CN111579626B - Preparation method and application of competitive immunosensor for detecting capsaicin - Google Patents
Preparation method and application of competitive immunosensor for detecting capsaicin Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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Abstract
A preparation method and application of competitive immunosensor for detecting capsaicin relate to a new preparation method of biosensor for detecting adulteration of edible vegetable oil, which is characterized in that firstly, ferroferric oxide-cerium oxide is used for modifying a glassy carbon electrode, so that the electron transfer rate can be accelerated, and the electric signal of the sensor can be improved; fixing capsaicin material antigen on the surface of the modified glassy carbon electrode, and dropwise adding BSA (bovine serum albumin) to seal non-specific sites; dripping mixed solution of capsaicin standard substances and antibodies with different concentrations, reducing the binding rate of the capsaicin antibody and the antigen along with the increase of the concentration of the capsaicin standard substances, and enhancing the electric signal, so that the content of the capsaicin in the standard solution is calculated and obtained by the obtained standard curve; the competitive immunosensor has high sensitivity and good stability, and can be used for quickly detecting adulteration marker capsaicin in edible vegetable oil.
Description
Technical Field
The invention relates to a preparation method of an immunosensor for detecting capsaicin and application of the immunosensor in edible vegetable oil identification, and belongs to the fields of food, immunoassay and electrochemical biosensors.
Background
Edible vegetable oil is widely applied to household cooking and food industry, and is one of important foods in our daily life; vegetable oils provide some important nutritional and health ingredients for humans, including but not limited to Essential Fatty Acids (EFAs), vitamins, minerals, etc.; with the increase of consumption demand of edible vegetable oil, the edible oil is mixed with inedible waste oil or impure edible oil, which causes serious food safety problem; in order to protect the benefits of consumers, many researchers have been looking for a fast and reliable method for detecting the adulteration of edible oil; the adulteration of the inedible waste oil is generally detected by adopting a sensory evaluation method, however, the sensory analysis depends on the experience of an analyst, and the subjective judgment may cause wrong negative results; under these circumstances, many analytical methods based on wet chemistry or chromatography detect and quantify the adulteration of edible vegetable oils by measuring and quantifying the free acid radical and fatty acid composition, such as high performance liquid chromatography, which can sensitively detect the adulteration of vegetable oils, and chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) combined with chemometrics, have been successfully used for the detection of adulteration of edible oils; these conventional methods are often used as official detection methods due to their high accuracy and sensitivity; but also has some obvious disadvantages, such as complex sample pretreatment, chemical hazardous articles, professional training, time consumption and the like; therefore, there is a need to develop a simple and sensitive method for detecting the adulteration of edible vegetable oil; the research develops an electrochemical immunosensor, and the edible vegetable oil is analyzed by taking capsaicin as a marker compound.
The pepper not only has spicy and aromatic sensory characteristics, but also is rich in nutrient substances such as carotene, vitamins and the like; the capsaicinoids contain capsaicin and dihydrocapsaicin at most, and account for nearly 90% of the total stimulation of pepper fruits; synthetic capsaicin has similar chemical structure and pungent property with dihydrocapsaicin, and is often used as a substitute for capsaicin and dihydrocapsaicin; research has shown that capsaicin and dihydrocapsaicin are lipophilic and stable in the refining process of high-boiling non-edible waste oil; thus, these compounds can be selected as scientific biomarkers for edible vegetable oils for detection and certification.
The existing capsaicin detection methods comprise an immunoassay method, a high performance liquid chromatography, a gas chromatography and the like; the liquid chromatography and the gas chromatography are mature methods for detecting capsaicin, have high accuracy and good sensitivity, but have higher requirements on test instruments and workers, long consumed time, strict requirements on comparison samples, high consumption of manpower and material resources and high cost; a method which can realize rapid detection and is simple and convenient to operate is needed, and the emerging electrochemical immunosensor can well meet the requirements of people.
The electrochemical immunosensor has good specificity, high sensitivity and short time consumption, and is widely applied to the fields of disease diagnosis, food sanitation, environmental monitoring and the like; the key of electrochemical detection is to generate a signal with high sensitivity and high intensity; therefore, in recent years, various nanomaterials are widely used to improve the performance of electrodes. Metal nanoparticles such as cerium oxide and iron oxide are widely concerned due to the characteristics of strong catalytic performance, large surface volume ratio, high surface activity, strong adsorption capacity and the like; if a competitive electrochemical immunosensor capable of specifically detecting capsaicin is prepared by utilizing the principle of antigen-antibody specific binding, a new way for detecting adulteration of edible vegetable oil is opened up.
Disclosure of Invention
The invention aims to overcome the defects, construct a competitive electrochemical immunosensor with high sensitivity, high stability and good selectivity, and be used for detecting three capsaicin substances, namely capsaicin, synthetic capsaicin and dihydrocapsaicin.
The technical scheme of the invention is as follows: a preparation method of a competitive immunosensor for detecting capsaicin is characterized in that: by mixing ferroferric oxide-cerium dioxide (Fe) 3O4-CeO 2) Modifying the surface of a glassy carbon electrode in a dripping way to enhance the conductivity of the electrode and fix capsaicin antigens, and then blocking nonspecific sites by BSA (bovine serum albumin) to realize the construction of the sensor, wherein the construction method comprises the following steps:
（1）Fe3O4-CeO 2preparation of composite materials
0.5 g of chitosan was weighed, dissolved in 50 mL of 1.0% acetic acid solution, stirred at room temperature for 3 hours to completely dissolve the chitosan, and then 10 mg of Fe was added3O4Adding the mixture into the solution, and ultrasonically stirring the mixture until yellow turbid liquid is obtained; finally, 10 mg of CeO was added to the mixed solution 2Continuing to perform ultrasonic dispersion until obtaining coffee liquid, and obtaining Fe3O4- CeO 2A composite material;
(2) preparation of electrochemical immunosensor for capsaicinoids
Firstly, Fe is mixed3O4-CeO 2Fixing the composite material on the surface of the pretreated glassy carbon electrode in a dripping way, and drying at room temperature to prepare a modified electrode; immersing the modified electrode into 9 mug/mL capsaicin antigen solution, and incubating overnight at 4 ℃; after washing with phosphate buffer solution with pH =7.5, continuously dropwise adding 5 μ L of BSA with mass fraction of 0.5%, and sealing at room temperature for 70 minutes; and washing the obtained electrode with a phosphate buffer solution with the pH =7.5, and airing to obtain the prepared electrochemical immunosensor.
The electrochemical immunosensing method for capsaicin substances comprises the following steps
(1) Preparation of electrochemical immunosensor for capsaicins
0.5 g of chitosan was weighed, dissolved in 50 mL of 1.0% acetic acid solution, stirred at room temperature for 3 hours to completely dissolve the chitosan, and then 10 mg of Fe was added3O4Adding the mixture into the solution, and ultrasonically stirring the mixture until yellow turbid liquid is obtained; finally, 10 mg of CeO was added to the mixed solution 2Continuing to perform ultrasonic dispersion until obtaining coffee liquid, and obtaining Fe3O4-CeO 2A composite material; mixing Fe3O4-CeO 2Fixing the composite material on the surface of the pretreated glassy carbon electrode in a dripping way, and drying at room temperature to prepare a modified electrode; immersing the modified electrode into 9 mug/mL capsaicin antigen solution, and incubating overnight at 4 ℃; after washing with phosphate buffer solution with pH =7.5, continuously dropwise adding 5 μ L of BSA with mass fraction of 0.5%, and sealing at room temperature for 70 minutes; and washing the obtained electrode with a phosphate buffer solution with the pH =7.5, and airing to obtain the prepared electrochemical immunosensor.
(2) Detection of capsaicinoids in standard solutions
Quickly dripping 5 mu L of mixed solution (ready-to-use) of capsaicin standard solution and capsaicin antibody with different concentrations on the surface of the electrochemical immunosensor prepared in the step (1), carrying out incubation reaction at 37 ℃ for 70 minutes, then washing out the antibody and the standard substance which are not combined with the antigen by adopting phosphate buffer solution with the pH =7.5, recording a current response value by using a differential pulse voltammetry in 5 mM potassium ferricyanide solution, and as the concentration of the capsaicin standard substance increases, the combination rate of the capsaicin antibody and the antigen decreases, and the electric signal increases, thereby calculating and obtaining the content of the capsaicin in the standard solution by using the obtained standard curve.
The invention also provides an application of an electrochemical immunosensing method established by the competitive immunosensor for detecting the capsaicinoids in the detection of adulteration of edible vegetable oil, which comprises the following steps:
adding an edible vegetable oil sample into 70% methanol-phosphate buffer solution (v/v), centrifuging at 4000 rpm for about 1 min, standing at 4 ℃ for 20 min, diluting the obtained methanol-phosphate buffer solution by 5 times, and preparing capsaicin standard solutions with different concentrations; dripping 5 mu L of mixed solution of capsaicin standard solution and capsaicin antibody with different concentrations on the surface of the electrochemical immunosensor, carrying out incubation reaction at 37 ℃ for 70 minutes, then washing out the antibody and the standard substance which are not combined with the antigen by adopting phosphate buffer solution with pH =7.5, and recording a current response value by using a differential pulse voltammetry method in 5 mM potassium ferricyanide solution, thereby calculating the content of the capsaicin in the edible vegetable oil sample solution.
The working principle of the invention is as follows: fe with stable performance and good conductivity is synthesized by utilizing the oxidation of cerium oxide and the high conductivity of ferroferric oxide3O4-CeO 2The compound is used for modifying a glassy carbon electrode to realize the preparation of an immunosensor; obtaining an electrochemical signal inhibition curve of the sensor through the competition of capsaicin antigens on the sensor and capsaicin standard substances with different concentrations in the solution for capsaicin antibodies; based on the current response of the signal, the current response can be established for food And (3) detecting the quantitative relation of capsaicinoids in the vegetable oil.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a Fe-based alloy3O4-CeO 2The preparation method of the competitive electrochemical immunosensor for detecting the capsaicin in the edible vegetable oil by using the composite material is used for detecting the capsaicin substances in the edible vegetable oil; with Fe3O4-CeO 2The glassy carbon electrode is modified, the antibody fixing effect is improved, the stability of the sensor is enhanced, the method can be used for detecting capsaicin substances in actual samples, and a new method is provided for the field rapid monitoring of supervision departments.
Drawings
FIG. 1 Assembly of an immunosensor.
Figure 2 DPV characterization of immunosensor.
Figure 3 antigen concentration optimization.
Figure 4 antibody concentration optimization.
Figure 5 incubation time optimization.
FIG. 6 base solution pH optimization.
Fig. 7 standard graph.
Detailed Description
The present invention will be described in further detail with reference to the following drawings and examples, which are not intended to limit the invention in any manner.
Example 1: preparation of immunosensor
(1) Preparation of nanomaterials
0.5 g of chitosan was weighed, dissolved in 50 mL of 1.0% acetic acid solution, stirred at room temperature for 3 hours to completely dissolve the chitosan, and then 10 mg of Fe was added 3O4Adding the mixture into the solution, and ultrasonically stirring the mixture until a yellow dispersion liquid is obtained; finally, 10 mg of CeO was added to the mixed solution 2Continuing to perform ultrasonic dispersion until obtaining coffee dispersion liquid, and obtaining Fe3O4-CeO 2A composite material;
(2) pretreatment of electrodes
The glassy carbon electrode is coated with 0.3 μm Al 2 0 3Polishing in slurry to mirror surfaceSequentially immersing in ultrapure water and absolute ethyl alcohol for 1 min by ultrasonic treatment, and adding N 2After drying, scanning and measuring the potential difference in 5 mM potassium ferricyanide by cyclic voltammetry to ensure that the potential difference is less than 80mV, thus obtaining the bare electrode with stable performance.
(3) Preparation of immunosensor
Firstly, Fe is mixed3O4-CeO 2Fixing the composite material on the surface of the pretreated glassy carbon electrode in a dripping way, and drying at room temperature to prepare a modified electrode; immersing the modified electrode into 9 mug/mL capsaicin antigen solution, and incubating overnight at 4 ℃; after washing with phosphate buffer solution with pH =7.5, continuously dropwise adding 5 μ L of BSA with mass fraction of 0.5%, and sealing at room temperature for 70 minutes; washing the obtained electrode with phosphate buffer solution with pH =7.5, and airing to obtain the prepared electrochemical immunosensor; finally, the prepared biosensor was stored in a refrigerator at a temperature of 4 ℃ for the following experiments; figure 1 shows an immunosensor assembly process.
(4) DPV characterization of immunosensors
As shown in fig. 2, the immunosensor was electrochemically characterized by differential pulse voltammetry techniques: the modified electrode (curve d) exhibited a higher peak current relative to the bare electrode (curve a), indicating Fe3O4-CeO 2Successful modification of the electrode surface and enhanced pre-electron transfer; after the capsaicin antigens are further modified on the electrode (curve b), the oxidation-reduction peak value is obviously reduced; when BSA blocked other active sites (curve c), the electrical signal was further reduced due to the increase in interface thickness; the material morphology characterization results show that the material is based on Fe3O4-CeO 2Immunosensors made from composite materials are feasible.
(5) Immunosensor condition optimization
FIG. 3 depicts the effect of antigen concentration on the sensor; the result shows that when the antigen concentration is 9 mug/mL, the difference value of the differential pulse voltammetry current reaches the maximum, but the current difference does not change along with the increase of the antigen concentration; because, the reaction of the antigen concentration with the target reaches saturation; therefore, the antigen concentration in the preparation of this immunosensor was 9. mu.g/mL.
FIG. 4 depicts the effect of antibody concentration on the sensor; the result shows that when the antibody concentration is 97.7 ng/mL, the difference value of the differential pulse voltammetry current reaches the maximum, but the current difference does not change along with the increase of the antibody concentration; because, the reaction of the antibody concentration with the target substance reaches saturation; therefore, the antibody concentration in the preparation of this immunosensor was 97.7 ng/mL.
The incubation time for specific binding of the target to the antibody is another important factor affecting the performance of the immunosensor, and studies conducted thereon found that 70 minutes was the optimal incubation time (fig. 5).
In addition, the pH of the test solution is an important factor for obtaining good analytical performance, and it was found that 7.5 at pH is the optimum test environment (FIG. 6).
Example 2: based on Fe3O4-CeO 2Composite material competitive electrochemical immunosensor for detecting capsaicin in edible vegetable oil
(1) Establishment of a Standard Curve
To monitor the electrochemical signal changes occurring on the immunosensor, a differential pulse voltammetric detection technique of 5.0mM [ Fe (CN)6 ] 3−/4−In solution (pH 7.5, containing 0.1M KCl) at a potential in the range of-0.4 to +0.6V (potential increment of 5 mV/s, amplitude of 50 mV); all immunosensors were prepared under optimal experimental conditions and their initial DPV responses (noted I) were measured; then, 5 μ L of a mixture of capsaicin standards and antibodies at different concentrations was dropped on the immunosensor, incubated at room temperature for 70 minutes, and the electrochemical response was again measured after rinsing and drying (recorded as I)0) Calculate Δ I value (Δ I = I-I)0) Analyzing and researching the relation between the concentration of the capsaicin and the concentration of a capsaicin standard substance; fig. 7 shows a linear relationship between Δ I and the log of capsaicin concentration Y = -0.21055X + 2.22705; the detection range is 0.001 ng/mL at the lowest detection limit of 0.001 ng/mL to 10 ng/mL.
(2) Detection of capsaicinoids in edible vegetable oil samples
Adding an edible vegetable oil sample into 70% methanol-phosphate buffer solution (v/v), centrifuging at 4000 rpm for about 1 min, standing at 4 ℃ for 20 min, diluting the obtained methanol-phosphate buffer solution by 5 times, and preparing capsaicin standard solutions with different concentrations; dripping 5 mu L of mixed solution of capsaicin standard solution and capsaicin antibody with different concentrations on the surface of the electrochemical immunosensor, carrying out incubation reaction at 37 ℃ for 40 minutes, then washing out the antibody and the standard substance which are not combined with the antigen by adopting phosphate buffer solution with pH =7.5, and recording a current response value by using a differential pulse voltammetry method in 5 mM potassium ferricyanide solution, thereby calculating the content of the capsaicin in the edible vegetable oil sample solution. The recovery rate of the experimental result is between 79.3% and 112.5%, which indicates that the immunosensor prepared by the method can be used for detection and analysis of actual samples.
Claims (1)
1. An application of a competitive electrochemical immunosensor for detecting capsaicin substances in edible vegetable oil adulteration detection is characterized by comprising the following steps:
adding an edible vegetable oil sample into 70% methanol-phosphate buffer solution (v/v), centrifuging at 4000 rpm for about 1 min, standing at 4 ℃ for 20 min, diluting the obtained methanol-phosphate buffer solution by 5 times, and preparing capsaicin standard solutions with different concentrations; dripping 5 mu L of mixed solution of capsaicin standard solution and capsaicin antibody with different concentrations on the surface of the electrochemical immunosensor, carrying out incubation reaction at 37 ℃ for 70 minutes, then washing out the antibody and the standard substance which are not combined with the antigen by adopting phosphate buffer solution with pH =7.5, and recording a current response value by using a differential pulse voltammetry method in 5 mM potassium ferricyanide solution, thereby calculating the content of the capsaicin in the edible vegetable oil sample solution;
The sensor is prepared by mixing ferroferric oxide-cerium dioxide (Fe)3O4-CeO2) Modifying the surface of a glassy carbon electrode in a dripping way to enhance the conductivity of the electrode and fix capsaicin antigens, and then blocking nonspecific sites by BSA (bovine serum albumin) to realize the construction of the sensor, wherein the construction method comprises the following steps:
(1) pretreating a glassy carbon electrode: firstly, polishing a bare glassy carbon electrode in 0.3 mu m alumina slurry to a mirror surface, then respectively carrying out ultrasonic treatment on the polished glassy carbon electrode in ultrapure water and absolute ethyl alcohol in sequence for 1 minute and drying the polished glassy carbon electrode by using nitrogen, finally, measuring the potential difference of the glassy carbon electrode in a potassium ferricyanide solution with the concentration of 5 mM by using a cyclic voltammetry to be less than 80 mV, and storing the glassy carbon electrode at 4 ℃ for later use;
(2) preparing a modified electrode: firstly, Fe3O4-CeO2Fixing the composite material on the surface of the electrode in a dripping way, and drying at room temperature to prepare a modified electrode;
(3) fixing capsaicin material antigen: immersing the electrode prepared in the step (2) into a capsaicin antigen solution with a proper concentration, and incubating overnight at 4 ℃ to fix the capsaicin antigen in Fe3O4-CeO2A composite material layer;
(4) dropping BSA blocking solution to block non-specific binding sites on the surface of the electrode, washing and drying to prepare the competitive electrochemical immunosensor for capsaicin substances;
Wherein, the Fe in the step (2)3O4-CeO2The preparation process of the composite material comprises the following steps: 0.5 g of chitosan was weighed, dissolved in 50 mL of 1.0% acetic acid solution, stirred at room temperature for 3 hours to completely dissolve the chitosan, and then 10 mg of Fe was added3O4Adding the mixture into the solution, and ultrasonically stirring the mixture until a yellow dispersion liquid is obtained; finally, 10 mg of CeO was added to the mixed solution2Continuing to perform ultrasonic dispersion until obtaining coffee dispersion liquid, and obtaining Fe3O4-CeO2A composite material;
the capsaicin antigen solution with the appropriate concentration in the step (3) is capsaicin antigen solution with the concentration of 9 mug/mL;
dropping BSA blocking solution to block the non-specific binding sites on the surface of the electrode, washing and drying, namely washing the electrode cleanly by using phosphate buffer solution with pH =7.5, then continuously dropping 5 mu L of BSA with the mass fraction of 0.5%, and blocking for 70 minutes at room temperature; the resulting electrode was rinsed clean with phosphate buffer pH =7.5 and blown dry.
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