A folate sensor integrated with calibration function and application thereof in sweat folate concentration detection

Abstract

The application belongs to the technical field of biomedical monitoring, and particularly relates to a folate sensor integrated with a calibration function and application thereof in sweat folate concentration detection; specifically, a high-sensitivity folate immunosensor is optimized, combined with a pH and ion strength sensor, a folate immunosensor with a calibration detection function is developed, and the folate immunosensor is applied to sweat folate detection; based on folate level information of sweat obtained by the sensor, a sweat and blood folate concentration correlation analysis method is established; through a human population cohort experiment, it is proved that the sensor can specifically and sensitively measure the folate concentration in sweat and has a high correlation with the folate concentration in serum.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical monitoring, and particularly relates to a folate sensor integrated with a calibration function and application thereof in sweat folate concentration detection, in particular, a method for realizing blood folate concentration level correlation analysis through non-invasive sweat detection and dynamic monitoring of sweat folate dynamic changes under the intervention of nutritional supplements. BACKGROUND

[0002] Folic acid (FA), also known as vitamin B9, is considered to be one of the essential biomolecules in the process of cell growth and division. Traditional human folate assessment mainly relies on blood detection. Blood detection requires venipuncture, and has problems such as invasiveness, difficulty in real-time dynamic monitoring due to sampling frequency limitation, high professional requirement for operation, etc. In addition, blood detection cannot capture the instantaneous fluctuation of folate concentration, and the above-mentioned problems restrict the precision and normalization of folate nutrition management. At present, non-invasive real-time monitoring of human folate has not been practically verified, and the core reason is that a suitable body fluid carrier showing strong correlation with blood folate level has not been found in non-invasive collected body fluids such as sweat and saliva.

[0003] Sweat is a kind of body fluid that can reflect human physiological information. In most cases, the average daily sweat output of an adult is hundreds of milliliters. Sweat contains rich biochemical information, such as electrolytes (e.g., Na + , K + , H + , Ca 2+ ) and metabolic molecules (e.g., glucose, lactic acid), etc. In addition, there are many vitamins such as folate and vitamin C in sweat. Recent reports show that there is a good correlation between many substances in sweat, such as uric acid, estradiol, amino acids, vitamin C, and the levels of corresponding substances in blood. However, as of now, the correlation between sweat folate and blood folate level has not been elucidated, and the sweat folate detection result cannot be used as a substitute indicator for blood folate level. In addition, the effects of folate intake, lifestyle habits (such as smoking) on sweat folate concentration level are not clear, and the above factors all limit the application of sweat folate analysis in the field of nutrition monitoring. Detecting the folate concentration level in sweat and establishing the correlation between it and the folate concentration level in blood, and exploring the effects of folate supplements, lifestyle habits (such as smoking) on sweat folate concentration level will promote the development of non-invasive sweat folate detection and the application of sweat folate analysis in the field of nutrition monitoring.

[0004] Currently, the application of folate biosensor technology in the field of nutrition monitoring is facing three technical bottlenecks: (1) The existing folate sensor is difficult to realize high-precision quantitative detection of folate in sweat matrix due to the limitation of sensitivity and insufficient anti-interference performance; (2) The correlation between sweat and blood folate concentrations has not been clearly explained; (3) There is a lack of systematic study on the influence of folate metabolism related factors (including folate intake, smoking and other lifestyles) on the dynamic changes of sweat folate concentration. The uncertainty of these key scientific issues makes it difficult to use sweat folate detection results as a reliable alternative biomarker for nutritional status assessment, which limits the application of sweat folate analysis in the field of nutrition monitoring. SUMMARY

[0005] In order to improve the accuracy of sweat folate detection, we further optimize the folate detection method, combine the folate sensor with pH and ion strength sensor, and improve the sensitivity of sweat folate detection through pH and ion strength calibration. The folate sensor with calibration function is applied to sweat folate detection, and its correlation with folate concentration in blood is established to explore the influence of factors such as folate supplements and lifestyle habits (such as smoking) on sweat folate concentration. The present application helps to promote the development of non-invasive sweat folate detection and promote the application of sweat folate analysis in the field of nutrition monitoring.

[0006] The application provides an application of a folate sensor integrated with calibration function in detection of folate concentration, characterized in that the sample for detection is sweat; wherein the sensor integrated with calibration function is prepared by the following method:

[0007] (1) An electrochemical electrode AuNFs / SNC composite material with gold nanoflower layer is prepared;

[0008] (2) The AuNFs / SNC composite material of step (1) is used as working electrode material of ion sensor;

[0009] (3) FA electrode preparation: folate binding protein FBP is fixed on the surface of the electrode of step (1); and non-specific blocking is performed;

[0010] (4) pH calibration electrode: the surface of the base carbon electrode is coated, and dried overnight;

[0011] Further, the operation of step (1) is as follows:

[0012] 1.1) A screen-printed electrode is used as a base electrode, which is cleaned and activated;

[0013] 1.2) The electrode is electrochemically doped to achieve sulfur / nitrogen co-doping to obtain an SNC electrode;

[0014] 1.3) Gold nanoflower layer is electrodeposited on the surface of the SNC electrode obtained above.

[0015] Preferably, the cleaning and activation in step 1.1) is performed by cyclic voltammetry (CV) in 0.1 M H2SO4 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 cycles; more preferably, the working electrode material and the counter electrode material of the base electrode SPE electrode are both carbon, and the reference electrode material is Ag / AgCl.

[0016] Preferably, the operation of step 1.2) is performed by cyclic voltammetry in a thiourea solution for electrochemical doping; the concentration of the thiourea is 0.5-0.7 M, and the cyclic voltammetry is continuous electro-doping at -1.2 V-0.2 V for 25-35 cycles at 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 uniformity.

[0017] Preferably, the operation of step 1.3) is performed by CV in a chloroauric acid (HAuCl4) solution to electrodeposited a gold nanoflower layer to obtain an AuNFs / SNC nanocomposite; preferably, the concentration of the chloroauric acid is 2-3 mM, and the solvent is 1×PBS; more preferably, the voltage range of the CV is -0.5 V-0.4 V; more preferably, the scan rate of the CV is 40-60 mV / s, and the electrodeposition of gold is 8-12 cycles;

[0018] Further, the AuNFs / SNC nanocomposite is further washed thoroughly with ultrapure water and dried with nitrogen.

[0019] Further, the operation of step (2) is that the AuNFs / SNC composite material of step (1) is used as the working electrode material of an ion sensor, the impedance response of the sensor is recorded in 0.05-1.6×PBS buffer, and the impedance response is converted into an admittance signal to establish a corresponding calibration graph;

[0020] Further, the operation of step (3) is:

[0021] 3.1) Carboxyl interface activation of the electrode;

[0022] 3.2) FBP solution is coated on the surface of the electrode and incubated;

[0023] Preferably, the carboxyl interface activation in step 3.1) is carried out by using MES buffer to configure 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to replace EDC / NHS solution, and then drop the solution on the surface of the SPE working electrode and react in the dark. After the reaction is completed, the electrode surface is gently washed with pre-cooled MES buffer for 3 times, and then dried with nitrogen; more preferably, the MES buffer is freshly prepared, and the concentration is 0.08-0.12M MES buffer; more preferably, the concentration of EDC and NHS in the EDC / NHS solution is 0.3-0.5M and 0.08-0.12M, respectively; more preferably, the activation reaction temperature is 20-30℃, and the reaction time is 20-40min.

[0024] More preferably, the operation of step 3.2) is to drop the FBP solution on the electrode surface, and then incubate at 35-40℃ for 30-50min; more preferably, the FBP solution is prepared by dissolving in 1×PBS with pH 7.4, and the concentration is 70-80mg / mL.

[0025] Further, the blocking operation in step (3) is to immerse the modified electrode into a 1% bovine serum albumin (BSA) (source leaf, S12012) solution, and then block at 37℃ for 30min, so as to shield the unbound active sites on the electrode surface and reduce the background noise.

[0026] Further, the operation of step (4) is to drop the mixed solution of the pH selective membrane on the surface of the carbon electrode and dry overnight, and then pretreat in 200μL of 10mM HCl solution for 30min before use. In the pH change range of 4-8, the potential response of the sensor is recorded, and the corresponding calibration graph is established.

[0027] Preferably, the pH selective mixed solution is prepared by adding 1wt% of hydrogen ion carrier I, 0.6wt% of Na-TFPB, 33wt% of PVC and 65.4wt% of DOS in tetrahydrofuran.

[0028] Further, the sampling site of the sweat for detection is the skin of the arm, back or leg; preferably, the sampling site is selected from the skin of the arm and back.

[0029] Further, the detection is a non-disease diagnosis application.

[0030] The second aspect of the present application provides an electrochemical immunosensor for specifically detecting folic acid, which is prepared in the application of the first aspect.

[0031] The beneficial effects of the present application include: the present application optimizes a high-sensitivity folic acid immunosensor, combines it with a pH and ionic strength sensor, develops a folic acid immunosensor with a calibration detection function and applies it to sweat folic acid detection; based on the sweat folic acid level information obtained by the sensor, a sweat and blood folic acid concentration correlation analysis method is established; through a human population cohort experiment, it is proved that after oral folic acid supplement, the sweat folic acid concentration presents a similar dynamic change rule as blood, and it is found that the sweat folic acid baseline value of the smoking population is significantly lower than that of the non-smoking group. The scheme at least partially solves the related technical problems. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Structure diagram of the sensor with calibration function;

[0033] Figure 2 Potential response (a) and corresponding calibration graph (c) of the pH sensor; impedance response (b) and corresponding calibration graph (d) of the ionic strength sensor;

[0034] Figure 3 Selectivity (a), reversible response ability (b) of the pH sensor and accuracy (c) of its analysis of the pH of sweat samples;

[0035] Figure 4 Performance of the folic acid electrochemical immunosensor under different pH (a) and ionic strength (b) levels;

[0036] Figure 5 a-h Comparison of sweat folic acid concentration levels of 8 subjects (S1-S8) before and after taking 5mg folic acid;

[0037] Figure 6 Vitamin folic acid was monitored for two consecutive days. (a) shows the time schedule of the 48-hour subject study, and the subjects took 5mg folic acid every day. (b) and (c) show the changes in sweat folic acid levels of subject 1 (S1) and subject 2 (S2);

[0038] Figure 7 Changes in sweat folic acid levels of a single subject for three consecutive days;

[0039] Figure 8 a-b: Comparison of sweat folic acid levels at different body parts of 2 subjects (S1, S2);

[0040] Figure 9 Relationship between folic acid concentration in sweat and folic acid supplement intake;

[0041] Figure 10 Relationship between sweat and blood folic acid levels. (r=0.849);

[0042] Figure 11Box-and-whisker plot of folate levels in sweat and serum of smoking (n=8) / non-smoking (n=7) human populations. DETAILED DESCRIPTION

[0043] The concept and the technical effects of the present application are further described below in conjunction with specific embodiments, so as to fully understand the objects, features and effects of the present application. The methods are all conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present application and the descriptions thereof are used to explain the present application and do not constitute an improper limitation on the present application. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0044] The research subjects and experimental methods in the specific embodiments of the present application are as follows:

[0045] Example 1 Construction and performance study of pH and ionic strength sensor

[0046] The sensor electrodes are all prepared by screen printing technology. The working electrode and the counter electrode are both carbon electrodes, and the reference electrode is Ag / AgCl.

[0047] (1) Sensor preparation: first, 0.1M H2SO4 solution is used as the electrolyte, and the carbon working electrode is activated by cyclic voltammetry in the potential window of -0.2V to 1.2V, and then washed with ultrapure water and dried after effectively removing the surface oxides. Then, 0.6M thiourea solution (Macklin, T819602) is prepared, ultrasonically treated, and 200μL is dropped on the surface of the activated electrode, and 30 cyclic voltammetry scans (60mV / s) are performed in the range of -1.2V to 0.2V, to obtain a sulfur and nitrogen co-doped carbon (SNC) electrode. Based on the SNC electrode, 2.5mM chloroauric acid solution (Shin-Etsu, G-19310) is further dropped on the surface, and electrochemical deposition is performed by cyclic voltammetry to successfully construct a gold nanoflower modified AuNFs / SNC composite material. Finally, the electrode is washed with ultrapure water and dried by nitrogen blowing for standby.

[0048] (2) The electrode modification process of the ionic strength sensor is as follows: the AuNFs / SNC composite material of step (1) is used as the working electrode material of the ion sensor, and the impedance response of the sensor is recorded in 0.05-1.6×PBS buffer, and the impedance response of the sensor is converted into the admittance signal, and the corresponding calibration chart is established.

[0049] (3) FA electrode preparation: 10 μΐ of FBP (Biomine, BM841004) solution (75 mg / mL, pH 7.4 1xPBS) was dropped on the surface of AuNFs / SNC composite prepared in step (1) and incubated at 37 °C for 40 min. The stable immobilization of FBP was achieved through amide bond; the modified electrode was immersed in 1% bovine serum albumin (BSA) (Source Leaf, S12012) solution and blocked at 37 °C for 30 min to shield the unbound active sites on the electrode surface and reduce background noise.

[0050] (4) pH calibration electrode: AuNFs / SNC composite prepared in step (1) was added with 1 wt% of hydrogen ion carrier I (Sigma-Aldrich, 95292), 0.6% wt of Na-TFPB (Sigma-Aldrich, 692360), 33% wt of PVC and 65.4% wt of DOS (Sigma-Aldrich, 84818) in 2 mL of tetrahydrofuran to prepare a pH selective membrane mixed solution, which was dropped on the surface of a carbon electrode and dried overnight, and pretreated in 200 μΐ of 10 mM HC1 solution for 30 min before use. The potential response of the sensor was recorded in the range of varying pH values from 4 to 8, and the corresponding calibration graph was established.

[0051] Figure 1 is a schematic diagram of the integrated electrochemical sensor prepared by screen printing technology. The sensor array is composed of FA immunosensor, ion strength sensor and pH sensor, with the left and right being the counter electrode and reference electrode, respectively.

[0052] Figure 2 a-d show the H + The pH sensor with ion selective membrane and the ion strength sensor based on impedance signal have good detection performance, with R 2 values of the standard curves being higher than 0.96.

[0053] Figure 3 a-c show that the developed pH sensor has good selectivity and reversible response ability, and the accuracy of the developed pH sensor in analyzing sweat samples is verified using a commercial pH meter.

[0054] Figure 4 a-b show the detection performance of the folate sensor at different pH values and ion strength levels, and the effects of pH and ion strength on the folate sensor can be analyzed according to the pH sensor and Na +The readings of the sensor enable calibration. Both pH and ionic strength can affect the readings of the FA sensor. To improve the accuracy of FA detection, we calibrated the FA sensor with the information of pH and ionic strength. With pH 7.4 and 1 * PBS as the reference condition, we quantified the effects of pH and ionic strength on the slope and intercept of the calibration curve of the FA sensor. Considering the cumulative effects of pH and ionic strength, we recalculated the slope and intercept of the calibration curve under different conditions using a first-order correction algorithm, and simultaneously corrected the cumulative effects of pH and temperature for accurate determination of the folate concentration in sweat

[0055] Example 2 Detection of folate concentration in sweat

[0056] 1. Subject recruitment

[0057] According to the protocol approved by the Human Research Ethics Committee of China Agricultural University (ID 020250306), subjects were recruited through advertisements to conduct a study on the correlation between sweat and blood folate concentration levels. All subjects had signed a written informed consent form before participating in the study. In order to conduct a study on the correlation between sweat and blood folate concentration levels and understand the potential of sweat as a non-invasive surrogate body fluid to reflect folate nutritional status, 9 healthy subjects aged 18-35 years, with a body mass index (BMI) of 18.5-24.9 kg / m 2 ; fasting blood glucose <100 mg / dL; in the study of the effect of smoking habits on sweat and blood folate concentration levels, the inclusion criteria included: 18≤age≤35; body mass index (BMI) of 18.5-24.9 kg / m 2 ; fasting blood glucose <100 mg / dl; smoking duration ≥1 year; smoking amount ≥10 cigarettes / day.

[0058] 2. Collection, processing and analysis of sweat and blood samples before folate supplement intervention

[0059] 2.1 Preparation of sweat stimulating gel

[0060] Microwave heating method was used to dissolve agarose (3%, w / w) (Sigma-Aldrich, type IV) in deionized water to prepare iontophoresis hydrogel containing carbachol (Sigma-Aldrich, C4382) for placement on iontophoresis electrodes. After the agarose was completely dissolved, the system was cooled to 165°C, and 1% carbachol (for anode) or 1% KCl (for cathode) was added respectively for stirring and mixing. Then the solution was injected into a molding mold, and the solidification process was completed at room temperature. The obtained hydrogel was stored in a 4°C refrigerated environment until experimental use.

[0061] 2.2 Collection of sweat and blood samples

[0062] Subjects arrived at the lab in the morning at 8:00 in the fasted state, and sweat and venous blood were collected at the designed time points. By placing two electrodes on the pre-cleaned forearm area of the subject, the use of II iontophoresis sweat stimulation. After the iontophoresis device was connected to the power supply, a current of 1.5 mA was applied for 5 minutes, and the secreted sweat was sampled for 40 minutes, and then the sweat samples were analyzed using the folic acid electrochemical immunosensor. Fresh blood samples were collected by venous blood sampling in the same time period as the sweat. The collected venous blood was centrifuged at 3000 rpm at 4°C for 10 minutes after the completion of the standardized blood clotting procedure, and the serum was immediately stored at -80°C.

[0063] 2.3 Sweat and blood sample analysis

[0064] The sweat sample was analyzed using an electrochemical immunosensor electrode, 10 μL of the sweat sample was dropped onto the working electrode with HRP-FA (Baiming Biological, BM841003) prepared in 1x PBS (pH 7.4), so that the FA and HRP-FA contained in the sample competed with the FBP on the surface of the electrode for 30 minutes. After the end of the competition binding, 200 μL of PBS buffer with 2.0 mM hydroquinone / 2.5 mM H2O2 was dropped on the electrode surface for DPV testing. The DPV voltage range was -0.3V-0.2V.

[0065] The integrated electrode can directly obtain the pH ionic strength of the sweat, the FA concentration related electrical signal, and the FA concentration calibration based on the pH ionic strength information to improve the accuracy of the detection results.

[0066] The electrochemical immunosensor electrode integrates the pH and ionic strength detection module, significantly improves the detection accuracy of folic acid by synchronous calibration, and has good consistency with the commercial ELISA method. The serum sample was analyzed using the chemiluminescence method folic acid detection kit provided by Jiangsu Baiming Biological Technology Co., Ltd. This method uses a chemiluminescence competition method to detect the folic acid content in the sample. The experimental steps are as follows: first, use the extraction agent to treat the sample to convert the bound folic acid to the free state; then add the treated sample, HRP-FA complex and biotinylated folic acid binding protein in turn in the streptavidin coated microplate, and form a solid phase complex (biotin-streptavidin binding system) after incubation; at this time, the folic acid in the sample competes with the enzyme label for binding to the folic acid binding protein; after washing to remove the unbound substances, add the chemiluminescence substrate, generate a luminescence signal under the catalysis of HRP, and realize the quantitative analysis of the folic acid concentration by detecting the relative luminescence intensity and referring to the calibration curve.

[0067] 3. Effect of folic acid supplements on sweat folic acid levels

[0068] (1) 8 healthy subjects were enrolled to investigate the effect of oral folic acid supplement on sweat folic acid level. Subjects arrived at the lab in the morning after a 12-hour fast and sweat samples were collected, then they took 5 mg folic acid preparation (Lisheng Pharmaceutical, GMP, H12020215) orally in a fasting state, and 3 hours later, sweat samples were collected again. To exclude interference factors, subjects were required to strictly abstain from alcohol and caffeine-containing substances 24 hours before the test. High-folic acid foods were avoided within 3 hours of oral folic acid supplement. Liquid intake was controlled throughout the experiment to maintain the subject's water intake within the range of 500 ± 50 mL / h, and a standardized measuring cup was used for accurate measurement.

[0069] Figure 5 a-h shows the results of a series of sweat folic acid analysis on 8 subjects. Among the 8 subjects, we found that the fasting folic acid concentration in sweat was within 10 nM, and after taking folic acid tablets, the folic acid concentration in sweat of all subjects showed a significant metabolic response, with an increase of about 10-20 nM.

[0070] (2) 2 healthy adult subjects (1 male and 1 female, aged 28) were enrolled for a 48-hour continuous folic acid monitoring study. During the experiment, 5 mg of folic acid supplement was taken orally at 9 o'clock every day, and sweat samples were collected at 3 time points (morning, noon, and evening) for folic acid concentration detection. The subject's water intake was strictly controlled throughout the experiment, and high-folic acid foods were prohibited.

[0071] 48-hour monitoring data show that Figure 6 a-c), the baseline sweat folic acid concentration of the two healthy subjects was low (<10 nM). 3 hours after taking 5 mg of folic acid, the sweat concentration increased to more than twice the baseline level; by 9 hours after intake, subject 2 returned to the baseline level, while subject 1 remained at about twice the baseline level, and the next day showed similar variation patterns.

[0072] (3) One of the subjects was further monitored longitudinally for 3 consecutive days: the first 2 days maintained normal diet (no additional folic acid supplement), and on the 3rd day, 5 mg of folic acid supplement was taken at 9 o'clock, and sweat samples were collected at 3 time points (morning, noon, and evening) for dynamic analysis. In addition, we also collected sweat from the left forearm, right forearm, and scapular region of the back of the subject using iontophoresis (constant current 1.5 mA, duration 5 minutes) to monitor the folic acid level in sweat from different parts of the body to explain the possible differences between different parts of the body.

[0073] In the 3-day continuous monitoring of 1 subject, Figure 7), we observed that the folate concentration in sweat was relatively low in the first 2 days without folate intake, and only showed a small fluctuation within a day. After folate intake on the 3rd day, the folate concentration in sweat increased significantly. After analyzing the sweat collected from different parts, we found that Figure 8 a-b, the folate concentration in sweat collected from different parts using iontophoresis showed only a small difference.

[0074] (4) To investigate how the folate concentration in sweat changes with different oral doses, a stepwise dosing design was used, in which 2 subjects took 0, 5, 10, 15, and 20 mg of folate, with a 5-day interval between each dose to eliminate the residual effect. Sweat was collected 3 hours after intake using iontophoresis and analyzed. The subjects were strictly controlled in terms of water intake to ensure the same amount of water intake during the measurement period, and the environmental temperature was maintained at about 24°C.

[0075] As shown in Figure 9 , when the results collected 3 hours after oral intake were plotted against the dose, we found that the folate level in sweat increased with the increase in folate intake. Specifically, the folate in sweat increased significantly as the dose increased from 0 to 10 mg, and only slightly increased when the dose increased to 20 mg.

[0076] Example 3 Correlation between sweat and blood folate concentration levels

[0077] This study further used a paired sample experimental design to systematically evaluate the correlation between sweat and blood folate concentrations. Specifically, by synchronously collecting sweat-blood paired samples from 31 healthy subjects using non-invasive microfluidic sweat patches and venipuncture. The chemiluminescence immunoassay kit was used to quantify the serum folate concentration, while the electrochemical immunosensor (detection range 0-100 nM) based on gold nanoflower / sulfur-nitrogen co-doped carbon modified screen-printed electrodes was used to determine the sweat folate content. To verify the correlation between the folate concentrations in the two biological matrices, a linear regression model was used to analyze the correlation between the sweat folate concentration and the blood folate concentration. By synchronously collecting paired samples, the blood and sweat folate concentrations obtained by chemiluminescence immunoassay and electrochemical immunosensing were subjected to Pearson correlation test.

[0078] As shown in Figure 10 , according to the results, the correlation coefficient between sweat and blood was 0.849.

[0079] Example 4 Effect of smoking on sweat folate concentration levels

[0080] Based on the previous study that smoking behavior has a significant impact on folate metabolism, 8 subjects with smoking habits were recruited as research subjects. The subjects completed the collection of sweat samples and venous blood samples simultaneously in the morning on the experimental day (8:00) in the fasting state. The concentrations of folate in blood and sweat were analyzed by chemiluminescence immunoassay and electrochemical immunoassay, respectively.

[0081] As Figure 11 shown, comparative analysis showed that compared with the non-smoking control group, the serum folate concentration of the smoking group was significantly reduced (6.37±3.22 vs 22.24±9.47 nM), and the sweat folate level also showed a synchronous downward trend (3.87±2.02 vs 10.448±5.00 nM).

[0082] The above-described embodiments are only some of the embodiments of the present application, not all the embodiments, and other embodiments obtained by those skilled in the art based on the embodiments of the present application without creative labor are within the protection scope of the present application.

Claims

1. Use of a folate sensor integrated with a calibration function in detecting a folate concentration, characterized in that, The detected sample is sweat; wherein the integrated calibration function sensor is prepared by the following method: (1) an electrochemical electrode AuNFs / SNC composite material with a gold nanoflower layer is prepared; (2) the AuNFs / SNC composite material of step (1) is used as a working electrode material of an ion sensor; (3) FA electrode preparation: folate binding protein FBP is fixed on the electrode surface of step (1); and non-specific blocking is performed; (4) pH calibration electrode: a pH selective membrane mixed solution is prepared, and the mixed solution is dropped on the carbon electrode surface and dried overnight; wherein the operation of step (1) is as follows: 1.1) a screen-printed electrode is used as a base electrode, which is cleaned and activated, and the working electrode material and the counter electrode material of the base electrode SPE electrode are both carbon, and the reference electrode material is Ag / AgCl; 1.2) electrochemical doping is performed on the above electrode to achieve sulfur / nitrogen co-doping to obtain an SNC electrode; 1.3) gold nanoflower layers are electrodeposited on the surface of the obtained SNC electrode to obtain an AuNFs / SNC nanocomposite material. The operation of step (3) is as follows: 3.1) carboxyl interface activation of the electrode; 3.2) FBP solution is applied to the electrode surface for incubation; wherein the carboxyl interface activation of step 3.1) is that EDC / NHS solution is prepared by using MES buffer to configure 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and the solution is dropped on the working electrode surface of the SPE, and the reaction is carried out in the dark; after the reaction is completed, the electrode surface is gently washed with pre-cooled MES buffer for 3 times, and dried with nitrogen; The operation of step 3.2) is that FBP solution is dropped on the electrode surface and incubated at 35~40°C for 30~50 min; The blocking operation in step (3) is that the electrode modified by FBP is immersed in a bovine serum albumin (BSA) solution for blocking; The pH selective mixed solution of step (4) is prepared as follows: 1wt% of hydrogen ion carrier I, 0.6%wt of Na-TFPB, 33%wt of PVC and 65.4%wt of DOS are added to tetrahydrofuran. The detection is a non-disease diagnosis application.

2. Use according to claim 1, characterized in that, The cleaning and activation operation in step 1.1) is that the electrode is activated in 0.1 M H2SO4 by cyclic voltammetry (CV) to remove surface oxides and enhance activity.

3. Use according to claim 1, characterized in that, The operation of step 1.2) is that electrochemical doping is performed in thiourea solution by cyclic voltammetry; the concentration of the thiourea is 0.5~0.7M, and the cyclic voltammetry is continuous electro-doping at −1.2 V~0.2 V for 25~35 cycles with a scan rate of 50~70 mV / s.

4. Use according to claim 1, characterized in that, The operation of step 1.3) is that gold nanoflower layers are electrodeposited in a chloroauric acid (HAuCl4) solution by CV to obtain an AuNFs / SNC nanocomposite material.

5. The use according to claim 1, characterized in that, The sampling site of the detection is selected from one or more of the arm, back, and leg skin.

6. The electrochemical immunosensor for specific detection of folic acid obtained by the preparation method according to any one of claims 1-5 for use in the method of any one of claims 1-5.

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