CN113203788B - Bimodal rapid detection method for acetamiprid in fruits and vegetables - Google Patents
Bimodal rapid detection method for acetamiprid in fruits and vegetables Download PDFInfo
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Abstract
The invention belongs to the technical field of food detection, and relates to a bimodal rapid detection method for acetamiprid in fruits and vegetables; the bimodal nanoprobe with both fluorescence characteristic and electrochemical characteristic is constructed by combining the fluorescence technology and the electrochemical technology by utilizing the dual-signal characteristic of the zirconium-based metal organic framework for the first time, so that the bimodal detection of the single nanomaterial on the acetamiprid is realized. The bimodal detection of acetamiprid provided by the invention can reduce or eliminate low detection accuracy or false positive results caused by external factors such as substrate concentration, external environment, instrument conditions and the like. The correlation coefficients of the electrochemical and fluorescence method correlation prediction models respectively reach 0.998 and 0.997, and the selectivity and accuracy of the nano probe in a complex system are greatly improved.
Description
Technical Field
The invention belongs to the technical field of food detection, and particularly relates to a method for quickly detecting acetamiprid in fruits and vegetables in an electrochemical/fluorescent bimodal manner.
Background
China is the first big producing country of fruits and vegetables in the world, and various functional pesticides are used in a large quantity in the planting process of the fruits and vegetables in order to reduce the influence of diseases, insects, grass and other harmful organisms on the yield of the fruits and vegetables. However, excessive pesticide applied in the agricultural production process can directly or indirectly remain on the surfaces of fruits and vegetables, and the eating quality of the fruits and vegetables and the physical health of consumers are seriously affected. Acetamiprid is a novel broad-spectrum insecticide with acaricidal activity, and is widely used for preventing and treating insect pests such as aphids, plant hoppers and the like in the process of planting fruits and vegetables. At present, acetamiprid detection methods with strong specificity and high sensitivity are all instrumental analysis methods with high detection cost, long period and complex operation. In view of the influence of the acetamiprid on the edibility of the fruits and vegetables and the defects of the existing detection method, the development of the method for quickly, conveniently and accurately detecting the content of the acetamiprid in the fruits and vegetables has important practical significance.
The acetamiprid detection method based on the fluorescence technology and the electrochemical technology is concerned about because of high detection precision, high speed, low cost and the like. However, the single fluorescence technology and the electrochemical technology both have respective advantages and defects, and in order to avoid the problem of false positive or false negative in the detection result, a bimodal detection strategy combining the fluorescence technology and the electrochemical technology becomes an important research direction in the field of rapid detection at present. The fluorescent technology and the electrochemical technology are combined, the bimodal nanoprobe with the fluorescent characteristic and the electrochemical characteristic is constructed, the limitation of a single technology can be overcome, the detection result can be corrected in a targeted manner, the accuracy of the detection result is improved, and the advantage complementation effect is played. The existing method for detecting acetamiprid based on the bimodal nanoprobe has the following two defects that firstly, the nanoprobe constructed for realizing bimodal detection adopts complex nanophase materials, and the manufacturing and synthesizing process is complicated; secondly, interaction of multiple nano materials is needed to realize fluorescent and electrochemical bimodal detection, the nano materials are mutually influenced, and the stability of the probe is poor.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention provides a method for rapidly detecting acetamiprid in fruits and vegetables based on a zirconium-based metal organic framework, which can realize electrochemical/fluorescent bimodal rapid and low-cost detection of acetamiprid in fruits and vegetables. The zirconium-based metal organic framework has the fluorescence characteristic and the electrochemical characteristic at the same time, and is simple to synthesize and low in cost.
In order to achieve the purpose, the specific technical scheme of the invention is as follows:
a bimodal rapid detection method for acetamiprid in fruits and vegetables comprises the following steps:
(1) Preparation of zirconium-based metal organic frameworks
S1, dissolving zirconium tetrachloride and 2-aminoterephthalic acid in N, N-dimethylformamide, carrying out ultrasonic oscillation and uniform mixing, adding glacial acetic acid, continuously stirring to obtain a mixed solution, transferring the mixed solution into a reaction kettle, carrying out reaction at a certain temperature, washing a product for several times by using N, N-dimethylformamide and methanol in sequence after the reaction, and drying in a vacuum drying oven to obtain a purified zirconium-based metal organic framework;
s2, modifying a zirconium-based metal organic framework by an acetamiprid aptamer: mixing the acetamiprid aptamer solution with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), stirring and incubating at normal temperature, adding the zirconium-based metal organic framework purified in the step S1, incubating at normal temperature, and centrifuging the product to obtain solid precipitate, namely the acetamiprid aptamer-modified zirconium-based metal organic framework;
(2) Acquisition of electrochemical and fluorescent signals
S1, electrode pretreatment: polishing and grinding the gold electrode on polishing flannelette by using aluminum oxide powder with different grain diameters, sequentially carrying out ultrasonic treatment in absolute ethyl alcohol and deionized water, and drying by using nitrogen; then placing the activated gold electrode in a sulfuric acid solution for cyclic potential scanning, and observing the oxidation and reduction peak potentials until the oxidation and reduction peaks are completely coincided to obtain an activated gold electrode;
s2, gold electrode acetamiprid aptamer complementary strand modification: dropwise adding the acetamiprid aptamer complementary strand solution to the surface of the activated gold electrode in the step (2) in the S1, and washing the unbound acetamiprid aptamer complementary strand solution on the surface with deionized water after incubation at normal temperature; then, adding Bovine Serum Albumin (BSA) solution dropwise, standing at room temperature for a period of time, and washing the surface of the electrode with deionized water to obtain the acetamiprid aptamer complementary chain modified gold electrode;
s3, measuring by using a pulse voltammetry method: preparing acetamiprid standard samples with different concentrations, and respectively marking as C 1 ,C 2 ,……,C n-1 ,C n N is a positive integer; then adding the acetamiprid aptamer modified zirconium-based metal organic framework obtained in the S2 in the step (1) into the acetamiprid standard sample to obtain a mixed solution; inserting the gold electrode modified by the complementary chain of the acetamiprid aptamer obtained in the step (2) into the mixed solution, stirring and enriching for a period of time, so that the zirconium-based metal organic framework modified by the acetamiprid aptamer is connected to the gold electrode modified by the complementary chain of the acetamiprid aptamer through base complementary pairing; after the enrichment is finished, stopping stirring, standing the solution for a period of time, and attaching the zirconium-based metal organic framework modified by the acetamiprid aptamer to the surface of the gold electrode modified by the complementary chain of the acetamiprid aptamer in the process to obtain a working electrode;
s4, electric signal acquisition stage: potential scanning is carried out on the working electrode in the step (2) S3, the potential is scanned from 1.2V to 0.5V, an electric signal of the working electrode is obtained, and acetamiprid standard samples (C) with different concentrations are measured and recorded by the working electrode 1 ,C 2 ,……,C n-1 ,C n ) Intensity of electric signal of (E) 1 ,E 2 ,……,E n-1 ,E n N is a positive integer;
s5, fluorescent signal acquisition stage: after measuring the electric signal intensity of each acetamiprid standard sample with one concentration, taking out the working electrode, washing the surface of the working electrode by using a phosphate buffer solution, collecting and washing the phosphate buffer solution with the concentration, and mixing the phosphate buffer solution with the acetamiprid standard sample with the concentration; after the operation, mixed liquor of acetamiprid standard samples with different concentrations and the washed phosphate buffer solution is obtained, the volumes are all fixed to a certain same volume, then the solutions with the fixed volumes are respectively put into a quartz cuvette, the excitation wavelength of a fluorescence spectrometer is set, the optimal fluorescence emission intensity is measured and recorded, and the optimal fluorescence emission intensity is recorded as F 1 ,F,……,F n-1 ,F n N is a positive integer; (3) Rapid prediction of acetamiprid concentration
S1, an electrochemical rapid detection prediction model: according to the electric signal intensity E in S4 of the step (2) 1 ,E 2 ,……,E n-1 ,E n In the standard sample C of acetamiprid with different concentrations 1 ,C 2 ,……,C n-1 ,C n Establishing a model to obtain an electrochemical rapid detection prediction model E = f (X) of acetamiprid, wherein E is electrochemical current, X is acetamiprid concentration, and n is a positive integer;
s2, a fluorescence rapid detection prediction model: subjecting the fluorescence emission intensity F described in S5 of step (2) 1 ,F,……,F n-1 ,F n With acetamiprid standard sample C of different concentrations 1 ,C 2 ,……,C n-1 ,C n Establishing a model to obtain a fluorescence rapid detection prediction model F = F (X) of acetamiprid, wherein F is fluorescence emission intensity, X is acetamiprid concentration, and n is a positive integer;
s3, rapidly predicting the acetamiprid concentration in the fruits and vegetables: shearing or stirring a fruit and vegetable sample to be detected, adding the universal sample extracting solution, and fully oscillating and uniformly mixing to obtain a liquid to be detected; respectively obtaining electric signal intensity and fluorescence emission intensity according to the methods for measuring the electrochemical signal and the fluorescence signal in the S4 and the S5 in the step (2), and respectively substituting the electric signal intensity and the fluorescence emission intensity into an electrochemical rapid detection prediction model and a fluorescence rapid detection prediction model to obtain a rapid prediction concentration value of the acetamiprid obtained by an electrochemical method and a fluorescence method; rapidly predicting a concentration value of acetamiprid obtained by an electrochemical method and a fluorescence method, and performing group difference analysis by using SPSS; if there is a significant difference between the two sets of data (p < 0.05), the data is discarded; if the two groups of data have no significant difference (p is greater than 0.05), taking the average value of the data as the acetamiprid concentration in the fruits and vegetables, and realizing the rapid detection of the acetamiprid concentration.
Preferably, the molar ratio of the zirconium tetrachloride to the 2-aminoterephthalic acid in S1 of step (1) is 1:1; the concentration of the zirconium tetrachloride and the 2-amino terephthalic acid in the mixed solution is both 8 mmol.L- 1 (ii) a The volume ratio of the N, N-dimethylformamide to the glacial acetic acid is 4:1; the time for uniformly mixing by ultrasonic oscillation is 5 to 10 minutes; the reaction temperature in the reaction kettle is 110-150 ℃, and the reaction time is 8-15 hours; the washing is carried out for 1 to 4 times.
Preferably, the ratio of the acetamiprid aptamer solution, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and purified zirconium-based metal-organic framework in S2 of step (1) is 1mL: 1-2 g; the concentration of the acetamiprid aptamer solution is 100 mu mol/L; the concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 0.1mmol/L; the concentration of the N-hydroxysuccinimide is 0.1mmol/L; the base sequence of the acetamiprid aptamer is as follows: 5'- (COOH) - (CH 2) 6-ACA GGC TAC GAG GGA AAT GCG GTG GGT GTG GGC GAT-3'.
Preferably, the incubation time in step (1) S2 is 1-2 hours; the incubation time after adding the purified zirconium-based metal organic framework is 10 to 12 hours.
Preferably, the alumina powders with different grain diameters in the S1 of the step (2) have the grain diameters of 1.0 μm, 0.3 μm and 0.05 μm in sequence, and the grinding time is 3-5 min; the ultrasonic treatment time is 3-5 min; the concentration of the sulfuric acid solution is 0.5mol/L.
Preferably, the base sequence of the complementary strand of the acetamiprid aptamer in S2 of step (2) is as follows: 5'- (SH) - (CH 2) 6-TGT CCG ATG CTC-3'; the concentration of the acetamiprid aptamer complementary strand solution is 100 mu mol/L, and the dosage of the acetamiprid aptamer complementary strand solution dripped on the surface of the activated gold electrode is 5-10 mu L; the incubation time at normal temperature is 2-4 hours.
Preferably, the mass concentration of the bovine serum albumin solution in the S2 in the step (2) is 1%, and the dosage of the bovine serum albumin solution dripped on the surface of the activated gold electrode is 10-20 mu L; the standing time at room temperature is 1-2 hours.
Preferably, the acetamiprid concentration in S3 in the step (2) is 0-10 mg/kg; the dosage ratio of the acetamiprid standard sample to the acetamiprid aptamer-modified zirconium-based metal organic framework is (10-15) mL: (1-2) g; the stirring and enriching time is 10 to 20 minutes; the standing time is 10-20 seconds.
Preferably, the pH of the phosphate buffer solution in the S4 of the step (2) is 7.2-7.5, and the volume of the washing working electrode is 1-2 ml; the volume after constant volume is 10mL.
Wherein W is a specific excitation wavelength, is greater than 0 and is a positive integer; preferably, the excitation wavelength is 365nm.
Preferably, the universal extracting solution in the S3 of the step (3) is a YRSPN sample universal extracting solution; the dosage ratio of the fruit and vegetable sample to be detected to the universal extracting solution of the sample is 2-2.5 g:5mL.
The invention has the beneficial effects that:
the invention firstly proposes that the double-signal characteristic of the zirconium-based metal organic framework is used for the electrochemical and fluorescent rapid detection of acetamiprid in fruits and vegetables, and the bimodal detection of acetamiprid by a single nano material is firstly realized.
The bimodal detection of acetamiprid provided by the invention can reduce or eliminate low detection accuracy or false positive results caused by external factors such as substrate concentration, external environment, instrument conditions and the like. The correlation coefficients of the electrochemical and fluorescence method correlation prediction models respectively reach 0.998 and 0.997.
The acetamiprid aptamer is used for specifically identifying the acetamiprid, so that the selectivity and the accuracy of the nano probe in a complex system are greatly improved.
The invention simplifies the manufacture of the bimodal nano-sensor sensing system and further reduces the detection error caused by the stability problem of the sensing system (the relative error of the detection result with the national standard method is less than 1 percent).
Drawings
FIG. 1 is a topographical feature diagram of a zirconium-based metal organic framework.
FIG. 2 is a graph showing an electrochemical signal (a) and a fluorescence signal (b) of a zirconium-based metal organic framework.
Detailed description of the preferred embodiments
The present invention will be described in detail below with reference to embodiments. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Furthermore, various changes or modifications may be made by one skilled in the art after reading the disclosure herein, and equivalents may fall within the scope of the invention as defined by the claims appended hereto.
The gold electrode used in the invention is purchased from Shanghai Lu major company, inc., and the other reagents are purchased from the national medicine group chemical reagent company, inc.
A bimodal rapid detection method for acetamiprid in fruits and vegetables comprises the following steps:
(1) Preparation of zirconium-based metal organic frameworks
S1, dissolving zirconium tetrachloride and 2-amino terephthalic acid in 20mL of N, N-dimethylformamide, wherein the use amounts of the zirconium tetrachloride and the 2-amino terephthalic acid are both 0.2mol; carrying out ultrasonic oscillation for 10 minutes, moving to a magnetic stirrer, adding 5mL of glacial acetic acid, continuously stirring and uniformly mixing for 10 minutes, and transferring the mixed solution to a 50mL reaction kettle; reacting for 15 hours at 130 ℃, washing the obtained zirconium-based metal organic framework with N, N-dimethylformamide for 2 times, washing with methanol for 2 times, and drying in a vacuum drying oven to obtain the purified zirconium-based metal organic framework.
S2, modifying a zirconium-based metal organic framework by using an acetamiprid aptamer, mixing 1mL of a 100 mu mol/L acetamiprid aptamer (5 '- (COOH) - (CH 2) 6-ACA GGC TAC GAG GGA AAT GCG GTG GGT GTG GGC GAT-3') solution with 5mL of 0.1mmol/L EDC solution and 5mL of 0.1mmol/L NHS solution, and stirring and incubating for 2 hours at normal temperature to activate carboxyl; and then adding 1.5g of the zirconium-based metal organic framework obtained above, and incubating for 12 hours at normal temperature to obtain the acetamiprid aptamer-modified zirconium-based metal organic framework.
(2) Acquisition of electrochemical signals
Electrode pretreatment: polishing and grinding the gold electrode by using alumina powder with the grain diameters of 1.0 mu m, 0.3 mu m and 0.05 mu m in sequence for 5min on polishing flannelette, then respectively carrying out ultrasonic treatment in absolute ethyl alcohol and deionized water for 5min to remove residual alumina, and drying by using nitrogen; carrying out cyclic potential scanning in 0.5mol/L dilute sulfuric acid, and observing the oxidation peak potential and the reduction peak potential until the oxidation peak and the reduction peak are completely coincided, which represents that the surface of the gold electrode is clean, so as to obtain an activated gold electrode;
modification of gold electrode acetamiprid aptamer complementary strand: dripping 5 mu L of 100 mu mol/L acetamiprid aptamer complementary chain (5 '- (SH) - (CH 2) 6-TGT CCG ATG CTC-3') solution on the surface of an activated gold electrode, incubating for 3 hours at normal temperature, and washing the unbound acetamiprid aptamer complementary chain on the surface by deionized water. Titrating 20 mu L of BSA with the mass concentration of 1%, standing at room temperature for 1 hour, and washing the surface of the electrode with deionized water to obtain the acetamiprid aptamer complementary chain modified gold electrode.
And (3) pulse voltammetry determination: preparing acetamiprid standard samples with different concentrations, wherein the concentrations of the acetamiprid are respectively 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10mg/kg, and adding 1.5g of the zirconium-based metal organic framework modified by the acetamiprid aptamer into the standard samples. Inserting the acetamiprid aptamer complementary chain modified gold electrode into the mixed solution, stirring to ensure that the acetamiprid aptamer complementary chain modified on the gold electrode is complementarily paired and combined with the acetamiprid aptamer base modified on the zirconium-based metal organic framework, enriching the zirconium-based metal organic framework on the surface of the gold electrode, and continuing the enrichment process for 15 minutes to obtain a working electrode; after the enrichment is finished, stopping stirring, and standing the solution for 15 seconds; an electric signal acquisition stage: the potential of the working electrode is scanned from 1.2V to 0.5V, a zirconium-based metal organic framework is attached to the surface of the gold electrode in the process, the dissolution current is formed, and the electric signal intensity of 21.782, 18.987, 17.056, 15.71, 13.227, 11.057, 9.315, 7.1352, 5.2454, 3.189 and 0.987mA under different acetamiprid concentrations is measured and recorded by an electrochemical workstation.
(3) Acquisition of fluorescence signals
Taking out the working electrode in the step (2), taking 1mL of phosphate buffer solution to wash the surface of the electrode, collecting the washing solution and the solution in the step (2), fixing the volume to 10mL, taking 2mL of the solution in a quartz cuvette, setting the excitation wavelength of a fluorescence spectrometer to be 365nm, measuring and recording the optimal fluorescence emission intensity of 199.74, 248.56, 315.71, 361.24, 428.79, 479.56, 516.91, 597.16, 668.25, 719.53 and 787.15 of the mixed solution under the acetamiprid standard samples with different concentrations.
(4) Rapid prediction of acetamiprid concentration
Electrochemical rapid detection prediction model: and (3) modeling the electrochemical signals 21.782, 18.987, 17.056, 15.71, 13.227, 11.057, 9.315, 7.1352, 5.2454, 3.189 and 0.987mA obtained in the step (2) on acetamiprid standard samples 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10mg/kg with different concentrations. To obtainElectrochemical rapid detection prediction model E = -2.033X +21.411 (R) of acetamiprid 2 ＝0.998)；
Fluorescence rapid detection prediction model: modeling the fluorescence signals 199.74, 248.56, 315.71, 361.24, 428.79, 479.56, 516.91, 597.16, 668.25, 719.53 and 787.15 obtained in the step (3) and acetamiprid standard samples 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10mg/kg with different concentrations to obtain a fluorescence rapid detection prediction model F =58.532X 191.21 (R) of acetamiprid 2 ＝0.997)。
Example 1: rapid prediction of acetamiprid concentration in apples
Weighing 5 parts of the apple sample to be detected, wherein the parts are respectively (2.05 g, 2.12g, 2.09g, 2.06g and 2.15 g), and adding the cut or crushed sample into a 10mL centrifuge tube; adding 5mL of the universal extracting solution of the YRSPN sample, fully oscillating and uniformly mixing to obtain the solution to be detected.
Respectively adding the acetamiprid aptamer-modified zirconium-based metal organic framework into 2mL of solution to be detected to respectively obtain a fluorescence signal and an electrochemical signal, repeatedly measuring for three times, and obtaining acetamiprid concentration results by using rapid prediction models of the fluorescence method and the electrochemical method as shown in Table 1. The SPSS analysis result shows that the fluorescence method and the electrochemical method in the group 4 sample have significant difference, which indicates that the data has problems, and the group data is determined as invalid data and discarded; if there is no significant difference among the data of groups 1, 2, 3 and 5, the average value of the data obtained is 0.466mg/kg, 1.074mg/kg, 1.553mg/kg and 2.471mg/kg respectively. In order to verify the detection accuracy of the method, acetamiprid concentration analysis is carried out on the samples of the groups 1, 2, 3 and 5 by using a national standard method (GB/T23584-2009) to obtain acetamiprid concentrations of 0.462mg/kg, 1.068mg/kg, 1.541mg/kg and 2.465mg/kg respectively. The relative error of the obtained detection result is less than 1%, which shows that the bimodal rapid detection method for acetamiprid in fruits and vegetables further reduces the detection error caused by the stability problem of a sensing system and improves the detection accuracy.
TABLE 1 measurement of acetamiprid content in apple by fluorescence and electrochemical methods
Example 2: rapid prediction of acetamiprid concentration in cherry tomato
Weighing 5 parts of cherry tomato sample to be detected (2.03 g, 2.02g, 2.06g, 2.09g and 2.11 g), respectively, and adding the sheared or stirred sample into a 10mL centrifuge tube; adding 5mL of the universal extracting solution of the YRSPN sample, fully oscillating and uniformly mixing to obtain the solution to be detected.
And (3) respectively adding a zirconium-based metal organic framework into 2mL of solution to be detected to respectively obtain a fluorescence signal and an electrochemical signal, repeatedly measuring for three times, and obtaining the acetamiprid concentration result by using rapid prediction models of a fluorescence method and an electrochemical method as shown in Table 2. The SPSS analysis result shows that the fluorescence method and the electrochemical method measurement result in the group 1 sample have significant difference analysis, which indicates that the data have problems, and the group data is determined as invalid data and discarded; and if the data of groups 2, 3, 4 and 5 have no significant difference, the concentrations of the acetamiprid in the cherry tomatoes are respectively 2.312mg/kg, 3.476mg/kg, 4.683mg/kg and 5.366mg/kg. In order to verify the detection accuracy of the method, acetamiprid concentration analysis is carried out on the samples of the groups 2, 3, 4 and 5 by using a national standard method (GB/T23584-2009) to obtain acetamiprid concentrations of 2.324mg/kg, 3.469mg/kg, 4.655mg/kg and 5.357mg/kg respectively. The relative error of the obtained detection result is less than 1%, which shows that the bimodal rapid detection method for acetamiprid in fruits and vegetables further reduces the detection error caused by the stability problem of a sensing system and improves the detection accuracy.
Table 2. Measuring the content of acetamiprid in cherry tomatoes by a fluorescence method and an electrochemical method
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, while 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 are intended to be included herein within the scope of this disclosure and the present invention and protected by the following claims.
Claims (10)
1. A bimodal rapid detection method for acetamiprid in fruits and vegetables is characterized by comprising the following specific steps:
(1) Preparation of zirconium-based metal organic framework
S1, dissolving zirconium tetrachloride and 2-amino terephthalic acid in N, N-dimethylformamide, uniformly mixing by ultrasonic oscillation, adding glacial acetic acid, continuously stirring to obtain a mixed solution, transferring the mixed solution into a reaction kettle, reacting at a certain temperature, sequentially washing a product for several times by using the N, N-dimethylformamide and methanol after reaction, and drying in a vacuum drying oven to obtain a purified zirconium-based metal organic framework;
s2, modifying a zirconium-based metal organic framework by an acetamiprid aptamer: mixing the acetamiprid aptamer solution with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, stirring and incubating at normal temperature, adding the zirconium-based metal organic framework purified in the step S1, incubating at normal temperature, and centrifuging the product to obtain solid precipitate, namely the acetamiprid aptamer-modified zirconium-based metal organic framework;
(2) Acquisition of electrochemical and fluorescent signals
S1, electrode pretreatment: polishing and grinding the gold electrode on polishing flannelette by using alumina powder with different grain diameters, sequentially carrying out ultrasonic treatment in absolute ethyl alcohol and deionized water, and drying by using nitrogen; then placing the activated gold electrode in a sulfuric acid solution for cyclic potential scanning, and observing the oxidation and reduction peak potentials until the oxidation and reduction peaks are completely coincided to obtain an activated gold electrode;
s2, gold electrode acetamiprid aptamer complementary strand modification: dropwise adding the acetamiprid aptamer complementary strand solution to the surface of the activated gold electrode in the step (2) in the S1, and washing the unbound acetamiprid aptamer complementary strand solution on the surface with deionized water after incubation at normal temperature; then, dropwise adding bovine serum albumin solution, standing at room temperature for a period of time, and washing the surface of the electrode with deionized water to obtain the acetamiprid aptamer complementary chain modified gold electrode;
s3, measuring by using a pulse voltammetry method: preparing acetamiprid standard samples with different concentrations, and respectively marking as C 1 ,C 2 ,……,C n-1 ,C n N is a positive integer; then adding the acetamiprid aptamer modified zirconium-based metal organic framework obtained in the S2 in the step (1) into the acetamiprid standard sample to obtain a mixed solution; inserting the gold electrode modified by the complementary chain of the acetamiprid aptamer obtained in the step (2) into the mixed solution, stirring and enriching for a period of time, so that the zirconium-based metal organic framework modified by the acetamiprid aptamer is connected to the gold electrode modified by the complementary chain of the acetamiprid aptamer through base complementary pairing; after the enrichment is finished, stopping stirring, standing the solution for a period of time, and attaching the zirconium-based metal organic framework modified by the acetamiprid aptamer to the surface of the gold electrode modified by the complementary chain of the acetamiprid aptamer in the process to obtain a working electrode;
s4, an electric signal acquisition stage: potential scanning is carried out on the working electrode in the step (2) S3, the potential is scanned from 1.2V to 0.5V, an electric signal of the working electrode is obtained, the working electrode is utilized to measure and record the intensity of the electric signal of the acetamiprid standard sample with different concentrations, and the intensity is marked as E 1 ,E 2 ,……,E n-1 ,E n N is a positive integer;
s5, fluorescent signal acquisition stage: after measuring the electric signal intensity of each acetamiprid standard sample with one concentration, taking out the working electrode, washing the surface of the working electrode by using a phosphate buffer solution, collecting and washing the phosphate buffer solution with the concentration, and mixing the phosphate buffer solution with the acetamiprid standard sample with the concentration; after the operation, mixed solution of the acetamiprid standard samples with different concentrations and the washed phosphate buffer solution is obtained, the volume is constant to a certain same volume, then the solution with constant volume is respectively put into a quartz cuvette, the excitation wavelength of a fluorescence spectrometer is set, the optimal fluorescence emission intensity is measured and recorded, and the optimal fluorescence emission intensity is recorded as F 1 ,F,……,F n-1 ,F n N is a positive integer;
(3) Rapid detection of acetamiprid concentration
S1, an electrochemical rapid detection prediction model: the intensity E of the electric signal according to the step (2) S4 1 ,E 2 ,……,E n-1 ,E n With acetamiprid standard sample C of different concentrations 1 ,C 2 ,……,C n-1 ,C n Establishing a model to obtain an electrochemical rapid detection prediction model E = f (X) of acetamiprid, wherein E is electrochemical current, X is acetamiprid concentration, and n is a positive integer;
s2, a fluorescence rapid detection prediction model: subjecting the fluorescence emission intensity F described in S5 of step (2) 1 ,F,……,F n-1 ,F n With acetamiprid standard sample C of different concentrations 1 ,C 2 ,……,C n-1 ,C n Establishing a model to obtain a fluorescence rapid detection prediction model F = F (X) of acetamiprid, wherein F is fluorescence emission intensity, X is acetamiprid concentration, and n is a positive integer;
s3, rapidly predicting the acetamiprid concentration in the fruits and vegetables: taking a fruit and vegetable sample to be detected, shearing or stirring, adding the universal sample extracting solution, and fully oscillating and uniformly mixing to obtain a liquid to be detected; respectively obtaining electric signal intensity and fluorescence emission intensity according to the methods for measuring the electrochemical signal and the fluorescence signal in the S4 and the S5 in the step (2), and respectively substituting the electric signal intensity and the fluorescence emission intensity into an electrochemical rapid detection prediction model and a fluorescence rapid detection prediction model to obtain the acetamiprid rapid prediction concentration value measured by an electrochemical method and a fluorescence method; rapidly predicting a concentration value of acetamiprid determined by an electrochemical method and a fluorimetry, and performing inter-group difference analysis by using SPSS; if there is significant difference between the two sets of data, discarding the data; if the two groups of data have no significant difference, taking the average value of the data as the acetamiprid concentration in the fruits and vegetables, and realizing the rapid detection of the acetamiprid concentration.
2. The bimodal rapid detection method of acetamiprid in fruits and vegetables according to claim 1, wherein the molar ratio of the zirconium tetrachloride to the 2-amino terephthalic acid in S1 of step (1) is 1:1; the concentrations of the zirconium tetrachloride and the 2-amino terephthalic acid in the mixed solution are both 8 mmol.L -1 (ii) a The N, N-dimethyl esterThe volume ratio of the benzamide to the glacial acetic acid is 4:1; the time for uniformly mixing by ultrasonic oscillation is 5 to 10 minutes; the reaction temperature in the reaction kettle is 110-150 ℃, and the reaction time is 8-15 hours; the washing is carried out for 1 to 4 times.
3. The bimodal rapid detection method for acetamiprid in fruits and vegetables according to claim 1, wherein the usage ratio of the acetamiprid aptamer solution, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and purified zirconium-based metal organic framework in S2 in step (1) is 1mL: 1-2 g; the concentration of the acetamiprid aptamer solution is 100 mu mol/L; the concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 0.1mmol/L; the concentration of the N-hydroxysuccinimide is 0.1mmol/L; the base sequence of the acetamiprid aptamer is as follows: 5'- (COOH) - (CH 2) 6-ACA GGC TAC GAG GGAAAT GCG GTG GGT GTG GGC GAT-3'.
4. The bimodal rapid detection method for acetamiprid in fruits and vegetables according to claim 1, wherein the time of stirring and incubation in S2 of step (1) is 1-2 hours; the incubation time after adding the purified zirconium-based metal organic framework is 10 to 12 hours.
5. The bimodal rapid detection method for acetamiprid in fruits and vegetables according to claim 1, wherein the grain sizes of the alumina powders with different grain sizes in S1 in step (2) are 1.0 μm, 0.3 μm and 0.05 μm in sequence, and the grinding time is 3-5 min; the ultrasonic treatment time is 3-5 min; the concentration of the sulfuric acid solution is 0.5mol/L.
6. The bimodal rapid detection method for acetamiprid in fruits and vegetables according to claim 1, wherein the base sequence of complementary strand of acetamiprid aptamer in S2 of step (2) is: 5'- (SH) - (CH 2) 6-TGT CCG ATG CTC-3'; the concentration of the acetamiprid aptamer complementary strand solution is 100 mu mol/L, and the dosage of the acetamiprid aptamer complementary strand solution dripped on the surface of the activated gold electrode is 5-10 mu L; the incubation time at normal temperature is 2-4 hours.
7. The bimodal rapid detection method of acetamiprid in fruits and vegetables according to claim 1, wherein in the step (2) S2, the mass concentration of the bovine serum albumin solution is 1%, and the dosage of the bovine serum albumin solution dripped on the surface of the activated gold electrode is 10-20 μ L; the standing time at room temperature is 1-2 hours.
8. The bimodal rapid detection method of acetamiprid in fruits and vegetables according to claim 1, wherein the acetamiprid concentration in S3 of step (2) is 0-10 mg/kg; the dosage ratio of the acetamiprid standard sample to the acetamiprid aptamer-modified zirconium-based metal organic framework is (10-15) mL: (1-2) g; the stirring and enriching time is 10 to 20 minutes; the standing time is 10-20 seconds.
9. The bimodal rapid detection method of acetamiprid in fruits and vegetables according to claim 1, wherein the pH of the phosphate buffer solution in the S4 of the step (2) is 7.2-7.5, and the volume of the washing working electrode is 1-2 ml; the volume after constant volume is 10mL.
10. The bimodal rapid detection method for acetamiprid in fruits and vegetables according to claim 1, characterized in that the universal extract in S3 of step (3) is a universal extract of YRSPN sample; the dosage ratio of the fruit and vegetable sample to be detected to the universal extracting solution of the sample is 2-2.5 g:5mL.
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