CN110632156A - Be used for detecting aflatoxin B1Aptamer sensor and preparation method thereof - Google Patents
Be used for detecting aflatoxin B1Aptamer sensor and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a method for detecting aflatoxin B1The aptamer sensor and the preparation method thereof belong to the technical field of electrochemical biosensors, and the sensor is a composite material which takes a boron-doped diamond film as a substrate, is compounded by an aptamer/gold nanoparticles/boron-doped diamond and is formed by occupying blank active sites on the gold nanoparticles by 6-mercaptohexane-1-ol; the preparation method comprises the steps of growing a boron-doped polycrystalline diamond film on the P-type silicon, sputtering a gold film, annealing, modifying and the like. The sensor prepared by the invention has the advantages of high sensitivity, specificity and practicability, simple process and low cost.
Description
Technical Field
The invention belongs to the technical field of electrochemical biosensors, and relates to a method for detecting aflatoxin by using mercaptohexanol, an aptamer, gold nanoparticles and a boron-doped diamond filmElement B1The composite material sensor and the preparation method thereof.
Background
Aflatoxin is one of the most relevant groups of mycotoxins found in food products, has strong toxicity and exists in a wide range. There are 4 aflatoxins found at present: aflatoxin B1Aflatoxins B2Aflatoxin G1And aflatoxin G2There are also two metabolites: aflatoxin M1And aflatoxin M2. Wherein, the aflatoxin B1Widely exists in moldy human food and animal feed, can cause liver cancer and even death, so the aflatoxin B1Is classified as a primary carcinogen. Eu countries specify: aflatoxin B in peanuts and nuts and their processed products and in all cereals and processed products1The limit was 2.0. mu.g/kg (6.4 nM). Aflatoxin B1The polluted food is mainly grain and oil food such as peanut, corn, rice, wheat and peanut oil, and is most seriously polluted in high-temperature and high-humidity areas in south China. Aflatoxin B1It is heat resistant and can be cleaved at 268 ℃, so it is difficult to destroy at the temperature of general cooking processing. Therefore, it is extremely important to detect the substance.
At present, against aflatoxin B1The detection method mainly comprises typical technologies such as thin-layer chromatography, high performance liquid chromatography, enzyme-linked immunosorbent assay and the like, and the detection limit is 10-9～10-7mol/L, but the detection process of the methods is complex, and the result cannot be quickly obtained.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects in the background technology, and by selecting functional materials and designing a special structure, a 6-mercaptohexane-1-ol/aptamer/gold nanoparticle/boron-doped diamond composite electrode structure is constructed by taking boron-doped diamond as a substrate, and an electrochemical sensor is manufactured, wherein the sensor is used for aflatoxin B1Has high sensitivity, good specificity and practicability.
The specific technical scheme is as follows:
a kind ofFor detecting aflatoxin B1The aptamer sensor is a composite material which is formed by compounding an aptamer/gold nanoparticles/boron-doped diamond and occupying blank active sites on the gold nanoparticles by 6-mercaptohexane-1-alcohol, wherein the boron-doped diamond film is used as a substrate; the structure of the aptamer is 5' -SH- (CH)2)6-GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CCA CA-3'; the average size of the gold nanoparticles is preferably 13nm, and the density is preferably 3.2X 1011cm-2。
Be used for detecting aflatoxin B1Firstly, growing a boron-doped polycrystalline diamond film on the P-type silicon by a microwave plasma chemical vapor deposition method; then sputtering a gold film on the surface of the boron-doped polycrystalline diamond film by using an ion sputtering method, wherein the sputtering time is 10 seconds; annealing the obtained gold film and the boron-doped polycrystalline diamond film in the air at 800 ℃ for 30 seconds to 1 minute to obtain gold nanoparticle and boron-doped diamond composite film structures; then, soaking the gold nanoparticles and the boron-doped diamond composite membrane in 5 mu mol/L aptamer solution, and modifying for 5 hours at constant temperature of 37 ℃ to obtain an aptamer/gold nanoparticles/boron-doped diamond composite electrode; then the structure is washed by phosphate buffer for 15 minutes and then is immersed into 1 mmol/L6-mercapto hexane-1-alcohol solution for modification for 30 minutes to 1 hour to occupy blank active sites on the gold nanoparticles, and the aflatoxin B for detection is obtained1The 6-mercaptohexan-1-ol/aptamer/gold nanoparticle/boron-doped diamond aptamer sensor.
Has the advantages that:
1. the invention is used for aflatoxin B1The aptamer sensor has high sensitivity and good specificity practicability. The test result shows that the aptamer sensor is 1 multiplied by 10-13～1×10-8The method has good linear relation in the detection range of mol/L, and the detection limit can reach 5.5 multiplied by 10-14The ultra-low level of mol/L verifies that the sensor has high sensitivity. The sensor has little response to the similar structure through detection of the similar structure, and the sensor is verified to have good specificity.
2. The sensor is applied to detecting the aflatoxin B-containing substance1The good recovery rate is obtained in the peanut sample, which shows that the sensor has better practicability.
3. Compared with the conventional detection method, the sensor detection method has the obvious advantages that: the traditional detection method has low detection sensitivity, generates a large amount of byproducts in the detection process, and has complex operation process and high cost; the novel aflatoxin B prepared by the invention1Aptamer sensor, using boron-doped diamond as substrate for the first time as aflatoxin B1The detection electrode material has high sensitivity, good specificity and practicability, simple electrode preparation process and low price, and has wide prospect in the field of practical application.
Drawings
FIG. 1 is a scanning electron microscope topographical view of a boron-doped diamond film prepared in example 1, with a partial enlarged view.
FIG. 2 is a scanning electron microscope topography of the gold nanoparticle/boron doped diamond film prepared in example 1, with an inset being a partial magnified view.
FIG. 3 is aflatoxin B of example 11Flow chart of the preparation process of the aptamer sensor.
FIG. 4 is aflatoxin B of example 11Impedance change of aptamer sensor and aflatoxin B1The concentration correspondence of (3).
FIG. 5 aflatoxin B of example 11Relative impedance change of aptamer sensor and aflatoxin B1The concentration correspondence of (3).
FIG. 6 aflatoxin B of example 11Aptamer sensor and aflatoxin B1Histogram of relative impedance changes of the common response of similar interferents.
Detailed Description
The present application is described in further detail below with reference to the accompanying drawings and examples, which are intended to facilitate the understanding of the present application and are not intended to limit the same in any way.
Example 1: for detecting aflatoxin B1Preparation of aptamer sensor
Growing a boron-doped polycrystalline diamond film on the P-type silicon by a microwave plasma chemical vapor deposition method (see fig. 1); then sputtering a gold film on the surface of the boron-doped polycrystalline diamond film by using an ion sputtering method, wherein the sputtering time is 10 seconds; annealing the obtained gold film and the boron-doped polycrystalline diamond film in a box-type furnace at 800 ℃ for 1 minute in the air atmosphere to obtain a gold nanoparticle/boron-doped diamond composite film structure (see figure 2); soaking the gold nanoparticles and the boron-doped diamond composite membrane in 5 mu mol/L aptamer solution for 5 hours at the constant temperature of 37 ℃ for modification to obtain an aptamer/gold nanoparticles/boron-doped diamond composite electrode; washing with phosphate buffer solution for 15 minutes, and then soaking in 1 mmol/L6-mercaptohexane-1-alcohol solution for modification for 1 hour to obtain the reagent for detecting aflatoxin B1The flow chart of the preparation of the 6-mercaptohexan-1-ol/aptamer/gold nanoparticle/boron-doped diamond aptamer sensor is shown in figure 3.
The aptamer specific structure selected in the example is 5' -SH- (CH)2)6-GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CCA CA-3′。
Example 2: for detecting aflatoxin B1Preparation of aptamer sensor
On the basis of example 1, the annealing time was changed to 30 seconds; the modification time in 6-mercaptohex-1-ol (MCH) solution was changed to 30 minutes, and the same results were obtained.
Example 3: for detecting aflatoxin B1Performance testing of aptamer sensors
As can be seen from the scanning electron microscope image of FIG. 1, the average size of the prepared conductive polycrystalline diamond film was about 1.8 μm, the surface of the electrode was deposited with very uniform gold nanoparticles, and the density of the gold particles was about 3.2X 1011cm-2(see fig. 2), the increased specific surface area may provide more active sites for the aptamer.
Aspergillus flavus at different concentrations using the sensor prepared in example 1Toxin B1The impedance test was performed and the results are shown in fig. 4. Aflatoxin B1Impedance value of aptamer sensor along with aflatoxin B1The concentration increases. Definition (R)ct-Rct0) Divided by Rct0As a relative impedance (i.e., (R)ct-Rct0)/Rct0，RctRepresents aflatoxin B1Aptamer sensor and aflatoxin B with certain concentration1Combined resistance value, Rct0Represents aflatoxin B1Initial impedance of aptamer sensor). Thus calculated relative impedance (%) and aflatoxin B1The log linear relationship of the concentrations is shown in FIG. 5. The linear equation is relative impedance (%) < 209.84+13.89logC, R20.997, C is aflatoxin B1R is a correlation coefficient. The detection limit of the sensor is 5.5 multiplied by 10 by calculating through triple signal-to-noise ratio-14mol L-1. Relative rate of change of impedance and aflatoxin B1The logarithm of the concentration is in linear relation, and the detection concentration range is 1.0 multiplied by 10-13～1.0×10-8mol/L。
Example 4: for detecting aflatoxin B1Specificity test of aptamer sensor
Selecting three interferents (aflatoxin B)2Aflatoxin G1And aflatoxin G2) For aflatoxin B1The specificity of the aptamer sensor was studied (FIG. 6) and the relative impedance response was measured at 0.5X 10-10mol L-1Aflatoxin B1Is carried out in the solution of (1). Three additive structures selected and aflatoxin B1Similarly, the concentration is aflatoxin B1100 times of the total weight of the powder. Aflatoxin B1The original impedance is set as 100 percent, and the impedance change of other mixed solution is between-2.1 percent and 2.8 percent, which indicates that the aflatoxin B1The aptamer sensor has good specificity and still has good application prospect in complex environment.
Example 5: sensor for detecting peanut sample
Selecting a fresh peanut sample to be ground,peanut crumbs (6 grams) were added to PBS buffer, then sonicated for 15 minutes, finally centrifuged at high speed for 10 minutes, the upper layer whey solution was filtered and made up to 20mL using PBS buffer. Addition of different concentrations of AFB1And putting the peanut seeds into the solution to be used as a peanut sample for later use. In the peanut sample, the sensor has higher detection sensitivity. For aflatoxin B with different concentrations in peanut samples1The recovery rates of (A) were investigated, and the results are shown in Table 1. In the experiment, aflatoxin B1In the concentration range of 1.0X 10-13～1.0×10-11mol L-1The recovery rate is 96-109%, and the relative standard deviation is less than 4.4%. The sensor has higher sensitivity and better practicability in peanut sample detection.
TABLE 1 peanut samples with varying concentrations of aflatoxin B1The recovery rate of (1).
Claims (3)
1. Be used for detecting aflatoxin B1The aptamer sensor is a composite material which is formed by compounding an aptamer/gold nanoparticles/boron-doped diamond and occupying blank active sites on the gold nanoparticles by 6-mercaptohexane-1-alcohol, wherein the boron-doped diamond film is used as a substrate; the structure of the aptamer is 5' -SH- (CH)2)6-GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CCA CA-3′。
2. The method for detecting aflatoxin B according to claim 11The aptamer sensor is characterized in that the average size of the gold nanoparticles is 13nm, and the density of the gold nanoparticles is 3.2 multiplied by 1011cm-2。
3. The method for detecting aflatoxin B of claim 11Firstly, growing a boron-doped polycrystalline diamond film on the P-type silicon by a microwave plasma chemical vapor deposition method(ii) a Then sputtering a gold film on the surface of the boron-doped polycrystalline diamond film by using an ion sputtering method, wherein the sputtering time is 10 seconds; annealing the obtained gold film and the boron-doped polycrystalline diamond film in the air at 800 ℃ for 30 seconds to 1 minute to obtain gold nanoparticle and boron-doped diamond composite film structures; then, soaking the gold nanoparticles and the boron-doped diamond composite membrane in 5 mu mol/L aptamer solution, and modifying for 5 hours at constant temperature of 37 ℃ to obtain an aptamer/gold nanoparticles/boron-doped diamond composite electrode; then the structure is washed by phosphate buffer for 15 minutes and then is immersed into 1 mmol/L6-mercapto hexane-1-alcohol solution for modification for 30 minutes to 1 hour to occupy blank active sites on the gold nanoparticles, and the aflatoxin B for detection is obtained1The 6-mercaptohexan-1-ol/aptamer/gold nanoparticle/boron-doped diamond aptamer sensor.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113358720A (en) * | 2021-06-09 | 2021-09-07 | 中国农业大学 | Polycrystalline gold immunosensor and application thereof in detection of novel coronavirus nucleocapsid protein |
| CN113702370A (en) * | 2021-09-16 | 2021-11-26 | 盐城工学院 | Method for detecting aflatoxin B1 by using glucose-gold nanoparticles |
| CN115266850A (en) * | 2022-07-26 | 2022-11-01 | 长春工业大学 | Preparation method of aptamer sensor for detecting cefquinome |
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| CN113702370A (en) * | 2021-09-16 | 2021-11-26 | 盐城工学院 | Method for detecting aflatoxin B1 by using glucose-gold nanoparticles |
| CN115266850A (en) * | 2022-07-26 | 2022-11-01 | 长春工业大学 | Preparation method of aptamer sensor for detecting cefquinome |
| CN115266850B (en) * | 2022-07-26 | 2024-04-12 | 长春工业大学 | Preparation method of aptamer sensor for detecting cefquinome |
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