied to optimization studies of an immunosensor, analysis characteristics of an immunosensor, and a real sample analysis section.

(5) Mouse serum leptin detection

20 healthy male C57/B16 mice, 6 weeks old, were placed in standard cages at room temperature and humidity with a light/dark cycle for 12 h. The experimental protocol for mice was approved by the animal protection and utilization committee of the university of east Shandong Master. The normal fat diet group (NFD) and the high fat diet group (HFD) were randomly divided. All mice were fed with normal fat diet for one week prior to diet adjustment. The HFD group mice were fed a high fat diet (60% fat function) and the NFD group mice were fed a normal diet (15% fat function). When fed to 12 weeks, 5 obese mice were selected as a standard for NFD standard deviation of more than +2 times the standard body weight in the experimental group, and body length and body weight were measured. In addition, eyeball blood and epididymal adipose tissue were collected. Serum was collected by centrifugation (4 ℃, 3000r/min,15 min). Serum leptin level was detected by electrochemical method and Elisa method, respectively. Meanwhile, the fat tissue of the epididymis of the mouse is placed in special fat fixing liquid, and hematoxylin-eosin staining (HE staining) is carried out after paraffin embedding. Tissue target areas were selected for 200 images using Eclipse CI-L camera microscopy. After imaging was completed, the cell diameter and total area of adipocytes were measured for each section using Image-Pro Plus 6.0 analysis software, respectively, and the total number and density of adipocytes in each Image were calculated.

(6) Serum leptin detection for different BMI populations

BMI is a commonly used international measure of obesity and health in humans. Severe obesity is defined as BMI greater than 35kg/m². Serum samples of 3 normal and 3 obese people were collected from sporadic community populations in denna city. The collection of samples was approved by the university of Shandong university medical ethics review Committee and signed by all volunteers for a study informed consent. Collecting blood vessel empty part by adopting blood collecting vessel without anticoagulant2-3 mL of abdominal venous blood, and collecting venous blood and centrifuging at 4 ℃ (4000r/min, 5 min). Collecting supernatant to obtain serum sample, sealing with sealing film, and storing in refrigerator at-80 deg.C. Prior to measurement, the samples were diluted 1:10 with 0.1M PBS to reduce interfering matrix effects. And finally, detecting by adopting DPV.

Example 2 Performance and characterization

(1) Characterization of the composite Material

Transmission Electron Microscopy (TEM) studies reveal the morphological structures of the synthesized graphene oxide, reduced graphene oxide, and reduced graphene oxide-gold nanocomposite. Pure graphene oxide appears as a transparent film, indicating that it completely exfoliated in aqueous solution (fig. 2A). It is clear from the image that the reduced graphene oxide is in a wrinkled structure (fig. 2B), and the spherical gold nanoparticles of 10-20nm are uniformly distributed on the reduced graphene oxide sheet (fig. 2C). This indicates that rGO-Au nanoparticles have been successfully synthesized.

FIG. 2D is a Raman spectrum showing that the graphene oxide particles are 1350cm in length^-1And 1600cm^-1And the D and G characteristic peaks are obvious nearby. The D/G peak intensity ratio (ID/IG) of rGO-Au (1.25) and rGO (1.23) is significantly improved compared to GO (0.91), similar to what Wu et al do. This indicates that the grain size and structure of graphene oxide undergoes a significant change in the functionalization reaction. Also, the successful reduction of graphene oxide to reduced graphene oxide is demonstrated.

(2) Electrochemical performance of composite materials

At 10mM K₃[Fe(CN)₆]The CV method is used for researching the electrochemical activity of naked GCE, rGO/GCE and rGO-Au/GCE. Each modified electrode had well-defined redox peaks at 224 and 123mV, which correlates with the quasi-reversible redox performance of ferricyanide ions in the system (FIG. 3A). The cathode peaks for bare GCE, rGO/GCE and rGO-Au/GCE were 90.49, 177 and 213.4. mu.A, respectively. Due to the synergistic effect of the bimetallic nanocomposite, the electrochemical performance of rGO-Au/GCE is optimal. The current response of the nanocomposite was increased by a factor of 2 (2.36) compared to bare GCE. The increase of the electric active area is probably combined with the excellent conductivity and the increase of the specific surface area of the rGO-Au nano composite materialAnd off. The results show that the rGO-Au nano composite material is suitable for preparing biosensors.

The kinetics of the modified electrode was explored by studying the effect of the scan rate on cyclic voltammetry. At 10mM K₃[Fe(CN)₆]In the medium, the electrochemical performance of rGO-Au/GCE is detected at a scanning rate of 10-200 mV/s. The maximum current of the redox reaction increases linearly with the increase of the scan rate and the distance between the redox peaks gets larger (fig. 3B). Based on the above results, we have shown that the peak current and the square root of the scan rate (v) for the oxidation peak (Ipa) and the reduction peak (Ipc)^1/2) A linear fit was performed (fig. 3C). The final linear equation is Ipa-22.39 × v^1/2-21.10(R²＝0.999)，Ipc＝-5.54×v^1/2-21.52(R²0.999). These calculations indicate that the immunosensor is a diffusion-controlled surface reaction.

(3) Characterization and optimization of antibody directed immobilization

And (3) detecting the binding capacity of ZZ-BNC and a leptin antibody by using an atomic force microscope. Leptin antibodies (figure 2E) and ZZ-BNC were added directly and with height and roughness of 74.02nm and 12.83nm, respectively, on rGO-Au modified cover slips (figure 2F). After ZZ-BNC modification, the height and the roughness are respectively increased by 38.31nm and 10.89nm, which is probably related to the directional immobilization of the antibody.

The influence of the concentration of the leptin antibody on the rGO-Au/GCE electrode is also researched, and the immunosensor system with high sensitivity and good reproducibility is obtained. Different concentrations of leptin antibodies (5, 15, 25, 35 and 45ng/mL) were designed for immunosensor assays with 1pg/mL leptin standards. When the concentration was below 25ng/mL, the loading of the antibody on the electrode was incomplete (fig. 6A). When the concentration is further increased, the electrochemical reaction does not change significantly. Therefore, 25ng/mL is the optimal concentration for constructing an immunosensor.

In addition, the modified electrodes were incubated with leptin antibodies for 30, 60, 90, 120 and 150min to optimize the incubation time of the immunosensor system. The incubation time of the leptin antibody greatly affected the reaction of the immunosensor (fig. 6B). At shorter incubation times, the leptin antibody may not be effectively immobilized on the electrode surface. However, if the time is too long, the quality of protein adsorbed on the surface of the electrode may be too high, and the surface of the electrode may be insulated or even damaged. Therefore, 120min was finally selected as the optimal incubation time for the leptin antibody.

To investigate the production of leptin immunosensors, at 10mM K₃[Fe(CN)₆]DPV measurements were performed (fig. 3D). After ZZ-BNC is dripped on the surface of rGO-Au/GCE, the peak current is reduced, which indicates that ZZ-BNC is successfully combined with Au nanoparticles. Upon addition of leptin antibody, the current further decreased, indicating that the ZZ terminus of ZZ-BNC successfully bound to the antibody, which may indicate that ZZ-BNC may act as a bridge. BSA was added dropwise to block non-specific sites and the current was further reduced. The additional protein in each step hindered the electron transfer, resulting in the observed decrease in conductivity. These results indicate that leptin biosensors have been successfully prepared.

(4) Research on detection performance of leptin immunosensor

Under optimal experimental conditions, at 10mM K₃[Fe(CN)₆]The assay performance was determined in solution using different concentrations of leptin immunosensors. DPV measurements showed that the peak current signal decreased with increasing leptin concentration (fig. 4A). This decrease in performance is due to the inhibition of the electron transfer capability of the electrode after the addition of leptin. The constructed leptin immunosensor showed a broad linear range from 0.001 to 1000pg/mL with a limit of detection of 0.00087 pg/mL. The linear regression equation was calculated as I ═ 3.94lg C (leptin) +38.15 (R)²0.999) (fig. 4B). Comparing the linear range, detection limit and sensitivity of the novel immunosensor with those of previous immunosensors, it was found that the prepared leptin immunosensor has a wider linear detection range (table 1).

TABLE 1 comparison of electrochemical sensors for tumor marker detection

(5) The selectivity, anti-interference performance, reproducibility and stability of the leptin electrochemical biosensor are complicated due to the composition of a biological sample, and the immunosensor must have excellent selectivity and anti-interference capability. And evaluating the selectivity and the anti-interference performance of the prepared immunosensor by using BSA, TNF-alpha and IL-6 as interference substances. The selectivity of the sensor was examined by observing the electrochemical reaction of 1pg/mL leptin, BSA, TNF-alpha and IL-6 (FIG. 4C). In addition, the anti-interference ability of the modified electrode was evaluated by detecting electrochemical reactions of 1pg/mL leptin and a mixture of 1pg/mL leptin with 1pg/mL BSA, TNF-alpha or IL-6 as an interferent (FIG. 4D). As can be seen from fig. 4A and 4B, the sensor constructed based on the directional antibody immobilization has good selectivity and anti-interference for detecting leptin.

The stability of the sensor was evaluated over a period of time. After one week of storage in a refrigerator at 4 ℃, the current value of rGO-Au/GCE decreased by only 3.15%, indicating good durability (fig. 4E). Furthermore, the reproducibility of the immunosensor was evaluated by detecting 5 identical leptin concentrations (1pg/mL), with a Relative Standard Deviation (RSD) between the measurements of 4.66% (fig. 4F). This indicates that the immunosensor has acceptable reproducibility.

(6) Detection of mouse serum leptin by different diet modes

The body weight and body length of the mice in the 12-week obese group were higher than those in the normal group, and the differences were statistically significant (Table 2). For the experimental group, in terms of body weight, body weight gain was detected after 2 weeks of NFD and became evident after 4 weeks, with the difference in body weight being more evident after 6 weeks (51.72%). However, the body weight gain was not significant in the control group (19.40%) (fig. 7). And meanwhile, HE staining is carried out on epididymis adipose tissues, so that the successful establishment of a mouse obesity model is proved, and the success of the obesity model is proved. And meanwhile, HE staining is carried out on epididymis adipose tissues, so that successful establishment of a mouse obesity model is proved.

TABLE 2 body weight, body length and Lee index of different groups of mice

Under the same magnification, based on the results of HE staining experiments, we found that the number of adipocytes in the 12-week obese group was much smaller than that in the normal group, and the cell area was significantly larger than that in the normal group (table 3). The diameter of adipose tissues in the obese group was about 2 times the diameter of normal adipose tissues. The cell number of the obese adipose tissue was about twice that of the normal adipose tissue (fig. 5A and 5B). The cell volume of fat tissue of the obese group is obviously increased, the number of fat vacuoles in unit area is reduced, and nucleus is extruded to be flat and rare; the individual adipocytes are disrupted and fused to each other.

TABLE 3 statistics of mean diameter, number, area and density of adipocytes in different groups of mice in HE staining

Electrochemical measurements (1:1 dilution) of serum diluted in PBS clearly showed that serum leptin levels (1213.43pg/mL) of HFD (high fat diet group) were much higher than serum leptin levels (456.02pg/mL) of NFD (normal fat diet group). And compared by ELISA method, the result is consistent with the electrochemical detection method (FIG. 5C).

(7) Detection of serum leptin for different BMI populations

BMI is a commonly used international measure of obesity and health in humans. Severe obesity is defined as BMI greater than 35kg/m². Electrochemical detection and statistical analysis were performed. As shown in FIG. 8, serum leptin levels were significantly higher in obese people than in normal people. It is clear that serum leptin levels vary with body mass index. In general, the higher the BMI, the higher leptin in serum (fig. 5D).

In conclusion, the embodiment of the invention develops an electrochemical platform for immobilizing the directional antibody, which is used for detecting the serum leptin content of mice with different obesity indexes and different BMI (human body interface) populations. The prepared electrode has good electrochemical performance: the linear detection range is wide, the sensitivity is high, and the detection limit is low. Overall, the immunosensor performs better than most leptin sensors described above. In addition, the electrochemical detection of the leptin content of the mice with different diet modes is consistent with the ELISA result. More importantly, the excellent detection performance of the biosensor lays a solid foundation for the application of the biosensor in clinical detection.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.