CN112557471A - Preparation method and detection method of modified electrode capable of simultaneously detecting vitamins B6 and B9 - Google Patents
Preparation method and detection method of modified electrode capable of simultaneously detecting vitamins B6 and B9 Download PDFInfo
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Abstract
The invention belongs to the technical field of vitamin detection, and particularly relates to a preparation method of a modified electrode capable of simultaneously detecting vitamin B6 and vitamin B9, 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; 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 composed of one or more of sulfuric acid, graphene, carbon nano tubes, vitamin B12, acetic acid, citric acid, gold nanoparticles, silver nitrate, sodium sulfate, sodium acetate, potassium chloride and amino black 10B, and the concentration range of each component is 0.0002-3 mol/L. The modified electrode overcomes the defects of the prior art, can detect vitamin B6 and B9 simultaneously, has high sensitivity, does not interfere with the peaks of two vitamins, has good reproducibility, and can greatly reduce the detection limit.
Description
Technical Field
The invention belongs to the technical field of vitamin detection, and particularly relates to a preparation method of a modified electrode capable of simultaneously detecting vitamin B6 and vitamin B9.
Background
With the progress of society, people pay more and more attention to their health. As a vitamin having important physiological functions of the body, its rational application, treatment and diagnostic monitoring are also gaining more and more attention. Wherein vitamin B6 is also called pyridoxine, which comprises pyridoxine, pyridoxal and pyridoxamine, and exists in the form of phosphate in vivo, and vitamin B6 is a component of certain coenzymes in human body, and is involved in various metabolic reactions, especially with amino acid metabolism. Vitamin B6 has functions of antibody synthesis, gastric acid production in the digestive system, fat and protein utilization (especially for weight loss), and sodium/potassium balance maintenance (stabilization of nervous system). General symptoms of the deficiency of vitamin B6 include inappetence, low food utilization rate, weightlessness, vomiting, diarrhea and other diseases. Severe deficiency can be manifested by acne, anemia, arthritis, childhood spasm, depression, headache, hair loss, easy inflammation, learning disability, asthenia, etc. Excessive vitamin B6 can cause lethargy, and the addiction can be caused after long-term administration.
Vitamin B9, also called folic acid, is a water-soluble vitamin, has the function of promoting the maturation of immature cells in bone marrow, and is especially important for pregnant women, who lack folic acid can cause megaloblastic anemia and leukopenia. Folic acid can maintain growth and intestinal function, and pregnant women can prevent congenital malformation such as premature birth of newborn and cleft palate. Meanwhile, folic acid also has the effects of resisting tumors and cancers and promoting the development of nerve cells and brain cells of infants. The lack of folic acid can cause fetal neural tube malformation, cause hyperhomocysteinemia and increase the risk of cardiovascular diseases; while excessive amount can induce convulsion of patients, influence the absorption of zinc and lead to zinc deficiency, and lead to damage of nervous system.
Therefore, the quantitative detection of the vitamins B6 and B9 has great clinical significance. The existing methods for detecting vitamin B6 and B9 are different, and mainly comprise a gravimetric method, a titration method, a fluorescence analysis method, a high performance liquid chromatography method, an electrochemical method 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 B6 and B9 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 B6 and B9 in a blood sample are detected simultaneously, the detection limit is high, and the peaks of the two substances cannot be separated, so that the concentration levels of the vitamins B6 and B9 in the blood sample cannot be reached.
Disclosure of Invention
The invention aims to provide a preparation method of a modified electrode capable of simultaneously detecting vitamin B6 and vitamin B9, which overcomes the defects of the prior art, the modified electrode not only can simultaneously detect vitamin B6 and vitamin B9, but also has higher sensitivity, the peaks of the two vitamins are not mutually interfered, the reproducibility is good, 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 capable of detecting vitamin B6 and vitamin B9 simultaneously 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 modification solution is composed of one or more of sulfuric acid, graphene, carbon nano tubes, vitamin B12, acetic acid, citric acid, gold nanoparticles, silver nitrate, sodium sulfate, sodium acetate, potassium chloride and amino black 10B, and the concentration range of each component is 0.0002-3 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 or potential step method.
Further, the amount of the modification solution dripped on the surface of the substrate electrode in the step (2) is 5-800 microliters.
The invention also provides a detection method of the modified electrode capable of simultaneously detecting vitamin B6 and vitamin B9, which comprises the following steps:
taking a blood sample to be detected to react with vitamin B6 and B9 releasing agents, so that vitamin B6 and B9 in a combined state in the blood sample to be detected are converted into vitamin B6 and B9 in a free state;
selecting the optimized modified electrode as a working electrode, and simultaneously detecting the redox currents generated on the working electrode by the vitamins B6 and B9 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 series of vitamin B6 and B9 standard samples with different concentrations are respectively compared, a standard curve is made with the corresponding concentrations of vitamin B6 and B9 standard solutions, and the content of vitamin B6 and the content of vitamin B9 in a blood sample to be detected are 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 contents of vitamin B6 and B9 in a blood sample are detected by using a convolution current method, the initial electrode potential of instrument parameters is-0.4-0.2V, the final electrode potential is 0.8-1.4V, the scanning speed is 30-200 mV/s, the working mode is a differential mode for 0.5 times, 1.5 times or 2.5 times, and the noise filtering is a low-pass mode; the vitamin B6, B9 release 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, acetic acid, phosphoric acid and boric acid, the concentration of the solvent is 0.00005-0.1 mol/L, the inert supporting electrolyte is one or more of sodium acetate, potassium chloride, sodium citrate, dipotassium hydrogen phosphate, sodium sulfate, disodium hydrogen phosphate and sodium dihydrogen phosphate, and the concentration of the inert supporting electrolyte is 0.001-5 mol/L.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention is modified on the basis of the existing glassy carbon electrode, gold electrode or copper electrode, not only can simultaneously detect vitamins B6 and B9, but also has higher sensitivity, the peaks of the two vitamins are not mutually interfered, the reproducibility is good, and the detection limit can be greatly reduced.
2. The method can simultaneously determine the contents of the vitamins B6 and B9 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 the vitamins B6 and B9 in the blood sample in the same system.
3. The invention adopts a standard addition method to simultaneously determine the contents of the vitamins B6 and B9 in the sample, eliminates the interference caused by the matrix effect of the sample, reduces the detection limits of the vitamins B6 and B9, and has accurate and reliable measurement results.
Drawings
FIG. 1 is a linear equation corresponding to the detection of vitamin B6 and vitamin B9 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 capable of detecting vitamin B6 and vitamin B9 simultaneously, 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 0.0002mol/L sulfuric acid, 0.001mol/L carbon nano tube and 0.005mol/L potassium chloride.
Wherein, the electroplating method in the step (2) is selected from one of cyclic voltammetry or potential step method; the amount of the modification solution dripped on the surface of the substrate electrode in the step (2) is 5-800 microliters.
Example 2
The embodiment discloses a preparation method of a modified electrode capable of detecting vitamin B6 and vitamin B9 simultaneously, 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 0.5mol/L acetic acid, 0.03mol/L gold nano-particles and 0.2mol/L sodium acetate.
Wherein, the electroplating method in the step (2) is selected from one of cyclic voltammetry or potential step method; the amount of the modification solution dripped on the surface of the substrate electrode in the step (2) is 5-800 microliters.
Example 3
The embodiment discloses a preparation method of a modified electrode capable of detecting vitamin B6 and vitamin B9 simultaneously, 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 1mol/L citric acid, 3mol/L sodium sulfate and 0.5mol/L amino black 10B.
Wherein, the electroplating method in the step (2) is selected from one of cyclic voltammetry or potential step method; the amount of the modification solution dripped on the surface of the substrate electrode in the step (2) is 5-800 microliters.
Detection method
One-standard addition method and one-standard addition method for simultaneously determining contents of vitamin B6 and B9 in blood samples
The specific implementation process comprises the following steps:
1. pretreatment of vitamin B6 and B9 modified electrodes: commercially available glassy carbon electrodes, gold electrodes or copper electrodes with the diameter of 2mm are selected as probes of the vitamin B6 and B9 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. Preparation of vitamin B6 and B9 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 the vitamin B6 and B9 sensor probes were modified in the modification solution prepared in example 1 by a potential step method. Setting instrument parameters: the initial electrode potential is-0.6-0.4V (relative to the reference electrode), the final electrode potential is 0.8-2V (relative to the reference electrode), the scanning speed is 20-200 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 B6 and B9 release agents: 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 0.001-5 mol/L.
4. Release of vitamins B6, B9 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 B6 and B9 specific release agents, covering a test tube plug, gently shaking uniformly, standing for 5 minutes, and determining by using an on-machine when vitamin B6 and B9 combined in the blood sample are released and become free micromolecules.
5. Simultaneous determination of vitamins B6, B9 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 B6 and B9 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-0.4-0.2V, the final electrode potential is 0.8-1.4V, the scanning speed is 30-200 mV/s, and the convolution currents of vitamin B6 and vitamin B9 obtained in the detection sample solution are recorded as h respectively.
Then quantitatively transferring Vs mL of standard solutions of vitamin B6 and B9 by using a micropipette, adding the standard solutions into the sample solution, uniformly stirring, and detecting under the same condition to obtain convolution currents of vitamin B6 and vitamin B9 which are recorded as H.
6. Calculation of vitamin B6, B9 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 CAH is a convolution current value obtained by detecting vitamin B6 and vitamin B9 in a sample solution, H is a convolution current value obtained by detecting after a standard solution is added, Vx is the volume of a diluent, Vs is the volume of the added standard solution, cs is the concentration of the standard solution, and k is the dilution multiple of the sample. The volume and concentration of the added vitamin B6 and B9 standard solutions meet the following requirements:
Vs<100Vx，cs>100cA
second, multi-standard addition method for determining vitamin B6 and B9 contents in blood sample
The specific implementation process comprises the following steps:
1. pretreatment of vitamin B6 and B9 modified electrodes: the same as in example 1.
2. Preparation of vitamin B6 and B9 modified electrodes: the same as in example 1.
3. Preparation of vitamin B6 and B9 release agents: the same as in example 1.
4. Release of vitamins B6, B9 in blood samples: the same as in example 1.
5. Determination of vitamins B6, B9 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 B6 and B9 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 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-0.4-0.2V, the final electrode potential is 0.8-1.4V, the scanning speed is 30-200 mV/s, and the convolution currents of vitamin B6 and vitamin B9 obtained in the detection sample solution are recorded as h0 respectively.
Then, vitamin B6 and B9 standard solutions V1mL, V2mL, V3mL, … and VnmL are sequentially removed by a micropipette, added to the sample solution, and sequentially detected under the same conditions, so as to obtain convolution currents respectively designated as h1, h2, h3, … and hn, and the concentrations of correspondingly added vitamin B6 and B9 are respectively c1, c2, c3, … and cn (which can be obtained by converting the volume of each added standard solution). FIG. 1 shows linear equations corresponding to the detection of vitamin B6 and vitamin B9 using the modified electrode prepared in example 1.
6. Calculation of vitamin B6, B9 concentration in blood samples:
minimum linear fitting was performed using the convolution current and concentration, respectively (concentration c0 of vitamin B6, B9 of the sample solution without standard addition is 0), resulting in the following linear relationship:
h＝kc+B (2)
thus, the concentration of vitamin B6, B9 in the blood sample can be obtained as
cA＝-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.
Claims (6)
1. A preparation method of a modified electrode capable of detecting vitamin B6 and vitamin B9 simultaneously 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 modification solution is composed of one or more of sulfuric acid, graphene, carbon nano tubes, vitamin B12, acetic acid, citric acid, gold nanoparticles, silver nitrate, sodium sulfate, sodium acetate, potassium chloride and amino black 10B, and the concentration range of each component is 0.0002-3 mol/L.
2. The preparation method of the modified electrode capable of simultaneously detecting vitamin B6 and vitamin B9 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 capable of simultaneously detecting vitamin B6 and vitamin B9 as claimed in claim 1, wherein the modified electrode comprises: the method for electroplating in the step (2) is selected from one of cyclic voltammetry or potential step method.
4. The preparation method of the modified electrode capable of simultaneously detecting vitamin B6 and vitamin B9 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 (2) is 5-800 microliters.
5. A method of detecting a modified electrode as claimed in any one of claims 1 to 7 capable of simultaneously detecting vitamin B6, B9, wherein: the method comprises the following steps:
taking a blood sample to be detected to react with vitamin B6 and B9 releasing agents, so that vitamin B6 and B9 in a combined state in the blood sample to be detected are converted into vitamin B6 and B9 in a free state;
selecting the optimized modified electrode as a working electrode, and simultaneously detecting the redox currents generated on the working electrode by the vitamins B6 and B9 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 series of vitamin B6 and B9 standard samples with different concentrations are respectively compared, a standard curve is made with the corresponding concentrations of vitamin B6 and B9 standard solutions, and the content of vitamin B6 and the content of vitamin B9 in a blood sample to be detected are respectively calculated through a linear equation according to the current signal values of the sample to be detected under the same condition.
6. A detection method of a modified electrode capable of simultaneously detecting vitamin B6 and vitamin B9 is characterized in that: when the contents of vitamin B6 and B9 in a blood sample are detected by a convolution current method, the initial electrode potential of instrument parameters is-0.4-0.2V, the final electrode potential is 0.8-1.4V, the scanning speed is 30-200 mV/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; the vitamin B6, B9 release 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, acetic acid, phosphoric acid and boric acid, the concentration of the solvent is 0.00005-0.1 mol/L, the inert supporting electrolyte is one or more of sodium acetate, potassium chloride, sodium citrate, dipotassium hydrogen phosphate, sodium sulfate, disodium hydrogen phosphate and sodium dihydrogen phosphate, and the concentration of the inert supporting electrolyte is 0.001-5 mol/L.
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