Construction and application of confinement-enhanced photoelectrochemistry-electrochemiluminescence dual-mode biosensor

Abstract

The invention discloses construction and application of a confinement enhanced photoelectrochemistry-electrochemiluminescence dual-mode biosensor, and relates to the technical field of biosensing and analytical chemistry. The preparation method of the biosensor comprises the following steps: taking a CdS (at) SiO2 (at) NaYF4: Yb / Tm nano composite material as a substrate to obtain a confinement enhanced photoelectrochemistry (PEC) and electrochemiluminescence (ECL) sensing interface; in combination with a DNA wheel nanostructure triggered by target acetamiprid, signal amplification is realized; and synchronous dual-mode quenching detection of PEC and ECL signals is realized by virtue of peroxidase-like catalytic precipitation and wide-spectrum absorption characteristics of the PtPd-CoSnO3 nanocube. The biosensor prepared by the invention has the advantages of high sensitivity, strong reliability, good selectivity and the like, can be applied to accurate detection of acetamiprid in samples such as agricultural products and the like, and has important application value in the aspects of environmental monitoring and food safety control.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biosensors, in particular to a construction and application of a confined-enhanced photoelectrochemical-electrochemiluminescence dual-mode biosensor. BACKGROUND

[0002] Pesticides play a vital role in the development of modern agriculture, but the corresponding pesticide residues are inevitable. Ecological environment and food safety problems are usually caused by pesticide residues, which can cause harm to human health even at low concentrations. In particular, as one of the most common organophosphorus and carbamate pesticides, acetamiprid residues are often found in water, soil, food and even human urine. The long-term accumulation of acetamiprid is usually associated with the pathogenesis of various neurological diseases by inhibiting cholinesterase activity and destroying peripheral blood lymphocytes. Therefore, it is very important to develop a sensitive and accurate method for detecting acetamiprid.

[0003] Thanks to different response mechanisms and relatively independent signal transduction modes, dual-mode biosensors can achieve high detection accuracy and reliability by cross-verification results. For example, photoelectrochemical (PEC) and electrochemiluminescence (ECL) dual-mode output sensing strategies not only have the inherent characteristics of their respective response modes, but also can verify the detection results with each other, showing excellent flexibility when dealing with various detection conditions. In order to improve the sensing efficiency of PEC and ECL, some substrate materials have been developed, such as inorganic hybrid compounds, fine strategy of core-shell structure, covalent organic framework microreactor and various nanocomposites. Among them, the confined enhancement strategy provides great potential for improving signal strength efficiency by concentrating the co-reactant and sensing substrate in a closed space. Therefore, the present application intends to utilize the confined effect and multiple light reflection between CdS nanoparticles, SiO2 and NaYF4:Yb / Tm to construct CdS@SiO2@NaYF4:Yb / Tm core-shell nanocomposites, realize fast electron transfer and high-efficiency light utilization, and thus significantly improve the PEC and ECL response performance to achieve sensitive detection of acetamiprid. SUMMARY

[0004] The purpose of the present application is to provide a construction and application of a confined-enhanced photoelectrochemical-electrochemiluminescence dual-mode biosensor to solve the problems existing in the prior art. The biosensor prepared by the present application has the advantages of high accuracy, strong sensitivity and good selectivity, and can be applied to the accurate detection of acetamiprid in food, having important application value in the field of food safety.

[0005] To achieve the above purpose, the present application provides the following solutions:

[0006] The application provides a preparation method of a confined enhanced photoelectrochemical-electrochemiluminescence dual-mode biosensor, and comprises the following steps:

[0007] After the indium tin oxide electrode (ITO) is cleaned, the CdS@SiO2@NaYF4:Yb / Tm composite is added dropwise to the surface of the electrode to obtain a CdS@SiO2@NaYF4:Yb / Tm / ITO electrode;

[0008] The CdS@SiO2@NaYF4:Yb / Tm / ITO electrode is sequentially immersed in H1:H2 double-stranded, acetamiprid transduced T-chain solution, T4 DNA ligase and Nt.CviPII endonuclease, and incubation reaction is carried out to initiate the formation of DNA rolling nanostructure;

[0009] The obtained sensing interface is immersed in PtPd-CoSnO3 nanocube solution to carry out incubation reaction, and the biosensor is obtained;

[0010] The CdS@SiO2@NaYF4:Yb / Tm nanomaterial is a core-shell structure formed by sequentially coating a SiO2 layer and CdS nanoparticles on NaYF4:Yb / Tm as the inner core;

[0011] The H1:H2 double-stranded solution is obtained by incubation reaction of H1 chain with 3' end labeled phosphate group and thiolated H2 chain with 3' end modified carboxyl group;

[0012] The nucleotide sequence of the H1 chain is shown as SEQ ID NO. 1, and the nucleotide sequence of the H2 chain is shown as SEQ ID NO. 2;

[0013] The acetamiprid transduced T chain is obtained by combining aptamer recognition and magnetic bead separation technology;

[0014] The nucleotide sequence of the aptamer chain is shown as SEQ ID NO. 3, and the nucleotide sequence of the T chain is shown as SEQ ID NO. 4;

[0015] The PtPd-CoSnO3 nanocube is synthesized by a hydrothermal method, and PtPd nanoparticles are in-situ grown on the CoSnO3 nanocube.

[0016] Further, the preparation method of the CdS@SiO2@NaYF4:Yb / Tm nanomaterial comprises the following steps:

[0017] YCl3·6H2O, YbCl3·6H2O, TmCl3·6H2O and NaF are sequentially added to an ethylenediaminetetraacetic acid solution, and reaction is carried out at 180 DEG C for 12 h to obtain NaYF4:Yb / Tm;

[0018] NaYF4:Yb / Tm was dispersed in ammonia-ethanol mixed solution, tetraethyl orthosilicate was added and reacted for 1h to obtain SiO2@NaYF4:Yb / Tm;

[0019] SiO2@NaYF4:Yb / Tm was mixed with sodium citrate, Cd(NO3)2.4H2O, ammonia and thiourea, and reacted at 80°C for 3h to obtain CdS@SiO2@NaYF4:Yb / Tm.

[0020] Further, the molar ratio of YCl3.6H2O, YbCl3.6H2O and TmCl3.6H2O is 20:8:1.

[0021] Further, the T chain of acetamiprid transduction is an aptamer, a T chain modified magnetic bead and an acetamiprid incubation reaction.

[0022] Further, the preparation method of the PtPd-CoSnO3 nanocube comprises the following steps:

[0023] 238mg CoCl2.6H2O and 294mg sodium citrate were dissolved in 30mL deionized water to obtain solution A;

[0024] 351mg SnCl4.5H2O was dissolved in 5mL ethanol to obtain solution B;

[0025] After mixing solution A and B, 5mL NaOH (2.0M) was added, and reacted for 1h, and further annealed at 500°C for 2h to obtain PtPd-CoSnO3 nanocube.

[0026] The application also provides a confined enhanced photoelectrochemical- electrochemiluminescence dual-mode biosensor prepared by the preparation method.

[0027] The application also provides the use of the above-mentioned biosensor in the preparation of acetamiprid detection in food.

[0028] Further, the biosensor further comprises a H1 chain labeled with a 3' end phosphate group, a thiolated H2 chain modified with a 3' end carboxyl group, an acetamiprid aptamer and a T chain.

[0029] The nucleotide sequence of the M chain is shown in SEQ ID NO. 1;

[0030] The nucleotide sequence of the telomerase primer is shown in SEQ ID NO. 2;

[0031] The nucleotide sequence of the S chain is shown in SEQ ID NO. 3;

[0032] The nucleotide sequence of the F chain is shown in SEQ ID NO. 4;

[0033] The sensing interface is applied to the detection of PEC and ECL signals in turn, and according to the changes of photocurrent (ΔI) and ECL intensity (ΔECL), the double-mode analysis of acetamiprid in the sample is realized.

[0034] The present application discloses the following technical effects:

[0035] The present application constructs a confined enhanced photoelectrochemical-electrochemiluminescence double-mode biosensor, and realizes the detection of acetamiprid. The biosensor uses CdS@SiO2@NaYF4:Yb / Tm nanocomposite material as a sensing substrate, and obtains a confined enhanced PEC and ECL sensing interface. With the help of magnetic bead separation technology, the detection of acetamiprid is converted into nucleic acid analysis. The DNA wheel nanostructure is introduced into the sensing interface to realize signal amplification. With the help of the peroxidase-like catalytic precipitation and wide spectrum absorption characteristics of PtPd-CoSnO3 nanocube, the synchronous double-mode quenching of PEC and ECL signals is realized, so that the accurate detection of acetamiprid in food is realized.

[0036] The present application uses CdS@SiO2@NaYF4:Yb / Tm as a double-mode signal substrate, and uses the multiple light reflection between the core-shell structure interfaces to exhibit enhanced photocurrent and ECL signals. The signal transduction of acetamiprid is carried out by combining the magnetic bead separation technology of aptamer, and the anti-interference performance of the sensing system is improved. Based on the signal amplification strategy of the DNA wheel nanostructure triggered by acetamiprid and the PtPd-CoSnO3 nanocube quenching, the sensitivity of the biosensor is improved. Through the cross verification of the PEC and ECL double signal output mode, the accuracy and reliability of the detection result are effectively improved. Therefore, the biosensor prepared by the present application has the advantages of high accuracy, strong sensitivity and good selectivity, and can be applied to the accurate detection of acetamiprid in food, and has important application value in the field of food safety. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0038] Figure 1 It is a preparation and application schematic diagram of the confined enhanced photoelectrochemical-electrochemiluminescence double-mode biosensor of the present application.

[0039] Figure 2 It is a scanning electron microscope (SEM) image of SiO2@NaYF4:Yb / Tm.

[0040] Figure 3 Low magnification (A) and high magnification (B) SEM images of CdS@SiO2@NaYF4:Yb / Tm;

[0041] Figure 4 X-ray photoelectron spectroscopy (XPS) image of CdS@SiO2@NaYF4:Yb / Tm;

[0042] Figure 5 SEM image and elemental mapping of CdS@SiO2@NaYF4:Yb / Tm;

[0043] Figure 6 SEM image of PtPd-CoSnO3 nanocubes;

[0044] Figure 7 Particle size distribution of PtPd-CoSnO3 nanocubes;

[0045] Figure 8 XPS image of PtPd-CoSnO3 nanocubes;

[0046] Figure 9 Monitoring results of DNA sequence involved in the reaction process in Example 1; (A) the signal transduction feasibility verification of acetamiprid: lane 1-3 represent DNA molecular mass standard, aptamer and T chain respectively, lane 4 represents the mixture of aptamer and T chain, and lane 5 represents the supernatant of aptamer:T / magnetic bead complex after incubation with acetamiprid and magnetic separation; (B) the feasibility verification of DNA wheel nanostructure signal amplification: lanes 1-3 represent DNA molecular mass standard, H1 chain and H2 chain respectively, lane 4 represents the mixture of H1 chain and H2 chain, lane 5 represents the mixture of H1 chain and acetamiprid transduced T chain, lane 6 represents the product of DNA wheel nanostructure after Nease treatment, lane 7 represents the product of H2 after Nease treatment, and lane 8 represents the product of H1:H2 double-stranded after Nease treatment;

[0047] Figure 10 The photocurrent-time curve of the sensing substrate interface construction process, a represents CdS@SiO2@NaYF4:Yb / Tm / ITO, b represents H1:H2 / CdS@SiO2@NaYF4:Yb / Tm / ITO, c represents the sensing substrate after the reaction of b with acetamiprid transduced T chain, d represents c after Nease treatment, and e represents the incubation of d with PtPd-CoSnO3 nanocubes;

[0048] Figure 11The photocurrent-time curve of the sensing substrate interface construction process, a represents CdS@SiO2@NaYF4:Yb / Tm / ITO, b represents H1:H2 / CdS@SiO2@NaYF4:Yb / Tm / ITO, c represents the sensing substrate after the T chain reaction of b and acetamiprid transduction, d represents c after Nease treatment, and e represents d incubated with PtPd-CoSnO3 nanocubes;

[0049] Figure 12 The photocurrent-time response curve (A) and the corresponding linear relationship graph (B) of different concentrations of acetamiprid; wherein a-h correspond to the concentrations of 0, 1fM, 5fM, 150fM, 1.5pM, 15pM, 150pM and 1nM, respectively;

[0050] Figure 13 The ECL-time curve (A) and the corresponding linear relationship graph (B) of different concentrations of acetamiprid; wherein a-h correspond to the concentrations of 0, 1fM, 5fM, 150fM, 1.5pM, 15pM, 150pM and 1nM, respectively;

[0051] Figure 14 The columnar graph of the selective response of the sensor to different pesticides; DETAILED DESCRIPTION

[0052] The various illustrative embodiments of the present application will now be described in detail below. This detailed description is not intended to limit the application, but rather to provide a full and enabling description of certain example aspects, features, and embodiments of the present application.

[0053] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, for a range of values of a variable being disclosed, each intervening value of the variable, to the extent that there are intervening values of the variable, between the upper and lower limits of that range is also disclosed. In addition, each individual value of the variable in the stated range and any other stated or intervening value of the variable are incorporated herein. The upper and lower limits of these intervening values are also disclosed. The same applies to ranges of values of other specified variables in the disclosed combinations.

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described therein. In the case of conflict between the present specification and any document incorporated herein by reference, the present specification will control.

[0055] Many modifications and variations to the illustrative embodiments described herein will be apparent to those of ordinary skill in the art from this specification, which is to be regarded in the light of the scope and spirit of the application. Other embodiments apparent to those of ordinary skill in the art from this specification are intended to be within the scope of the present application. The specification and examples are illustrative only.

[0056] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0057] The present application uses CdS@SiO2@NaYF4:Yb / Tm as a dual-mode signal substrate, and utilizes the multiple light reflection between the core-shell structure interfaces to exhibit enhanced photocurrent and ECL signals; the signal transduction of acetamiprid is performed by combining the magnetic bead separation technology of aptamer, thereby improving the anti-interference performance of the sensing system; based on the signal amplification strategy of DNA nanometer structure and PtPd-CoSnO3 nanometer cube quenching triggered by acetamiprid, the sensitivity of the biosensor is improved; through the cross verification of the PEC and ECL dual signal output modes, the accuracy and reliability of the detection results are effectively improved (the sensing mechanism diagram of the biosensor is shown in Figure 1 ), and the specific details are as follows:

[0058] Example 1

[0059] The construction and application process of a confined enhanced photoelectrochemical-electrochemiluminescence dual-mode biosensor are as follows:

[0060] First, an indium tin oxide electrode (ITO) (2 cm x 1 cm) is ultrasonically cleaned in acetone, ethanol and deionized water for 5 min in sequence and dried by nitrogen blowing. 20 μL of CdS@SiO2@NaYF4:Yb / Tm nanomaterials are added to the surface of the electrode to obtain CdS@SiO2@NaYF4:Yb / Tm / ITO.

[0061] Then, 100 μL of H2 chain (5.0 μM) is mixed with 2 μL of tris(2-carboxyethyl)phosphine hydrochloride solution (10 mM) to obtain thiol-activated H2 chain, which is then incubated with an equal volume of H1 chain (5.0 μM) at 37°C for 1 h to form H1:H2 double-stranded chain. The CdS@SiO2@NaYF4:Yb / Tm / ITO is immersed in the H1:H2 double-stranded chain solution and incubated at 37°C for 2 h to obtain H1:H2 / CdS@SiO2@NaYF4:Yb / Tm / ITO.

[0062] The preparation method of the CdS@SiO2@NaYF4:Yb / Tm nanomaterial is as follows: 1.488 g of ethylenediaminetetraacetic acid (EDTA) and 2.1 g of sodium fluoride are sequentially added into 10 mL of a mixture containing 0.2 mol / L YCl3·6H2O, 0.08 mol / L YbCl3·6H2O and 0.01 mol / L TmCl3·6H2O, and reaction is carried out at 180 °C for 12 h to obtain NaYF4:Yb / Tm. Subsequently, 200 mg of NaYF4:Yb / Tm is dispersed in a mixed solution of ammonia water (24 mL)-ethanol (81 mL), 3.0 mL of tetraethyl orthosilicate is added, and reaction is carried out for 1 h to obtain SiO2@NaYF4:Yb / Tm. Then, 200 mg of SiO2@NaYF4:Yb / Tm is mixed with 2 mL of sodium citrate (1 mol / L), 1 mL of Cd(NO3)2·4H2O (1 mol / L), 3 mL of ammonia water and 4 mL of thiourea (1 mol / L), stirred for 10 min, and reaction is carried out at 80 °C for 3 h to obtain the CdS@SiO2@NaYF4:Yb / Tm nanomaterial.

[0063] The nucleotide sequence of the H1 strand (SEQ ID NO. 1) is 5'-P-CGACTTCATAATAGCATTGAGATCAGTACACATGAACACCATATTATGAAGCCATGTGTAGATGGTAGCTTA-3';

[0064] The nucleotide sequence of the H2 strand (SEQ ID NO. 2) is 5'-COOH-TACTGATCTCAATGCTATTAAGCTGTCGTAAGCTACCATCTACACATGGTAGCTTATCAGACTGATGTTGAAGTCTGTATCACA-SH-3'.

[0065] The method for using the biosensor is as follows: pyridaben is extracted from cucumbers and peaches and transferred to the aptamer:T double-stranded / magnetic bead complex to conduct signal transduction, and the released T strand is collected after magnetic separation; then, the H1:H2 / CdS@SiO2@NaYF4:Yb / Tm / ITO is incubated with a mixture containing the released T strand, T4 DNA ligase and Nt.CviPII endonuclease to initiate the formation of DNA rolling nanometer structures; thereafter, the obtained sensing interface is activated by N-hydroxysuccinimide (10 mg / mL) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (20 mg / mL), and then is placed into 100 μL of PtPd-CoSnO3 nanocubes (0.1 mg / mL) to react at 25°C for 2 h; finally, the sensing substrate after incubation is used as a working electrode, an Ag / AgCl electrode is used as a reference electrode, and a platinum wire is used as an auxiliary electrode to form a three-electrode system. After the sensing interface is placed into a mixture containing 1 mg / mL 3'-diaminobenzidine (DAB) and 10 mM H2O2 to react for 30 min, the photocurrent signal (PEC mode) is detected in an ascorbic acid solution (0.1 M). The test instrument is a CHI760e electrochemical workstation equipped with a 500W xenon lamp, and the applied potential is 0.1 V. The interval time for turning on and off the xenon lamp is 10 seconds. At the same time, the sensing interface is placed into a solution containing 0.1 M potassium persulfate to detect the ECL signal (ECL mode). During the detection process, the voltage of the photomultiplier tube is set to 700 V, the potential scanning range is 0 to -1.8 V, and the scanning rate is 100 mV / s. The specific details are as follows:

[0066] (1) Signal transduction of pyridaben.

[0067] The specific operation is as follows: the carboxylated magnetic beads (1 mg / mL) are treated with a mixture of N-hydroxysuccinimide (10 mg / mL) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (20 mg / mL) at 25°C for 1 h to obtain carboxyl-activated magnetic beads; the activated magnetic beads are added to 500 μL of 5' amino-modified aptamer (5 μM) to react at 25°C for 1 h, and the aptamer is assembled onto the surface of the magnetic beads through an amide bond to form an aptamer / magnetic bead complex. The surface of the magnetic beads is blocked with 2% mass fraction of bovine serum albumin to block the non-specific sites. After the aptamer / magnetic bead is separated by a magnet and washed with a buffer solution, it is further placed in 5 μM T strand to react at 25°C for 1 h to form an aptamer:T / magnetic bead complex; after being separated by a magnet and washed with a buffer solution, 500 μL of pyridaben with different concentrations is added to react at 37°C for 2 h, and the signal transduction T strand of pyridaben is obtained after magnetic separation.

[0068] The nucleotide sequence of the aptamer (SEQ ID NO. 3) is 5'-NH2-TTTCTGACACCATATTATGAAGA-3';

[0069] The nucleotide sequence of the T strand (SEQ ID NO. 4) is 5'-TTTCTTCATAATATGGTGTCATGTG-3';

[0070] (2) Assembling DNA wheel nanostructure at the interface of H1:H2 / CdS@SiO2@NaYF4:Yb / Tm / ITO electrode.

[0071] Incubating the H1:H2 / CdS@SiO2@NaYF4:Yb / Tm / ITO electrode with the released T strand at 37℃ for 150 min to form the DNA wheel nanostructure. Then, the modified electrode is immersed in 100 μL of Nt.CviPII NEase (0.5 unit / μL) at 37℃ for 1 h.

[0072] (3) After the electrode obtained in step (2) is washed with buffer solution, the 5' end carboxyl group of H2 is activated by a mixture of N-hydroxysuccinimide (10 mg / mL) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (20 mg / mL), and then the modified electrode is placed in 100 μL of PtPd-CoSnO3 nanocubes (0.1 mg / mL) at 25℃ for 2 h to introduce the quencher PtPd-CoSnO3 nanocubes into the electrode interface through amide bond. After the incubation, the sensing substrate is collected; finally, the incubated sensing substrate is used as the working electrode, the Ag / AgCl electrode is used as the reference electrode, and the platinum wire is used as the auxiliary electrode to form a three-electrode system, and the photocurrent and ECL signal are detected, and the concentration of acetamiprid residues in the food is calculated according to the standard curve.

[0073] The PtPd-CoSnO3 nanocubes are synthesized by a hydrothermal method, and PtPd nanoparticles are in-situ grown on the CoSnO3 nanocubes to obtain: 238 mg of CoCl2·6H2O and 294 mg of sodium citrate are dissolved in 30 mL of deionized water to obtain solution A. 351 mg of SnCl4·5H2O is dissolved in 5 mL of ethanol to obtain solution B; finally, after mixing solution A and B, 5 mL of NaOH (2.0 M) is added, and the reaction is carried out for 1 h, and further annealing at 500℃ for 2 h to obtain PtPd-CoSnO3 nanocubes.

[0074] Example 2

[0075] The performance of the biosensor prepared in Example 1 is detected, and the specific process is as follows:

[0076] (1) The biosensor of the present application utilizes CdS@SiO2@NaYF4:Yb / Tm with core-shell structure as PEC and ECL sensing substrate, and PtPd-CoSnO3 nanocube as quenching agent. In order to verify the feasibility of synthesizing nanomaterials, the CdS@SiO2@NaYF4:Yb / Tm and PtPd-CoSnO3 nanocube prepared in Example 1 are characterized. As shown in Figure 2 It can be seen that the SiO2@NaYF4:Yb / Tm has a spherical structure with smooth surface. Subsequently, with the addition of sodium citrate and Cd(NO3)2·4H2O, CdS nanoparticles are in-situ aggregated on the surface of SiO2@NaYF4:Yb / Tm Figure 3 A and 3B), forming CdS@SiO2@NaYF4:Yb / Tm core-shell nanocomposite. Figure 4 and Figure 5 It is shown that the material contains Si, Y, Tm, Yb, C, O, F and Na elements. In addition, Figure 6 and 7 It is shown that the PtPd-CoSnO3 nanomaterial has a cubic structure, and the particle size is about 380 nm. Figure 8 It is shown that the material contains Cd, F, Na, O, S, Tm, Si, Y and Yb elements.

[0077] (2) The biosensor of the present application further improves the detection sensitivity through signal transduction and DNA wheel nanostructure signal amplification reaction. Therefore, the construction process of the sensing interface, signal transduction and the feasibility of DNA wheel nanostructure are monitored and verified. As shown in Figure 9 A, lanes 1-3 represent DNA molecular weight standard, aptamer and T chain respectively, lane 4 represents the mixture of aptamer and T chain, and the new band of lag represents the formation of aptamer:T double strand. Lane 5 represents the band of supernatant after incubation of aptamer:T double strand / magnetic bead complex and acetamiprid, and it can be seen that acetamiprid is specifically combined with aptamer, and T chain can be released. In Figure 9 B, lanes 1-3 represent DNA molecular weight standard, H1 chain and H2 chain respectively. Lane 4 represents the mixture of H1 chain and H2 chain, and the new band of lag represents the formation of H1:H2 double strand. Lane 5 represents the mixture of H1 chain and acetamiprid transduced T chain, and the new band represents the formation of H1:T double strand. Lane 6 represents the product of DNA wheel nanostructure after Nease treatment, and the structure of DNA wheel nanostructure does not change. Lane 7 represents the product of H2 after Nease treatment, and lane 8 represents the product of H1:H2 double strand after Nease treatment, and new bands are formed, indicating the presence of dissociated DNA fragments.

[0078] Then, the construction of the sensing interface is characterized by photocurrent and ECL, and the results are as follows Figure 10and Figure 11 As shown in curve a, CdS@SiO2@NaYF4:Yb / Tm / ITO exhibits strong photocurrent and ECL signals. When CdS@SiO2@NaYF4:Yb / Tm / ITO is sequentially incubated with H1:H2 double strands (curve b) and the T strand transduced by acetamiprid (curve c), both photocurrent and ECL signals decrease, attributed to the weak conductivity of the DNA layer. Subsequently, after Nease treatment, the modified electrode shows enhanced photocurrent and ECL signals, indicating that after Nease cleavage of the H1:H2 double strands that did not form DNA ring nanostructures, some DNA fragments are moved away from the electrode surface. Finally, further reaction of the assembled electrode within PtPd-CoSnO3 nanocubes significantly reduces both photocurrent and ECL signals, demonstrating the quenching effect of PtPd-CoSnO3 nanocubes on photocurrent and ECL signals. The above photocurrent and ECL results effectively demonstrate the construction process of the electrode sensing interface.

[0079] (3) The detection performance of the biosensor was verified by using different concentrations of acetamiprid (0, 1 fM, 5 fM, 150 fM, 1.5 pM, 15 pM, 150 pM and 1 nM) according to the usage method described in Example 1.

[0080] Photocurrent and ECL response curves of different concentrations of acetamiprid are shown below. Figure 12 and 13 As shown, it can be seen that with the increase of acetamiprid concentration, the photocurrent ( Figure 12 A) and ECL signal ( Figure 13 A) The intensity gradually decreases. The linear relationship between the logarithmic concentration of acetamiprid and the change in photocurrent intensity (the difference in photocurrent values ​​before and after target incubation) is as follows: Figure 12 As shown in B, the linear regression equation is ΔI = 157.4lg c + 2296.2(R²). 2 =0.997). Based on the rule of 3 times relative standard deviation, the detection limit is calculated to be 0.31 fM. Similarly, in Figure 13 In B, the linear relationship between the logarithmic concentration of acetamiprid and the change in ECL signal intensity is ΔECL=1.03lg c+14.81(R 2 =0.995), the detection limit is 0.42 fM (S / N = 3). From Figure 14 It was observed that, compared to the response of the blank phosphate buffer in both PEC and ECL modes, the introduction of individual interfering substances (chlorpyrifos, trichlorfon, imidacloprid, and atrazine) did not lead to significant changes. Conversely, only acetamiprid and mixtures of the aforementioned interfering substances with acetamiprid significantly reduced the photocurrent and ECL signal. The specific recognition of acetamiprid by the aptamer and the magnetic bead separation technology ensured the anti-interference capability of this biosensor.

[0081] Example 3

[0082] This example used cucumbers and peaches (No. 1-3) to verify the detection performance of the biosensor of Example 1. The specific process is as follows:

[0083] The surface of cucumbers and peaches was cut into 1cm x 1cm square specimens, and 10μL of 100pM acetamiprid was evenly sprayed on the surface of the specimens. After 30min of reaction at 25℃, 1mL of acetonitrile was added, ultrasonic treatment was performed for 30min, 0.22μm membrane filtration was performed, and rotary evaporation was performed at 65℃ to obtain concentrated acetamiprid for PEC and ECL method detection. As a control, the acetamiprid sample was also tested by high performance liquid chromatography (HPLC). The results are shown in Table 1, and compared with the results of standard method HPLC detection, the concentration error is less than 5%. It shows that the biosensor designed in the application has good detection ability and application for acetamiprid in food.

[0084] Table 1. Analysis of acetamiprid residues on the surface of cucumbers and peaches.

[0085]

[0086] The above-described examples are only to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the spirit of the design of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A method for the preparation of a confined enhanced photoelectrochemical- electrochemiluminescence dual mode biosensor, characterized in that, The method comprises the following steps: After the indium tin oxide electrode (ITO) is cleaned, CdS@SiO2@NaYF4:Yb / Tm nanomaterials are added dropwise to the surface of the electrode to obtain a CdS@SiO2@NaYF4:Yb / Tm / ITO electrode; The CdS@SiO2@NaYF4:Yb / Tm / ITO electrode is sequentially immersed in H1:H2 double-stranded, acetamiprid-transduced T-chain solution, T4 DNA ligase and Nt.CviPII endonuclease, and incubated to initiate the formation of DNA rolling nanometer structures; The obtained sensing interface is immersed in a PtPd-CoSnO3 nanocube solution for incubation to obtain the biosensor; The CdS@SiO2@NaYF4:Yb / Tm nanomaterials have a core-shell structure, in which NaYF4:Yb / Tm is the core, and SiO2 and CdS nanoparticles are sequentially coated on the core; The H1:H2 double-stranded solution is obtained by incubation of H1 chains labeled with 3' phosphate groups and H2 chains modified with 3' carboxyl groups; The nucleotide sequence of the H1 chain is shown in SEQ ID NO. 1, and the nucleotide sequence of the H2 chain is shown in SEQ ID NO. 2; The acetamiprid-transduced T chain is obtained by combining aptamer recognition and magnetic bead separation technology; The nucleotide sequence of the aptamer chain is shown in SEQ ID NO. 3, and the nucleotide sequence of the T chain is shown in SEQ ID NO. 4; The PtPd-CoSnO3 nanocube is synthesized by a hydrothermal method, and PtPd nanoparticles are grown in situ on the CoSnO3 nanocube.

2. The production method according to claim 1, characterized by, The preparation method of the CdS@SiO2@NaYF4:Yb / Tm nanomaterials comprises the following steps: YCl3·6H2O, YbCl3·6H2O, TmCl3·6H2O and NaF are sequentially added to an ethylenediaminetetraacetic acid solution, and reacted at 180℃ for 12 hours to obtain NaYF4:Yb / Tm; NaYF4:Yb / Tm is dispersed in an ammonia-ethanol mixed solution, and tetraethyl orthosilicate is added and reacted for 1 hour to obtain SiO2@NaYF4:Yb / Tm; SiO2@NaYF4:Yb / Tm is mixed with sodium citrate, Cd(NO3)2·4H2O, ammonia and thiourea, and reacted at 80℃ for 3 hours to obtain CdS@SiO2@NaYF4:Yb / Tm.

3. The preparation method according to claim 2, characterized in that, The molar ratio of YCl3·6H2O, YbCl3·6H2O and TmCl3·6H2O is 20:8:

1.

4. The production method according to claim 1, characterized by, The acetamiprid-transduced T chain is obtained by incubation of aptamer, T chain modified magnetic beads and acetamiprid.

5. The preparation method according to claim 1, characterized in that, The preparation method of the PtPd-CoSnO3 nanocube comprises the following steps: 238mg of CoCl2·6H2O and 294mg of sodium citrate are dissolved in 30mL of deionized water to obtain solution A; 351mg of SnCl4·5H2O is dissolved in 5mL of ethanol to obtain solution B; After mixing solution A and B, 5 mL NaOH (2.0 M) was added, and the reaction was carried out for 1 h, and then the reaction was further annealed at 500 ℃ for 2 h to obtain PtPd-CoSnO3 nanocubes.

6. The method of claim 1, wherein the biological sensor is a glucose sensor. The method comprises the following steps: After cleaning the ITO electrode, 20 μL of CdS@SiO2@NaYF4:Yb / Tm nanosolution was added dropwise, and CdS@SiO2@NaYF4:Yb / Tm / ITO was obtained after drying. The CdS@SiO2@NaYF4:Yb / Tm / ITO was immersed in H1:H2 double-stranded DNA, and the reaction was carried out at 37 ℃ for 2 h to obtain H1:H2 / CdS@SiO2@NaYF4:Yb / Tm / ITO. The H1:H2 / CdS@SiO2@NaYF4:Yb / Tm / ITO was incubated with the T chain transduced by acetamiprid to form a DNA wheel nanostructure, T4 DNA ligase was added to connect the gap, and Nt.CviPII NEase was used for treatment. The obtained electrode was incubated with PtPd-CoSnO3 nanocubes, the quencher was introduced into the sensing interface through an amide bond, and the biosensor was obtained.

7. The production method according to claim 6, wherein The concentration of the CdS@SiO2@NaYF4:Yb / Tm is 0.5 mg / mL, and the concentration of the Nt.CviPII Nease is 0.5 unit / μL.

8. The preparation method according to claim 6, characterized in that, The H1:H2 double-stranded DNA is obtained by mixing H1 and H2 in equimolar amounts and incubating at 37 ℃ for 1 h; and the H2 chain is activated by 10 mM tris(2-carboxyethyl)phosphine hydrochloride for 1 h before use.

9. The biosensor prepared according to the preparation method of any one of claims 1-8 for detecting the content of acetamiprid, characterized in that, The sensing interface is applied to the detection of PEC and ECL signals in sequence, and according to the changes in photocurrent (ΔI) and ECL intensity (ΔECL), the double-mode analysis of acetamiprid in food is realized.

10. Use according to any one of claims 6 to 9, characterized in that, The sample to be detected includes fruit and vegetable acetamiprid residue samples.

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