CN115753936A - Electrochemical luminescence biosensor for detecting organophosphorus pesticide, preparation method and application - Google Patents
Electrochemical luminescence biosensor for detecting organophosphorus pesticide, preparation method and application Download PDFInfo
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- CN115753936A CN115753936A CN202211479660.3A CN202211479660A CN115753936A CN 115753936 A CN115753936 A CN 115753936A CN 202211479660 A CN202211479660 A CN 202211479660A CN 115753936 A CN115753936 A CN 115753936A
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Abstract
The invention discloses an electrochemical luminescence biosensor for detecting organophosphorus pesticides, and a preparation method and application thereof, wherein the electrochemical luminescence biosensor comprises a glassy carbon electrode, and a cadmium compound quantum dot @ mesoporous polydopamine nano material, a nafion film layer, a molecular probe and complementary DNA which are sequentially modified on the surface of the glassy carbon electrode from inside to outside; the molecular probe comprises an aptamer with a 5' end modified amino group and a 3' end modified disulfide group, and a noble metal nanoparticle coupled with the 3' end of the aptamer, wherein the secondary structure of the aptamer is a hairpin structure; the complementary DNA and the molecular probe can be subjected to base complementary pairing, so that the organophosphorus compound can be rapidly and sensitively detected.
Description
Technical Field
The invention relates to the field of analyzing pesticide residue by using a novel functional nano material and an electrochemical biosensor, in particular to an electrochemical luminescence biosensor for detecting organophosphorus pesticide, a preparation method and application thereof.
Background
Organophosphorus pesticides (Ops) are one of the main types of pesticides, have broad spectrum, high efficiency and low price, and are widely applied to agricultural product planting of fruits and vegetables. However, in actual production, an unscientific application method or strict control of the use amount is not provided, a plurality of medicaments are often mixed for use, random mixing, increase of the use amount, increase of the use frequency and the like frequently occur according to the use condition of pesticide labels, and the medicament residue is more serious. The long-term intake of fruits and vegetables with overproof organophosphorus pesticide residues can cause serious influence on human health, such as weakening of phagocytic function of human leukocytes, poor liver capability, central nervous disorder and the like, and even cause the problems of canceration, variation and the like of cells. Therefore, the method has important and profound significance for strengthening the supervision of organophosphorus pesticide residues and ensuring food safety and human health.
At present, methods for detecting organophosphorus pesticides in fruit and vegetable agricultural products mainly comprise high performance liquid chromatography mass spectrometry (HPLC), liquid chromatography-mass spectrometry (HPLC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the like. Although the methods have the advantages of high sensitivity and accuracy, capability of measuring various components at one time and the like, large and expensive instruments and equipment are needed, the analysis procedure is complex, the detection period is long, the detection cost is high, professional technicians are needed for completing the detection, and the methods are difficult to be applied to the rapid detection and the field analysis of the pesticide residues.
The ECL (Electrochemiluminescence) analysis method combining electrochemistry and chemiluminescence has many unique advantages, such as rapidness, high sensitivity, wide linear range, low background signal, no need of any external light source, simplified optical device and the like, and is a rapidly developing technology. Therefore, the construction of a rapid and sensitive electrochemiluminescence biosensor for detecting organophosphorus pesticide residues has important research significance and market value.
Disclosure of Invention
In view of this, the present application provides an electrochemical luminescence biosensor for detecting organophosphorus pesticides, and a preparation method and applications thereof, which can rapidly and sensitively detect organophosphorus compounds.
In order to achieve the technical purpose, the following technical scheme is adopted in the application:
in a first aspect, the application provides an electrochemical luminescence biosensor for detecting organophosphorus pesticides, which comprises a glassy carbon electrode, and a cadmium compound quantum dot @ mesoporous polydopamine nano material, a nafion film layer, a molecular probe and complementary DNA which are sequentially modified on the surface of the glassy carbon electrode from inside to outside; the molecular probe comprises an aptamer with a 5' end modified amino group and a 3' end modified disulfide group, and a noble metal nanoparticle coupled with the 3' end of the aptamer, wherein the secondary structure of the aptamer is a hairpin structure; complementary DNA and molecular probes through base complementary pairing.
Preferably, the cadmium compound quantum dot @ mesoporous polydopamine nanomaterial is formed by growing an aqueous cadmium compound quantum dot in a porous of the mesoporous polydopamine nanomaterial.
Preferably, the cadmium compound quantum dots comprise one of cadmium telluride quantum dots, cadmium selenide quantum dots and cadmium sulfide quantum dots.
Preferably, the noble metal nanoparticles include one of gold nanoparticles, platinum nanoparticles, silver nanoparticles, gold-platinum bimetallic nanoparticles, and gold-silver bimetallic nanoparticles.
Preferably, the particle size of the cadmium compound quantum dots is 5-10nm.
Preferably, the preparation method of the cadmium compound quantum dot @ mesoporous polydopamine nano material comprises the following steps:
s1, mixing and dispersing block polyether F-127 and aqueous cadmium compound quantum dots in an ethanol water solution to obtain a uniform solution;
s2, sequentially adding dopamine hydrochloride and 1,3, 5-trimethylbenzene into the uniform solution, then adding ammonia water, and stirring for reaction to obtain an intermediate product;
s3, centrifuging and washing the intermediate product, dispersing the intermediate product in an ethanol water solution for ultrasonic treatment, and heating the treated product at 100 ℃ overnight under a sealed condition to obtain the cadmium compound quantum dot @ mesoporous polydopamine nano material.
In a second aspect, the present application provides a method for preparing an electrochemiluminescence biosensor for detecting organophosphorus pesticides, comprising the following steps:
K1. polishing and cleaning the glassy carbon electrode;
K2. dropwise adding the cadmium compound quantum dot @ mesoporous polydopamine nano-material solution on the surface of the polished and cleaned glassy carbon electrode, modifying the electrode, and airing at room temperature to obtain the cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode;
K3. dripping nafion solution on the surface of the cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode, airing at room temperature, and washing with water to obtain a nafion/cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode;
K4. dripping the molecular probe on the surface of the nafion/cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode, and incubating overnight to obtain the molecular probe/nafion/cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode;
K5. the sealing agent is dripped on the surface of the molecular probe/nafion/cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode, complementary DNA is dripped after the incubation overnight and the washing, and the incubation and the washing are carried out to obtain the complementary DNA/molecular probe/nafion/cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode, namely the electrochemical luminescence biosensor for detecting the organophosphorus pesticide.
Preferably, the blocking agent is a solution of 6-mercapto-1-hexanol.
In a third aspect, the application provides an application of an electrochemical luminescence biosensor for detecting an organophosphorus pesticide in detecting the organophosphorus pesticide, wherein a detected reference electrode is a silver/silver chloride electrode, a counter electrode is a platinum sheet electrode or a platinum wire electrode, and a working electrode is a complementary DNA/molecular probe/nafion/cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode.
Preferably, the excitation voltage is detected at 2.0 to 0V and the scan rate is 50-100mV/s.
The beneficial effect of this application is as follows:
the application applies the cadmium compound quantum dot @ mesoporous polydopamine composite material to the preparation of the electrochemical luminescence biosensor, the analysis performance of the sensor is obviously improved, and a novel method for detecting organophosphorus pesticide by the electrochemical luminescence biosensor is constructed, and the method can be easily realized in most laboratories without complex and fussy large-scale equipment support; the sensor prepared by the invention has the advantages of low cost, simple preparation steps and high detection speed, and can be applied to portable detection;
according to the method, the porous structure characteristics and the large specific surface area of the mesoporous polydopamine are utilized, so that the cadmium telluride quantum dots are wrapped and loaded in situ in the pores of the mesoporous polydopamine, the load of the cadmium telluride quantum dots is favorably improved, the cadmium telluride quantum dot @ mesoporous polydopamine composite material is prepared, the composite material is modified on the surface of an electrode as a substrate material, a nafion solution is used for fixing the composite material, the luminous signal is stable, and the composite material is applied to the construction of an electrochemical luminescence biosensor;
the molecular probe is an aptamer, has high specificity and affinity to organophosphorus pesticides, has a secondary structure of a hairpin structure, and is complementary-paired with a complementary DNA base, so that the detection linear range and sensitivity of the sensor are improved.
Drawings
FIG. 1 is a process for preparing an electrochemiluminescence biosensor;
FIG. 2 is a schematic diagram of the detection mechanism of the electrochemiluminescence biosensor;
FIG. 3 is an X-ray diffraction pattern of cadmium telluride quantum dots, mesoporous polydopamine, and cadmium telluride quantum dots @ mesoporous polydopamine;
FIG. 4 is a transmission electron micrograph of a cadmium telluride quantum dot;
FIG. 5 is a transmission electron micrograph of mesoporous polydopamine;
FIG. 6 is an EDS elemental analysis chart of cadmium telluride quantum dots @ mesoporous polydopamine;
FIG. 7 is a UV-visible spectrum of gold nanoparticles;
FIG. 8 is a graph of cyclic voltammetry and Electrochemiluminescence (ECL) cyclic stability of a nafion/cadmium telluride quantum dot @ mesoporous polydopamine/glassy carbon electrode in a 0.1mol/L potassium persulfate solution;
FIG. 9 is a plot of cyclic voltammetry characterization for the fabrication of an electrochemiluminescence biosensor;
FIG. 10 is a diagram of the feasibility verification of the prepared electrochemiluminescence biosensor for detecting organophosphorus pesticide.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The term abbreviation:
a glassy carbon electrode: GCE;
6-mercapto-1-hexanol solution: MCH;
carboxyl cadmium telluride quantum dots: cdTe-COOH QDs;
mesoporous polydopamine: MPDA;
sodium tellurite: na (Na) 2 TeO 3 ；
Hydrazine hydrate: n is a radical of hydrogen 2 H 4 ·H 2 O；
Thioglycolic acid: TGA;
1,3, 5-trimethylbenzene: TMB:
complementary DNA: ctDNA.
As shown in fig. 1, the electrochemical luminescence biosensor for detecting organophosphorus pesticides comprises a glassy carbon electrode, and a cadmium compound quantum dot @ mesoporous polydopamine nano material, a nafion film layer, a molecular probe and complementary DNA which are sequentially modified on the surface of the glassy carbon electrode from inside to outside.
In the scheme, the cadmium compound quantum dot @ mesoporous polydopamine nano material is formed by growing aqueous cadmium compound quantum dots in the pores of the mesoporous polydopamine nano material; the cadmium compound quantum dots comprise one of cadmium telluride quantum dots, cadmium selenide quantum dots and cadmium sulfide quantum dots, the particle size of the cadmium compound quantum dots is 5-10nm, and preferably, the cadmium compound quantum dots are water-soluble cadmium telluride.
The preparation method of the cadmium compound quantum dot @ mesoporous polydopamine nano material comprises the following steps:
s1, mixing and dispersing block polyether F-127 and aqueous cadmium compound quantum dots in an ethanol water solution to obtain a uniform solution;
s2, sequentially adding dopamine hydrochloride and 1,3, 5-trimethylbenzene into the uniform solution, then adding ammonia water, and stirring for reaction to obtain an intermediate product;
s3, centrifuging and washing the intermediate product, dispersing the intermediate product in an ethanol water solution for ultrasonic treatment, and heating the intermediate product at 100 ℃ overnight under a sealed condition to obtain the cadmium compound quantum dot @ mesoporous polydopamine nano material.
Taking cadmium telluride quantum dots @ mesoporous polydopamine as a cadmium compound quantum dots @ mesoporous polydopamine nano material as an example, the preparation method comprises the following steps:
1.0g of block polyether F-127 and 0.36-0.144 g of water-soluble cadmium telluride quantum dot are added into 50-100 mL of ethanol-water mixed solution and stirred vigorously to obtain a uniform solution of CdTe quantum dot dispersed in block polyether F-127. Then, 1.0g of dopamine hydrochloride was dissolved in the above solution, and 2.0mL of 1,3, 5-trimethylbenzene was added to the mixed solution. Then, 1.0-2.5 mL of ammonia water is added into the mixed solution of the reaction, and the mixture is stirred and reacted for 4-12 h. And finally, centrifuging and washing the mixed solution of ethanol and water for several times to obtain a product of mesoporous polydopamine particles, dispersing the product in a certain amount of mixed solution of water and ethanol again, performing ultrasonic treatment for a period of time, transferring the product into a sealed Teflon lining autoclave, and heating the product at 100 ℃ for 24 hours to obtain the cadmium telluride quantum dot @ mesoporous polydopamine composite material.
The preparation method of the water-soluble cadmium telluride quantum dot comprises the following steps: cadmium chloride semi- (pentahydrate) (45.672mg, 0.2mmol) and thioglycolic acid (46.06mg, 0.5mmol) are sequentially dispersed into 50mL of distilled water, and then NaOH solution is dropwise added to adjust the pH value of the Cd-TGA compound solution to 7.0-11.0. By mixing high purity N 2 Bubbling for 30min to deoxygenate the solution. Under the condition of violent stirring, sequentially adding sodium tellurite solution (0.01-0.04 mmol) and 50-85% hydrazine hydrate solution (0.1-0.5 mL), then refluxing, stirring and heating in an oil bath kettle (80-100 ℃ for 1-4 h) to obtain the thioglycolic acid end-capped cadmium telluride quantum dot. Finally, the crude water-soluble carboxyl cadmium telluride quantum dots (CdTe-COOH QDs) are purified by excessive ethanol centrifugation (8000-10000rpm, 5-10 min).Dispersing the washed water-soluble carboxyl cadmium telluride quantum dots (CdTe-COOH QDs) in deionized water, and storing at 4 ℃ in the dark.
The outermost layer of the cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode is modified with nafion so as to prevent the cadmium telluride quantum dot @ mesoporous polydopamine composite material from falling off from the surface of the glassy carbon electrode; the nafion solution is prepared by ethanol-water mixed solution, and the volume ratio of deionized water to ethanol is 1/9-9/1.
The aptamer (Aptmer, apt) is an oligonucleotide sequence (RNA or DNA) obtained by screening through a systematic evolution technology of exponential enrichment ligands, has strict recognition capability and high affinity with corresponding ligands, and has the advantages of wide range of target molecules, easy artificial synthesis, good stability, convenient modification and the like, the molecular probe of the scheme comprises the aptamer of which the 5 'end is modified with amino and the 3' end is modified with disulfide, the aptamer has high specificity on organophosphorus pesticides, the 5 'end of amino can form a covalent bond with a sulfonic group of nafion, and the aptamer is coupled with the 3' end of noble metal nanoparticles, and the secondary structure of the aptamer is a hairpin structure; preferably, the molecular probe is hairpin-DNA-AuNPs, the aptamer is a section of DNA, and the 5' end of the aptamer is modified with amino (-NH) 2 ) The 3' end is modified with sulfydryl (-SH), the amino at the 5' end is fixed on the surface of the electrode through a covalent bond formed by the amino and a sulfonic group of nafion, and the gold nanoparticle coupled aptamer forms an Au-S covalent bond through the sulfydryl at the 3' end of the aptamer; the noble metal nanoparticles comprise one of gold nanoparticles, platinum nanoparticles, silver nanoparticles, gold-platinum bimetallic nanoparticles and gold-silver bimetallic nanoparticles, and preferably, the noble metal nanoparticles are gold nanoparticles; auNPs are prepared by using chloroauric acid as a gold source, 200mL of ultrapure water is measured, 3mL of 1% chloroauric acid solution is added, and the mixture is cooled to 4 ℃ in a refrigerator. After 0.5mL of potassium carbonate solution (0.2 mol/L) is added at 4 ℃, stirring is continued and vigorous, 9mL of sodium borohydride (0.5 mg/mL) is added rapidly and vigorously and stirred for 5min. The resulting wine red solution was stored in a 4 ℃ freezer for future use. FIG. 7 is the ultraviolet-visible spectrum of gold nanoparticles, which has the ultraviolet characteristic absorption peak of gold nanoparticles at about 520nm, and the preparation of gold nanoparticlesThe preparation was successful.
The complementary DNA and the molecular probe form double-stranded DNA through base complementary pairing, and the double-stranded DNA has a rigid structure; complementary DNA and a molecular probe aptamer can be modified on the surface of an electrode through base complementary pairing, double-stranded DNA has a rigid structure, the existence of the complementary DNA is beneficial to reducing blank background signals of the sensor, the linear range of the sensor is wider, the detection flexibility of the sensor is improved, and finally, the preparation of the electrochemical luminescence biosensor is completed and the electrochemical luminescence biosensor is used for detecting organophosphorus pesticides.
The electrochemical luminescence biosensor for detecting the organophosphorus pesticide realizes detection of the organophosphorus pesticide based on a resonance energy transfer mechanism, and specifically comprises the steps that when a target exists, the spatial structure of a nucleic acid aptamer is changed, the nucleic acid aptamer and the target organophosphorus pesticide form a compound, complementary DNA (deoxyribonucleic acid) falls off from the surface of an electrode, gold nanoparticles at the 3' end are drawn close to the surface of the electrode, energy transfer (fluorescence quenching) occurs, and the luminous intensity of a luminous body cadmium telluride quantum dot is reduced.
As shown in the preparation process of FIG. 1, the application provides a preparation method of an electrochemical luminescence biosensor for detecting organophosphorus pesticides, which comprises the following steps:
K1. polishing and cleaning the glassy carbon electrode, which comprises the following specific steps: using Al 2 O 3 Slurry polish glassy carbon electrodes on chamois to a mirror surface, 1:1 nitric acid, 1:1, respectively ultrasonically cleaning the electrode by using ethanol and distilled water for 5-10 min, activating the glassy carbon electrode in 0.5-1.0 mol/L H2SO4 solution by using cyclic voltammetry after completely cleaning, repeatedly scanning until a stable cyclic voltammogram is reached, cleaning the surface of the electrode by using ultrapure water, and drying by using nitrogen;
K2. transferring 5-10 mu L (0.05-0.5 mg/mL) of cadmium telluride quantum dot @ mesoporous polydopamine composite material to vertically drip and coat on the surface of a working electrode, modifying the electrode, and airing at room temperature to obtain a cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode;
K3. dripping 5-10 mu L (0.5 wt.%) of nafion solution on the surface of the cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode to fix the composite material, drying the composite material at room temperature, washing the composite material with water, and removing redundant nafion solution on the surface of the electrode to obtain a nafion/cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode;
K4. transferring 5-10 mu L (0.1-1.0 mu mol/L) of the molecular probe to the surface of the nafion/cadmium telluride quantum dot @ mesoporous polydopamine/glassy carbon electrode, incubating overnight, and forming a covalent bond through a sulfonic group and an amino group to fix the molecular probe to a sensing interface of the nafion/cadmium telluride quantum dot @ mesoporous polydopamine/glassy carbon electrode to obtain the molecular probe/nafion/cadmium telluride quantum dot @ mesoporous polydopamine/glassy carbon electrode;
K5. dropping 5-10 mu L (0.1 mol/L) of a sealing agent on the surface of a molecular probe/nafion/cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode, sealing non-specific binding sites, incubating for 0.5-1.0 h, slightly washing the surface of the electrode with PBS buffer solution (pH7.4), removing redundant 6-sulfydryl-1-hexanol solution on the surface of the electrode, dropping 5-10 mu L (0.1-1.0 mu mol/L) of complementary DNA, incubating for 1-3 h, and slightly washing with PBS buffer solution (pH7.4) to obtain the complementary DNA/molecular probe/nafion/cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode, namely the electrochemical luminescence biosensor for detecting organophosphorus pesticides.
Preferably, the blocking agent is 6-mercapto-1-hexanol solution, and the blocking agent 6-mercapto-1-hexanol solution (MCH) blocks non-specific binding site conditions: and dripping MCH solution (0.1 mol/L) on the surface of the nafion/cadmium telluride quantum dot @ mesoporous polydopamine/glassy carbon electrode on the modified electrode hairpin-DNA-AuNPs, incubating at room temperature for 0.5-1.0 h, slightly washing by using PBS buffer solution (pH7.4), and removing the redundant MCH solution on the surface of the electrode.
As shown in the detection process of FIG. 2, the application provides an application of an electrochemical luminescence biosensor for detecting organophosphorus pesticides in detecting organophosphorus pesticides, the detection method is an electrochemical luminescence method, a reference electrode for detection is a silver/silver chloride electrode, a counter electrode is a platinum sheet electrode or a platinum wire electrode, a working electrode is a complementary DNA/molecular probe/nafion/cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode, the test steps are that organophosphorus pesticides with different concentrations are incubated in the prepared electrochemical luminescence biosensor, incubation reaction is carried out at 37 ℃ for 1.0-3.0 h, PBS buffer solution is slightly washed, the electrochemical luminescence method is carried out for detection, the luminescence intensity is recorded, and a working curve between the luminescence intensity and the concentration is obtained, preferably, the excitation voltage for detection is 2.0-0V, and the scanning rate is 50-100mV/s.
The present application is further illustrated by the following specific examples.
Example 1
Preparing raw materials:
the preparation of the water-soluble carboxyl cadmium telluride quantum dots (CdTe-COOH QDs) comprises the following specific synthesis steps:
cadmium chloride semi- (pentahydrate) (45.672mg, 0.2mmol) and thioglycolic acid (46.06mg, 0.5mmol) are sequentially dispersed into 50mL of distilled water, then NaOH solution is dropwise added to adjust the pH of the Cd-TGA compound solution to 7.0-11.0, and high-purity N is added 2 Bubbling for 30min to deoxygenate the solution. Under vigorous stirring, sequentially adding sodium tellurite solution (4.4316mg, 0.02mmol) and 85% hydrazine hydrate (0.377 mL), then heating under reflux in an oil bath kettle (100 ℃,4 h) to obtain CdTe-COOH QDs capped by TGA, finally, purifying crude CdTe-COOH QDs by excessive ethanol centrifugation (8000rpm, 10min), dispersing the washed CdTe-COOH QDs in deionized water, and storing under 4 ℃ dark condition, as shown in a transmission electron microscope image of cadmium telluride quantum dots in a figure 4, wherein the particle diameter is 5-10 nm;
the preparation of the cadmium telluride quantum dot @ mesoporous polydopamine composite material (CdTe @ MPDA) comprises the following specific synthesis steps:
1g of block polyether F-127 and 0.144g of water-soluble carboxyl cadmium telluride quantum dot are added into 50mL of ethanol-water mixed solution and stirred vigorously to obtain a uniform solution of water-soluble CdTe quantum dot dispersed in the block polyether F-127. Then, 1.0g of dopamine hydrochloride was dissolved in the above solution, and 2.0mL of 1,3, 5-trimethylbenzene was added to the mixed solution. Subsequently, 2.5mL of aqueous ammonia was added to the reaction mixture, and the reaction was stirred for 4 hours. And finally, centrifuging and washing the mixed solution of ethanol and water for several times to obtain a product of mesoporous polydopamine particles, dispersing the product in a certain amount of mixed solution of water and ethanol again, carrying out ultrasonic treatment for a period of time, transferring the product into a sealed Teflon lining autoclave, and heating the product at 100 ℃ for 24 hours to obtain the cadmium telluride quantum dot @ mesoporous polydopamine composite material. Fig. 3 is an X-ray diffraction spectrum of cadmium telluride quantum dots, mesoporous polydopamine and cadmium telluride quantum dots @ mesoporous polydopamine, and after cadmium telluride and mesoporous polydopamine are compounded, compared with cadmium telluride and mesoporous polydopamine alone, a characteristic peak shifts to the right and is weakened. FIG. 5 is a transmission electron microscope image of mesoporous polydopamine prepared in the absence of cadmium telluride quantum dots, with a spheroidal porous structure and uniform particle size; FIG. 6 is an EDS elemental analysis chart of cadmium telluride quantum dot @ mesoporous polydopamine, the composite material is composed of five elements of C, N, O, cd and Te; fig. 8 is a graph of cyclic voltammetry and Electrochemiluminescence (ECL) cyclic stability of nafion/cadmium telluride quantum dot @ mesoporous polydopamine/glassy carbon electrode in 0.1mol/L potassium persulfate solution, at an excitation voltage: 2.0-0V and scanning rate: under the condition of 100mV/s, the cadmium telluride quantum dots react with potassium persulfate, and the generated electrochemiluminescence signal is strong and stable. The experimental characterization results are in line with expectations, and the cadmium telluride quantum dot @ mesoporous polydopamine is successfully prepared.
By using the raw materials, the embodiment provides an electrochemical luminescence biosensor for detecting organophosphorus pesticides, which comprises a glassy carbon electrode, and a cadmium telluride quantum dot @ mesoporous polydopamine nano material, a nafion film layer, a hairpin-DNA-AuNPs molecular probe and complementary DNA which are sequentially modified on the surface of the glassy carbon electrode from inside to outside; the hairpin-DNA-AuNPs molecular probe comprises a nucleic acid aptamer with amino modified at the 5' end and a disulfide group modified at the 3' end, and a gold nanoparticle coupled with the 3' end of the nucleic acid aptamer, wherein the secondary structure of the nucleic acid aptamer is a hairpin structure; complementary DNA and molecular probes are base complementary paired.
The preparation method of the electrochemical luminescence biosensor for detecting the organophosphorus pesticide comprises the following steps:
K1. pretreating a glassy carbon electrode, polishing the glassy carbon electrode on a chamois leather to a mirror surface by using Al2O3 slurry, 1:1 nitric acid, 1:1, respectively ultrasonically cleaning the ethanol and the distilled water for 5min, after thorough cleaning, activating the glassy carbon electrode in 0.5mol/L H2SO4 solution by using a cyclic voltammetry method, and repeatedly scanning until a stable cyclic voltammogram is reached;
K2. transferring 10 mu L (0.5 mg/mL) of cadmium telluride quantum dot @ mesoporous polydopamine composite material to vertically drip-coat the pretreated electrode surface, modifying the electrode, and naturally airing at room temperature;
K3. dripping 5 mu L (0.5 wt.%) of nafion solution to fix the composite material, slightly washing with distilled water, naturally drying at room temperature, slightly washing with distilled water, and removing the redundant nafion solution on the surface of the electrode;
K4. fixing a molecular probe hairpin-DNA-AuNPs on a nafion/cadmium telluride quantum dot @ mesoporous polydopamine/glassy carbon electrode, transferring 10 mu L (1.0 mu mol/L) of hairpin-DNA-AuNPs solution, dripping the solution on the surface of a modified electrode, forming a covalent bond through a sulfonic group and an amino group to fix a sensing interface of the molecular probe to the nafion/cadmium telluride quantum dot @ mesoporous polydopamine/glassy carbon electrode, and incubating overnight;
K5. dripping 5 mu L (0.1 mol/L) of 6-mercapto-1-hexanol solution on the surface of the modified electrode to block non-specific binding sites, incubating for 1.0h, slightly washing the surface of the electrode with PBS buffer solution (pH 7.4), and removing the redundant 6-mercapto-1-hexanol solution on the surface of the electrode; and (3) dripping 10 mu L (1.0 mu mol/L) of complementary DNA on the surface of the molecular probe/nafion/cadmium telluride quantum dot @ mesoporous polydopamine/glassy carbon electrode modified electrode, incubating for 3h, completing the preparation of the sensor, detecting the organophosphorus pesticide, and storing in a refrigerator at 4 ℃ for later use when not in use. The electrochemiluminescence biosensor fabrication process was characterized using cyclic voltammetry, and the sensor was successfully fabricated by characterizing the fabrication process for each step of the sensor (fig. 9).
Application example
Feasibility analysis of organophosphorus pesticide detection using the electrochemiluminescence biosensor prepared in example 1:
electrochemical luminescence biosensor test: a classical three-electrode system is used, an Ag/AgCl electrode (saturated calomel electrode) is used as a reference electrode, a platinum sheet electrode (5 multiplied by 0.2 mm) is used as a counter electrode, a 6-sulfydryl-1-hexanol/nafion/cadmium telluride quantum dot @ mesoporous polydopamine/glassy carbon electrode modified electrode is used as a working electrode, then 5 mu L of organic phosphorus pesticides with different concentrations are incubated and reacted for 1 hour at room temperature, and PBS buffer solution (pH7.4) is used for slight washing. An electrochemiluminescence test was performed in a potassium persulfate solution (0.1 mol/L), and a certain voltage was applied using cyclic voltammetry, excitation voltage: 2.0-0V, scanning rate: 100mV/s, the luminous intensity obtained by testing different concentrations of organophosphorus pesticide is recorded. As a result, as shown in FIG. 10, the conformation of the aptamer was changed by the presence of the organophosphorus pesticide, the complementary DNA was released from the electrode surface, and the gold nanoparticles modified at the 3' -end of the molecular probe aptamer were pulled to the electrode surface, thereby enhancing the quenching effect, and the higher the concentration of the organophosphorus pesticide, the lower the luminescence intensity.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are also included in the scope of the present invention.
Claims (10)
1. An electrochemical luminescence biosensor for detecting organophosphorus pesticides is characterized by comprising a glassy carbon electrode, and a cadmium compound quantum dot @ mesoporous polydopamine nano material, a nafion film layer, a molecular probe and complementary DNA which are sequentially modified on the surface of the glassy carbon electrode from inside to outside; the molecular probe comprises an aptamer with an amino group modified at the 5' end and a disulfide group modified at the 3' end, and a noble metal nanoparticle coupled with the 3' end of the aptamer, wherein the secondary structure of the aptamer is a hairpin structure; the complementary DNA and the molecular probe are paired through base complementation.
2. The electrochemical luminescence biosensor for detecting organophosphorus pesticides as claimed in claim 1, wherein the cadmium compound quantum dot @ mesoporous polydopamine nano material is formed by growing aqueous cadmium compound quantum dots in the porous of mesoporous polydopamine nano material.
3. The electrochemiluminescence biosensor for detecting organophosphorus pesticides of claim 1, wherein the cadmium compound quantum dots comprise one of cadmium telluride quantum dots, cadmium selenide quantum dots and cadmium sulfide quantum dots.
4. The electrochemiluminescence biosensor for detecting organophosphorus pesticides of claim 1, wherein the noble metal nanoparticles comprise one of gold nanoparticles, platinum nanoparticles, silver nanoparticles, gold-platinum bimetallic nanoparticles and gold-silver bimetallic nanoparticles.
5. The electrochemiluminescence biosensor for detecting organophosphorus pesticides according to claim 1, wherein the particle size of the cadmium compound quantum dots is 5-10nm.
6. The electrochemical luminescence biosensor for detecting organophosphorus pesticides as claimed in claim 1, wherein the preparation method of the cadmium compound quantum dot @ mesoporous polydopamine nano-material comprises the following steps:
s1, mixing and dispersing block polyether F-127 and aqueous cadmium compound quantum dots in an ethanol water solution to obtain a uniform solution;
s2, sequentially adding dopamine hydrochloride and 1,3, 5-trimethylbenzene into the uniform solution, then adding ammonia water, and stirring for reaction to obtain an intermediate product;
s3, centrifuging and washing the intermediate product, dispersing the intermediate product in an ethanol water solution for ultrasonic treatment, and heating the intermediate product at 100 ℃ overnight under a sealed condition to obtain the cadmium compound quantum dot @ mesoporous polydopamine nano material.
7. The preparation method of the electrochemical luminescence biosensor for detecting organophosphorus pesticides as described in any one of claims 1 to 6, comprising the following steps:
K1. polishing and cleaning the glassy carbon electrode;
K2. dropwise adding the cadmium compound quantum dot @ mesoporous polydopamine nano-material solution on the surface of the polished and cleaned glassy carbon electrode, modifying the electrode, and airing at room temperature to obtain the cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode;
K3. dripping nafion solution on the surface of the cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode, airing at room temperature, and washing with water to obtain a nafion/cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode;
K4. dripping a molecular probe on the surface of the nafion/cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode, and incubating overnight to obtain the molecular probe/nafion/cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode;
K5. and dripping a sealant on the surface of the molecular probe/nafion/cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode, after incubation overnight and washing, dripping complementary DNA, and then incubating and washing to obtain the complementary DNA/molecular probe/nafion/cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode, namely the electrochemical luminescence biosensor for detecting the organophosphorus pesticide.
8. The method for preparing the electrochemiluminescence biosensor for detecting organophosphorus pesticide according to claim 7, wherein the blocking agent is 6-mercapto-1-hexanol solution.
9. The application of the electrochemical luminescence biosensor for detecting organophosphorus pesticide according to any one of claims 1 to 6, wherein the reference electrode for detection is a silver/silver chloride electrode, the counter electrode is a platinum sheet electrode or a platinum wire electrode, and the working electrode is a complementary DNA/molecular probe/nafion/cadmium compound quantum dot @ mesoporous polydopamine/glassy carbon electrode.
10. Use according to claim 9, characterized in that the excitation voltage detected is between 2.0 and 0V and the scan rate is between 50 and 100mV/s.
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