An electrochemical immunosensor for high-sensitivity detection of folic acid and a preparation method and application thereof

Abstract

The application belongs to the technical field of electrochemical sensors, and particularly relates to an electrochemical immunosensor for high-sensitivity detection of folic acid as well as a preparation method and application thereof. The application introduces FBP as a specific recognition element into a folic acid detection system for the first time. Compared with a traditional immunosensor based on an antibody, the system innovatively utilizes high affinity and specific binding characteristics between FBP and folic acid, constructs a sensing interface with dual functions of molecular recognition and electrochemical signal conversion by modifying FBP on the surface of an AuNFs / SNC electrode. Experimental results show that the sensor presents excellent response characteristics for 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] This invention belongs to the field of electrochemical sensor technology, specifically relating to a highly sensitive electrochemical immunosensor for detecting folic acid, its preparation method, and its application. Background Technology

[0002] Folic acid (FA), also known as vitamin B9, is considered 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 green vegetables, egg yolks, liver, and citrus fruits. FA deficiency can lead to a range of diseases, such as neural tube defects (NTDs), cardiovascular disease, and may also be associated with congenital malformations and low birth weight. However, the low levels of FA in some samples, its poor stability, and the complexity of sample composition pose significant challenges to FA detection. To improve FA detection accuracy, a sensitive, robust, simple, and reliable detection method is urgently needed.

[0003] In recent years, various methods for the quantitative detection of folic acid (FA) have been reported. Commonly used methods for FA detection mainly include chromatography, microbiological methods, immunoassay, and electrochemical methods. Chromatography is a widely used technique in laboratories. High-performance liquid chromatography (HPLC) has high sensitivity and is suitable for drug quality control and clinical research, but the pretreatment steps are complex and dependent on specialized equipment. Liquid chromatography-mass spectrometry (LC-MS) combines chromatographic separation and mass spectrometry detection, significantly improving sensitivity and specificity, and can simultaneously analyze multiple forms of folic acid. However, the pretreatment steps are complex, the equipment cost is high, and it requires professional operation. Microbiological methods are the most traditional detection method, indirectly determining folic acid content by observing the growth of folic acid-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 operation. Immunoassay methods, including ELISA and immunochromatographic strips, are based on antigen-antibody reactions. This method is simple and rapid, suitable for batch screening and on-site testing, but the kits are expensive and suffer from insufficient reproducibility and numerous interfering factors. Electrochemical methods utilize the redox properties of folic acid, offering the potential for speed, low cost, and portability. However, they are susceptible to interference from other electroactive substances, resulting in poor specificity and sensitivity.

[0004] Electrochemical immunosensing is a novel detection technology that combines the high specificity of immunoassay with the high sensitivity of electrochemical detection. Its core principle is the conversion of biorecognition into a measurable electrical signal through the specific binding of antigen and antibody, thereby achieving quantitative detection of the target analyte. Compared to traditional detection methods, the most significant advantages of electrochemical immunosensing are its high sensitivity and specificity. The immune reaction enables precise identification, while electrochemical signal amplification technology enhances sensitivity. Simultaneously, the specific recognition protein selectively binds to the target analyte, effectively avoiding interference from other substances. It is particularly suitable for scenarios requiring highly sensitive and selective detection. Due to its higher sensitivity, lower cost, easier operation, and real-time analysis capabilities, this method has been applied to the detection of various vitamins. However, very few folic acid biosensors based on electrochemical immunosensing have been reported so far. Lermo et al. (2009) proposed an electrochemical immunoassay for folic acid based on a magnetic sensor with a detection limit as low as 13.1 nM. Although this method is fast, inexpensive and environmentally friendly, it cannot detect samples with low folic acid content because the content of FA in many samples is extremely low (below 10 nM). Therefore, it is crucial to develop electrochemical immunosensors with higher sensitivity. Summary of the Invention

[0005] To address the current technical deficiencies in the electrochemical detection of folic acid, this invention provides the following technical solution:

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

[0007] (1) Preparation of an electrochemical electrode with gold nanoflower layers;

[0008] (2) Immobilize folic acid-binding protein FBP on the electrode surface;

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

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

[0011] 1.1) A screen-printed electrode was used as the base electrode, and it was then cleaned and activated;

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

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

[0014] Preferably, the cleaning and activation operation in step 1.1) is as follows: the electrode is activated in 0.1 M H₂SO₄ 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 for activating the electrode is 8-12; even 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, step 1.2) involves electrochemical doping in a thiourea solution using cyclic voltammetry; the concentration of the thiourea solution is 0.5–0.7 M, and the cyclic voltammetry is performed by continuous electrodoping at -1.2 V to 0.2 V for 25–35 cycles with a scan rate of 50–70 mV / s; more preferably, the concentration of the thiourea solution is 0.6 M, and ultrasonic dispersion is performed for 10 min to maintain homogeneity.

[0016] Preferably, step 1.3) involves electrodepositing gold nanoflowers in a chloroauric acid (HAuCl4) solution using a CV method to obtain AuNFs / SNC nanocomposite materials; 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 to 0.4V; even more preferably, the scan rate of the CV is 40–60 mV / s, and the gold electrodeposition cycle is 8–12.

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

[0018] Furthermore, 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 in step 2.1) involves preparing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) using MES buffer, dropping the EDC / NHS solution onto the surface of the SPE working electrode, and reacting in the dark. After the reaction, the electrode surface is immediately and gently rinsed three times with pre-cooled MES buffer and dried with nitrogen. More preferably, the MES buffer is freshly prepared with a concentration of 0.08–0.12 M MES buffer. More preferably, the concentrations of EDC and NHS in the EDC / NHS solution are 0.3–0.5 M and 0.08–0.12 M, respectively. More preferably, the activation reaction temperature is 20–30 °C, and the reaction time is 20–40 min.

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

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

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

[0025] A third aspect of the invention is to provide the application of the method described in the first aspect or the sensor described in the second aspect in the detection of folic acid; wherein the detection is a non-disease diagnostic detection.

[0026] The beneficial effects of this invention include:

[0027] 1) A highly electrochemically active nanocomposite material, AuNFs / SNC, was prepared.

[0028] The SN co-doped carbon substrate (SNC) significantly enhances the electrochemical activity of carbon materials by introducing sulfur and nitrogen heteroatoms. 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 load of gold nanoflowers. This yields the AuNFs / SNC nanocomposite material, which serves as the working electrode material, further improving the electrochemical performance of the sensing material. Morphological and physical analysis methods (such as XRD, SEM, TEM, EDS, and XPS) confirmed the successful preparation of AuNFs / SNC. The hydrophilic surface of AuNFs / SNC (contact angle = 58.57°) synergistically promotes high-density immobilization of folate-binding protein (FBP), amplifying the electrochemical signal perturbation caused by target binding and further enhancing the sensor's sensitivity.

[0029] 2) This is the first time that folic acid binding protein has been applied to the electrochemical immunosensing detection of folic acid.

[0030] The constructed electrochemical immunosensor was validated using 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 detection of FA

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

[0033] 4) The sensor has high 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), and vitamin B... 12 (VB 12 The selectivity of FA sensing was evaluated by assessing the changes in the DPV signal caused by interference. The results showed that the changes in the electrical signal caused by interference were negligible, indicating excellent selectivity for FA sensing.

[0035] 5) The sensor has good storage stability.

[0036] The sensor retains more than 90% of its initial response signal after being stored at 4℃ for 25 days. Stability tests confirm that the sensor has a shelf life of ≥25 days when stored in a sealed container at 4℃, which can meet the stability requirements of clinical testing and other scenarios. Attached Figure Description

[0037] Figure 1 The fabrication of the FA electrochemical immunosensor and the detection principle diagram of the method described therein;

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

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

[0040] Figure 4 4a is the XPS full spectrum of AuNPs / SNC, and 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 angle of nanomaterials on the surface of working electrodes at different preparation stages;

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

[0043] Figure 7 Optimization of conditions for electrochemical FA immunosensor. Figure 7 ac represents the effects of HRP-FA dilution ratio, FBP concentration, and incubation time on the electrochemical signal, respectively.

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

[0045] Figure 9 FA electrochemical immunosensor specific detection;

[0046] Figure 10 Storage stability testing of FA electrochemical immunosensor. Detailed Implementation

[0047] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0048] Example 1: Construction of an electrochemical immunosensor

[0049] 1. Construction of electrochemical immunosensors: The process is as follows Figure 1 As shown.

[0050] 1.1 Electrode pretreatment and nanomaterial modification

[0051] (1) Electrode cleaning and activation

[0052] A screen-printed electrode (SPE) (Borean 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 within 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) Fabrication of sulfur-nitrogen co-doped carbon (SNC) electrode

[0054] A solution containing 0.6 M thiourea (Maclean, T819602) was prepared. 200 μL of the thiourea solution was dropped onto the activated SPE surface, and cyclic voltammetry was used to continuously dope the carbon electrode for 30 cycles at a sweep rate of 60 mV / s from -1.2 V to 0.2 V, co-doping sulfur (S) and nitrogen (N) onto the carbon electrode surface. The electrode was then rinsed with ultrapure water and dried.

[0055] (3) Gold nanoparticle electrodeposition

[0056] A 2.5 mM chloroauric acid solution was prepared by dissolving chloroauric acid (HAuCl4) (Heinz, G-19310) in 1×PBS. 200 μL of the chloroauric acid solution was dropped onto the surface of the SNC electrode, and gold was electrodeposited for 10 cycles using cyclic voltammetry (scan rate 50 mV / s) within the range of -0.5 V to 0.4 V, forming a uniform gold nanoflower layer on the SNC electrode surface, thus obtaining the AuNFs / SNC nanocomposite material. 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) Activation of the electrode carboxyl interface

[0059] Prepare a fresh 0.1M MES buffer solution. Using the MES buffer, prepare an EDC / NHS solution containing 0.4M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Yuan Ye, S30053) and 0.1M N-hydroxysuccinimide (NHS) (Yuan Ye, S20179). Drop 20 μL of the EDC / NHS solution onto the surface of the SPE working electrode and react at 25°C in the dark for 30 minutes. After the reaction, immediately and gently rinse the electrode surface three times with pre-cooled MES buffer and dry it with nitrogen gas.

[0060] (2) FBP immobilization

[0061] Subsequently, 10 μL of FBP (Baiming Biotechnology, BM841004) solution (75 mg / mL, pH 7.4, 1×PBS) was drop-coated onto the electrode surface and incubated at 37°C for 40 min. FBP was stably immobilized via amide bonds.

[0062] (3) Non-specific site blocking

[0063] The modified electrode was immersed in a 1% bovine serum albumin (BSA) (Yuan Ye, S12012) solution and sealed 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, appearance and composition characterization: SEM, TEM, XPS and GIXRD were used to characterize the morphology and material properties of the sensing electrode surface before and after modification. TEM was used in conjunction with energy dispersive spectroscopy (EDS) to achieve accurate analysis of the types and contents of various elements in the micro-regions of the material.

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

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

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

[0069] X-ray photoelectron spectroscopy (XPS) analysis was performed. Figure 4 The total spectrum proves the presence of C, N, O, S, and Au in AuNFs / SNCs. Figure 4 The high-resolution C1s peak in b can be fitted as four peaks centered at 284.8, 285.9, 286.7, and 289.2 eV, attributed to C, C, CN / C=N, and CO bonds. Figure 4 In the c O 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 the metal 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 / 2The N 1s peak is composed of the S=O subpeak, located at binding energies of 161.7 eV, 162.9 eV, and 164.1 eV, respectively. The N 1s peak can be unconvolved into four peaks, belonging to pyridine N (398.7 eV), pyrrole N (400.1 eV), graphitic N (401.5 eV), and oxygen-containing N (404.1 eV), respectively. 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 measuring instrument (JY-82C). The measurement was performed at room temperature using a 16μL water droplet on a flat electrode surface using the seat drop method.

[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, and the contact angle decreases to 87.50°. The contact angle of AuNFs / SNC obtained by electrodeposition on the SNC surface is only 58.57°. Its hydrophilic surface synergistically promotes the high-density fixation of FBP, amplifies the electrochemical signal perturbation caused by target binding, and further improves the sensitivity of the sensor.

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

[0073] The results are as follows Figure 6 The figures show the CV(a) and EIS(b) graphs of the AuNFs / SNC electrode in 0.01M PBS solution containing 2.0mM 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 solution and the electrode surface is hindered; the resistance increases and the redox current decreases after each modification step. This confirms the successful immobilization of biomacromolecules such as FBP and BSA on the surface of the AuNFs / SNC electrode.

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

[0075] 10 μL of FA standard solution (Maclean's, F809516) or the analyte biofluid and HRP-FA (Baiming Biotechnology, BM841003) prepared in 1×PBS (pH 7.4) were dripped onto the working electrode, allowing the FA and HRP-FA in the sample to compete for binding with FBP on the electrode surface for 30 minutes. After the competitive binding was completed, 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 -0.3V to 0.2V.

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

[0077]

[0078] The benzoquinone generated in the reaction is reduced on the electrode surface, producing a characteristic reduction peak current. The reduction peak current value is extracted using DPV curves.

[0079] Example 3: Optimization of Electrochemical Immunosensing Conditions

[0080] The concentration of HRP-FA was optimized by comparing the reduction peak currents obtained from 0 and 50 nM FA with HRP-FA diluted at 1 / 100, 1 / 200, and 1 / 300. The concentration of FBP was optimized by comparing the reduction peak currents obtained from 0 and 50 nM FA on immunosensing electrodes immobilized with FBP at concentrations of 25, 50, 75, and 100 mg / mL. The incubation time was optimized by comparing the reduction peak currents obtained from 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 detecting the peak current change using DPV. The optimal HRP-FA dilution ratio (1 / 100) was selected. Figure 7 (a)), FBP concentration (75 mg / mL), Figure 7 (b)), incubation time (30 min, Figure 7 (c)).

[0082] Under optimal detection conditions, DPV was used in pH 7.4 PBS buffer to achieve an ultra-low detection limit of 0.33 nM (S / N = 3). Figure 8 (a) shows a significantly lower detection limit compared to the previously reported electrochemical folic acid detection immunoassay based on a magnetic sensor (13.1 nM). The sensor exhibits excellent response characteristics to FA within a linear range of 1-100 nM. Figure 8 (b)).

[0083] Example 4: Selective Evaluation

[0084] To evaluate the selectivity of the sensor, seven interfering substances that may coexist with the target analyte were selected. Specifically, these were compared among different potential interfering substances, such as glucose (Glu), uric acid (UA), lactic acid (Lac), ascorbic acid (AA), vitamin B1 (VB1), vitamin B7 (VB7), and vitamin B6. 12 (VB 12 The selectivity of FA sensing is evaluated by the change in DPV signal caused by )

[0085] Based on the typical concentration range of interfering substances in actual samples, the selectivity was verified by comparing the signal deviation when 10 nM FA existed alone and coexisted with interfering substances. Competitive binding was performed under optimized conditions, and the current response was recorded by DPV. The specific binding was verified by calculating the difference in reduction peak current. All tests were repeated 3 times (n=3).

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

[0087] Example 5, Stability Assessment

[0088] The modified electrodes (n=36) were stored at 4°C. Six electrodes were taken every 5 days, and the DPV current response generated by 1×PBS and 1×PBS containing 10 nM FA was detected under optimized conditions. The monitoring was carried out continuously for 25 days, and the signal attenuation 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, the sensor retains more than 90% of its initial response signal after being stored at 4℃ for 25 days. The stability test confirms that the sensor has a shelf life of ≥25 days when stored in a sealed container at 4℃, which can meet the stability requirements of clinical testing and other scenarios.

[0090] Example 6 Application Case

[0091] This study systematically evaluated the recovery rate of folic acid (FA) in human serum using a standard addition method. Following standard operating procedures, venous blood samples were coagulated and centrifuged at 3000 rpm for 10 minutes at 4°C to obtain clear serum. The serum samples were diluted 100-fold, and three equal volumes were used as experimental samples. Different concentrations of FA standard solution were added to each sample, and three parallel assays were performed using differential pulse voltammetry (DPV). The experimental data shown in Table 1 indicate that the spiked recoveries were within a reasonable range of 96.07% to 106.07%, with a relative standard deviation (RSD) of less than 4.69%. These data validate that the DPV detection system based on AuNFs / SNCs possesses excellent accuracy and repeatability, meeting the requirements for detecting folic acid content in real biological samples.

[0092] Table 1. Spiked recovery experiment for the detection of FA in human serum.

[0093]

[0094]

[0095] The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. A method for preparing an electrochemical immunosensor specifically for detecting folic acid, characterized in that, The method includes the following steps: (1) Preparation of electrochemical electrode with gold nanoflower layer: gold nanoflower / sulfur-nitrogen co-doped carbon AuNFs / SNC nanocomposite material; (2) Immobilize folic acid-binding protein (FBP) on the electrode surface; (3) Seal the electrode surface that is not bound to FBP; The operation of step (1) is as follows: 1.1) Using screen-printed electrode SPE as the base electrode, it was cleaned and activated: the electrode was activated in 0.1 M H2SO4 by cyclic voltammetry (CV) to remove surface oxides and enhance activity; 1.2) Electrochemical doping of the above electrodes to achieve sulfur / nitrogen co-doping and obtain SNC electrodes: Electrochemical doping was performed in thiourea solution using cyclic voltammetry. 1.3) Gold nanoparticles were electrodeposited onto the surface of the SNC electrode obtained above to obtain a gold nanoflower layer: AuNFs / SNC nanocomposite material was obtained by electrodepositing the gold nanoflower layer in chloroauric acid (HAuCl4) solution using the CV method; The operation of step (2) is as follows: 2.1) Activation of the electrode carboxyl interface: Prepare EDC and NHS solutions using MES, drop them onto the surface of the SPE working electrode, and react in the dark; 2.2) Apply FBP solution to the electrode surface and incubate.

2. The preparation method according to claim 1, characterized in that, 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 activation electrode is 8~12. The concentration of thiourea in step 1.2) is 0.5~0.7M, and the cyclic voltammetry method is... Continuous electrodoping at 1.2 V to 0.2 V for 25 to 35 cycles, with a scan rate of 50 to 70 mV / s; In step 1.3), the concentration of chloroauric acid is 2-3 mM, and the solvent is 1×PBS; the voltage range of CV is -0.5 V to 0.4 V; the scan rate of CV is 40-60 mV / s, and the gold electrodeposition is performed for 8-12 cycles.

3. The preparation method according to claim 2, characterized in that, The AuNFs / SNC nanocomposite material was also thoroughly rinsed with ultrapure water and dried with nitrogen.

4. The preparation method according to claim 3, characterized in that, In step 2.1), the MES is freshly prepared with a concentration of 0.08~0.12 M; the concentrations of EDC and NHS in the EDC and NHS solutions are 0.3~0.5 M and 0.08~0.12 M, respectively; the activation reaction temperature is 20~30℃ and the reaction time is 20~40 min.

5. The preparation method according to claim 3, characterized in that, The incubation conditions described in step 2.2) are 35-40°C for 30-50 min; the FBP solution is prepared by dissolving in pH 7.4 1×PBS with a concentration of 70-80 mg / mL.

6. The method according to claim 1, characterized in that, Step (3) involves immersing the modified electrode from step (2) in a bovine serum albumin (BSA) solution and sealing it.

7. An electrochemical immunosensor for specific detection of folic acid obtained by the preparation method according to any one of claims 1 to 6.

8. The application of the preparation method according to any one of claims 1 to 6 or the sensor according to claim 7 in the detection of folic acid; wherein the detection is a non-disease diagnostic detection.

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