CN111175364B - Preparation method of ratiometric electrochemical aptamer sensor for simultaneously detecting aflatoxin B1 and ochratoxin A - Google Patents
Preparation method of ratiometric electrochemical aptamer sensor for simultaneously detecting aflatoxin B1 and ochratoxin A Download PDFInfo
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Abstract
The invention belongs to the technical field of electrochemical sensing, and relates to a preparation method of a ratio electrochemical aptamer sensor for simultaneously detecting AFB1 and OTA; the sensor was prepared by sequentially assembling AQ-hDNA, Fc-Apt1 and MB-Apt2 on gold electrodes, and binding of the target to the aptamer caused Fc-Apt1 and MB-Apt2 to peel off from the electrode surface, resulting in a ratio signal IFc/IAQAnd IMB/IAQThe AFB1 and the OTA are quantized and detected respectively; the linear range of detection of the sensor to AFB1 is 10pg/mL-3ng/mL, and the detection limit is 3.3 pg/mL; the detection linear range of the OTA is 30pg/mL-10ng/mL, and the detection limit is 10.0 pg/mL; and 2 substances are detected simultaneously, and the method has the characteristics of high sensitivity, good selectivity, high precision and the like.
Description
Technical Field
The invention belongs to the technical field of electrochemical sensing, and relates to a preparation method of a ratio electrochemical aptamer sensor for simultaneously detecting aflatoxin B1 and ochratoxin A.
Background
Aflatoxin B1(AFB1) and ochratoxin a (ota) are teratogenic, nephrotoxic and oncogenic mycotoxins produced by specific fungi under high temperature and humidity conditions in agricultural products. AFB1 and OTA can enter the food chain through contaminated food and animal feed, causing serious health hazards to humans. International agency for research on cancer IARC has classified AFB1 as a class 1 carcinogen (strongly carcinogenic to humans), while OTA is classified as a class 2B carcinogen (potentially carcinogenic to humans). Due to the simultaneous presence of multiple fungi, AFB1 and OTA are often present in the same agricultural product, such as moldy wheat or corn. The coexistence of mycotoxins increases their likelihood of being taken by mistake and small intakes may also be a significant hazard to humans due to the synergistic effect between toxins. Therefore, there is a need to develop effective detection techniques to achieve simultaneous detection and analysis of AFB1 and OTA in agricultural products.
At present, the simultaneous detection technology for AFB1 and OTA mainly comprises high performance liquid chromatography-mass spectrometry, high performance liquid chromatography-fluorescence method and the like, but the detection technologies have the defects of expensive instruments, complex operation, long time consumption and the like, and a simple and quick detection means needs to be developed.
The electrochemical sensing method is expected to become a promising method for simultaneously detecting various mycotoxins with the advantages of high sensitivity, quick detection, simple operation and the like. At present, electrochemical aptamer sensors for single mycotoxins have good sensitivity and selectivity, but the detection accuracy needs to be further improved. And the ratio electrochemical aptamer sensor based on the ratio strategy has an internal correction function, so that the accuracy of the electrochemical sensor can be greatly improved. Currently, there is no report on the simultaneous detection of AFB1 and OTA using ratiometric electrochemical aptamer sensors. Therefore, the development of the ratiometric electrochemical aptamer sensor capable of realizing high-sensitivity and high-accuracy simultaneous detection of AFB1 and OTA has important research significance.
Disclosure of Invention
Aiming at the problems in the prior art, the invention utilizes the sequential assembly of the complementary hairpin DNA marked by AQ, the aflatoxin B1 aptamer marked by Fc and the ochratoxin A aptamer marked by MB on a gold electrode to construct a ratiometric electrochemical aptamer sensor, thereby realizing the high-sensitivity and high-precision simultaneous detection of the aflatoxin B1 and the ochratoxin A.
A method of making a ratiometric electrochemical aptamer sensor for simultaneous detection of aflatoxin B1 and ochratoxin a, comprising the steps of:
(1) sequentially polishing the gold electrode AuE with aluminium oxide powder with different particle sizes, sequentially ultrasonically cleaning in ethanol and water to remove surface residues, and then electrochemically cleaning the electrode in sulfuric acid solution by using cyclic voltammetry;
(2) modifying the complementary hairpin DNA marked by anthraquinone-2-carboxylic acid, namely AQ-hDNA, onto the electrode treated in the step (1), storing for a period of time at room temperature, and fixing the AQ-hDNA on the surface of the gold electrode by using a gold-sulfur bond;
(3) modifying Mercaptohexanol (MCH) on the electrode treated in the step (2), and incubating for a period of time at room temperature to block non-specific binding sites of gold;
(4) modifying the electrode treated in the step (3) with an aptamer of aflatoxin B1 marked by ferrocene, marking as Fc-Apt1, reacting for a period of time at a certain temperature, and assembling Fc-Apt1 on the electrode by utilizing complementary hybridization of the aptamer and hDNA;
(5) modifying the ochratoxin A aptamer marked by methylene blue as MB-Apt2 on the electrode treated in the step (4), and further assembling MB-Apt2 on the electrode by utilizing complementary hybridization to obtain the ratiometric electrochemical aptamer sensor which is marked as Fc-Apt1 and MB-Apt 2/MCH/AQ-hDNA/AuE.
Preferably, in the step (1), the diameter of the gold electrode is 2 or 3 mm; the grain sizes of the aluminum oxide powder are 0.3 μm and 0.05 μm in turn; the scanning rate of the cyclic voltammetry is 50 or 100mV/s, the scanning potential range is-0.2-1.6V, and the concentration of sulfuric acid is 0.01-0.1M.
Preferably, in the step (2), the AQ-hDNA modification dosage is 4-8 μ L, the concentration is 1-8 μ M, and the AQ-hDNA is stored for 4-12 hours at room temperature.
Preferably, in the step (3), the MCH modification dosage is 4-8 muL, and the concentration is 0.01-10 mM; and incubating for 0.5-2 hours at room temperature.
Preferably, in the step (4), the dosage of the Fc-Apt1 modification is 4-8 muL, and the concentration is 1-8 muM; the certain temperature condition is 37 ℃, and the reaction time is 1-2 hours.
Preferably, in the step (5), the consumption of the MB-Apt2 modification is 4-8 muL, the concentration is 1-8 muM, the reaction time is 1-2 hours, and the temperature is 37 ℃.
The sensor prepared by the invention is used for detecting aflatoxin B1 and ochratoxin A, and comprises the following steps:
(1) the prepared plurality of sensors Fc-Apt1&Sequentially dripping aflatoxin B1 and ochratoxin A standard solutions with different concentrations into MB-Apt2/MCH/AQ-hDNA/AuE, wherein each sensor corresponds to one concentration; after incubation at room temperature for a certain period of time, washing was performed with Tris-HCl (pH 7.4) solution. In a three-electrode system, the washed electrode is used as a working electrode, an Ag/AgCl (saturated KCl) electrode is used as a reference electrode, a platinum wire is used as a counter electrode, a phosphate buffer solution (pH 7.4) is used as an electrolyte, an electrochemical Alternating Current Voltammetry (ACV) curve is scanned, and a ratio signal I is measuredFc/IAQAnd IMB/IAQEstablishing a ratio signal I corresponding to the concentrations of aflatoxin B1 and ochratoxin A respectivelyFc/IAQAnd IMB/IAQA standard linear curve of (d);
wherein, IFcIs Fc oxidation current, IMBFor MB oxidation current, IAQIs AQ oxidation current;
(2) dropwise adding the solution to be detected in the Fc-Apt1 sensor&MB-Apt2/MCH/AQ-hDNA/AuE, incubated at room temperature for a certain period of time, and washed with Tris-HCl (pH 7.4) solution. In a three-electrode system, the washed electrode is used as a working electrode, an Ag/AgCl (saturated KCl) electrode is used as a reference electrode, a platinum wire is used as a counter electrode, a phosphate buffer solution (pH 7.4) is used as an electrolyte, an electrochemical Alternating Current Voltammetry (ACV) curve is scanned, and a ratio signal I is measuredFc/IAQAnd IMB/IAQAnd substituting the standard linear curve to realize high-sensitivity and high-accuracy simultaneous detection of the aflatoxin B1 and the ochratoxin A.
Preferably, in the step (1), the concentration range of the aflatoxin B1 is 1 × 10-11～3×10-9g/mL, OTA concentration range of 3X 10-11～1×10-8g/mL。
Preferably, in the steps (1) and (2), the incubation is carried out for 0.5-2 hours at room temperature.
The working principle of the prepared sensor is as follows:
first, AQ-hDNA, Fc-Apt1 and MB-Apt2 modified sequentially are assembled on the surface of a gold electrode to form a hairpin DNA-based double-stranded DNA structure, and a large Fc oxidation current (I) is generated at the timeFc) MB Oxidation Current (I)MB) And AQ oxidation current (I)AQ). In the presence of aflatoxin B1 or ochratoxin A, its specific binding to the aptamer results in stripping of the electrode surface by Fc-Apt1 or MB-Apt2, leading to IFcAnd IMBIs reduced byAQRemain unchanged. Thus, by measuring the ratio signal IFc/IAQAnd IMB/IAQThe simultaneous detection of target aflatoxin B1 and ochratoxin A is realized.
The invention has the beneficial effects that:
(1) the method utilizes the AQ-labeled hDNA, the Fc-labeled Apt1 and the MB-labeled Apt2 to form a double-rate strategy with 3 response current signals, thereby improving the accuracy of electrochemical detection.
(2) The invention constructs the sensor by utilizing the specific complementary hybridization of the hairpin DNA and the 2 aptamers, and the cross influence between the target objects can not be generated.
(3) The ratio electrochemical aptamer sensor for simultaneously detecting aflatoxin B1 or ochratoxin A, which is prepared by the invention, has the characteristics of high sensitivity, good selectivity, high accuracy and the like, and has a wide application prospect in the field of agricultural product quality safety.
Drawings
FIG. 1 is a schematic diagram of the construction and detection process of the ratiometric aptamer sensor.
FIG. 2 (A) shows ACV responses corresponding to different AFB1 concentrations and OTA, wherein the AFB1 concentration is 1 × 10-11，2×10-11，5×10-11，1×10-10，2×10-10，1×10-9And 3X 10-9g/mL; the concentration of OTA is 3 × 10-11，1×10-10，2×10-10，5×10-10，1×10-9，5×10-9And 1X 10-8g/mL; (B) is a ratio signal IFc/IAQAnd IMB/IAQStandard line between the logarithm of the concentration of AFB1 and OTA respectivelyAnd (4) a sexual curve.
Detailed Description
The invention is further described with reference to the drawings and examples.
Example 1
A method for preparing a ratiometric electrochemical aptamer sensor for simultaneously detecting aflatoxin B1 and ochratoxin a, wherein the construction and detection process of the ratiometric electrochemical aptamer sensor is shown in figure 1;
the method specifically comprises the following steps:
(1) polishing a gold electrode (AuE) with the diameter of 3mm by using aluminum oxide powder with the diameter of 0.3 mu M and the diameter of 0.05 mu M in sequence, performing ultrasonic treatment in ethanol and water in sequence to remove surface residues, and then performing electrochemical cleaning on the electrode by using cyclic voltammetry at the sweep rate of 100mV/s and the potential of-0.2-1.6V in a 0.1M sulfuric acid solution;
(2) modifying 6 mu L of AQ-hDNA with the concentration of 2 mu M on the electrode treated in the step (1), standing for 8 hours at room temperature, and fixing the AQ-hDNA on the surface of the gold electrode by using a gold-sulfur bond;
(3) modifying Mercaptohexanol (MCH) at a concentration of 1mM at 6. mu.L on the electrode treated in step (2), and incubating at room temperature for 1 hour to block non-specific binding sites of gold;
(4) modifying 6 mu L of ferrocene-labeled aflatoxin B1 aptamer (Fc-Apt1) with the concentration of 4 mu M on the electrode treated in the step (3), reacting at 37 ℃ for 1 hour, and assembling the Fc-Apt1 on the electrode by utilizing complementary hybridization of the aptamer and hDNA;
(5) and (3) modifying 6 mu L of methylene blue labeled ochratoxin A aptamer (MB-Apt2) with the concentration of 4 mu M on the electrode treated in the step (4), reacting at 37 ℃ for 1 hour, and further assembling MB-Apt2 on the electrode by utilizing complementary hybridization to obtain a ratiometric electrochemical aptamer sensor which is marked as Fc-Apt1& MB-Apt 2/MCH/AQ-hDNA/AuE.
The prepared sensor Fc-Apt1&Sequentially dripping aflatoxin B1 and ochratoxin A standard solution with different concentrations into MB-Apt2/MCH/AQ-hDNA/AuE, wherein the concentration of AFB1 is 1 × 10-11，2×10-11，5×10-11，1×10-10，2×10-10，1×10-9And 3X 10-9g/mL, OTA concentration of 3X 10-11，1×10-10，2×10-10，5×10-10，1×10-9，5×10-9And 1X 10-8g/mL; after incubation at room temperature for 40 minutes, washing was performed using a Tris-HCl (pH 7.4) solution. In a three-electrode system, the washed electrode is used as a working electrode, an Ag/AgCl (saturated KCl) electrode is used as a reference electrode, a platinum wire is used as a counter electrode, a phosphate buffer solution (pH 7.4) is used as an electrolyte, an electrochemical Alternating Current Voltammetry (ACV) curve is scanned, and a ratio signal I is measuredFc/IAQAnd IMB/IAQEstablishing a ratio signal I corresponding to the concentrations of aflatoxin B1 and ochratoxin A respectivelyFc/IAQAnd IMB/IAQA standard linear curve of (d);
from (A) of FIG. 2, it can be seen that as the concentration of AFB1 and OTA increases, IFcAnd IMBRespectively, are gradually decreased.
From (B) of FIG. 2, it can be seen that the ratio IFc/IAQThe standard linear curve between the logarithm of the concentration and AFB1 is IFc/IAQ＝-0.733-0.171Log CAFB1[g/mL](R20.997), the linear range is 10pg/mL-3ng/mL, and the detection limit is 3.3 pg/mL; ratio IMB/IAQThe standard linear curve between the logarithm of the OTA concentration is IMB/IAQ＝-0.611-0.129Log COTA[g/mL](R20.998), linear range 30pg/mL-10ng/mL, detection limit 10.0 pg/mL.
The obtained sensor is applied to standard-adding detection of aflatoxin B1 and ochratoxin A with different concentrations in actual sample corn, and the detection process is as follows: the sample solution is dripped on the sensor Fc-Apt1&MB-Apt2/MCH/AQ-hDNA/AuE, incubated at room temperature for 40 minutes, and then washed with Tris-HCl (pH 7.4) solution. In a three-electrode system, the washed electrode is used as a working electrode, an Ag/AgCl (saturated KCl) electrode is used as a reference electrode, a platinum wire is used as a counter electrode, a phosphate buffer solution (pH 7.4) is used as an electrolyte, an electrochemical Alternating Current Voltammetry (ACV) curve is scanned, and a ratio signal I is measuredFc/IAQAnd IMB/IAQSubstituting into the standard linear curve IFc/IAQ＝-0.733-0.171Log CAFB1[g/mL]And IMB/IAQ＝-0.611-0.129LogCOTA[g/mL]And calculating to obtain the concentration information of aflatoxin B1 and ochratoxin A in the corn sample.
To verify the detection accuracy of the constructed sensors, we tested the same corn samples using AFB1 and OTA's national standard detection method, high performance liquid chromatography-mass spectrometry (HPLC-MS, GB 5009.22-2016 and 5009.96-2016). The detection results of the two methods are shown in table 1;
table 1: the sensor and the high performance liquid chromatography-mass spectrometry method which are constructed by the invention respectively detect the detection results of AFB1 and OTA in the corn sample
The results in table 1 show that the constructed sensor can realize high-sensitivity and high-accuracy simultaneous detection of aflatoxin B1 and ochratoxin A.
Description of the drawings: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; thus, although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted; all such modifications and variations that do not depart from the spirit and scope of the invention are intended to be included within the scope of the appended claims.
Claims (9)
1. A preparation method of a ratiometric electrochemical aptamer sensor for simultaneously detecting aflatoxin B1 and ochratoxin A, which is characterized by comprising the following steps:
(1) sequentially polishing the gold electrode AuE with aluminium oxide powder with different particle sizes, sequentially ultrasonically cleaning in ethanol and water, and then electrochemically cleaning the electrode in sulfuric acid solution by using cyclic voltammetry;
(2) modifying the complementary hairpin DNA (AQ-hDNA) marked by anthraquinone-2-carboxylic acid onto the electrode treated in the step (1), storing for a period of time at room temperature, and fixing the AQ-hDNA on the surface of the gold electrode by using a gold-sulfur bond;
(3) modifying mercaptohexanol on the electrode treated in the step (2), and incubating for a period of time at room temperature to block the nonspecific binding sites of gold;
(4) modifying an aptamer Fc-Apt1 of the aflatoxin B1 marked by ferrocene on the electrode treated in the step (3), reacting for a period of time under a certain temperature condition, and assembling the Fc-Apt1 on the electrode by utilizing complementary hybridization of the aptamer and hDNA;
(5) modifying an ochratoxin A aptamer MB-Apt2 marked by methylene blue on the electrode treated in the step (4), reacting for a period of time at a certain temperature, and further assembling MB-Apt2 on the electrode by utilizing complementary hybridization of the ochratoxin A aptamer and hDNA to obtain a ratio electrochemical aptamer sensor which is marked as Fc-Apt1& MB-Apt 2/MCH/AQ-hDNA/AuE.
2. The method for preparing a ratiometric electrochemical aptamer sensor for simultaneously detecting aflatoxin B1 and ochratoxin A according to claim 1, wherein in the step (1), the diameter of the gold electrode is 2 or 3 mm; the grain sizes of the aluminum oxide powder are 0.3 mu m and 0.05 mu m in turn; the scanning rate of the cyclic voltammetry is 50 or 100mV/s, and the scanning potential range is-0.2-1.6V; the concentration of the sulfuric acid solution is 0.01-0.1M.
3. The preparation method of the ratiometric electrochemical aptamer sensor for simultaneously detecting aflatoxin B1 and ochratoxin A according to claim 1, wherein in the step (2), the AQ-hDNA modification amount is 4-8 muL, the AQ-hDNA concentration is 1-8 muM, and the sensor is stored for 4-12 hours at room temperature.
4. The preparation method of the ratiometric electrochemical aptamer sensor for simultaneously detecting aflatoxin B1 and ochratoxin A according to claim 1, wherein in the step (3), the amount of MCH is 4-8 muL, and the concentration is 0.01-10 mM; and incubating for 0.5-2 hours at room temperature.
5. The method for preparing the ratiometric electrochemical aptamer sensor for simultaneously detecting aflatoxin B1 and ochratoxin A according to claim 1, wherein in the step (4), the dosage of Fc-Apt1 is 4-8 muL, and the concentration is 1-8 muM; the certain temperature condition is 37 ℃, and the reaction time is 1-2 hours.
6. The preparation method of the ratiometric electrochemical aptamer sensor for simultaneously detecting aflatoxin B1 and ochratoxin A according to claim 1, wherein in the step (5), the dosage of MB-Apt2 is 4-8 muL, and the concentration is 1-8 muM; the certain temperature condition is 37 ℃, and the reaction time is 1-2 hours.
7. Use of a sensor prepared by the method of any one of claims 1 to 6 for simultaneous detection of aflatoxin B1 and ochratoxin a, comprising the steps of:
(1) the prepared plurality of sensors Fc-Apt1&Sequentially dripping aflatoxin B1 and ochratoxin A standard solutions with different concentrations into MB-Apt2/MCH/AQ-hDNA/AuE, wherein each sensor corresponds to one concentration; incubating for a certain time at room temperature, and washing by using a Tris-HCl solution; in a three-electrode system, the electrode after washing is used as a working electrode, an Ag/AgCl electrode is used as a reference electrode, a platinum wire is used as a counter electrode, phosphate buffer solution is used as electrolyte, an electrochemical AC volt-ampere curve is scanned, and a ratio signal I is measuredFc/IAQAnd IMB/IAQEstablishing a ratio signal I corresponding to the concentrations of aflatoxin B1 and ochratoxin A respectivelyFc/IAQAnd IMB/IAQA standard linear curve of (d); wherein, IFcIs Fc oxidation current, IMBFor MB oxidation current, IAQIs AQ oxidation current;
(2) dropwise adding the sample solution to be detected in the Fc-Apt1 sensor&MB-Apt2/MCH/AQ-hDNA/AuE, incubating for a certain time at room temperature, and washing with Tris-HCl solution; in a three-electrode system, the electrode after washing is used as a working electrode, an Ag/AgCl electrode is used as a reference electrode, a platinum wire is used as a counter electrode, phosphate buffer solution is used as electrolyte, an electrochemical AC volt-ampere curve is scanned, and a ratio signal I is measuredFc/IAQAnd IMB/IAQAnd (4) substituting the standard linear curve in the step (1) to realize the simultaneous detection of the aflatoxin B1 and the ochratoxin A of the samples to be detected.
8. The use according to claim 7, wherein the aflatoxin B1 concentration in step (1) is in the range of 1 x 10-11~3×10-9g/mL, OTA concentration range of 3X 10-11~1×10-8 g/mL。
9. The use according to claim 7, wherein the incubation at room temperature in steps (1) and (2) is carried out for a period of time of 0.5 to 2 hours.
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| CN115165995B (en) * | 2022-06-09 | 2024-05-28 | 西北大学 | Detection method of ochratoxin A and preparation method of electrochemical OTA aptamer sensor |
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