CN113624826B - Polypeptide-nano gold modified glassy carbon electrode and application thereof - Google Patents

Polypeptide-nano gold modified glassy carbon electrode and application thereof Download PDF

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CN113624826B
CN113624826B CN202111061685.7A CN202111061685A CN113624826B CN 113624826 B CN113624826 B CN 113624826B CN 202111061685 A CN202111061685 A CN 202111061685A CN 113624826 B CN113624826 B CN 113624826B
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aflatoxin
glassy carbon
carbon electrode
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毛烨炫
张西亚
党梦
黄现青
谢新华
宋莲军
张平安
王田林
杨缓
丁明月
王尤一
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Henan Agricultural University
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Abstract

The invention belongs to the technical field of detection of total aflatoxin in peanuts and products thereof in foods, and discloses a polypeptide-nanogold (PP-AuNPs) modified glassy carbon electrode and a method for detecting the total aflatoxin in the peanuts and products thereof by using the electrode. The modified material is prepared by the following steps: (1) HAuCl 4 Adding the mother solution into HEPES buffer solution containing polypeptide, and stirring at room temperature to obtain PP-AuNPs composite material; (2) Polishing the bare glassy carbon electrode on alumina powder, sequentially carrying out ultrasonic treatment in absolute ethanol and ultrapure water, washing with water, and placing in potassium ferricyanide solution for cyclic voltammetric scanning; (3) And (3) dripping the PP-AuNPs composite material obtained in the step (1) on the surface of the bare glassy carbon electrode treated in the step (2) and drying at constant temperature to obtain the PP-AuNPs modified glassy carbon electrode (PP-AuNPs/GCE for short). The modification material is dripped on the surface of the activated bare glassy carbon electrode, dried, and then antigen, blocking Solution (BSA) and a sample to be detected are dripped in sequence, incubated, and then electrochemical signal detection is carried out. The detection method can improve the response signal of the total amount of aflatoxin, improve the reliability of aflatoxin detection, and realize qualitative or quantitative detection of the residual amount of aflatoxin in peanuts and products thereof.

Description

Polypeptide-nano gold modified glassy carbon electrode and application thereof
Technical Field
The invention relates to the technical field of residual detection of total aflatoxin in peanuts and products thereof, in particular to a glassy carbon electrode modified by polypeptide-nanogold (PP-AuNPs) and a method for detecting the total aflatoxin by using the glassy carbon electrode to construct an electrochemical immunosensor.
Background
Aflatoxin is a secondary metabolite of toxin-producing strains such as fungi aspergillus flavus and parasitic koji, is also a natural substance with the highest recognized carcinogenicity, and mainly comprises aflatoxin B 1 (AFB 1 ) Aflatoxin B 2 (AFB 2 ) Aflatoxin G 1 (AFG 1 ) Aflatoxin G 2 (AFG 2 ) Aflatoxin M 1 (AFM 1 ) And aflatoxin M 2 (AFM 2 ) 20 kinds of peanuts are mainly the first 4 kinds of peanuts. The physical and chemical properties of aflatoxin are particularly stable, and aflatoxin existing in food cannot be discharged through decomposition after being digested and absorbed by human and animal bodies, but can be accumulated in the body, and various toxic metabolites are generated through the actions of hydroxylation, epoxidation, demethylation and the like, so that adverse diseases such as cancers, mutations, deformity and the like are easily caused. Not only severely affect foodSensory quality also severely threatens the life health of humans.
According to national food safety standard of food mycotoxin amount and solicited opinion manuscript: aflatoxin B in peanuts and cooked peanuts 1 The limit amount of (C) is 20 mug.kg -1 Aflatoxin B in peanuts (directly eaten or used as raw material) is taken by European Union (EU) 1 The limit standard is set to 8 mug.kg -1 The method comprises the steps of carrying out a first treatment on the surface of the The European Food Security Agency (EFSA) will be ready to eat and use as a food ingredient the total amount of aflatoxins (including AFB) in peanuts and products thereof 1 、AFB 2 、AFG 1 、AFG 2 ) The maximum limit is 4 mug.kg -1 To 10 mug.kg -1 And performing risk assessment. Therefore, the residual quantity of the aflatoxin needs to be monitored and controlled in time; at present, the detection methods of aflatoxin in peanuts and products thereof mainly comprise Thin Layer Chromatography (TLC), high Performance Liquid Chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), immunoaffinity column purification, fluorescence photometry and the like. It can be seen that the existing detection methods have limitations, and in order to ensure the health of eaters and meet import and export trade requirements, a detection method of aflatoxin with the advantages of safety, simple processing, easy operation, rapid result, and certain sensitivity and accuracy needs to be developed.
Aflatoxin B 1 、B 2 、G 1 、G 2 、M 1 、M 2 Chemical structure of (2)
Disclosure of Invention
The invention aims to provide a PP-AuNPs modified glassy carbon electrode, which is applied to the detection of total aflatoxin, improves the response signal of aflatoxin and improves the sensitivity and reliability of aflatoxin detection.
In order to achieve the purpose of the invention, the technical scheme is as follows:
the PP-AuNPs modified glassy carbon electrode is prepared by the following method:
1. HAuCl 4 Adding the mother solution into HEPES buffer solution containing polypeptide, stirring at room temperature to obtain PP-AuNPs composite material.
2. Polishing the bare glassy carbon electrode on alumina powder, sequentially carrying out ultrasonic treatment in absolute ethanol and ultrapure water, flushing with ultrapure water, and placing in potassium ferricyanide solution for cyclic voltammetric scanning; (preferably, the peak potential difference is kept about 75 mV).
3. And (3) dripping the PP-AuNPs composite material obtained in the step (1) on the surface of the bare glassy carbon electrode treated in the step (2) and drying at constant temperature to obtain the PP-AuNPs modified glassy carbon electrode (PP-AuNPs/GCE for short).
Preferably, all glass instruments required for synthesizing the composite material are soaked in an acid tank for one day, taken out, thoroughly rinsed with ultrapure water, rinsed clean and dried in an incubator at 37 ℃.
Preferably, HAuCl 4 The concentration of the mother solution is 10 mg.mL -1 The polypeptide is dissolved in HEPES solution to prepare 0.04 mmol.L -1 Is a polypeptide solution of (a). HAuCl 4 The mass concentration ratio of the mother solution to the polypeptide solution is as follows: 1:250.
Preferably, in step 1, 80. Mu.L of HAuCl is added 4 The mother solution is added into 20mL of HEPES solution of polypeptide, and stirred for 1-2h at room temperature, thus obtaining the PP-AuNPs composite material.
The modification material is dripped on the surface of the activated bare glassy carbon electrode, dried, and then antigen, blocking Solution (BSA) and a sample to be detected are dripped in sequence, incubated, and then electrochemical signal detection is carried out. The voltage applied to the glassy carbon electrode modified by the polypeptide-nano gold (PP-AuNPs) presents different response signals to the catalytic action of the solution in the electrolytic cell, so that the concentration of the sample to be detected is reflected.
Considering that other factors possibly influence the sensitive detection of aflatoxin, the experimental parameters such as the concentration of antigen, the concentration of antibody, the temperature, the pH, the reaction time and the like are required to be optimized, and the detection of aflatoxin is carried out under the optimal experimental conditions; and the concentration is in the range of 0 ng.mL -1 、0.00005ng·mL -1 、0.0001ng·mL -1 、0.0005ng·mL -1 、0.0008ng·mL -1 、0.001ng·mL -1 、0.005ng·mL -1 、0.008ng·mL -1 、0.01ng·mL -1 、0.05ng·mL -1 、0.08ng·mL -1 、0.1ng·mL -1 Aflatoxin B of (a) 1 、B 2 、G 1 、G 2 、M 1 、M 2 The standard substance is detected under the electrochemical immunosensor, and a corresponding standard curve is drawn.
And after the sample waiting to be detected is processed, the sample waiting to be detected is dripped on the electrochemical immunosensor, and is incubated for a certain time and then is put into potassium ferricyanide solution for electrochemical detection. The electrochemical workstation outputs voltage of-0.2V to 0.6V to be loaded on the PP-AuNPs modified glassy carbon electrode prepared by the invention, and the peak current of a sample to be detected is measured by detecting aflatoxin through the catalytic action of the PP-AuNPs modified glassy carbon electrode on a solution in an electrolytic cell. And (3) qualitatively or quantitatively detecting the aflatoxin in the sample to be detected by standard curve comparison.
And setting blank control for each detection and screening of the experimental parameters such as antigen concentration, antibody concentration, temperature, pH, reaction time, modified materials and the like, and repeating the experiment to obtain the optimal experimental parameters by multiple comprehensive comparisons. Obtaining optimal experimental parameters: antigen concentration 5.0. Mu.g.mL -1 Antibody concentration 2.5. Mu.g.mL -1 At room temperature, ph=7.4, reaction time 5min.
The actual sample is detected after being processed, and the actual sample is matched with the drawn standard curve B 1 、B 2 、G 1 、G 2 、M 1 、M 2 By comparison, the concentration of aflatoxin in the sample to be detected is known, and the immunosensor has almost identical and better detection effect on 6 aflatoxins as can be seen from the superposition graph of 6 standard curves and the cross reaction rate.
And (3) performing stability and reproducibility experiments, wherein the data has certain reproducibility and reliability.
The invention has the innovation points and advantages that: the invention utilizes the unique geometry of nano gold (AuNPs),good electrochemical stability, stronger conductivity, large specific surface area, certain electrocatalytic activity and the like; the polypeptide is used as an excellent biological material, has various side chain functional groups, can be self-assembled into ordered one-dimensional, two-dimensional and three-dimensional structures, and is widely used for templates, ligands, protective agents, surface modification elements and the like. In addition, studies have shown that the growth of nano-metal particles is affected by the polypeptide and is regularly arranged around the polypeptide, thereby achieving the ordering of nano-metal particle arrangement. Therefore, the two materials are compounded together to prepare the polypeptide-nanogold (PP-AuNPs) modified glassy carbon electrode, and the electrochemical behavior of aflatoxin is researched by adopting the electrode, so that the PP and AuNPs materials show higher synergistic effect when detecting the aflatoxin. The composite modified electrode improves the electrochemical response signal of aflatoxin, thereby improving the sensitivity of aflatoxin detection. Experiments prove that the linear range of the aflatoxin detection method is 0.05 pg.mL -1 -100pg·mL -1 The detection limit was 0.012 pg/mL -1 The anti-interference capability is strong, and the stability and the reproducibility are good. Therefore, the glassy carbon electrode modified by the PP-AuNPs can be well applied to detection of an actual sample, complex pretreatment of the actual sample is not needed, and the glassy carbon electrode modified by the polypeptide-nano gold composite material has good application potential.
Drawings
FIG. 1 shows that the different modified electrodes are at 1mmol L -1 K 3 Fe(CN) 6 (containing 0.1mol L) -1 Cyclic voltammogram in KCl supporting electrolyte solution (sweep rate: 0.1. 0.1V s) -1 )；
FIG. 2 is a bare electrode, PP-AuNPs/GCE modified electrode, antigen/PP-AuNPs/GCE modified electrode, BSA/antigen/PP-AuNPs/GCE and to-be-tested/BSA/antigen/PP-AuNPs/GCE modified electrode at 5mmol L -1 Fe(CN) 6 4-/3- (1:1) in solution (containing 0.10mol L) -1 KCl) with a frequency range of 100000-0.01Hz;
FIG. 3 is a differential pulse voltammogram of aflatoxin detection by different concentrations of antigen-modified electrode pair (a, concentration of antigen 10. Mu.g·mL -1 Antibody 5. Mu.g.mL -1 The method comprises the steps of carrying out a first treatment on the surface of the b, antigen concentration of 5. Mu.g.mL -1 Antibody 2.5. Mu.g.mL -1 The method comprises the steps of carrying out a first treatment on the surface of the c, the concentration of antigen is 2.5 mug.mL -1 Antibody 1.25. Mu.g.mL -1 The method comprises the steps of carrying out a first treatment on the surface of the d, the concentration of antigen is 1.0 mug.mL -1 Antibody 0.5. Mu.g.mL -1 . Room temperature (25 ℃), ph=7.4, incubation time 20min, aflatoxin B 1 The concentration is 0.1 ng.mL -1 Potential range: -0.2-0.6V, amplitude 0.05V, sweep rate 0.10V s -1 .)；
FIG. 4 is a differential pulse voltammetry (a, antibody 5. Mu.g.mL) of aflatoxin detection by different concentrations of antibody modified electrode pairs -1 The method comprises the steps of carrying out a first treatment on the surface of the b, antibody 2.5. Mu.g.mL -1 The method comprises the steps of carrying out a first treatment on the surface of the c, antibody 1.25. Mu.g.mL -1 The method comprises the steps of carrying out a first treatment on the surface of the d, antibody 0.5. Mu.g.mL -1 The antigen concentration was 5. Mu.g.mL -1 Room temperature (25 ℃), ph=7.4, reaction time 20min, aflatoxin B 1 The concentration is 0.1 ng.mL -1 Potential range: -0.2-0.6V, amplitude 0.05V, sweep rate 0.10V s -1 .)；
FIG. 5 is a differential pulse voltammetry of aflatoxin detection by modified electrodes at different temperatures (a 4 ℃, b room temperature, c 37 ℃, antigen concentration 5. Mu.g.mL) -1 The concentration of the antibody was 2.5. Mu.g.mL -1 Aspergillus flavus toxin B 1 Is 0.1 ng.mL -1 Ph=7.4, reaction time of 20min, potential range: -0.2-0.6V, amplitude 0.05V, sweep rate 0.10V s -1 .)；
FIG. 6 is a differential pulse voltammogram (antigen concentration 5. Mu.g.mL) of aflatoxin detection by different pH modified electrodes -1 The concentration of the antibody was 2.5. Mu.g.mL -1 Aflatoxin B 1 Is 0.1 ng.mL -1 A, ph=6.5; b, ph=7.4; c, ph=8.0; reaction time 20min, potential range: -0.2-0.6V, amplitude 0.05V, sweep rate 0.10V s -1 .)；
FIG. 7 is a differential pulse voltammetry (a, 20min, b,10min, c,5min, d,2min; antigen concentration 5. Mu.g.mL) of aflatoxin detection with electrode repair at different reaction times -1 The concentration of the antibody was 2.5. Mu.g.mL -1 Aspergillus flavus toxinElement B 1 Is 0.1 ng.mL -1 Ph=7.4 potential range: -0.2-0.6V, amplitude 0.05V, sweep rate 0.10V s -1 .)；
FIG. 8 shows various modified materials (four modified materials of C14R2-AuNPs, C14R3-AuNPs, C14R4-AuNPs, C14R5-AuNPs in case of optimal experimental parameters);
FIG. 9 is a graph showing the effect of organic reagents on an electrode of the present invention;
FIG. 10 shows different concentrations of aflatoxin B 1 (A) Aflatoxin B 2 (B) Aflatoxin G 1 (C) And aflatoxin G 2 (D) The concentration of a-h aflatoxin is gradually increased and is respectively 0 ng.mL -1 、0.00005ng·mL -1 、0.0001ng·mL -1 、0.000nμg·mL -1 、0.0008ng·mL -1 、0.001ng·mL -1 、0.005ng·mL -1 、0.008ng·mL -1 、0.01ng·mL -1 、0.05ng·mL -1 、0.08ng·mL -1 、0.1ng·mL -1 ；
FIG. 11 shows the effect of scan rate on the electrochemical response signal of aflatoxin, with a.fwdarw.h scan rates of 10 mV.s, respectively -1 、25mV·s -1 、50、100mV·s -1 、200mV·s -1 、300mV·s -1 、400mV·s -1 、500mV·s -1 ；
Fig. 12 is a graph showing the effect of the number of scan turns on electrode stability.
Detailed Description
The present invention will be described in detail with reference to specific examples and experimental procedures and conclusions.
The reagents and instruments used in the invention are as follows:
C14R5-AuNPs, aflatoxin antigen, aflatoxin antibody, PBS, PBST prepared by Henan agricultural university laboratory; aflatoxin standard, potassium ferricyanide, potassium ferrocyanide were purchased from ala Ding Shiji (Shanghai) limited. The analytical pure KCl, peanut, corn and peanut milk are all commercial products, and the experimental water is ultrapure water.
CHI660E electrochemical workstation (Shanghai Chen Hua instruments Co., ltd.); three electrode system: saturated Calomel Electrode (SCE) is reference electrode, pt wire is counter electrode, and Glassy Carbon Electrode (GCE) is working electrode. Conditions for measuring ac impedance: 0.01-100 kHz. All pH values were adjusted by PHS-3C precision pH meter (Shanghai torpedo magnetic equipment works) and daily calibrated with standard buffer solutions prior to use. Sample: peanut, corn flour and peanut milk.
Example 1 preparation of polypeptide-nanogold modified glassy carbon electrode comprising the steps of:
step 1, soaking all glass instruments required by composite material synthesis in an acid tank for one day, taking out the glass instruments, thoroughly rinsing with ultrapure water, and drying the rinsed glass instruments in an incubator at 37 ℃ for later use.
Step 2, haucl 4 The solid of (C) was dissolved in ultrapure water to prepare a solution having a concentration of 10 mg/mL -1 Is then dissolved in 20mL HEPES solution to prepare 0.04 mmol.L -1 Is a polypeptide solution of (a).
Step 3, 80. Mu.L of HAuCl is taken 4 Adding the mother solution into 20mL of HEPES solution of polypeptide, and stirring for 1-2h at room temperature to obtain the PP-AuNPs composite material.
Step 4, polishing the bare glassy carbon electrode on alumina powder, sequentially carrying out ultrasonic treatment in absolute ethyl alcohol and water, washing with water and placing in potassium ferricyanide solution (1 mmol.L) -1 ) Performing cyclic voltammetry scanning; the peak potential difference was kept around 75 mV. And 5, dripping the PP-AuNPs solution obtained in the step 3 on the surface of the bare glassy carbon electrode treated in the step 4, and drying the bare glassy carbon electrode in an electrothermal constant temperature oven at 37 ℃ to obtain the PP-AuNPs modified glassy carbon electrode (PP-AuNPs/GCE for short).
Example 2 characterization of modified electrode performance:
in order to verify whether the electrochemical immunosensor was successfully constructed, the five electrodes GCE, PP-AuNPs/GCE, antigen/PP-AuNPs/GCE, BSA/antigen/PP-AuNPs/GCE and to-be-detected/BSA/antigen/PP-AuNPs/GCE were compared in potassium ferricyanide solution (1 mmol L) -1 ) Is a signal of electrochemical response. As shown in FIG. 1, each of these five modified electrodes had a pair of good redox peaks in the potassium ferricyanide solution. On the side of the GCE,the potential difference between the redox peaks is less than 75mV, which indicates that the electrode activation is better and the conductivity is better. The curve of the electrode modified by the PP-AuNPs is better, the oxidation peak value is slightly reduced, the reduction peak value is obviously enhanced, the peak potential difference is reduced, and the PP-AuNPs is successfully modified on the GCE, and the modified material has good catalytic action on the reduction of potassium ferricyanide, so that the response signal is amplified. The electrode modified by aflatoxin antigen/PP-AuNPs has round peak shape, reduced reduction peak compared with modified GCE, and increased peak potential difference. This is because aflatoxin has a relatively large molecular weight, and the fixation on GCE has a certain blocking effect on electron movement, thus reducing the reduction reaction of potassium ferricyanide. The reduction peak of GCE modified by BSA/aflatoxin antigen/PP-AuNPs is further reduced, which indicates that the BSA successfully blocks redundant active sites, and the response signal of current is further reduced. And finally, when the to-be-detected solution after the incubation of the aflatoxin antibody and the aflatoxin is finished is modified on the surface of the electrode, the antibody is a protein macromolecule and is specifically combined with an antigen fixed on the GCE to form an electron blocking layer, so that CV response is reduced, and peak potential difference is increased.
The alternating current impedance spectrum (Electrochemical Impedance Spectroscopy, EIS) is used for researching the blocking effect of the modified electrode on the current in the circuit, the charge transfer resistivity is related to the diameter of a semicircular arc in the impedance diagram, and the arc part is smaller, so that the electron transfer speed is higher. As can be seen from fig. 2, compared with GCE, the conductivity is reduced due to the modification of PP-AuNPs, so that the semicircle diameter of the pattern is reduced, and the conductivity is correspondingly enhanced; the semicircle diameter of the pattern of the BSA/antigen/PP-AuNPs modified is obviously increased, which indicates that antigen molecules obstruct the transfer of charges on the surface of the electrode, the resistance of the sensor is increased, the BSA modification seals redundant active sites on the surface of the GCE, and the semicircle diameter of the impedance spectrum is further increased. The alternating current impedance spectrum and the characterization result of the electrode cyclic voltammetry behavior are consistent, and the fact that the immunosensor is constructed successfully is also indicated.
Example 3 effect of optimization of experimental parameters on aflatoxin detection:
optimization of antigen concentration (fig. 3): the electrochemical signal (potential range: -0.2-0.6V, amplitude: 0.05V) was detected by differential pulse voltammetry technique. The concentration ratio of antigen to antibody was set to 2:1 at room temperature (25 ℃) at pH=7.4 for 20min, and the detection concentration was 5.0 ng.mL -1 The electrochemical response intensity of aflatoxin is influenced by the concentration of antigen and antibody, so as to generate different response signals. And meanwhile, comparing the measured current with blank liquid to be measured, defining the current to be measured-blank current=delta I, and judging whether the detection effect is good or not according to the delta I. When the concentration of the Aspergillus flavus antigen is 10.0 mug.mL -1 And 1.0. Mu.g.mL -1 When the reduction peak current is enhanced obviously, the peak potential is shifted negatively to a certain extent, and the detection enhancement of aflatoxin signals is not obvious. When the concentration of aflatoxin antigen is 5.0 mug.mL -1 And 2.5. Mu.g.mL -1 The reduction peak is obviously enhanced, the peak shape is better, and the concentration is 5.0 mug.mL -1 When Δi=0.575 μa, the reduction peak moves forward to some extent, indicating that the reduction of potassium ferricyanide can be promoted when the potential is relatively positive.
Optimization of antibody concentration (fig. 4): the detection concentration was 0.1ng mL at room temperature (25 ℃ C.), pH=7.4, and reaction time of 20min -1 The concentration of immobilized antigen in the solution to be tested of aflatoxin and antibody of (2) is 5.0 ng.mL -1 And a blank control was set. When the concentration of the antibody was 5.0. Mu.g.mL -1 Two reduction peaks appear, the peak current is obviously weakened, the peak potential is shifted negatively, and the peak shape is more unsightly; when the concentration of the antibody was 2.5. Mu.g.mL -1 、1.0μg·mL -1 And 0.5. Mu.g.mL -1 The peak current was significantly increased, but the antibody concentration was 1.0. Mu.g.mL -1 And 0.5. Mu.g.mL -1 When the peak shape is not good, and the peak potential of the two is negatively shifted to a certain extent, only the concentration of the antibody is 2.5 mug.mL -1 Δi=0.575 μa, not only is the peak shape relatively good looking, but also has a degree of forward shift that can catalyze the reduction of potassium ferricyanide at a relatively positive potential.
Optimization of temperature (fig. 5): continuing the optimized conditions: the antigen concentration was 5. Mu.g.mL -1 The concentration of the antibody was 2.5. Mu.g.mL -1 Aspergillus flavus toxin B 1 Is 0.1 ng.mL -1 Ph=7.4, reaction time was 20min, and a blank control for the test solution was set. Compared with the blank, the temperature is 4 ℃ and 37 ℃, the peak current is not changed obviously, and the peak shape at 37 ℃ is not good. In contrast, at room temperature, the peak shape became clearly better and the peak current enhancement was evident, with Δi=0.575 μa peak potential slightly shifted forward, and the detected signal was better than at other temperatures.
Optimization of pH (fig. 6): antigen concentration at room temperature 5. Mu.g.mL -1 The concentration of the antibody was 2.5. Mu.g.mL -1 Aflatoxin B 1 Is 0.1 ng.mL -1 Ph=6.5, ph=7.4, ph=8.0, reaction time 20min, and blank. The difference between peak currents at ph=6.5 and ph=8.0 is small compared with the blank, and the change of response signals is not obvious, so that the detection of aflatoxin at the pH is not suitable; at ph=7.4, the enhancement of peak current is more pronounced and peak shape also becomes better looking, being more suitable for aflatoxin detection.
The reaction time was optimized when the other detection conditions were optimal (fig. 7). It can be seen that the peak current was relatively low and the detected signal was not apparent at a time of 20 min. When the time is 10min and 2min, the peak current difference is not obvious enough, and the detection time is not optimal; when the time is 5min, the peak current difference is obvious, the detected signal is enhanced, the peak potential difference is reduced, and the peak shape becomes very good, which belongs to the ideal detection time.
Optimization of the finishing material: four polypeptides CH are selected in this study 3 -(CH 2 ) 6 -CO-NH-R-R-NH 2 (C14R2)、CH 3 -(CH 2 ) 6 -CO-NH-R-R-R-NH 2 (C14R3)CH 3 -(CH 2 ) 6 -CO-NH-R-R-R-R-NH 2 (C14R4)CH 3 -(CH 2 ) 6 -CO-NH-R-R-R-R-R-NH 2 (C14R 5) Synthesis and preparation of four polypeptide-nanogold materials (C14R 2-AuNPs, C14R 3-Au)NPs, C14R4-AuNPs, C14R 5-AuNPs), the prepared modification material was modified on an electrode, and detection of response signals to the test solution was performed using electrochemical DPV (fig. 8). It can be seen that the peak currents of C14R3 and C14R4 are reduced compared to GCE, and the corresponding reduction in response signals results in longer peak shapes, which is more unsightly. The peak current of C14R2 is slightly enhanced, the peak current of C14R5 is obviously enhanced, the peak shape is quite good, the strength of the detection signal is obviously enhanced, and the method can be used for modifying a bare electrode, detecting aflatoxin and enhancing a response signal.
Example 4 detection analysis of total aflatoxin in actual samples:
the modified electrode prepared by the invention is used for detecting actual samples, including peanuts, corn flour and peanut milk. The accuracy of the method was judged using standard addition methods and the measurement results are listed in table 1. No electrochemical response signal was observed when no aflatoxin standard was added, indicating that the sample contained no aflatoxin, or that the aflatoxin content was too low to be detected. When the corresponding amount of aflatoxin standard substance is added, obvious reduction peaks can be seen, and the recovery rate can reach 84% -93%, which shows that the method can be used for detecting the total amount of aflatoxin in peanuts and products thereof.
4.1 detection of aflatoxins in peanut and corn meal samples
The preparation of peanut and corn powder samples comprises the steps of crushing a proper amount of samples by a crusher, sieving the crushed samples by a 20-mesh sieve, placing 2.0g of the sieved samples into a glass bottle with a plug, adding 8mL of ethanol (the volume percentage content is 95%) and 2mL of ultrapure water (the ratio of ethanol to water is 4:1), adding 1g of sodium chloride, screwing a glass bottle cap, shaking the glass bottle cap uniformly, and putting the glass bottle into an ultrasonic instrument for ultrasonic treatment for one hour. Taking out the glass bottle, standing for a moment to separate the supernatant from the solid sample, sucking the supernatant into a centrifuge tube by a pipetting gun, and taking out the sample liquid for dilution.
4.2 detection of aflatoxin in peanut milk samples
Peanut milk: and a proper amount of fresh peanut milk is taken, and the peanut milk can be directly diluted for 50 times for detecting aflatoxin.
4.3 Effect of organic Agents
Since the process of aflatoxin extraction uses organic reagent ethanol, whether the organic reagent has influence on aflatoxin detection needs to be detected, the addition amount of the ethanol is respectively 4%, 8%, 16% and 20%, and the influence of the organic reagent on aflatoxin detection is almost insignificant (figure 9) or is extremely small.
TABLE 1 labeled recovery of actual samples
The invention synthesizes a polypeptide-nano gold composite material, and constructs a high-sensitivity electrochemical sensor for detecting aflatoxin by utilizing the synergistic effect of AuNPs and-pp. The experimental parameters are optimized, the standard curve is measured under the optimal experimental parameters, the influence of the organic reagent ethanol is eliminated, and the stability test such as the number of sweeping turns is carried out, so that the immunosensor has good stability and reproducibility, and the obtained experimental data has certain reliability. And the experimental sample does not need a complex pretreatment process, thereby being beneficial to popularization and application of detection of aflatoxin in peanuts and products thereof.
In addition to the above embodiments, the methods of the present invention were also analyzed or tested as follows:
1. establishment of aflatoxin standard curve
Under the optimized optimal parameters, constructing a BSA/antigen/PP-AuNPs/GCE sensor; then with B containing 1 After completion of the DPV scan (potential range: -0.2-0.6V, amplitude: 0.05V). Without changing other experimental conditions, only the incubated B is changed 1 Different DPV profiles were obtained for concentration (fig. 10). When aflatoxin B 1 The concentration of (C) is from 0 pg.mL -1 Increase to 100 pg.mL -1 The reduction peak value of DPV is linearly increased, the positive correlation is presented, the greater the concentration of aflatoxin in the liquid to be tested is, the greater the response current value is, the wide detection line and the wide detection line are presentedLow detection range. The standard curves for four aflatoxins obtained based on this are shown below:
B 1 is I (μA) =5.76 x (ng.mL) -1 )+16.52(R 2 ＝0.9999)；
B 2 Is I (μA) =5.63 x (ng.mL) -1 )+19.55(R 2 ＝0.9995)；
G 1 Is I (μA) =6.19x (ng.mL) -1 )+17.09(R 2 ＝0.9998)；
G 2 Is I (μA) =5.84 x (ng.mL) -1 )+17.32(R 2 ＝0.9992)，
The detection limit is as low as 0.012 pg.mL -1 . The four curves are almost completely coincident in combination with the superimposed graph of the standard curve, all show a tendency to appear gradually increasing, almost identical relatively obvious response signals, combined with the cross-reactivity of Table 2, in B 1 Calculating the corresponding concentration when the signal intensity is normal as a standard, B 1 Is considered as 100%, B 2 Has a cross-reactivity of c (B) 2 )/c(B 1 ) By analogy, the results reach over 95%, which shows that the detection method can realize accurate and sensitive detection of the total aflatoxin.
TABLE 2
2. Influence of the scanning speed on the electrochemical behaviour of the electrode
The detection adopts CV to study the influence of scanning speed on electrode stability. As shown in FIG. 11, the scanning speed was from 10 mV.s -1 To 500 mV.s -1 The peak current value of the reduction peak of aflatoxin is also gradually increased during the change. In order to study whether the electrochemical reaction of aflatoxin is adsorption-controlled or diffusion-controlled, further analysis of the relationship between peak current value and sweep rate is required: if the peak current value of the aflatoxin reduction peak is in linear relation with the scanning speedThe adsorption control is performed, and the diffusion control is performed if the peak current value of the yellow koji toxin reduction peak and the 1/2 power of the scanning speed are in a linear relationship. As can be seen, the peak current of the electrochemical response reduction peak of aflatoxin has positive correlation with the scanning speed, and the corresponding linear regression equation is I= -0.0552-15.09V (R 1 2 = 0.9911). The peak current value of the electrochemical response reduction peak of aflatoxin is positively correlated with the 1/2 th power of the scanning speed, and the corresponding linear regression equation is I= -1.486-7.642V (R 2 2 ＝0.9905)。R 1 2 Greater than R 2 2 And is closer to 1, so that the electrochemical behavior of aflatoxin on the sensor is controlled by adsorption and diffusion, and adsorption control is dominant.
3. Experiment of electrode stability
At a sweeping speed of 0.05V/s -1 During the scanning, 30 circles are scanned, the stability of the CV detection electrode is achieved, along with the increase of the number of scanning circles (figure 12), the CV spectrum has no obvious displacement change, more lines are repeated together, the electrode property is stable, and the detection result is stable and reliable. And the experimental optimization and detection processes are repeated for a plurality of times, and the experimental result has accuracy and repeatability.

Claims (3)

1. The polypeptide-nano gold (PP-AuNPs) modified glassy carbon electrode is characterized by being prepared by the following method:
(1) HAuCl 4 Adding the mother solution into HEPES buffer solution containing C14R5 polypeptide, and stirring at room temperature to obtain PP-AuNPs composite material;
(2) Polishing the bare glassy carbon electrode on alumina powder, sequentially carrying out ultrasonic treatment in absolute ethanol and ultrapure water, washing with water, and placing in potassium ferricyanide solution for cyclic voltammetric scanning;
(3) And (3) dripping the PP-AuNPs composite material obtained in the step (1) on the surface of the bare glassy carbon electrode treated in the step (2) and drying at constant temperature to obtain the PP-AuNPs modified glassy carbon electrode, namely PP-AuNPs/GCE.
2. The polypeptide-nanogold (PP-AuNPs) -modified glassy carbon electrode of claim 1, wherein HAuCl 4 The concentration of the mother solution is 10mg mL -1 Dissolving C14R5 polypeptide in HEPES solution to prepare 0.04 mmol.L -1 Has a polypeptide solution of HAuCl 4 The mass concentration ratio of the mother solution to the substance of the C14R5 polypeptide is as follows: 1:250.
3. The use of a polypeptide-nanogold (PP-AuNPs) -modified glassy carbon electrode as claimed in claim 1 or 2 in the detection of aflatoxins, characterized by being realized by: after a sample to be detected is processed, dropwise adding the sample to be detected onto a glassy carbon electrode modified by polypeptide-nano gold (PP-AuNPs), incubating, then placing the sample into potassium ferricyanide solution for electrochemical detection, loading voltage of-0.2V to 0.6V output by an electrochemical workstation onto the glassy carbon electrode modified by polypeptide-nano gold (PP-AuNPs), detecting peak current of the sample to be detected through differential pulse voltammetry scanning, and comparing the peak current with a standard curve to realize qualitative or quantitative detection of aflatoxin in the sample to be detected;
antigen concentration 5.0. Mu.g.mL -1 Antibody concentration 2.5. Mu.g.mL -1 At room temperature, ph=7.4, reaction time 5min.
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