Electrochemical luminescence sensor and application of electrochemical luminescence sensor in multiple detection of malathion and phorate

Abstract

The invention belongs to the technical field of food safety detection, and particularly relates to an electrochemical luminescence sensor and application of the electrochemical luminescence sensor in multiple detection of malathion and phorate. In order to detect a target object with high sensitivity and high selectivity, the preparation method comprises the following steps: firstly, synthesizing Co-PTC MOF by taking perylene tetracarboxylic acid potassium as a ligand, and preparing an Ag-Co-PTC composite material on the surface of the Co-PTC MOF through photo-reduction of AgNO3; the electrochemical luminescence biosensor based on the Ag-Co-PTC composite material is constructed by taking Ag-Co-PTC as an electrochemical luminescence signal probe and combining CRISPR / Cas12a and a spherical nucleic acid conversion technology, so that the dual detection of malathion and phorate is realized, and the detection limits are 1.08 * 10 <-13 > M and 1.01 * 10 <-12 > M.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety detection, and particularly relates to an electrochemiluminescence sensor and its application in multiple detection of malathion and phorate. Background Art

[0002] Malathion and phorate are two common organophosphorus pesticides, often used in agricultural production to control pests and increase crop yields. Excessive use of organophosphorus pesticides can easily lead to their residues in agricultural products, which accumulate in the food chain and ultimately enter the human body, posing a serious threat to food safety and public health. Toxicological studies have shown that these pesticides, upon entering organisms, irreversibly bind to cholinesterase, inhibiting its physiological function of catalyzing the hydrolysis of acetylcholine. This in turn triggers abnormal accumulation of neurotransmitters, ultimately leading to dysfunction of the central and peripheral nervous systems and affecting vital functions. In actual agricultural practices, mixed pesticides are often used to ensure maximum crop yields. This inevitably leads to multiple residues of organophosphorus pesticides in food and the environment, making multiplex detection of organophosphorus pesticides crucial. Commonly used multiplex organophosphorus determination methods, such as chromatography and immunoassays, require expensive instrumentation, complex procedures, and specialized personnel, limiting their application in certain scenarios. Therefore, there is a need to develop a rapid, sensitive, and accurate analytical method for the multiplex detection of organophosphorus pesticides in food.

[0003] Electrochemiluminescence (ECL) sensors are an emerging detection technology that generates light signals through electrochemical triggering. The intensity of the light signal is proportional to the concentration of the target, thereby enabling the determination of the target concentration. Compared with traditional analytical methods, ECL offers many unique advantages, such as low detection limits, wide detection range, fast detection speed, low cost, and easy integration with molecular labeling technologies. Furthermore, it does not require excitation from an external light source, reducing interference from light scattering and luminescent impurities. Therefore, ECL analysis has very low optical background noise. Currently, ECL has been widely used in clinical biomolecule detection, immunoassays, and food safety.

[0004] The present invention aims to construct an electrochemiluminescence biosensor to achieve dual detection of trace amounts of malathion and phorate in food samples. Summary of the Invention

[0005] To address these challenges, the present invention provides an electrochemiluminescent biosensor for the dual detection of malathion and phorate. This sensor utilizes the CRISPR / Cas12a system and a spherical nucleic acid conversion strategy to achieve highly sensitive and specific detection of malathion and phorate. The method first prepares potassium perylenetetracarboxylate (K4PTC) by KOH hydrolysis of 3,4,9,10-perylenetetracarboxylic dianhydride (PTCDA), which is then used as a ligand to synthesize a Co-PTC metal-organic framework (MOF). Next, AgNPs are synthesized on the MOF surface via photoreduction. Finally, AgNPs@Co-PTC is used as the substrate and layer-by-layer modified with Ag@Co-PTC / GCE to construct an electrochemiluminescent biosensor platform.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0007] The present invention provides an electrochemiluminescence sensor, which uses a Pt electrode as a counter electrode, Ag / AgCl as a reference electrode, and a modified glassy carbon electrode as a working electrode. The modified glassy carbon electrode is prepared by the following method:

[0008] Step 1, preparing AgNPs@Co-PTC nanocomposite materials;

[0009] Step 2, dispersing the prepared AgNPs@Co-PTC nanocomposite material in ultrapure water to obtain an AgNPs@Co-PTC composite material dispersion;

[0010] Step 3: The AgNPs@Co-PTC composite material dispersion was droplet-coated on the clean GCE surface and dried to obtain AgNPs@Co-PTC / GCE.

[0011] Step 4: The cDNA@Mal-Apt solution was drop-coated on the surface of AgNPs@Co-PTC / GCE, capped, and incubated to obtain cDNA@Mal-Apt / AgNPs@Co-PTC / GCE.

[0012] Step 5: Take the MCH solution and drop it onto the surface of cDNA@Mal-Apt / AgNPs@Co-PTC / GCE. After incubation, rinse it and dry it to obtain MCH / cDNA@Mal-Apt / AgNPs@Co-PTC / GCE.

[0013] Furthermore, the method for preparing the AgNPs@Co-PTC nanocomposite material in step 1 is as follows:

[0014] Step 1.1, dissolve K4PTC and Co(NO3)2·6H2O in ultrapure water respectively;

[0015] Step 1.2, adding the Co(NO3)2·6H2O aqueous solution to the K4PTC aqueous solution under stirring to carry out the reaction;

[0016] Step 1.3, collecting the product obtained in step 1.2 by centrifugation, washing with ultrapure water, and vacuum drying to obtain Co-PTC;

[0017] Step 1.4, disperse Co-PTC in anhydrous ethanol and dissolve AgNO3 in DMF;

[0018] Step 1.5, add the DMF solution containing AgNO3 to the ethanol solution of Co-PTC, irradiate with white light under stirring, and let it stand after the reaction is completed;

[0019] In step 1.6, the product was collected by centrifugation, washed with ultrapure water, and dried to obtain AgNPs@Co-PTC composite material.

[0020] Furthermore, the concentration of the AgNPs@Co-PTC composite material dispersion in step 2 is 0.5 mg / mL.

[0021] Furthermore, the concentration of the cDNA@Mal-Apt solution in step 4 is 1 μM; the incubation temperature is 37° C., and the time is 20 to 70 minutes;

[0022] The sequence of cDNA is: 5'-SH-ATACGGGAGCCAACACCAGC-3'; the sequence of Mal-Apt is: 5'-AACTTCTTGACTGCTGGTGTTGGCTCCCGTAT-3'.

[0023] Furthermore, the concentration of the MCH solution in step 5 is 2 mM; the incubation temperature is room temperature, and the incubation time is 50 min.

[0024] The present invention also provides an application of an electrochemiluminescence sensor for detecting malathion and phorate.

[0025] Furthermore, the method for detecting malathion is specifically:

[0026] 1) Malathion solutions of different concentrations were dropped onto the surface of MCH / cDNA@Mal-Apt / AgNPs@Co-PTC / GCE, rinsed, and dried;

[0027] 2) Drop TNAK buffer onto the electrode surface obtained in step 1) and incubate, rinse and dry;

[0028] 3) Using a three-electrode system, the Pt electrode serves as the counter electrode, the Ag / AgCl electrode serves as the reference electrode, and the electrode obtained in step 2) serves as the working electrode. Based on the ECL responses corresponding to different target concentrations, malathion and α-aminobutyric acid in the sample are quantitatively analyzed.

[0029] Furthermore, in step 2), the concentration of TNAK buffer is 20 mM, pH=7.4, and contains 1.5 μM BHQ1-DNA, and the sequence of the BHQ1-DNA is: 5'-BHQ1-TTTTTTTTGCTGGTGTTGGCTCCCGT AT-3';

[0030] The incubation time is 60 min.

[0031] Furthermore, the method for detecting phorate is specifically:

[0032] 1) Malathion solution was dropped onto the surface of MCH / cDNA@Mal-Apt / AgNPs@Co-PTC / GCE, incubated, rinsed, and dried;

[0033] 2) TNAK buffer was dripped onto the electrode surface obtained in step 1) for incubation, rinsed and dried to obtain MCH / cDNA@BHQ1-DNA / AgNPs@Co-PTC / GCE;

[0034] 3) Adding EDC and NHS to the carboxyl-modified Fe3O4 magnetic nanoparticles to activate the carboxyl groups on the surface of the magnetic beads to obtain carboxylated magnetic beads;

[0035] 4) Incubate Pho-Apt with activated DNA and shake LPho-Apt@aDNA with carboxylated magnetic beads to form SNA;

[0036] 5) Add different concentrations of phorate to the SNA dispersion, incubate, and obtain the aDNA-containing supernatant by magnetic separation;

[0037] 6) Activation of Cas12a: Cas12a, crRNA, NEB buffer, and RNA-free water were mixed and incubated to form a Cas12a / crRNA complex. The complex was mixed with the supernatant containing aDNA, and the mixture was dropped onto MCH / cDNA@BHQ1-DNA / AgNPs@Co-PTC / GCE, incubated, rinsed, and dried.

[0038] 7) Using a three-electrode system, a Pt electrode was used as the counter electrode, an Ag / AgCl electrode was used as the reference electrode, and the electrode obtained in step 6) was used as the working electrode. The phorate in the sample was quantitatively analyzed based on the ECL response corresponding to different target concentrations.

[0039] Furthermore, the concentration of the malathion solution in step 1) is 10 -6 M, incubation time was 60 min;

[0040] In step 2), the concentration of TNAK buffer is 20 mM, pH = 7.4, and contains 1.5 μM BHQ1-DNA, wherein the sequence of the BHQ1-DNA is: 5'-BHQ1-TTTTTTTTGCTGGTGTTGGCTCCCGT AT-3', and the incubation time is 60 min;

[0041] In step 4), the sequence of Pho-Apt is: 5'-NH2-AGCTTGCTGCAGCGATTCTTGATCGCCACAGAGCT-3'; the sequence of aDNA is: 5'-CCCAGGTAAACACACAAACCTTAATCGCTGCAGCAAGCT-3'. The incubation temperature is 37°C and the incubation time is 2 h.

[0042] In step 5), the incubation temperature is 37°C and the incubation time is 2 hours;

[0043] The sequence of crRNA in step 6) is: 5'-UAAUUUCUACUAAGUGUAGAUAAGGUUUGUGUGUUUACCUG-3', the volume ratio of Cas12a, crRNA, NEB buffer, RNA-free-water and aDNA-containing supernatant is 6:7.2:6:10.8:90, the temperature of the first incubation is 37 ° C, the incubation time is 10 min, and the cutting time is 80 min.

[0044] Compared with the prior art, the present invention has the following advantages:

[0045] (1) This invention is the first to use Ag@Co-PTC materials in the field of electrochemiluminescent biosensors, and construct an electrochemiluminescent sensor based on CRISPR / Cas12a reaction to detect malathion and phorate. It focuses on solving the shortcomings of the current chromatography-based detection of organophosphorus pesticides, such as cumbersome sample pretreatment process, expensive instruments, and complex operations. It enriches the application of MOF in the field of sensing and provides new ideas and methods for the construction of highly sensitive and specific Ag@Co-PTC MOF electrochemiluminescent biosensors.

[0046] (2) This invention combines CRISPR / Cas12a technology with electrochemiluminescence for the first time and is used in the field of multiplex detection of non-nucleic acid targets. Using Ag@Co-PTC as a single signal probe avoids the mutual interference between multiple signals in multiplex detection. At the same time, combined with SNA technology, target conversion is achieved, and biological signals are converted into readable optical signals, realizing the analysis and determination of trace targets in samples. This provides ideas and methods for electrochemiluminescence biosensors. This invention can achieve high sensitivity for malathion and phorate in actual samples (detection limits are 1.08×10 -13 M and 1.01×10 -12 M. Highly specific detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Fluorescence (curve a) and electrochemiluminescence spectra (curve b) of Co-PTC;

[0048] Figure 2 The effect of cDNA incubation time;

[0049] Figure 3 is the effect of BHQ1-DNA incubation concentration;

[0050] Figure 4 The effect of Cas12a cleavage time;

[0051] Figure 5 Cyclic voltammograms of different modified electrodes, including (a) bare GCE, (b) Ag@Co-PTC / GCE, (c) cDNA-Mal-Apt / Ag@Co-PTC / GCE, (d) MCH / cDNA@Mal-Apt / Ag@Co-PTC / GCE, (e) MCH / cDNA / Ag@Co-PTC / GCE, (f) MCH / cDNA@ BHQ1-DNA / Ag@Co-PTC / GCE, and (g) MCH / cDNA@ BHQ1-DNA / Ag@Co-PTC / GCE after Cas12 cleavage;

[0052] Figure 6The electrochemical impedance spectroscopy (EEIS) of different modified electrodes are shown in Figure 3, where (a) is bare GCE, (b) is Ag@Co-PTC / GCE, (c) is cDNA-Mal-Apt / Ag@Co-PTC / GCE, (d) is MCH / cDNA@Mal-Apt / Ag@Co-PTC / GCE, (e) is MCH / cDNA / Ag@Co-PTC / GCE, (f) is MCH / cDNA@ BHQ1-DNA / Ag@Co-PTC / GCE, and (g) is MCH / cDNA@ BHQ1-DNA / Ag@Co-PTC / GCE after cleavage by Cas12.

[0053] Figure 7 Electrochemiluminescence signals of different modified electrodes, including (a) bare GCE, (b) Co-PTC / GCE, (c) Ag@Co-PTC / GCE, (d) GCE introduced with BHQ1, and (e) GCE loaded with BHQ1 after cleavage by Cas12a.

[0054] Figure 8 is the electrochemiluminescence response of the sensor to different concentrations of malathion, where a~g represent concentrations of 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM, respectively;

[0055] Figure 9 The calibration curve corresponding to the electrochemiluminescence response of the sensor to different concentrations of malathion;

[0056] Figure 10 is the electrochemiluminescence response of the sensor to different concentrations of phorate, where a~g represent concentrations of 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM, respectively;

[0057] Figure 11 is the calibration curve corresponding to the electrochemiluminescence response of the sensor to different concentrations of phorate;

[0058] Figure 12 The experimental results of the sensor's selectivity to malathion are shown;

[0059] Figure 13 These are the experimental results of the sensor's selectivity for phorate. DETAILED DESCRIPTION

[0060] In order to further illustrate the technical solution of the present invention, the present invention is further described below through examples.

[0061] Example 1

[0062] Step 1, preparing AgNPs@Co-PTC nanocomposite materials;

[0063] Preparation of K4PTC: Weigh 0.394 g of PTCDA and ultrasonically dissolve it in 5 mL of ultrapure water. Add 0.280 g of KOH to the above solution and continue ultrasonicating until it is completely dissolved. Transfer the reaction system to a magnetic heating stirrer and stir at 55°C, 1000 rpm for 12 h. Then add 30 mL of ethanol, collect the product by centrifugation, wash it three times with ethanol, and dry it at 40°C to obtain a brown-yellow powder.

[0064] Preparation of Co-PTC: Weigh 0.186 g of K4PTC and 0.117 g of Co(NO3)2·6H2O, dissolve them in 25 mL and 5 mL of ultrapure water, respectively. Add the Co(NO3)2·6H2O aqueous solution to the K4PTC aqueous solution under stirring conditions, and stir the reaction at room temperature for 12 h. The resulting product is collected by centrifugation, washed five times with ultrapure water, and finally dried in vacuum at 50°C to obtain Co-PTC.

[0065] Preparation of Ag@Co-PTC: 10 mg of Co-PTC was weighed and dispersed in 9 mL of anhydrous ethanol. Simultaneously, 0.168 g of AgNO₃ was dissolved in 1 mL of dimethylformamide (DMF). The AgNO₃-DMF solution was added to the Co-PTC ethanol solution and irradiated with 9 W white light for 1 h while stirring. The product was collected by centrifugation, washed three times with ultrapure water, and dried at 50°C to obtain the Ag@Co-PTC composite.

[0066] Step 2, dispersing the prepared AgNPs@Co-PTC nanocomposite material in ultrapure water to obtain an AgNPs@Co-PTC composite material dispersion;

[0067] 0.5 mg of the prepared Ag@Co-PTC composite material was placed in a centrifuge tube, 1 mL of ultrapure water was added, and ultrasonic dispersion was performed for 5 min to obtain a 0.5 mg / mL Ag@Co-PTC dispersion.

[0068] Step 3: The AgNPs@Co-PTC composite material dispersion was droplet-coated on the clean GCE surface and dried at 45°C to obtain AgNPs@Co-PTC / GCE.

[0069] Use a small amount of aluminum oxide polishing powder to sprinkle on the suede, wet the suede with distilled water, and rinse the electrode surface with ultrapure water after polishing. 3+ / 4+ Measure CV and control ΔEp between 80 and 100 mV.

[0070] 5 μL of the prepared 0.5 mg / mL Ag@Co-PTC dispersion was pipetted with a pipette and drop-coated on the polished GCE electrode surface and dried at 45°C to obtain the Ag@Co-PTC modified electrode.

[0071] Step 4: 5 μL of 1 μM cDNA@Mal-Apt solution was drop-coated on the surface of AgNPs@Co-PTC / GCE, capped, and incubated at 37°C for 20–70 min. Unbound cDNA@Mal-Apt on the surface was washed with TNaK buffer to obtain cDNA@Mal-Apt / AgNPs@Co-PTC / GCE.

[0072] Step 5: 5 μL of 2 mM MCH solution was dropped onto the surface of cDNA@Mal-Apt / AgNPs@Co-PTC / GCE. After incubation for 40 min, the surface was rinsed and dried to obtain MCH / cDNA@Mal-Apt / AgNPs@Co-PTC / GCE.

[0073] Step 6: Malathion (Mal) solutions (diluted by Tri-HCl) with different concentrations were dropped onto the surface of MCH / cDNA@Mal-Apt / AgNPs@Co-PTC / GCE for 60 min, rinsed, and dried;

[0074] Step 7, drop 5 μL of TNAK buffer (20 mM, pH = 7.4, containing 1.5 μM BHQ1-DNA) onto the electrode surface obtained in step 1) and incubate for 60 min, rinse and dry. The sequence of the BHQ1-DNA is: 5'-BHQ1-TTTTTTTTGCTGGTGTTGGCTCCCGTAT-3';

[0075] Step 8, using a three-electrode system for testing, with a Pt electrode as the counter electrode, an Ag / AgCl electrode as the reference electrode, and the electrode obtained in step 7 as the working electrode, to quantitatively analyze malathion and in the sample based on the ECL response corresponding to different target concentrations;

[0076] Step 9, 10 -6 M malathion solution as a cofactor was dropped onto the surface of MCH / cDNA@Mal-Apt / AgNPs@Co-PTC / GCE and incubated for 60 min, then rinsed and dried;

[0077] Step 10: 5 μL of TNAK buffer (20 mM, pH = 7.4, containing 1.5 μM BHQ1-DNA) was dropped onto the electrode surface obtained in step 9 and incubated for 60 min. The mixture was then rinsed and dried to obtain MCH / cDNA@BHQ1-DNA / AgNPs@Co-PTC / GCE. The sequence of the BHQ1-DNA was: 5'-BHQ1-TTTTTTTTGCTGGTGTTGGCTCCCGT AT-3';

[0078] Step 11, activation of the surface groups of the magnetic beads: add 10 mM EDC and 20 mM NHS to 1 mL of carboxyl-modified Fe3O4 magnetic nanoparticles (MB) to activate the carboxyl groups on the surface of the magnetic beads to obtain carboxylated magnetic beads;

[0079] Step 12, preparation of spherical nucleic acids: 25 μL of Pho-Apt and 25 μL of activated DNA (aDNA) were incubated at 37°C for 2 h to form a dsDNA structure. 50 μL of 4 μM LPho-Apt@aDNA and carboxylated magnetic beads were shaken at room temperature for 3 h to form SNAs. The sequence of the Pho-Apt is: 5'-NH2-AGCTTGCTGCAGCGATTCTTGATCGCCACAGAGCT-3'; the sequence of the aDNA is: 5'-CCCAGGTAAACACACAAACCTTAATCGCTGCAGCAAGCT-3';

[0080] Step 13, aDNA enrichment: different concentrations of phorate were added to 50 μL of SNA dispersion, incubated at 37°C for 2 h, and magnetic separation was performed to obtain the aDNA-containing supernatant;

[0081] Step 14, activation of Cas12a: 6 μL Cas12a, 7.2 μL CrRNA, 6 μL NEB buffer and 10.8 μL RNA-free-water were mixed and incubated at 37 ° C for 10 min to form a Cas12a / CrRNA complex. The Cas12a / CrRNA complex was mixed with 90 μL of supernatant containing aDNA, and the mixture was dropped onto MCH / cDNA@BHQ1-DNA / AgNPs@Co-PTC / GCE. After cutting for 0-100 min and rinsing and drying, the sequence of the CrRNA was: 5'-UAAUUUCUACUAAGUGUAGAUAAGGUUUGUGUGUUUACCUG-3';

[0082] Step 15: Use a three-electrode system to perform the test, with the Pt electrode as the counter electrode, the Ag / AgCl electrode as the reference electrode, and the electrode obtained in step 14 as the working electrode. Quantitative analysis of phorate in the sample is performed based on the ECL response corresponding to different target concentrations.

[0083] Example 2

[0084] The optical properties of the nanomaterials were characterized. The results showed that ( Figure 1 ), under 365 nm excitation light, the fluorescence emission peak of Co-PTC appears at 483 nm (curve a), while its ECL emission peak is located at 535 nm (curve b).

[0085] Example 3

[0086] In a Chenhua CHI 830D electrochemical workstation, a three-electrode system was constructed with a platinum electrode as the counter electrode, an Ag / AgCl electrode as the reference electrode, and the modified electrode as the working electrode. CV (A) and EIS (B) tests were performed on different modification processes. In the presence of 5.0 mM [Fe(CN)6] 3- / 4- The test results in 0.1 M PBS solution are as follows Figure 5 and Figure 6 , where (a) is bare GCE, (b) is Ag@Co-PTC / GCE, (c) is cDNA-Mal-Apt / Ag@Co-PTC / GCE, (d) is MCH / cDNA@Mal-Apt / Ag@Co-PTC / GCE, (e) is MCH / cDNA / Ag@Co-PTC / GCE, (f) is MCH / cDNA@ BHQ1-DNA / Ag@Co-PTC / GCE, and (g) is MCH / cDNA@ BHQ1-DNA / Ag@Co-PTC / GCE after cleavage by Cas12. The results show that ( Figure 5 ), the current of Ag@Co-PTC loaded on the GCE electrode surface decreased accordingly, which is caused by the poor conductivity of MOF. Ag also improved the current response, but it still did not exceed that of bare GCE; cDNA@Mal-Apt was loaded on the Ag@Co-PTC / GCE electrode and bound to the electrode through Au-S, which hindered the electron transfer on the electrode surface; after the introduction of malathion, Mal-Apt was taken away from the electrode surface and the peak current increased; the introduction of BHQ1-DNA, due to its non-electroactive substance, reduced the peak current; after BHQ1 was removed, the current increased slightly. EIS ( Figure 6 ) were consistent with the results of CV, indicating the successful construction of the sensor.

[0087] Example 4

[0088] In a BPCL-ECL 150 electrochemiluminescence analyzer, a three-electrode system consisting of a platinum electrode as a counter electrode, an Ag / AgCl electrode as a reference electrode, and a modified electrode as a working electrode was used to perform ECL tests on different modification processes. The detection results in a 0.1 M PBS solution containing 20 mM K2SO4 are shown in Figure 2. Figure 7 As shown, (a) is bare GCE, (b) is Co-PTC / GCE, (c) is Ag@Co-PTC / GCE, (d) is cDNA@BHQ1-DNA / Ag@Co-PTC / GCE, and (e) is cDNA@BHQ1-DNA / Ag@Co-PTC / GCE treated with activated Cas12a. The results show that ( Figure 7 ): Bare GCE produces almost no ECL signal due to the lack of ECL active substances; Co-PTC / GCE produces a strong ECL signal due to the presence of ECL active substances on the electrode surface; Ag on the Ag@Co-PTC / GCE electrode participates in the reaction as a co-reactant accelerator, thereby enhancing the ECL signal; when BHQ1 is introduced into the electrode, an ECL-RET reaction occurs between Co-PTC and Ag@Co-PTC, resulting in quenching of the ECL signal of Ag@Co-PTC; after being cut by Cas12a, BHQ1 detaches from the electrode, restoring the ECL signal; these processes all indicate the successful construction of the sensor.

[0089] Example 5

[0090] ECL test was performed on Ag@Co-PTC / GCE with different incubation times. The results showed that ( Figure 2 ), within 20-70 min, the ECL signal gradually weakened with the increase of incubation time, and remained basically stable after 60 min. Therefore, 60 min was selected as the optimal incubation time for cDNA@Mal-Apt.

[0091] Example 6

[0092] ECL test was performed on BHQ1-DNA with different concentrations. The results showed that ( Figure 3 ), within the range of 0.3~1.5 μM, with the increase of the added BHQ1-DNA concentration, the ECL gradually weakened and remained almost stable after 1.5 μM. Therefore, 1.5 μM BHQ1-DNA was selected as the optimal dropwise coating concentration.

[0093] Example 7

[0094] ECL test was performed on different side cutting times of Cas12a. The results showed that ( Figure 4), when the cutting time was 80 min, the ECL signal reached the highest, so 80 min was adopted as the optimal cutting time of Cas12a.

[0095] Example 8

[0096] A standard curve for an electrochemiluminescent biosensor for detecting malathion and phorate using CRISPR / Cas12a was established, as follows:

[0097] (1) Prepare the concentration of 10 -12 M, 10 -11 M, 10- 10 M, 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M of malathion solution, add step 6 of Example 1 respectively, and adopt steps 7~8.

[0098] (2) A three-electrode system was used for testing, with Pt electrode as the counter electrode, Ag / AgCl as the reference electrode, and BHQ1-DNA@cDNA / MCH / Ag@Co-PTC / GCE electrode as the working electrode.

[0099] ECL measurements were performed in 0.1 M PBS (pH = 7.4) buffer solution containing 20 mM K2SO4 at a scanning voltage of -1.8 V to 0 V, a scanning rate of 0.1 v / s, and a PMT of 850 V. The electrochemiluminescence values ​​of 1×10 -12 M~1×10 -6 A standard curve was drawn using the logarithmic values ​​of malathion concentration within the range of M.

[0100] (3) The results are as follows Figure 8 and 9 As shown in the figure, the ECL response value of the constructed sensor is proportional to the logarithm of the malathion concentration at 1×10 -12 M~1×10 -6 There is a good linear relationship within the range of M, and the linear equation is: y=-1559.7 lgc-7506.8, and the correlation coefficient R 2 =0.995.

[0101] (4) Prepare the concentration of 10 -11 M, 10 -10 M, 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, 10 -5M of phorate solution, add step 9 of Example 1 respectively, and adopt steps 10~15.

[0102] (5) A three-electrode system was used for the test, with Pt electrode as the counter electrode, Ag / AgCl as the reference electrode, and BHQ1-DNA@cDNA / MCH / Ag@Co-PTC / GCE electrode after Cas12a cleavage as the working electrode. ECL measurements were performed in a 0.1 M PBS (pH = 7.4) buffer solution containing 20 mM K2SO4 at a scanning voltage of -1.8 V to 0 V, a scanning rate of 0.1 v / s, and PMT = 850 V. The electrochemiluminescence value of 1×10 -11 M~1×10 -5 A standard curve was drawn using the logarithmic values ​​of phorate concentrations within the M range.

[0103] (6) The results are as follows Figure 10 and 11 As shown in the figure, the ECL response value of the constructed sensor is proportional to the logarithm of the phorate concentration in the range of 1×10 -11 M~1×10 -5 There is a good linear relationship within the range of M, and the linear equation is: y=1228.1 lgc+16352.1, and the correlation coefficient R 2 =0.992.

[0104] Example 8

[0105] In order to study the selectivity of the sensor to malathion, phorate, methamidophos, chlorpyrifos and dichlorvos (all at 10 -7 The malathion concentration selected was 10 -9 M) and other organophosphorus pesticides were used as interfering substances in ECL testing. The results showed that ( Figure 12 ), a lower ECL response signal can only be obtained when malathion exists in the environment, while the four interferents have little effect on the ECL signal of the sensor, which indicates that the constructed biosensor has a specific recognition function for malathion and can achieve specific detection of malathion among many organophosphorus pesticides.

[0106] Example 9

[0107] In order to study the selectivity of the sensor for phorate, malathion, methamidophos, chlorpyrifos and dichlorvos (all at 10 -6 M, the selected phorate concentration was 10 -8 M) and other organophosphorus pesticides were used as interferences in ECL testing. The results showed that ( Figure 13), the ECL signal can be restored only when phorate exists in the environment, while the four interferents have little effect on the ECL signal of the sensor, which indicates that the constructed biosensor has a specific recognition function for malathion and can achieve specific detection of malathion among many interferents.

[0108] The foregoing shows and describes the principal features and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the present invention is defined by the appended claims, not the foregoing description, and all variations that come within the meaning and range of equivalents of the claims are intended to be embraced therein.

[0109] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. An electrochemiluminescence sensor, characterized in that A Pt electrode was used as a counter electrode, Ag / AgCl was used as a reference electrode, and a modified glassy carbon electrode was used as a working electrode. The modified glassy carbon electrode was prepared by the following method: Step 1, preparing AgNPs@Co-PTC nanocomposite materials; Step 2, dispersing the prepared AgNPs@Co-PTC nanocomposite material in ultrapure water to obtain an AgNPs@Co-PTC composite material dispersion; Step 3: The AgNPs@Co-PTC composite material dispersion was droplet-coated on the clean GCE surface and dried to obtain AgNPs@Co-PTC / GCE. Step 4: The cDNA@Mal-Apt solution was drop-coated on the surface of AgNPs@Co-PTC / GCE, capped, and incubated to obtain cDNA@Mal-Apt / AgNPs@Co-PTC / GCE. Step 5: Take the MCH solution and drop it onto the surface of cDNA@Mal-Apt / AgNPs@Co-PTC / GCE. After incubation, rinse it and dry it to obtain MCH / cDNA@Mal-Apt / AgNPs@Co-PTC / GCE.

2. An electrochemiluminescence sensor according to claim 1, characterized in that The method for preparing the AgNPs@Co-PTC nanocomposite material in step 1 is as follows: Step 1.1, dissolve K4PTC and Co(NO3)2·6H2O in ultrapure water respectively; Step 1.2, adding the Co(NO3)2·6H2O aqueous solution to the K4PTC aqueous solution under stirring to carry out the reaction; Step 1.3, collecting the product obtained in step 1.2 by centrifugation, washing with ultrapure water, and vacuum drying to obtain Co-PTC; Step 1.4, disperse Co-PTC in anhydrous ethanol and dissolve AgNO3 in DMF; Step 1.5, add the DMF solution containing AgNO3 to the ethanol solution of Co-PTC, irradiate with white light under stirring, and let it stand after the reaction is completed; In step 1.6, the product was collected by centrifugation, washed with ultrapure water, and dried to obtain AgNPs@Co-PTC composite material.

3. The electrochemiluminescence sensor according to claim 1, characterized in that The concentration of the AgNPs@Co-PTC composite material dispersion in step 2 is 0.5 mg / mL.

4. The electrochemiluminescence sensor according to claim 1, characterized in that The concentration of the cDNA@Mal-Apt solution in step 4 is 1 μM; the incubation temperature is 37° C., and the time is 20 to 70 minutes; The sequence of the cDNA is: 5′-SH-ATACGGGAGCCAACACCAGC-3′; The sequence of Mal-Apt is: 5'-AACTTCTTGACTGCTGGTGTTGGCTCCCGTAT-3'.

5. The electrochemiluminescence sensor according to claim 1, characterized in that: The concentration of the MCH solution in step 5 is 2 mM; the incubation temperature is room temperature, and the incubation time is 50 min.

6. Use of an electrochemiluminescence sensor according to any one of claims 1 to 5, characterized in that: Used to detect malathion and phorate.

7. The use of an electrochemiluminescence sensor according to claim 6, characterized in that: The method for detecting malathion is specifically: 1) Malathion solutions of different concentrations were dropped onto the surface of MCH / cDNA@Mal-Apt / AgNPs@Co-PTC / GCE, rinsed, and dried; 2) Drop TNAK buffer onto the electrode surface obtained in step 1) and incubate, rinse and dry; 3) Using a three-electrode system, the Pt electrode serves as the counter electrode, the Ag / AgCl electrode serves as the reference electrode, and the electrode obtained in step 2) serves as the working electrode. Based on the ECL responses corresponding to different target concentrations, malathion and α-aminobutyric acid in the sample are quantitatively analyzed.

8. The use of an electrochemiluminescence sensor according to claim 7, characterized in that: In step 2), the concentration of TNAK buffer is 20 mM, pH=7.4, and contains 1.5 μM BHQ1-DNA, wherein the sequence of the BHQ1-DNA is: 5'-BHQ1-TTTTTTTTGCTGGTGTTGGCTCCCGTAT-3'; The incubation time is 60 min.

9. The use of an electrochemiluminescence sensor according to claim 6, characterized in that: The method for detecting phorate is specifically: 1) Malathion solution was dropped onto the surface of MCH / cDNA@Mal-Apt / AgNPs@Co-PTC / GCE, incubated, rinsed, and dried; 2) TNAK buffer was dripped onto the electrode surface obtained in step 1) for incubation, rinsed and dried to obtain MCH / cDNA@BHQ1-DNA / AgNPs@Co-PTC / GCE; 3) Adding EDC and NHS to the carboxyl-modified Fe3O4 magnetic nanoparticles to activate the carboxyl groups on the surface of the magnetic beads to obtain carboxylated magnetic beads; 4) Incubate Pho-Apt with activated DNA and shake LPho-Apt@aDNA with carboxylated magnetic beads to form SNA; 5) Add different concentrations of phorate to the SNA dispersion, incubate, and obtain the aDNA-containing supernatant by magnetic separation; 6) Cas12a, crRNA, NEB buffer, and RNA-free water were mixed and incubated to form a Cas12a / crRNA complex, which was then mixed with the supernatant containing aDNA. The mixture was dropped onto the MCH / cDNA@BHQ1-DNA / AgNPs@Co-PTC / GCE, cut, rinsed, and dried. 7) Using a three-electrode system, a Pt electrode was used as the counter electrode, an Ag / AgCl electrode was used as the reference electrode, and the electrode obtained in step 6) was used as the working electrode. The phorate in the sample was quantitatively analyzed based on the ECL response corresponding to different target concentrations.

10. The use of an electrochemiluminescence sensor according to claim 9, characterized in that: The concentration of the malathion solution in step 1) is 10 -6 M, incubation time was 60 min; In step 2), the concentration of TNAK buffer is 20 mM, pH = 7.4, and contains 1.5 μM BHQ1-DNA, wherein the sequence of the BHQ1-DNA is: 5'-BHQ1-TTTTTTTTGCTGGTGTTGGCTCCCGT AT-3', and the incubation time is 60 min; In step 4), the sequence of Pho-Apt is: 5'-NH2-AGCTTGCTGCAGCGATTCTTGATCGCCACAGAGCT-3'; the sequence of aDNA is: 5'-CCCAGGTAAACACACAAACCTTAATCGCTGCAGCAAGCT-3'. The incubation temperature is 37°C and the incubation time is 2 h. In step 5), the incubation temperature is 37°C and the incubation time is 2 hours; The sequence of crRNA in step 6) is: 5'-UAAUUUCUACUAAGUGUAGAUAAGGUUUGUGUGUUUACCUG-3', the volume ratio of Cas12a, crRNA, NEB buffer, RNA-free-water and aDNA-containing supernatant is 6:7.2:6:10.8:90, the temperature of the first incubation is 37 ° C, the incubation time is 10 min, and the cutting time is 80 min.

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