CN107576702B - Preparation method of electrochemical sensor for detecting concentration of galectin-3 in serum - Google Patents

Abstract

The invention relates to a preparation method and application of an electrochemical sensor for detecting the blood concentration level of a biomarker-galectin-3 (Gal-3) for early diagnosis of early diagnosis biomarker-galectin-3 (Gal-3) caused by the decrease in insulin sensitivity of type 2 diabetes, and belongs to the technical field of electrochemical detection. It is characterized by: firstly synthesizing N‑GNRs‑Fe‑MOFs@AuPNs sensor substrate modified nanocomposite material and AuPt‑MB nanocomposite signal material, and then mixing detection antibody that can specifically bind to Gal‑3 and AuPt‑MB composite material , to prepare biosignal probes. Then, the Gal-3-specific capture antibody was attached to the substrate platform modified by the N-GNRs-Fe-MOFs@AuPNs composite material to facilitate the specific capture of the target Gal-3. Finally, through the specific binding of Gal-3 to its detection antibody and capture antibody, an electrochemical sensor for detecting the blood concentration level of Gal-3 was prepared, and the sensor was successfully used for the detection of Gal-3 in serum. The invention has the advantages of high sensitivity, strong specificity, rapid detection and convenience. The invention provides early diagnosis basis for the insulin resistance causing type 2 diabetes mellitus.

Description

Preparation of an electrochemical sensor for detection of galectin-3 concentration in serum method

Technical field:

The invention relates to a preparation method and application of an electrochemical sensor for detecting the blood concentration level of the early diagnosis biomarker-galectin-3 (Gal-3) caused by the decrease in insulin sensitivity of type 2 diabetes mellitus, in particular based on AuPt-MB nanocomposite material as a The sandwich-type immunobiosensor prepared by a novel signal probe is used for detecting the concentration of galectin-3 (Gal-3) in serum, and belongs to the field of electrochemical detection.

Background technique:

Diabetes is growing rapidly worldwide and has posed a serious threat to human health. In particular, type 2 diabetes (T2D), caused by decreased insulin sensitivity, can lead to fatal cardiovascular complications and account for 90% of the prevalence of diabetes. In recent studies, it was found that galectin-3 (Gal-3) can directly bind to insulin receptors to inhibit downstream insulin receptor (IR) signaling and link inflammation to reduce insulin sensitivity, ultimately leading to insulin resistance and glucose intolerance, lead to type 2 diabetes. Therefore, monitoring the changes of Gal-3 concentration in blood is particularly important for the prevention of T2D and its cardiovascular complications.

The main traditional methods for the detection of Gal-3 at present are: enzyme-linked immunosorbent assay (ELISA), immunohistochemistry and "conventional Gal-3" assay (Abbott Diagnostics). However, these traditional methods are relatively expensive, time-consuming, semi-quantitative, require skilled operators, limited sensitivity and narrow linear range. In particular, it cannot meet the clinical needs of early detection. Therefore, it is very important to explore a simple, rapid and sensitive detection method for Gal-3. Compared with traditional detection methods, electrochemical immunosensor detection methods have attracted more and more attention in the detection of clinical protein biomarkers in recent years due to their advantages of high sensitivity, strong specificity and rapid detection. Meanwhile, electrochemical biosensors based on nanomaterials are widely used in the detection of biological samples due to their simplicity, rapidity, low cost, and high sensitivity. Therefore, the construction of a sandwich electrochemical immunobiosensor based on nanocomposites provides a new idea for the realization of low-concentration and high-sensitivity detection of Gal-3 in serum.

Methylene blue (MB), a derivative of phenothiazine dyes, has been widely used as an emerging electrochemical redox active species due to the presence of electron-rich sulfur and nitrogen heteroatoms in its structure for catalysis, sensing and Redox indication and other fields. However, its application in immunosensor signaling probes is limited due to its heterogeneous morphology. In order to solve this problem, the present invention innovatively introduces AuPt bimetallic nanomaterials with large specific surface area, strong electrical conductivity and good biomolecular compatibility to undergo oxidative polymerization with MB, and obtain AuPt-MB nanocomposite with uniform rod shape. material and showed remarkable electrochemical activity. In the present invention, the AuPt-MB nanocomposite material is combined with the Gal-3 specific detection antibody Ab2, and BSA is used as a blocking agent to prepare a novel nanometer signal probe.

In order to further increase the sensitivity and stability of the sandwich immunosensor. Introduced a novel nanocomposite of nitrogen-doped graphene nanoribbons (N-GNRs)-iron metal organic frameworks (Fe-MOFs)-gold nanoparticles (AuNPs)-(N-GNRs-Fe-MOFs@AuNPs) As a sensor substrate modification material. Metal-organic frameworks (MOFs) are a kind of zeolite-like crystalline porous materials, they have the advantages of large surface area, flexible porosity, easy to customize composition composition, and many active sites, etc. As new multifunctional materials, they have been widely used. It is widely used in the fields of gas storage/separation, catalysis and sensing. The Fe-MOFs introduced in the present invention have the characteristics of good stability, large specific surface area and low toxicity. Because of its large specific surface area, a large number of AuNPs nanoparticles can be bound on its surface through Au-N chemical bonds to increase its conductivity, and at the same time, it is helpful to immobilize and bind Gal-3 specific capture antibody Ab ₁ in the next step. To further increase the electrical conductivity, N-GNRs-Fe-MOFs@AuNPs nanocomposites were obtained by mixing N-GNRs with Fe-MOFs@AuNPs. N-GNRs are synthesized from N-doped multi-walled carbon nanotubes (N-MWCNTs), which are widely used to improve the electrical conductivity of modified electrode surfaces. Compared with carbon nanotubes, N-GNRs possess reactive boundaries and inherent terraced and stepped structures, which can enhance chemical activity and adsorption capacity. Surprisingly, the final N-GNRs-Fe-MOFs@AuNPs nanocomposite not only possesses the advantages of N-GNRs, Fe-MOFs, and AuNPs, but also shows a significant combined effect in electrical conductivity. Therefore, the novel nanocomposite material is applied in the substrate modification of the electrochemical immunosensor to further enhance the sensitivity and stability of the sensor.

In the present invention, a novel nano-signal probe is constructed based on AuPt-MB nanocomposite materials and N-GNRs-Fe-MOFs@AuNPs nanocomposite materials are used as substrate modification materials for immunosensors, and a new type of nano-signal probe for galectin-3 (Gal- 3) The preparation method and application of an electrochemical sensor for concentration detection provide an early diagnosis basis for insulin resistance of type 2 diabetes.

Invention content:

The object of the present invention is to provide a kind of electrochemical sensor preparation method and application for the detection of galectin-3 (Gal-3) concentration in serum, it is characterized in that comprising the following steps:

(1) Preparation of nitrogen-doped graphene nanoribbons (N-GNRs)-iron metal organic frameworks (Fe-MOFs)-gold nanoparticles (AuNPs) sensor substrate modified nanocomposites.

(2) Preparation of gold-platinum bimetallic (AuPt)-methylene blue (MB)-Gal-3 specific detection antibody (Ab ₂ ) nano-signal probe.

(3) Establish an electrochemical immunobiosensor, detect Gal-3 in serum, and draw a standard curve.

The preparation process of the N-GNRs-Fe-MOFs@AuNPs composite of the present invention specifically includes the following steps, and is characterized in that it includes the following steps:

(1) Preparation of N-GNRs materials:

Weigh 0.1 g of nitrogen-doped multi-walled carbon nanotubes (N-MWCNTs) into 10 mL of a mixed solution of H ₂ SO ₄ and H ₃ PO ₄ (H ₂ SO ₄ : H ₃ PO ₄ =9:1) and mix well . The above mixed solution was placed in an autoclave and heated to 140 °C for 10 min under magnetic stirring, and then the reaction mixture was transferred to an oil bath at 65 °C, and 0.25 g of KMnO was added under stirring to continue the reaction for ₈ min. After the reaction, the solution was controlled to cool down to room temperature, and the obtained solution was centrifuged at 10,000 r/min for 5 min, and washed with ultrapure water for 3 times. The obtained precipitate was dried in vacuum for 24 h and stored at 4°C for later use.

(2) Preparation of Fe-MOFs

0.187g FeCl ₃ ·6H ₂ O and 0.126g 2-aminoterephthalic acid were respectively weighed and added to 15 mL of dimethylformamide (DMF) solution, and mixed well. The above mixed solution was heated in silicone oil at 120° C. for 4 hours, and 200 μL of glacial acetic acid was added to the mixed solution 15 minutes after the start of heating. After heating, the solution was controlled to cool down to room temperature, and the obtained solution was centrifuged at 10,000 r/min for 5 min, and washed three times with DMF, absolute ethanol and ultrapure water, respectively. The obtained precipitate was dried in vacuum for 24 h and stored at 4°C for later use.

(3) Preparation of Fe-MOFs@AuNPs nanocomposites

Take 2 mL of HAuCl ₄ ·4H ₂ O (1%) solution and add it to the prepared 2 mL Fe-MOFs (1 mg mL ^-1 ) solution. After vigorously sonicating for 30 minutes, slowly add 4 mL of NaBH ₄ (0.1 M) to the above solution dropwise. ) at the same time on a magnetic stirrer to carry out stirring reaction for 30min, the obtained solution was centrifuged at 10000r/min for 5min, and washed 3 times with ultrapure water. The obtained precipitate was redispersed in 4 mL of deionized water and stored at 4°C for later use.

(4) Preparation of N-GNRs-Fe-MOFs@AuNPs nanocomposites

0.2 mg of N-GNRs were weighed and dissolved in 2 mL of deionized water by ultrasonic, which was mixed with 4 mL of Fe-MOFs@AuNPs complex solution by ultrasonic for 30 min, and stirred overnight under the condition of magnetic stirring at 500 r/min. The resulting solution was centrifuged at 8000 r/min for 5 min, and washed three times with ultrapure water. The obtained precipitate was redispersed in 4 mL of deionized water and stored at 4°C for later use.

The preparation process of the AuPt-MB-Ab ₂ nano-signal probe of the present invention specifically includes the following steps, and is characterized in that it includes the following steps:

(1) Preparation of AuPt-MB nanocomposites:

600 μL of HCL (0.1 M) and 6 mL of dodecyltrimethylammonium bromide (DTAB 1.14 mM) were added to 1 mL of MB (9.8 mM) solution and stirred vigorously for 10 min. 100 μL of HAuCl ₄ (4 wt %,) and 100 μL of H ₂ PtCl ₆ (4 wt %, ) were added simultaneously, and stirring was continued at room temperature for 6 h. The obtained solution was centrifuged at 8000 r/min for 10 min, and washed three times with ultrapure water. The obtained precipitate was redispersed into 2 mL of PBS buffer solution (0.1 M pH 7.0), and stored at 4°C for later use.

(2) Preparation of AuPt-MB-Ab ₂ nano-signal probe:

Add 100 μL of Gal-3-specific detection antibody Ab ₂ to 2 mL of AuPt-MB nanocomposite solution, and stir gently at 4°C for 12 h. 40 μL of bovine serum albumin BSA (1 wt%) was added and gentle stirring was continued for 6 h at 4°C. The obtained solution was centrifuged at 8000 r/min for 10 min, and the obtained precipitate was redispersed into 2 mL of PBS buffer solution (0.1 M pH 7.0), and stored at 4° C. for later use.

The establishment of an electrochemical immune biosensor according to the present invention, detecting Gal-3 in serum, and drawing a standard curve, is characterized in that the following steps are included:

(1) Polish the electrode into a mirror surface with Al ₂ O ₃ powder of 0.3 and 0.05 μm, respectively, then ultrasonically sonicate the electrode in the order of ultrapure water, absolute ethanol, and ultrapure water for 5 minutes each, and dry at room temperature for use;

(2) 6 μL of the prepared N-GNRs-Fe-MOFs@AuNPs nanocomposite solution was dropped on the electrode surface and dried at room temperature.

(3) Add 6 μL of Gal-3 specific capture antibody Ab ₁ solution dropwise to the modified electrode surface, and incubate at 4° C. for 12 h.

(4) Rinse the incubated electrode with double distilled water, drop 6 μL of BSA (1 wt %) blocking agent on the surface of the electrode, and block at room temperature for 30 min.

(5) The above-mentioned BSA-blocked electrode was rinsed with washing buffer (10 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ , 37 mM NaCl, 2.7 mM KCl, pH 7.4) and dried in nitrogen.

(6) 6 μL of different concentrations of target Gal-3 were added dropwise to the electrode surface, incubated at 37° C. for 1 h, rinsed with washing buffer, and dried in nitrogen.

(7) Drop 10 μL of the prepared AuPt-MB-Ab ₂ nano-signal probe solution on the electrode surface after drying, incubate at 37°C for 1 h, rinse with washing buffer and dry in nitrogen.

(8) The electrode was placed in 5mL, 0.1M PBS (PH 7.0) for characterization, and the peak value of its current change current response was measured.

(9) According to the obtained current change, the current response peak value and the Gal-3 concentration have a linear relationship, and the working curve is drawn.

Compared with the prior art, a preparation method and application of an electrochemical sensor for the detection of galectin-3 (Gal-3) concentration in serum of the present invention, its outstanding features are:

(1) The N-GNRs-Fe-MOFs@AuNPs nanocomposite was used for electrochemical immunobiosensor substrate modification, which not only provided a large number of active sites to immobilize the capture antibody, but also promoted the electron conduction rate, thereby improving the electrical conductivity. Sensitivity and biocompatibility of chemoimmunobiosensors;

(2) The AuPt-MB-based nanocomposite construction signal probe was introduced into the preparation of electrochemical immunobiosensors, in which the AuPt bimetallic nanomaterial not only has the function of amplifying its electrochemical signal, but also increased its ability to detect antibodies. Biocompatibility.

(3) The electrochemical immunobiosensor prepared by this method can provide a new idea for the detection of Gal-3 blood concentration level which reduces insulin sensitivity, and provide an early diagnosis basis for the prevention of type 2 diabetes.

(4) Using the exact same nanomaterials and modification methods, using the capture antibody, the specific recognition of the signal probe and the target protein, it is only necessary to change the detection antibody of the probe and the capture antibody immobilized on the substrate to achieve a variety of Specific and highly sensitive detection of protein biomarkers, in addition, this method is simple, fast, and easy to realize commercialization, thereby promoting the development of translational medicine.

Description of drawings:

FIG. 1 is a schematic diagram of the construction of the electrochemical immunobiosensor of the present invention.

Figure 2 is the scanning electron microscope image of N-GNRs, Fe-MOFs, Fe-MOFs@AuNPs and N-GNRs-Fe-MOFs@AuNPs of the present invention, and the transmission electron microscope and EDS images of AuPt-MB.

Figure 3 shows the EDS image of Fe-MOFs@AuNPs

FIG. 4 is the linear relationship between the current response peak value of differential pulse voltammetry and the logarithm of concentration obtained by the electrochemical immunobiosensor of the present invention when Gal-3 is detected.

Figure 5 shows the repeatability, specificity and stability of the sensor.

Detailed ways:

The present invention will be further described below with reference to specific embodiments, and it should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

Example 1

Step 1. Weigh 0.187g FeCl ₃ ·6H ₂ O and 0.126g 2-aminoterephthalic acid, respectively, into 15 mL of dimethylformamide (DMF) solution, and mix well. The above mixed solution was heated in silicone oil at 120° C. for 4 hours, and 200 μL of glacial acetic acid was added to the mixed solution 15 minutes after the start of heating. After heating, the solution was controlled to cool down to room temperature, and the obtained solution was centrifuged at 10,000 r/min for 5 min, and washed three times with DMF, absolute ethanol and ultrapure water, respectively. The obtained precipitate was dried in vacuum for 24 h to obtain Fe-MOFs, which were stored at 4°C for later use.

Step 2. Take 2 mL of HAuCl ₄ ·4H ₂ O (1%) solution and add it to the prepared 2 mL Fe-MOFs (1 mg mL ^-1 ) solution. After vigorously sonicating for 30 minutes, slowly add 4 mL of NaBH dropwise to the above solution ₄ (0.1M) was simultaneously placed on a magnetic stirrer for stirring reaction for 30min, the obtained solution was centrifuged at 10000r/min for 5min, and washed 3 times with ultrapure water. To obtain Fe-MOFs@AuNPs, the obtained Fe-MOFs@AuNPs precipitate was redispersed in 4 mL of deionized water, and stored at 4 °C for later use.

Step 3. Weigh 0.2 mg of N-GNRs and dissolve them in 2 mL of deionized water by ultrasonic, mix them with 4 mL of Fe-MOFs@AuNPs complex solution by ultrasonic for 30 min, and stir overnight under the condition of magnetic stirring at 500 r/min. The resulting solution was centrifuged at 8000 r/min for 5 min, and washed three times with ultrapure water. To obtain N-GNRs-Fe-MOFs@AuNPs, the obtained N-GNRs-Fe-MOFs@AuNPs precipitate was redispersed into 4 mL of deionized water, and stored at 4 °C for later use.

Step 4. Add 600 μL of HCL (0.1 M) and 6 mL of dodecyltrimethylammonium bromide (DTAB 1.14 mM) to 1 mL of MB (9.8 mM) solution, respectively, and stir vigorously for 10 min. 100 μL of HAuCl ₄ (4 wt %,) and 100 μL of H ₂ PtCl ₆ (4 wt %, ) were added simultaneously, and stirring was continued at room temperature for 6 h. The obtained solution was centrifuged at 8000 r/min for 10 min, and washed three times with ultrapure water. The obtained AuPt-MB precipitate was redispersed into 2 mL of PBS buffer solution (0.1 M pH 7.0), and stored at 4°C for later use.

Step 5. Add 100 μL of Gal-3-specific detection antibody Ab ₂ to 2 mL of the AuPt-MB nanocomposite solution, and stir gently at 4° C. for 12 h. 40 μL of bovine serum albumin BSA (1 wt%) was added and gentle stirring was continued for 6 h at 4°C. The obtained solution was centrifuged at 8000 r/min for 10 min to obtain AuPt-MB-Ab ₂ signal probe. The obtained signal probe precipitate was redispersed into 2 mL of PBS buffer solution (0.1M pH 7.0), and stored at 4°C for later use.

Step 6. Polish the electrode into a mirror surface with Al ₂ O ₃ powder of 0.3 and 0.05 μm, respectively, then ultrasonically sonicate the electrode in the order of ultrapure water, absolute ethanol, and ultrapure water for 5 minutes each, and dry at room temperature for use;

Step 7. Drop 6 μL of the prepared N-GNRs-Fe-MOFs@AuNPs nanocomposite solution on the electrode surface and dry at room temperature.

Step 8. Add 6 μL Gal-3 specific capture antibody Ab ₁ solution dropwise to the modified electrode surface, and incubate at 4°C for 12h.

Step 9. Rinse the incubated electrode with double distilled water, drop 6 μL BSA (1 wt %) blocking agent on the surface of the electrode, and block at room temperature for 30 min.

Step 10. The above BSA-blocked electrodes were rinsed with wash buffer (10 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ , 37 mM NaCl, 2.7 mM KCl, pH 7.4) and dried in nitrogen.

Step 11. Drop 6 μL of different concentrations of target Gal-3 onto the electrode surface, incubate at 37°C for 1 h, rinse with washing buffer and dry in nitrogen.

Step 12. Drop 10 μL of the prepared AuPt-MB-Ab ₂ nano-signal probe solution on the electrode surface after drying, incubate at 37° C. for 1 h, rinse with washing buffer and dry in nitrogen.

Step 13. The electrode was placed in 5mL, 0.1M PBS (PH 7.0) for characterization, and its current change peak value of current response was measured.

Step 14. According to the obtained current change, the current response peak value and the Gal-3 concentration have a linear relationship, and draw a working curve. The results showed that the concentration of Gal-3 had a linear relationship in the range of 100fg mL ^-1 -50ng mL ^-1 , the linear correlation coefficient was 0.99502, and the detection limit was 33.33fg mL ^-1 (S/N=3).

Step 15. Store the above-mentioned sensor of the present invention at 4°C, and intermittently use differential pulse voltammetry to detect the current response of the sensor. After 3 weeks of storage, the current response is still 95.17% of the initial current, and the surface sensor has good stability;

Step 16. The present invention takes 5 immunobiosensors prepared in the same batch, and measures 1 ng mL ^-1 of Gal-3 under the same conditions. Each electrode is measured 3 times, and the relative standard deviation of the current response value is obtained. It is 2.75%, indicating that the repeatability of the sensor is good.

Step 17. The above-mentioned sensor of the present invention is used to detect the mixture of 100ng mL ^-1 of different interfering substances and 1 ng mL ^-1 Gal-3, and the relative standard deviation of the detection result is 1.55%, indicating that the interfering substances have a great interference effect on the detection result of the sensor. Small, the specificity of the sensor is good.

The above are only the preferred embodiments of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and retouching should also be regarded as the protection scope of the present invention.

Claims (2)

1. A preparation method of an electrochemical sensor for detecting the concentration of galectin-3 in serum is characterized by comprising the following steps:

(1) preparing a nitrogen-doped graphene nanoribbon:

0.1g of nitrogen-doped multi-walled carbon nanotubes are weighed inTo 10mL of H₂SO₄And H₃PO₄Uniformly mixing the mixed solution, placing the mixed solution in a high-pressure reaction kettle, heating to 140 ℃ for 10min under the condition of magnetic stirring, transferring the reaction mixed solution into a 65 ℃ oil bath, adding 0.25g of potassium permanganate under the condition of stirring, continuously reacting for 8min, controlling the solution to cool to room temperature after the reaction is finished, carrying out 10000r/min on the obtained solution, centrifuging for 5min, washing for 3 times by using ultrapure water, carrying out vacuum drying on the obtained precipitate for 24h, and storing at 4 ℃ for later use;

(2) preparation of iron metal organic framework:

weighing 0.187g of ferric chloride hexahydrate and 0.126g of 2-amino terephthalic acid, adding into 15mL of dimethylformamide solution, uniformly mixing, placing the mixed solution into 120 ℃ silicone oil, heating for 4h, adding 200 mu L of glacial acetic acid into the mixed solution at the 15 th min after the heating is started, controlling the solution to cool to room temperature after the heating is finished, centrifuging the obtained solution for 5min at 10000r/min, respectively washing precipitates obtained by centrifuging for 3 times with DMF (dimethyl formamide), absolute ethyl alcohol and ultrapure water, finally drying the obtained precipitates in vacuum for 24h, and storing at 4 ℃ for later use;

(3) preparing an iron metal organic framework @ gold nanoparticle composite material:

2mL of 1% chloroauric acid tetrahydrate solution was added to 2mL,1mg mL of the prepared^-1After intense ultrasonic treatment is carried out for 30 minutes, 4mL of 0.1M sodium borohydride solution is slowly added into the iron metal organic framework solution dropwise, the solution is placed on a magnetic stirrer to be stirred and reacted for 30 minutes, the obtained solution is subjected to 10000r/min and centrifugation for 5 minutes, is washed for 3 times by ultrapure water, and the obtained precipitate is re-dispersed into 4mL of deionized water and is stored for later use at 4 ℃;

(4) preparing a nitrogen-doped graphene nanoribbon-iron metal organic framework @ gold nanoparticle composite material:

weighing 0.2mg of nitrogen-doped graphene nanoribbon, ultrasonically dissolving the nitrogen-doped graphene nanoribbon in 2mL of deionized water, ultrasonically mixing the nitrogen-doped graphene nanoribbon with 4mL of prepared iron metal organic framework @ gold nanoparticle compound solution for 30min, stirring overnight under the condition of magnetic stirring of 500r/min, centrifuging the obtained solution for 5min at 8000r/min, cleaning the solution for 3 times by using ultrapure water, re-dispersing the obtained precipitate in 4mL of deionized water, and storing the precipitate at 4 ℃ for later use;

(5) preparation of gold platinum-methylene blue:

adding 600 mu L of 0.1M hydrochloric acid and 6mL of 1.14mM dodecyl trimethyl ammonium bromide into 1mL of 9.8mM methylene blue solution, stirring vigorously for 10min, adding 100 mu L of 4 wt% chloroauric acid solution and 100 mu L of 4 wt% chloroplatinic acid solution, stirring continuously at room temperature for 6h, subjecting the obtained solution to 8000r/min, centrifuging for 10min, washing with ultrapure water for 3 times, re-dispersing the obtained precipitate into 2mL of 0.1M PBS buffer solution with pH of 7.0, and storing at 4 ℃ for later use;

(6) preparation of platinum-methylene blue-secondary antibody:

adding 100 mu L of galectin-3 specificity detection secondary antibody into 2mL of gold platinum-methylene blue nano composite material solution, stirring gently at 4 ℃ for 12 hours, adding 40 mu L of bovine serum albumin with the concentration of 1 wt%, stirring gently at 4 ℃ for 6 hours, centrifuging the obtained solution at 8000r/min for 10 minutes, re-dispersing the obtained precipitate into 2mL of PBS (phosphate buffer solution) with the concentration of 0.1M and the pH value of 7.0, and storing at 4 ℃ for later use;

(7) establishing an electrochemical sensor:

a. polishing the electrodes into mirror surfaces by using 0.3 and 0.05 mu m aluminum oxide powder respectively, then carrying out ultrasonic treatment on the electrodes for 5min respectively according to the sequence of ultrapure water, absolute ethyl alcohol and ultrapure water, and drying at room temperature for later use;

b. dripping 6 mu L of the prepared nitrogen-doped graphene nanoribbon-iron metal organic framework @ gold nanoparticle composite material solution on the surface of an electrode, and drying at room temperature;

c. dripping 6 mu L of galectin-3 specific capture first antibody solution to the surface of the modified electrode, and incubating for 12h at 4 ℃;

d. washing the incubated electrode with double distilled water, dripping 6 mu L of 1 wt% BSA on the surface of the electrode, and sealing at room temperature for 30 min;

e. the BSA solution was blocked and the electrode was used with 10mM Na₂HPO₄,2mM KH₂PO₄37mM NaCl,2.7mM KCl, pH7.4 wash buffer and dry under nitrogen.

2. The method for the quantitative detection of galectin-3 by the sensor obtained by the preparation method according to claim 1, comprising the steps of:

(1) dripping 6 mu L of target galectin-3 with different concentrations on the surface of an electrode, incubating at 37 ℃ for 1h, washing with a cleaning buffer solution, and drying in nitrogen;

(2) dripping 10 μ L of the prepared gold platinum-methylene blue-second antibody solution on the surface of the dried electrode, incubating at 37 ℃ for 1h, washing with a washing buffer solution, and drying in nitrogen;

(3) placing the electrode in 5mL of PBS (phosphate buffer solution) with the concentration of 0.1M and the pH value of 7.0 for characterization, and measuring the current change current response peak value of the electrode;

(4) and drawing a working curve according to the linear relation between the current change current response peak value and the concentration of the galectin-3.

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