阅读说明：本技术 一种同时检测维生素b2、c的修饰电极制备方法及电化学检测方法 (Preparation method of modified electrode for simultaneously detecting vitamins B2 and C and electrochemical detection method ) 是由 潘攀 吴雪景 何流 唐涛 于 2020-12-22 设计创作，主要内容包括：本发明属于维生素检测技术领域,具体涉及一种同时检测维生素B2、C的修饰电极制备方法,包括以下步骤：选取玻碳电极、金电极或者铜电极作为基体电极,对基体电极表面进行抛光清洁处理后,备用；制备修饰电极：在步骤(1)制备的基体电极的表面滴涂或者电镀修饰溶液,用红外灯烘干制得所述修饰电极；其中,修饰溶液由硫酸、硝酸、醋酸、柠檬酸、硝酸铋、硝酸银、硝酸钾、硫酸钠、醋酸钠、氯化钾、氯化钠、氨基黑10B中的一种或几种组成,各组分的浓度范围是0.0005～5mol/L。克服了现有技术的不足,利用该修饰电极不仅可以同时检测维生素B2、C,而且具有较高灵敏度,且两种维生素的出峰互不干扰,具有良好的重现性,能大大降低检出限。(The invention belongs to the technical field of vitamin detection, and particularly relates to a preparation method of a modified electrode for simultaneously detecting vitamin B2 and vitamin C, which comprises the following steps: selecting a glassy carbon electrode, a gold electrode or a copper electrode as a substrate electrode, and polishing and cleaning the surface of the substrate electrode for later use; preparing a modified electrode: dripping or electroplating a modification solution on the surface of the matrix electrode prepared in the step (1), and drying by using an infrared lamp to prepare the modified electrode; the modifying solution is composed of one or more of sulfuric acid, nitric acid, acetic acid, citric acid, bismuth nitrate, silver nitrate, potassium nitrate, sodium sulfate, sodium acetate, potassium chloride, sodium chloride and amino black 10B, and the concentration range of each component is 0.0005-5 mol/L. The modified electrode overcomes the defects of the prior art, can detect vitamin B2 and vitamin C simultaneously, has high sensitivity, does not interfere with the peaks of two vitamins mutually, has good reproducibility, and can greatly reduce the detection limit.)

1. A preparation method of a modified electrode for simultaneously detecting vitamin B2 and vitamin C is characterized by comprising the following steps: the method comprises the following steps:

(1) pretreatment of a substrate electrode: selecting a glassy carbon electrode, a gold electrode or a copper electrode as a substrate electrode, and polishing and cleaning the surface of the substrate electrode for later use;

(2) preparing a modified electrode: dripping or electroplating a modification solution on the surface of the matrix electrode prepared in the step (1), and drying by using an infrared lamp to prepare the modified electrode;

the modifying solution is composed of one or more of sulfuric acid, nitric acid, acetic acid, citric acid, bismuth nitrate, silver nitrate, potassium nitrate, sodium sulfate, sodium acetate, potassium chloride, sodium chloride and amino black 10B, and the concentration range of each component is 0.0005-5 mol/L.

2. The preparation method of the modified electrode for simultaneously detecting vitamin B2 and vitamin C as claimed in claim 1, wherein the modified electrode comprises: the polishing and cleaning treatment of the substrate electrode specifically comprises the following steps: firstly, polishing a matrix electrode on metallographic abrasive paper of different models to ensure that the surface of the matrix electrode is smooth like a mirror surface; then mirror polishing is carried out on the polished substrate electrode on the alumina polishing powder with the grain diameter of 1.0 mu m, 0.3 mu m and 0.05 mu m in sequence; and finally, respectively placing the polished and bright substrate electrode in ethanol and deionized water for 2 minutes by ultrasonic treatment to remove organic matters and adsorbed alumina powder remained on the surface of the substrate electrode, and drying the substrate electrode by using nitrogen for later use.

3. The preparation method of the modified electrode for simultaneously detecting vitamin B2 and vitamin C as claimed in claim 1, wherein the modified electrode comprises: the electroplating method in the step (3) is selected from one of cyclic voltammetry, potential step method or chronoamperometry.

4. The preparation method of the modified electrode for simultaneously detecting vitamin B2 and vitamin C as claimed in claim 1, wherein the modified electrode comprises: the amount of the modification solution dripped on the surface of the substrate electrode in the step (3) is 10-2000 microliters.

5. An electrochemical detection method of the modified electrode for simultaneously detecting vitamin B2, C according to claims 1-4, characterized in that: the method comprises the following steps:

taking a blood sample to be detected to react with vitamin B2 and a C releasing agent, so that the combined vitamin B2 and C in the blood sample to be detected is converted into free vitamin B2 and C;

selecting the optimized modified electrode as a working electrode, and simultaneously detecting the redox currents generated on the working electrode by the vitamins B2 and C in the blood sample to be detected by using a convolution current voltammetry method; the two vitamins respectively peak at specific peak potentials, and the peak shapes are good, so that the peak current corresponding to each vitamin can be conveniently measured, and the two peaks do not interfere with each other;

the current signal values of a series of vitamin B2 and C standard samples with different concentrations are respectively compared, standard curves are made with the corresponding concentrations of the vitamin B2 and C standard solutions, and the content of vitamin B2 and C in a blood sample to be detected is respectively calculated through a linear equation according to the current signal values of the sample to be detected under the same condition.

6. The electrochemical detection method for the modified electrode for simultaneously detecting the vitamins B2 and C according to claim 5, wherein the electrochemical detection method comprises the following steps: when the content of vitamin B2 and C in a blood sample is detected, the initial electrode potential of instrument parameters is-1.5 to-0.6V, the final electrode potential is 0.4 to 1.2V, the scanning speed is 20 to 500mV/s, the working mode is a differential mode of 0.5 times, 1.5 times or 2.5 times, and the noise filtering is a low-pass mode.

7. The electrochemical detection method for the modified electrode for simultaneously detecting the vitamins B2 and C according to claim 5, wherein the electrochemical detection method comprises the following steps: the vitamin B2, C releasing agent consists of a solvent and an inert supporting electrolyte; the solvent is one or more aqueous solutions of sodium hydroxide, potassium hydroxide, citric acid, hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid and boric acid, the concentration of the solvent is 0.00001-1 mol/L, the inert supporting electrolyte is one or more of sodium acetate, potassium chloride, sodium citrate, ammonium sulfate, sodium sulfate, disodium hydrogen phosphate, sodium dihydrogen phosphate, silver nitrate and lead nitrate, and the concentration of the inert supporting electrolyte is 0.005-10 mol/L.

Technical Field

The invention belongs to the technical field of vitamin detection, and particularly relates to a preparation method of a modified electrode for simultaneously detecting vitamin B2 and vitamin C.

Background

As is well known, vitamins are a kind of trace organic substances that maintain normal physiological functions of the body, and play an important role in regulating metabolism of substances, maintaining physiological functions and other functions. Wherein vitamin B2 is called riboflavin, is a water-soluble vitamin, is easy to digest and absorb, and the amount of excretion is increased or decreased according to the needs of the body and the possible loss degree of protein. Vitamin B2 has the physiological functions of promoting metabolism as coenzyme, maintaining normal visual function, maintaining normal functions of skin, nervous system and cells, promoting blood circulation, and lowering blood pressure. Deficiency of vitamin B2 can lead to skin diseases and nervous disorders, while excess can cause renal tubule obstruction and cause renal dysfunction such as oliguria.

Vitamin C, also known as ascorbic acid, is an acidic polyol containing 6 carbon atoms. Naturally occurring vitamin C has 2 types, L-type and D-type, the latter being biologically inactive. Vitamin C in food is mainly present in fresh vegetables and fruits and cannot be synthesized by human body. Vitamin C is an important component of the body's antioxidant defense system and is involved in collagen formation, preventing cardiovascular diseases and nutritional anemia, and blocking the formation of N-nitroso compounds. Vitamin C deficiency can lead to scurvy and decreased resistance, while excess can lead to gastric bleeding, stones, gout, infant dependence, pediatric orthopedic disorders, infertility, decreased immunity, and the like. Therefore, the quantitative detection of the vitamins B2 and C has great significance.

The existing methods for detecting vitamin B2 and vitamin C are different, and mainly comprise gravimetric methods, titration methods, fluorescence analysis methods, high performance liquid chromatography, electrochemical methods and the like. The gravimetric method and the titration method are complex to operate, the measurement error of low-content components is large, if the terminal point judgment is not sharp, a large error is caused, and the simultaneous detection of vitamins B2 and C cannot be carried out; the pretreatment processes of the spectral analysis method and the chromatography method are too complicated and time-consuming, more organic solvents are used, and the required amount of blood samples is large; the electrochemical method is sensitive and rapid, needs less samples, does not need pretreatment, and is particularly suitable for detecting the vitamins in the biological samples.

The existing electrochemical analysis technology for detecting vitamins is mainly realized by the traditional linear sweep voltammetry, the conventional pulse voltammetry, the differential pulse voltammetry, the square wave voltammetry, the stripping voltammetry and the like, although the methods can be used for detecting vitamins, the sensitivity is low, particularly when the vitamins B2 and C in a blood sample are detected simultaneously, the detection limit is high, and the peaks of the two substances are not separated, so that the concentration levels of the vitamins B2 and C in the blood sample cannot be reached.

Disclosure of Invention

The invention aims to provide a preparation method of a modified electrode for simultaneously detecting vitamin B2 and vitamin C, which overcomes the defects of the prior art, the modified electrode can be used for simultaneously detecting vitamin B2 and vitamin C, and has high sensitivity, peaks of the two vitamins are not interfered with each other, good reproducibility is realized, and the detection limit can be greatly reduced.

In order to solve the problems, the technical scheme adopted by the invention is as follows:

a preparation method of a modified electrode for simultaneously detecting vitamin B2 and vitamin C comprises the following steps:

(1) pretreatment of a substrate electrode: selecting a glassy carbon electrode, a gold electrode or a copper electrode as a substrate electrode, and polishing and cleaning the surface of the substrate electrode for later use;

(3) preparing a modified electrode: dripping or electroplating a modification solution on the surface of the matrix electrode prepared in the step (1), and drying by using an infrared lamp to prepare the modified electrode;

the modifying solution is composed of one or more of sulfuric acid, nitric acid, acetic acid, citric acid, bismuth nitrate, silver nitrate, potassium nitrate, sodium sulfate, sodium acetate, potassium chloride, sodium chloride and amino black 10B, and the concentration range of each component is 0.0005-5 mol/L.

Further, the polishing and cleaning treatment of the substrate electrode specifically comprises: firstly, polishing a matrix electrode on metallographic abrasive paper of different models to ensure that the surface of the matrix electrode is smooth like a mirror surface; then mirror polishing is carried out on the polished substrate electrode on the alumina polishing powder with the grain diameter of 1.0 mu m, 0.3 mu m and 0.05 mu m in sequence; and finally, respectively placing the polished and bright substrate electrode in ethanol and deionized water for 2 minutes by ultrasonic treatment to remove organic matters and adsorbed alumina powder remained on the surface of the substrate electrode, and drying the substrate electrode by using nitrogen for later use.

Further, the method for electroplating in the step (2) is selected from one of cyclic voltammetry, a potential step method or a chronoamperometry.

Further, the amount of the modification solution dripped on the surface of the substrate electrode in the step (2) is 10-2000 microliters.

The invention also provides an electrochemical detection method of the modified electrode for simultaneously detecting vitamin B2 and vitamin C, which comprises the following steps:

taking a blood sample to be detected to react with vitamin B2 and a C releasing agent, so that the combined vitamin B2 and C in the blood sample to be detected is converted into free vitamin B2 and C;

selecting the optimized modified electrode as a working electrode, and simultaneously detecting the redox currents generated on the working electrode by the vitamins B2 and C in the blood sample to be detected by using a convolution current voltammetry method; the two vitamins respectively peak at specific peak potentials, and the peak shapes are good, so that the peak current corresponding to each vitamin can be conveniently measured, and the two peaks do not interfere with each other;

the current signal values of a series of vitamin B2 and C standard samples with different concentrations are respectively compared, standard curves are made with the corresponding concentrations of the vitamin B2 and C standard solutions, and the content of vitamin B2 and C in a blood sample to be detected is respectively calculated through a linear equation according to the current signal values of the sample to be detected under the same condition.

Further, when the content of vitamin B2 and C in a blood sample is detected, the initial electrode potential of instrument parameters is-1.5 to-0.6V, the final electrode potential is 0.4 to 1.2V, the scanning speed is 20 to 500mV/s, the working mode is a differential mode of 0.5 times, 1.5 times or 2.5 times, and the noise filtering is a low-pass mode.

Further, the vitamin B2, C releasing agent consists of a solvent and an inert supporting electrolyte; the solvent is one or more aqueous solutions of sodium hydroxide, potassium hydroxide, citric acid, hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid and boric acid, the concentration of the solvent is 0.00001-1 mol/L, the inert supporting electrolyte is one or more of sodium acetate, potassium chloride, sodium citrate, ammonium sulfate, sodium sulfate, disodium hydrogen phosphate, sodium dihydrogen phosphate, silver nitrate and lead nitrate, and the concentration of the inert supporting electrolyte is 0.005-10 mol/L.

Compared with the prior art, the invention has the following beneficial effects:

1. the modified electrode can be used for simultaneously detecting the vitamins B2 and C, has high sensitivity, does not interfere with the peaks of the two vitamins, has good reproducibility and can greatly reduce the detection limit.

2. The method can simultaneously determine the contents of vitamin B2 and vitamin C in the blood sample by adopting the convolution current voltammetry, has the advantages of high sensitivity, good reproducibility, more convenient peak shape measurement of current signals, convenient and quick operation, shorter detection time and the like, and can simultaneously and accurately detect the contents of vitamin B2 and vitamin C in the blood sample in the same system.

3. The invention adopts a standard addition method to simultaneously determine the content of vitamin B2 and C in the sample, eliminates the interference caused by the matrix effect of the sample, reduces the detection limit of vitamin B2 and C, and has accurate and reliable measurement result.

Drawings

FIG. 1 shows the linear equation corresponding to the detection of vitamin B2, C using the modified electrode prepared in example 1.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

The embodiment discloses a preparation method of a modified electrode for simultaneously detecting vitamin B2 and vitamin C, which comprises the following steps:

(1) pretreatment of a substrate electrode: selecting a glassy carbon electrode, a gold electrode or a copper electrode as a substrate electrode, and polishing and cleaning the surface of the substrate electrode for later use;

(2) preparing a modified electrode: dripping or electroplating a modification solution on the surface of the matrix electrode prepared in the step (1), and drying by using an infrared lamp to prepare the modified electrode; the modification solution is prepared by mixing 0.0005mol/L sulfuric acid, 0.002mol/L bismuth nitrate and 0.005mol/L potassium chloride.

Wherein, the electroplating method in the step (2) is selected from one of cyclic voltammetry, potential step method or chronoamperometry; and the amount of the modifying solution dripped on the surface of the matrix electrode is 10-2000 microliter.

Example 2

The embodiment discloses a preparation method of a modified electrode for simultaneously detecting vitamin B2 and vitamin C, which comprises the following steps:

(1) pretreatment of a substrate electrode: selecting a glassy carbon electrode, a gold electrode or a copper electrode as a substrate electrode, and polishing and cleaning the surface of the substrate electrode for later use;

(2) preparing a modified electrode: dripping or electroplating a modification solution on the surface of the matrix electrode prepared in the step (1), and drying by using an infrared lamp to prepare the modified electrode; the modification solution was prepared by mixing 0.02mol/L sulfuric acid, 0.5mol/L sodium sulfate and 0.2mol/L aminoblack 10B.

Wherein, the electroplating method in the step (2) is selected from one of cyclic voltammetry, potential step method or chronoamperometry; and the amount of the modifying solution dripped on the surface of the matrix electrode is 10-2000 microliter.

Example 3

The embodiment discloses a preparation method of a modified electrode for simultaneously detecting vitamin B2 and vitamin C, which comprises the following steps:

(1) pretreatment of a substrate electrode: selecting a glassy carbon electrode, a gold electrode or a copper electrode as a substrate electrode, and polishing and cleaning the surface of the substrate electrode for later use;

(2) preparing a modified electrode: dripping or electroplating a modification solution on the surface of the matrix electrode prepared in the step (1), and drying by using an infrared lamp to prepare the modified electrode; the modification solution is prepared by mixing 2mol/L of citric acid, 1mol/L of potassium sulfate and 5mol/L of sodium chloride.

Wherein, the electroplating method in the step (2) is selected from one of cyclic voltammetry, potential step method or chronoamperometry; and the amount of the modifying solution dripped on the surface of the matrix electrode is 10-2000 microliter.

Detection method

One-standard addition method for simultaneously determining vitamin B2 and C content in blood sample

The specific implementation process comprises the following steps:

1. pretreatment of vitamin B2 and C modified electrodes: commercially available glassy carbon electrodes, gold electrodes or copper electrodes with the diameter of 2mm are selected as probes of the vitamin B2 and C sensors, and the electrodes are firstly ground on metallographic abrasive paper of different types, so that the surfaces of the electrodes are smooth like a mirror surface. And then mirror polishing is carried out on the polished electrode on alumina polishing powder with the grain diameter of 1.0 mu m, 0.3 mu m and 0.05 mu m in sequence. And finally, respectively placing the polished and bright electrodes in ethanol and deionized water for 2 minutes by ultrasonic treatment to remove organic matters and adsorbed alumina powder remained on the surfaces of the electrodes, and drying the electrodes by using nitrogen for later use.

2. Preparing vitamin B2 and C modified electrodes: the treated electrode was used as a working electrode, a platinum wire electrode as an auxiliary electrode, and a silver/silver chloride electrode as a reference electrode, and in the plating solution prepared in example 1, the vitamin B2 and C sensor probes were modified by a potential step method. Setting instrument parameters: the initial electrode potential is-0.8-0.2V (relative to the reference electrode), the final electrode potential is 0.4-2V (relative to the reference electrode), the scanning speed is 50-100 mV/s, the scanning is carried out for 6-15 circles in a circulating mode under the stirring condition, then the cleaning is carried out by deionized water, and the nitrogen is dried for standby.

3. Preparation of vitamin B2 and C releasing agent: and (2) taking a proper amount (100-1000 mL) of the single solvent or the mixed solvent into a volumetric flask, weighing the single or composite supporting electrolyte according to the proportion, adding the single or composite supporting electrolyte into the volumetric flask, covering a bottle stopper, slightly shaking, adding the same solvent to the scale mark of the volumetric flask after the supporting electrolyte is dissolved, wherein the concentration of the supporting electrolyte in the solution is 0.005-10 mol/L.

4. Release of vitamins B2, C in blood samples: quantitatively transferring a blood sample (10-20 mu L) into a 5.0mL test tube by using a micropipette, quantitatively adding 0.1-2.0 mL of vitamin B2 and C specific releasing agent, covering a test tube plug, gently shaking uniformly, standing for 5 minutes, and detecting on a machine after vitamin B2 and C combined in the blood sample are released and become free micromolecules.

5. Simultaneous determination of vitamins B2, C in blood samples:

firstly, fixing a test tube containing a sample solution to be detected on a workbench of a vitamin detector, taking the preferable vitamin B2 and C modified electrodes as working electrodes, taking a platinum wire electrode as an auxiliary electrode, taking a silver/silver chloride electrode as a reference electrode, and inserting the electrodes into the sample solution to be detected together for detection. The working mode of the vitamin detector can be selectively set to be 0.5, 1.5 or 2.5 differential modes, and the noise filtering is a low-pass mode. The test parameters are: the initial electrode potential of the instrument parameters is-1.5 to-0.6V, the final electrode potential is 0.4 to 1.2V, the scanning speed is 20 to 500mV/s, and the convolution currents of the vitamin B2 and the vitamin C obtained in the detection sample solution are recorded as h respectively.

Then quantitatively transferring VsmL of standard solutions of vitamin B2 and vitamin C by using a micropipette, adding the VsmL into the sample solution, uniformly stirring, and detecting under the same condition to obtain the convolution currents of vitamin B2 and vitamin C respectively as H.

6. Calculation of vitamin B2, C concentration in blood samples:

the concentrations of vitamin B2 and vitamin C in the blood sample were calculated according to equation (1):

in the formula C_AThe concentration of vitamin B2, C in the blood sample, h is the test sample solutionThe convolution current values obtained by vitamin B2 and vitamin C, H is the convolution current value detected after the addition of the standard solution, Vx is the volume of the diluent, Vs is the volume of the added standard solution, cs is the concentration of the standard solution, and k is the dilution factor of the sample. The volume and concentration of the added vitamin B2 and C standard solution meet the following requirements:

Vs<100Vx，cs>100cA

second, multi-standard addition method for determining vitamin B2, C content in blood sample

The specific implementation process comprises the following steps:

1. pretreatment of vitamin B2 and C modified electrodes: the same as in example 1.

2. Preparing vitamin B2 and C modified electrodes: the same as in example 1.

3. Preparation of vitamin B2 and C releasing agent: the same as in example 1.

4. Release of vitamins B2, C in blood samples: the same as in example 1.

5. Determination of vitamins B2, C in blood samples:

firstly, fixing a test tube containing a sample solution to be detected on a workbench of a vitamin detector, taking modified vitamin B2 and C sensors as working electrodes, taking a platinum wire electrode as an auxiliary electrode, taking a silver/silver chloride electrode as a reference electrode, and inserting the two electrodes into the sample solution to be detected together for detection. The working mode of the vitamin detector can be selectively set to be 0.5, 1.5 or 2.5 differential modes, and the noise filtering is a low-pass mode. The test parameters are: the test parameters are: the initial electrode potential of the instrument parameters is-1.5 to-0.6V, the final electrode potential is 0.4 to 1.2V, the scanning speed is 20 to 500mV/s, and the convolution currents of vitamin B2 and vitamin C obtained in the detection sample solution are recorded as h0 respectively.

Then, a micropipette is used for quantitatively transferring standard solutions V1mL, V2mL, V3mL,. cndot.and VnmL of the vitamins B2 and C in sequence, the standard solutions are respectively added into the sample solution, the obtained convolution currents are respectively recorded as h1, h2, h3,. cndot.and hn under the same condition, and the concentrations of the correspondingly added vitamins B2 and C are respectively C1, C2, C3,. cndot.and cn (the concentration can be obtained by converting the volume of the standard solution added each time). Fig. 1 shows the linear equations corresponding to vitamin C and vitamin B2.

6. Calculation of vitamin B2, C concentration in blood samples:

minimum linear fitting was performed using the convolution current and concentration, respectively (concentration C0 of vitamin B2, C of the sample solution without standard addition is 0), resulting in the following linear relationship:

h＝kc+B (2)

thus, the concentration of vitamin B2, C in the blood sample can be obtained as

c_A＝-B (3)

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.