High-sensitivity electrochemical immunosensor for detecting folic acid as well as preparation method and application of high-sensitivity electrochemical immunosensor

Abstract

The invention belongs to the technical field of electrochemical sensors, and particularly relates to a high-sensitivity electrochemical immunosensor for detecting folic acid as well as a preparation method and application of the high-sensitivity electrochemical immunosensor. The FBP is introduced into a folic acid detection system as a specific recognition element for the first time, compared with a traditional immunosensor based on an antibody, the system innovatively utilizes the high affinity and specific binding characteristic between the FBP and folic acid, the surface of an AuNFs / SNC electrode is modified with the FBP, and the folic acid detection sensitivity is improved. And a sensing interface with integration of double functions of molecular recognition and electrochemical signal conversion is constructed. Experimental results show that the sensor has excellent response characteristics to FA in a linear range of 1-100 nM, and the detection limit is as low as 0.33 nM (S / N = 3).

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemical sensors, and in particular relates to an electrochemical immunosensor for detecting folic acid with high sensitivity, and a preparation method and application thereof. Background Art

[0002] Folic acid (FA), also known as vitamin B9, is considered to be one of the essential biomolecules for cell growth and division. FA cannot be stored in the human body and must be obtained through dietary supplements or other sources. It is widely found in leafy vegetables, egg yolks, liver, and citrus fruits. FA deficiency can lead to a range of diseases, such as neural tube defects (NTDs) and cardiovascular disease, and may also be associated with congenital malformations and low birth weight. However, factors such as the low content of FA in some samples, poor stability, and complex sample composition pose significant challenges to FA detection. In order to improve the level of FA detection, a sensitive, robust, simple and reliable detection method is urgently needed.

[0003] In recent years, a variety of methods for quantitative FA detection have been reported. Common methods for FA detection include chromatography, microbiology, immunoassays, and electrochemical methods. Chromatography is a widely used technique in laboratories. High-performance liquid chromatography (HPLC) offers high sensitivity and is suitable for drug quality control and clinical research, but the pretreatment steps are complex and require specialized equipment. Liquid chromatography-mass spectrometry (LC-MS), which combines chromatographic separation with mass spectrometry detection, significantly improves sensitivity and specificity and can simultaneously analyze multiple forms of folate. However, the pretreatment steps are complex, the equipment is expensive, and specialized personnel are required. Microbiology, the most traditional detection method, indirectly determines folate content by monitoring the growth of folate-dependent bacteria. It is low-cost and particularly suitable for complex matrices such as food. However, this method is time-consuming (requiring 24-48 hours of incubation), has poor specificity, and requires strict aseptic techniques. Immunoassays, including ELISA and immunochromatographic strips, are based on antigen-antibody reactions. They are simple and rapid to operate, suitable for batch screening and on-site testing, but the kits are expensive, and there are issues such as poor reproducibility and a high number of interfering factors. The electrochemical method utilizes the redox properties of folic acid and has the potential to be fast, low-cost and portable, but it is easily interfered by other electroactive substances and has poor specificity and sensitivity.

[0004] Electrochemical immunosensor is a new detection technology that combines the high specificity of immunoassay with the high sensitivity of electrochemical detection. Its core principle is to convert biological recognition into a measurable electrical signal through the specific binding of antigens and antibodies, thereby achieving quantitative detection of the target. Compared with traditional detection methods, the most significant advantages of electrochemical immunosensor are high sensitivity and specificity. The immune response achieves accurate recognition, and electrochemical signal amplification technology can improve sensitivity. At the same time, specific recognition proteins selectively bind to the target, effectively avoiding interference from other substances. It is especially suitable for scenarios that require highly sensitive and highly selective detection. Due to its higher sensitivity, lower cost, easier operation, and strong real-time analysis, this method has been applied to the detection of multiple vitamins. However, at present, very few folic acid biosensors based on electrochemical immunosensor have been reported. Lermo et al. (2009) proposed an electrochemical detection immunoassay for folic acid based on a magnetic sensor, with a detection limit as low as 13.1nM. Although this method is fast, cheap and environmentally friendly, due to the extremely low content of FA in many samples (less than 10nM), this method cannot detect low-content folic acid samples. It is crucial to develop electrochemical immunosensors with higher sensitivity. Summary of the Invention

[0005] In order to fill the technical deficiencies in the current electrochemical detection of folic acid, the present invention provides the following technical solutions:

[0006] The first aspect of the present invention is to provide a method for preparing an electrochemical immunosensor for specifically detecting folic acid, the method comprising the following steps:

[0007] (1) preparing an electrochemical electrode for obtaining a gold nanoflower layer;

[0008] (2) Immobilizing folate binding protein (FBP) on the electrode surface;

[0009] (3) Block the electrode surface that is not bound to FBP.

[0010] Furthermore, the operation of step (1) is:

[0011] 1.1) Use the screen-printed electrode as the base electrode, clean and activate it;

[0012] 1.2) electrochemically doping the above electrodes to achieve sulfur / nitrogen co-doping to obtain SNC electrodes;

[0013] 1.3) Electrodepositing gold nanoparticles on the surface of the SNC electrode obtained above to obtain a gold nanoflower layer.

[0014] Preferably, the cleaning and activation operations in step 1.1) are: activating the electrode in 0.1MH2SO4 by cyclic voltammetry (CV) to remove surface oxides and enhance activity; more preferably, the scan rate of the cyclic voltammetry is 80-120 mV / s; the number of cycles of activating the electrode is 8-12 cycles; more preferably, the working electrode material and the counter electrode material of the substrate electrode SPE electrode are both carbon, and the reference electrode material is Ag / AgCl.

[0015] Preferably, the operation of step 1.2) is: electrochemical doping in a thiourea solution using cyclic voltammetry; the concentration of the thiourea is 0.5 to 0.7 M, and the cyclic voltammetry is continuous electrochemical doping for 25 to 35 cycles at -1.2 V to 0.2 V, with a scan rate of 50 to 70 mV / s; more preferably, the concentration of the thiourea solution is 0.6 M, and ultrasonic dispersion is performed for 10 minutes to maintain uniformity.

[0016] Preferably, the operation of step 1.3) is: electrodepositing a gold nanoflower layer in a chloroauric acid (HAuCl4) solution by CV to obtain an AuNFs / SNC nanocomposite material; preferably, the concentration of chloroauric acid is 2-3 mM, and the solvent is 1×PBS; more preferably, the voltage range of the CV is -0.5V-0.4V; more preferably, the scan rate of the CV is 40-60mV / s, and the gold is electrodeposited for 8-12 cycles;

[0017] Furthermore, the AuNFs / SNC nanocomposite material was thoroughly rinsed with ultrapure water and dried with nitrogen.

[0018] Furthermore, the step (2) is performed as follows:

[0019] 2.1) Activation of the electrode carboxyl interface;

[0020] 2.2) Apply FBP solution to the electrode surface and incubate;

[0021] Preferably, the carboxyl interface activation described in step 2.1) is to prepare 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) with MES buffer, take the EDC / NHS solution and drop it on the surface of the SPE working electrode, and react in the dark. After the reaction is completed, the electrode surface is immediately and gently rinsed 3 times with pre-cooled MES buffer and blown dry with nitrogen; more preferably, the MES buffer is freshly prepared and its concentration is 0.08-0.12M MES buffer; more preferably, the concentrations of EDC and NHS in the EDC / NHS solution are 0.3-0.5M and 0.08-0.12M, respectively; more preferably, the activation reaction temperature is 20-30°C and the reaction time is 20-40min;

[0022] More preferably, the operation of step 2.2) is to drop the FBP solution onto the electrode surface and incubate at 35-40° C. for 30-50 min; more preferably, the FBP solution is prepared by dissolving it in 1×PBS at pH 7.4 to a concentration of 70-80 mg / mL.

[0023] Furthermore, the step (3) is performed by immersing the modified electrode in step (2) in a bovine serum albumin (BSA) solution for blocking. Preferably, the concentration of the BSA is 1-5%, and the blocking is performed at 35-40°C for 25-40 minutes, or at 4°C overnight.

[0024] The second aspect of the present invention is to provide an electrochemical immunosensor for specifically detecting folic acid prepared by the method described in the first aspect.

[0025] The third aspect of the present invention is to provide the use of the method described in the first aspect or the sensor described in the second aspect in detecting folic acid; the detection is a non-disease diagnosis detection.

[0026] The beneficial effects of the present invention include:

[0027] 1) Preparation of AuNFs / SNC nanocomposites with high electrochemical activity

[0028] By introducing sulfur and nitrogen heteroatoms onto a SN co-doped carbon substrate (SNC), the electrochemical activity of the carbon material is significantly enhanced. The abundant defect sites on the SNC surface effectively regulate the nucleation and growth of gold nanoflowers (AuNFs), resulting in a more uniform and dense surface loading of AuNFs. The resulting nanocomposite AuNFs / SNC serves as a working electrode material, further enhancing the electrochemical performance of the sensing material. Morphological and image analysis methods (e.g., XRD, SEM, TEM, EDS, and XPS) confirmed the successful preparation of the AuNFs / SNC. The hydrophilic surface of the AuNFs / SNC (contact angle = 58.57°) synergistically promotes the high-density immobilization of folate-binding protein (FBP), amplifying the electrochemical signal perturbation caused by target binding and further improving the sensor's sensitivity.

[0029] 2) First application of folate binding protein in electrochemical immunosensor detection of folate

[0030] The constructed electrochemical immunosensor was verified by CV and EIS. The results showed that the electrode modified with the composite material had the highest redox peak current. After each modification step, the corresponding CV redox peak current decreased and the impedance increased, proving the successful construction of the electrochemical immunosensor.

[0031] 3) Achieve ultra-sensitive FA detection

[0032] The optimal HRP-FA dilution ratio (1 / 100), FBP concentration (75 mg / mL), and incubation time (30 min) were selected. Under these optimal detection conditions, an ultra-low detection limit of 0.33 nM (S / N = 3) was achieved using DPV in pH 7.4 PBS buffer. This significantly lower limit of detection (LOD) than the 13.1 nM limit reported in a previous literature review for a magnetic sensor-based electrochemical immunoassay for folate detection. The sensor exhibited excellent response characteristics to FA within a linear range of 1-100 nM.

[0033] 4) The sensor has highly selective detection performance

[0034] By comparing different potential interfering substances, such as glucose (Glu), uric acid (UA), lactic acid (Lac), ascorbic acid (AA), vitamin B1 (VB1), vitamin B7 (VB7) 12 (VB 12 The selectivity of FA sensing was evaluated by measuring the DPV signal changes caused by interfering substances. The results showed that the changes in the electrical signal caused by interfering substances were negligible, indicating excellent selectivity for FA sensing.

[0035] 5) The sensor has good storage stability

[0036] The sensor still retains more than 90% of the initial response signal after being stored at 4°C for 25 days. Stability experiments have confirmed that the sensor has a shelf life of ≥25 days when sealed and stored at 4°C, which can meet the stability requirements in scenarios such as clinical testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Preparation of FA electrochemical immunosensor and detection principle diagram of the method;

[0038] Figure 2 XRD patterns of nanomaterials on the working electrode surface at different preparation stages;

[0039] Figure 3 3a, 3b, and 3c are the SEM images of C, SNC, and AuNPs / SNC on the working electrode surface, respectively; 3d is the TEM image of AuNPs / SNC; and 3e is the EDS element distribution map of AuNPs / SNC.

[0040] Figure 4 4a is the full XPS spectrum of AuNPs / SNC, bf are the XPS spectra of C1s, O 1s, Au 4f, S2p, and N 1s of AuNPs / SNC, respectively;

[0041] Figure 5 Static water contact angles of nanomaterials on the working electrode surface at different preparation stages;

[0042] Figure 6 CV (a) and EIS (b) plots of the AuNFs / SNC electrode in 0.01 M PBS solution containing 2.0 mM K4Fe(CN)6 / K3Fe(CN)6 after each surface modification step;

[0043] Figure 7 Optimization of electrochemical FA immunosensor conditions. Figure 7 ac are the effects of HRP-FA dilution ratio, FBP concentration, and incubation time on electrochemical signals;

[0044] Figure 8 DPV signals of the electrochemical immunosensor with 1–100 nM FA in 1× PBS (pH 7.4) (a) and the corresponding standard curve (b);

[0045] Figure 9 FA electrochemical immunosensor specific detection;

[0046] Figure 10 Storage stability test of FA electrochemical immunosensor. DETAILED DESCRIPTION

[0047] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods described are all conventional methods unless otherwise specified. The materials described can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute undue limitations of the present invention. It should be noted that, unless there is a conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0048] Example 1 Construction of electrochemical immunosensor

[0049] 1. Construction of electrochemical immunosensor: the process is as follows Figure 1 shown.

[0050] 1.1 Electrode pretreatment and nanomaterial modification

[0051] (1) Electrode cleaning and activation

[0052] A screen-printed electrode (SPE) (Botan Technology Co., Ltd.) was used as the substrate electrode. Both the working and counter electrodes were made of carbon, and the reference electrode was Ag / AgCl. 200 μL of 0.1 M H₂SO₄ solution was dropped onto the SPE surface. Cyclic voltammetry (scan rate 100 mV / s) was used to activate the electrode for 10 cycles in the range of -0.2 V to 1.2 V to remove surface oxides and enhance electrode activity. The electrode was then rinsed with ultrapure water and dried.

[0053] (2) Preparation of sulfur-nitrogen co-doped carbon (SNC) electrodes

[0054] Prepare a solution containing 0.6 M thiourea (McLean, T819602). Add 200 μL of thiourea solution to the activated SPE surface. Perform cyclic voltammetry at a scan rate of 60 mV / s for 30 cycles between -1.2 V and 0.2 V to co-dope the carbon electrode surface with sulfur (S) and nitrogen (N). Rinse the electrode with ultrapure water and air dry.

[0055] (3) Gold nanoparticle electrodeposition

[0056] A 2.5 mM HAuCl₄ solution was prepared by dissolving HAuCl₄ (Hynes, G-19310) in 1× PBS. 200 μL of HAuCl₄ solution was dropped onto the surface of a SNC electrode. Cyclic voltammetry (scan rate 50 mV / s) was used to electrodeposit gold for 10 cycles between -0.5 V and 0.4 V, forming a uniform layer of gold nanoflowers on the SNC surface. This resulted in the AuNFs / SNC nanocomposite. After deposition, the electrode surface was thoroughly rinsed with ultrapure water and dried with nitrogen gas for later use.

[0057] 1.2 FBP immobilization

[0058] (1) Electrode carboxyl interface activation

[0059] Freshly prepare 0.1 M MES buffer. Use the MES buffer to prepare an EDC / NHS solution containing 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Source Leaf, S30053) and 0.1 M N-hydroxysuccinimide (NHS) (Source Leaf, S20179). Apply 20 μL of the EDC / NHS solution to the SPE working electrode surface and allow to react at 25°C in the dark for 30 minutes. Immediately after the reaction, gently rinse the electrode surface three times with pre-chilled MES buffer and blow dry with nitrogen.

[0060] (2) FBP immobilization

[0061] Then, 10 μL of FBP (Baiming Biotechnology, BM841004) solution (75 mg / mL, pH 7.4 1× PBS) was dropped onto the electrode surface and incubated at 37°C for 40 min. Stable immobilization of FBP was achieved through amide bonds.

[0062] (3) Non-specific site blocking

[0063] The modified electrode was immersed in 1% bovine serum albumin (BSA) solution (Yuanye, S12012) and blocked at 37°C for 30 min to shield the unbound active sites on the electrode surface and reduce background noise.

[0064] 2. Electrode Characterization

[0065] (1) Morphology, image, and composition characterization: SEM, TEM, XPS, and GIXRD were used to characterize the morphology and material properties of the sensing electrode before and after surface modification. TEM was combined with an energy dispersive spectrometer (EDS) to accurately analyze the types and contents of various elements in the material micro-region composition.

[0066] The results are as follows Figure 2-4 ,in:

[0067] Figure 2 The XRD patterns of the products from each step of the SPE working electrode surface are shown. As shown, all diffraction peaks of AuNFs / SNCs correspond well to those of C (JCPDS No. 41-1487) and Au (JCPDS No. 04-0784), and no other peaks are observed.

[0068] Scanning electron microscopy (SEM, Figure 3 ac) images show that after electrical doping, the surface roughness of the C material is significantly improved, and uniform and dense gold nanoflowers (AuNFs) are generated by electrodeposition on its surface. The surface is rough and the diameter is about 50-200nm. Figure 3 de shows the TEM image and the corresponding EDS elemental distribution map, which clearly reveals its flower-like structure and the uniform distribution of C, N, O, S, and Au throughout the composite.

[0069] X-ray photoelectron spectroscopy (XPS) analysis was performed ( Figure 4 ). The overall spectrum proves the presence of C, N, O, S, and Au in AuNFs / SNC. Figure 4 The high-resolution C1s peak in b can be fitted into four peaks centered at 284.8, 285.9, 286.7, and 289.2 eV, which are attributed to CC, CS, CN / C=N, and CO bonds. Figure 4 In the CO 1s spectrum, the peaks at 531.8, 532.7, and 533.9 eV correspond to the three oxygen-containing groups C=O, OCO, and O=CO, respectively. Figure 4 The high-resolution XPS Au 4f spectrum in d shows that the valence state of Au is 0 + The two peaks with binding energies (BE) of 88.1 and 84.4 eV are attributed to metallic Au. 0 4f 5 / 2 and Au 0 4f 7 / 2 . Figure 4 e shows the S2p spectrum, which is composed of S2p 3 / 2 、S2p 1 / 2and S=O subpeaks, located at binding energies of 161.7eV, 162.9eV, and 164.1eV, respectively. The N 1s peak can be deconvoluted into four peaks, belonging to pyridinic N (398.7eV), pyrrolic N (400.1eV), graphitic N (401.5eV), and oxygen-containing N (404.1eV) ( Figure 4 f).

[0070] (2) Contact angle measurement: The contact angle between the working electrode surface material and water was measured using a contact angle meter (JY-82C). The measurement was performed on a flat electrode surface using a 16 μL water droplet using the sessile drop method at room temperature.

[0071] The results are as follows Figure 5 As shown, the contact angle of the AC material is 103.46°. After sulfur and nitrogen doping, the hydrophilicity is significantly improved, decreasing to 87.50°. The contact angle of AuNFs / SNC electrodeposited on the SNC surface is only 58.57°. The hydrophilic surface synergistically promotes the high-density immobilization of FBP, amplifying the electrochemical signal perturbation caused by target binding, further improving the sensitivity of the sensor.

[0072] (3) Electrochemical characterization: All electrochemical tests, including cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV), were performed on a Metrohm Autolab PGSTAT204 electrochemical workstation. To electrochemically characterize each surface modification step, we used CV and EIS to verify the construction of the immunosensor in 0.01 M PBS (pH 7.4) containing 2.0 mM K4Fe(CN)6 / K3Fe(CN)6.

[0073] The results are as follows Figure 6 Shown: CV (a) and EIS (b) plots of the AuNFs / SNC electrode in 0.01 M PBS solution containing 2.0 mM K₄Fe(CN)₆ / K₃Fe(CN)₆ after each surface modification step. The figures show that after each modification step, interfacial electron transfer between the redox probe in the solution and the electrode surface is hindered. With each modification step, the resistance increases and the redox current decreases. This confirms the successful immobilization of biomacromolecules such as FBP and BSA on the AuNFs / SNC electrode surface.

[0074] Example 2: FA Detection Process and Signal Analysis

[0075] 10 μL of FA standard solution (McLean, F809516) or the biofluid to be analyzed and HRP-FA (BioMing Biotechnology, BM841003) prepared in 1× PBS (pH 7.4) was dripped onto the working electrode. FA contained in the sample and HRP-FA competed for binding to FBP on the electrode surface for 30 minutes. After the competitive binding, 200 μL of PBS buffer containing 2.0 mM hydroquinone / 2.5 mM H₂O₂ was dripped onto the electrode surface for DPV testing. The DPV voltage range was -0.3 V to 0.2 V.

[0076] The reaction of HRP catalyzing H2O2 oxidation of HQ is as follows:

[0077]

[0078] The benzoquinone generated by the reaction is reduced on the electrode surface, generating a characteristic reduction peak current. The reduction peak current value is extracted from the DPV curve.

[0079] Example 3: Optimization of electrochemical immunosensing conditions

[0080] The HRP-FA concentration was optimized by comparing the reduction peak currents obtained with 0 and 50 nM FA with 1 / 100, 1 / 200, and 1 / 300 dilutions of HRP-FA. The FBP concentration was optimized by comparing the reduction peak currents obtained with 0 and 50 nM FA on immunosensor electrodes immobilized with 25, 50, 75, and 100 mg / mL FBP. The incubation time was optimized by comparing the reduction peak currents obtained with 0 and 50 nM FA at different incubation times (15, 30, 45, and 60 min).

[0081] In the experiment, the optimal combination was determined by DPV detection of peak current changes. The optimal HRP-FA dilution ratio (1 / 100, Figure 7 (a)), FBP concentration (75 mg / mL, Figure 7 (b)), incubation time (30 min, Figure 7 (c)).

[0082] Under the optimal detection conditions, an ultra-low detection limit of 0.33 nM (S / N=3, Figure 8 (a)) The detection limit of the electrochemical detection immunoassay of folic acid based on magnetic sensors (13.1 nM) was significantly lower than that of the previous literature, and the sensor showed excellent response characteristics to FA in the linear range of 1-100 nM ( Figure 8 (b)).

[0083] Example 4: Selectivity Evaluation

[0084] In order to evaluate the selectivity of the sensor, seven interfering substances that may coexist with the target analyte were selected. Specifically, by comparing different potential interfering substances, such as glucose (Glu), uric acid (UA), lactic acid (Lac), ascorbic acid (AA), vitamin B1 (VB1), vitamin B7 (VB7) 12 (VB 12 ) to evaluate the selectivity of FA sensing.

[0085] Selectivity was verified by comparing the signal deviation when 10 nM FA was present alone and in the presence of interfering substances, based on the typical concentration range of interfering substances in real samples. Competition binding was performed under optimized conditions, and the current response was recorded by DPV. Specific binding was verified by calculating the difference in the reduced peak current. All tests were repeated three times (n=3).

[0086] The results are as follows Figure 9 As shown, the results showed that the changes in electrical signals caused by interferents were negligible, indicating excellent specificity for FA sensing.

[0087] Example 5: Stability evaluation

[0088] The modified electrodes (n=36) were stored at 4°C. Six electrodes were collected every five days and the DPV current responses generated by 1×PBS and 1×PBS containing 10 nM FA were measured under optimized conditions. The monitoring was continued for 25 days, and the signal decay rate was calculated based on the response value detected on the day of electrode preparation.

[0089] The results are as follows Figure 10 As shown in the figure, the sensor still retains more than 90% of the initial response signal after being stored at 4°C for 25 days. The stability experiment confirms that the sensor has a shelf life of ≥25 days when sealed and stored at 4°C, which can meet the stability requirements in scenarios such as clinical testing.

[0090] Example 6 Application Example

[0091] In this study, the recovery rate of folic acid (FA) in human serum was systematically evaluated by the standard addition method. According to the standard operating procedure, venous blood samples were coagulated and centrifuged at 3000 rpm for 10 minutes at a low temperature of 4°C to obtain clarified serum. After the serum samples were diluted to a 100-fold concentration, three equal groups were taken as experimental samples, and different concentrations of FA standard solutions were added to them, and three parallel tests were performed using differential pulse voltammetry (DPV). As shown in Table 1, the experimental data show that the spiked recovery rate is distributed in a reasonable range of 96.07% to 106.07%, and the relative standard deviation (RSD) is less than 4.69%. These data verify that the DPV detection system constructed based on AuNFs / SNC has excellent accuracy and repeatability, and can meet the detection requirements of folic acid content in actual biological samples.

[0092] Table 1. Spike recovery experiment for detecting FA in human serum.

[0093]

[0094]

[0095] The embodiments described above are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing an electrochemical immunosensor for specific detection of folic acid, characterized in that: The method comprises the following steps: (1) Preparation of electrochemical electrodes for obtaining gold nanoflower layers: AuNFs / SNC nanocomposites; (2) Immobilizing folate binding protein (FBP) on the electrode surface; (3) Block the electrode surface that is not bound to FBP.

2. The preparation method according to claim 1, characterized in that The operation of step (1) is: 1.1) Using a screen-printed electrode as the base electrode, clean and activate it: Activate the electrode in 0.1 M H2SO4 by cyclic voltammetry (CV) to remove surface oxides and enhance activity; 1.2) Electrochemically doping the above electrodes to achieve sulfur / nitrogen co-doping to obtain SNC electrodes: electrochemical doping was performed in thiourea solution using cyclic voltammetry; 1.3) Electrodepositing gold nanoparticles on the surface of the SNC electrode obtained above to obtain a gold nanoflower layer: Electrodepositing a gold nanoflower layer in a chloroauric acid (HAuCl4) solution by the CV method to obtain an AuNFs / SNC nanocomposite material.

3. The preparation method according to claim 2, characterized in that Wherein in step 1.1), the working electrode material and the counter electrode material of the substrate electrode SPE electrode are both carbon, and the reference electrode material is Ag / AgCl; the scan rate of the cyclic voltammetry is 80-120 mV / s; and the number of cycles of the activated electrode is 8-12 cycles; The concentration of thiourea in step 1.2) is 0.5-0.7 M, and the cyclic voltammetry is performed by continuous electrical doping at -1.2 V to 0.2 V for 25-35 cycles at a scan rate of 50-70 mV / s; The concentration of chloroauric acid in step 1.3) is 2-3 mM, and the solvent is 1×PBS; the voltage range of the CV is -0.5 V to 0.4 V; the scan rate of the CV is 40-60 mV / s, and the electrodeposition of gold is 8-12 cycles.

4. The preparation method according to claim 3, characterized in that The AuNFs / SNC nanocomposite material was also thoroughly rinsed with ultrapure water and dried with nitrogen.

5. The preparation method according to claim 1, characterized in that The step (2) is as follows: 2.1) Electrode carboxyl interface activation: Prepare EDC and NHS solution in MES and drop it on the surface of the SPE working electrode in the dark. 2.2) Apply FBP solution to the electrode surface and incubate.

6. The method according to claim 5, characterized in that The MES buffer described in step 2.1) is freshly prepared, and its concentration is 0.08-0.12M MES buffer; the concentrations of EDC and NHS in the EDC / NHS solution are 0.3-0.5M and 0.08-0.12M, respectively; the activation reaction temperature is 20-30°C, and the reaction time is 20-40min.

7. The method according to claim 5, characterized in that The incubation in step 2.2) is performed at 35-40° C. for 30-50 min. The FBP solution is prepared by dissolving it in 1×PBS at pH 7.4 to a concentration of 70-80 mg / mL.

8. The method according to claim 1, characterized in that The step (3) is to immerse the electrode modified in the step (2) in a bovine serum albumin (BSA) solution for sealing.

9. An electrochemical immunosensor for specifically detecting folic acid obtained by the method according to any one of claims 1 to 8.

10. Use of the method according to any one of claims 1 to 8 or the sensor according to claim 9 in detecting folic acid; said detection is a non-disease diagnosis detection.

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