CN112505024A - Electrochemiluminescence aptamer sensor for detecting enrofloxacin, preparation method thereof and method for detecting enrofloxacin - Google Patents
Electrochemiluminescence aptamer sensor for detecting enrofloxacin, preparation method thereof and method for detecting enrofloxacin Download PDFInfo
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- G01N21/76—Chemiluminescence; Bioluminescence
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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Abstract
The invention discloses an electrochemiluminescence aptamer sensor for detecting enrofloxacin and a preparation method and application thereof.A titanium carbide-silver bromide nano composite material is prepared by a wet chemical method, then the titanium carbide-silver bromide nano composite material is modified on the surface of a glassy carbon electrode, then an aptamer containing a 3 '-CCC ATC AGG GGG CTA GGC TAA CAC GGT TCG GCT CTC TGA GCC CGG GTT ATT TCA GGG GGA-5' COOH base sequence is loaded, the electrochemiluminescence aptamer sensor is constructed, and the electrochemiluminescence aptamer sensor is used for detecting enrofloxacin.
Description
Technical Field
The invention belongs to the field of electrochemiluminescence detection, and relates to an electrochemiluminescence aptamer sensor for detecting enrofloxacin, a preparation method thereof and a method for detecting enrofloxacin.
Background
Enrofloxacin (Enrofloxacin, ENR for short), also known as ethyl ciprofloxacin and Enrofloxacin, belongs to fluoroquinolone chemical synthesis bacteriostats. Enrofloxacin is specified as a special animal medicine by the nation, and is widely applied to the animal and aquaculture industry due to the characteristics of strong antibacterial property, quick action, wide distribution in the body, no cross resistance with other antibiotics and the like. Currently, enrofloxacin is listed as the key point of veterinary drug residue monitoring, the maximum residue limit of muscle and fat of all food animals is 100 mug/kg, the maximum residue limit of liver and kidney of all food animals is 200 mug/kg, and the maximum residue detection limit of ENR in animal-derived food is 0.3 mug/g, which is specified by No. 235 published by Ministry of agriculture in 2002 of 12 months in 2002. Due to excessive use of the medicine, the medicine is accumulated and remained in a large amount in the animal body, so that the substance also exists in the excrement of the animal body and influences the surrounding environment. Wherein water resources are subject to severe pollution. However, once water resources are polluted, human health, public safety and safety of field environments are threatened, and thus an efficient and accurate method for monitoring the safety of water resources is required.
At present, the reported methods for determining enrofloxacin mainly comprise: high Performance Liquid Chromatography (HPLC), Capillary Electrophoresis (CEP), fluorescence photometry (FL), chemiluminescence immunoassay (CLIA), enzyme-linked immunosorbent assay (ELISA), etc. However, these analysis methods have many limitations, for example, high performance liquid chromatography and capillary electrophoresis have high accuracy, but the instruments are expensive and the analysis sensitivity is low; although the fluorescence photometry and the chemiluminescence method have high sensitivity, special reagents are required and the selectivity of the method is poor; the enzyme-linked immunosorbent assay is not suitable for being used as a single detection method because the prepared antibody reacts with various residual drugs, and is only suitable for rapidly screening and detecting a large number of samples; the immunoassay method is established based on antigen-antibody specificity recognition reaction, and has the advantages of high production cost of raw material antibodies and high immune activity easily influenced by environmental factors such as ionic strength, pH and the like. The Aptamer (Aptamer), also called as a mimic antibody, has the advantages of simple and rapid synthesis, low cost, good selectivity, stable property and easy modification and marking, and is a novel excellent antibody substitution recognition element. Therefore, it is necessary and extremely important to develop a simple, rapid and highly selective method for detecting enrofloxacin.
Electrochemiluminescence (ECL) is also called electrochemiluminescence, is an analysis technology for converting electric energy into light energy, does not need to introduce an external light source, collects a luminous intensity spectrum under the assistance of optical instruments such as a photomultiplier and the like, and establishes a relation between the luminous intensity spectrum and an object to be detected so as to realize microanalysis. The method is a combination of chemiluminescence and an electrochemical method, and has the advantages of simple operation, high sensitivity, low background, short detection time and the like. The electrochemical luminescence method is combined with the biological recognition molecules, so that the analysis and detection sensitivity and selectivity can be obviously improved, and the method is particularly suitable for environmental samples with low concentration of the detected object, complex components and more interferents.
Disclosure of Invention
The invention aims to provide an electrochemiluminescence aptamer sensor for detecting enrofloxacin, a preparation method thereof and a method for detecting enrofloxacin, aiming at the defects of the prior art for detecting enrofloxacin. The invention adopts a wet chemical method to prepare titanium carbide-silver bromide nano composite material (Ti)3C2-AgBr nano composite material) and modifying the AgBr nano composite material on the surface of a glassy carbon electrode, so that the sensitivity and stability of electrochemiluminescence are obviously improved, and then an Aptamer is loaded through electrostatic adsorption to obtain an electrochemiluminescence Aptamer sensor (Aptamer/Ti for short)3C2An AgBr/GCE sensor) which can specifically identify the target molecule enrofloxacin and improve the selectivity of enrofloxacin.
In order to realize the purpose of the invention, the adopted technical scheme is as follows:
an electrochemiluminescence aptamer sensor for detecting enrofloxacin, wherein the electrochemiluminescence aptamer sensor is formed by loading Ti (titanium dioxide) with an aptamer containing a 3 '-CCC ATC AGG GGG CTA GGC TAA CAC GGT TCG GCT CTC TGA GCC CGG GTT ATT TCA GGG GGA-5' COOH base sequence3C2The surface of the glassy carbon electrode modified by the-AgBr nano composite material.
The preparation method of the electrochemiluminescence aptamer sensor for detecting enrofloxacin comprises the following steps:
(1) preparation of Ti by wet chemical method3C2-AgBr nanocomposite, followed by Ti3C2Dispersing the-AgBr nano-composite in DMF to obtain a dispersion liquid;
(2) polishing the glassy carbon electrode, sequentially ultrasonically cleaning the glassy carbon electrode by using a nitric acid solution, an ethanol solution and ultrapure water, and drying the glassy carbon electrode at room temperature to obtain a pretreated glassy carbon electrode for later use; using a liquid-transfering gun to transfer the dispersion liquid prepared in the step (1) to be coated on the surface of the pretreated glassy carbon electrode, and naturally drying to obtain the Ti3C2A glassy carbon electrode modified by an AgBr nano composite material, and then the aptamer is loaded on Ti through electrostatic adsorption3C2And (4) naturally airing the surface of the glassy carbon electrode modified by the AgBr nano composite material to prepare the electrochemical luminescence aptamer sensor.
Further, Ti in the dispersion liquid obtained in the step (1)3C2The concentration of the-AgBr nanocomposite is 2 +/-1 mg/mL; and/or the transfer amount of the dispersion liquid in the step (2) is 6 +/-2 mu L.
Further, the step (2) loads the aptamer on Ti3C2The method for modifying the surface of the glassy carbon electrode by the-AgBr nano composite material comprises the following steps: firstly, KCl, NaCl and MgCl are added2Adding the aptamer into Tris-HCl buffer solution of EDTA to prepare aptamer solution with aptamer concentration of 0.5-10 mu mol/L, transferring 6 +/-2 mu L of the aptamer solution, and dripping the aptamer solution on the Ti3C2-a glassy carbon electrode surface modified by AgBr nanocomposite.
Further, the concentration of the aptamer in the aptamer solution is 2.0-5.0 mu mol/L, and more preferably 2.5 mu mol/L.
A method of detecting enrofloxacin, comprising the steps of:
the electrochemiluminescence aptamer sensor for detecting enrofloxacin is used as a working electrode, Ag/AgCl is used as a reference electrode, a platinum wire electrode is used as a counter electrode to form a three-electrode system, enrofloxacin in a sample to be detected is quantitatively captured on the surface of the sensor, and detection is carried out through a generated luminescence signal.
Further, the method also comprises the following steps:
0.05-0.1 mol/L K is prepared by using 0.1mol/L PBS buffer solution with pH of 7.0-8.02S2O8PBS buffer solution of (4);
placing the electrochemiluminescence aptamer sensor in the enrofloxacin standard solutions with different concentrations prepared in the step 2, soaking for the same time to enable the electrochemiluminescence aptamer sensor to be combined with enrofloxacin, taking out the electrochemiluminescence aptamer sensor, leaching to serve as a working electrode, taking Ag/AgCl as a reference electrode, taking a platinum electrode as a counter electrode to form a three-electrode system, and taking the K-containing standard solution in the step 1 as a K-containing standard solution2S2O8The PBS buffer solution is electrolyte, within an electrochemical window range of-1.8-0V, the photomultiplier tube has a high voltage of 600-800V and a sweep rate of 0.05-0.125V/s, cyclic voltammetry scanning is carried out, a luminous intensity-time curve is recorded, a linear relation between a luminous intensity difference value before and after the combination of an electrochemiluminescence aptamer sensor and enrofloxacin and a logarithm value of enrofloxacin concentration in an enrofloxacin standard solution is established, and a corresponding linear regression equation is obtained;
step 4, detecting enrofloxacin in the sample to be detected
Filtering the sample and then using the K-containing sample obtained in the step 12S2O8Adjusting the pH of the PBS buffer solution to the value containing K2S2O8The PBS buffer solution is consistent, then the electrochemical luminescence aptamer sensor is placed into the electrochemical luminescence aptamer sensor to be soaked for the same time, so that the electrochemical luminescence aptamer sensor is combined with enrofloxacin, and thenAnd taking out and leaching the sample, using the sample as a working electrode, detecting the luminous intensity by adopting the method in the step 3, and calculating the concentration of the enrofloxacin in the sample according to a linear regression equation.
Further, the soaking time in the step 3 is 20-40 min, and more preferably 30 min.
Further, the pH of the PBS buffer solution in the step 1 is 7.4, and the prepared K-containing solution2S2O8The concentration of the PBS buffer solution is 0.1 mol/L;
the soaking time in the step 3 is 30min, the photomultiplier high pressure is 800V, and the sweeping speed is 0.1V/s.
Compared with the prior art, the invention has the beneficial effects that:
the invention designs a Ti based on a two-dimensional layered material3C2The electrochemical luminescence aptamer sensor loaded with the inorganic photosensitizer AgBr combines the advantages of an aptamer and an electrochemical luminescence sensor, the sensitive detection of enrofloxacin is successfully realized through the mechanism of enhancing the ECL signal intensity of the enrofloxacin to the system, and the sensing platform can specifically identify the enrofloxacin as a detection object and has high selectivity. The detection range of the invention is 1 multiplied by 10-12mol/L～1×10-6mol/L, the lowest detection limit is 5.92 multiplied by 10-13mol/L. The method for detecting enrofloxacin has the advantages of simple operation, good selectivity, low detection cost and high sensitivity. The invention has important significance for popularizing the practical application of the aptamer sensor in the aspects of environment, food safety and the like.
Drawings
FIG. 1 is a graph of ECL response of an electrochemiluminescent aptamer sensor constructed in example 1 after binding with different concentrations of enrofloxacin, wherein the concentrations of enrofloxacin are, from left to right: (a) 0; (b) 1X 10-12mol/L；(c)1×10-11mol/L；(d)1×10-10mol/L；(e)1×10-9mol/L；(f)1×10-8mol/L；(g)1×10-7mol/L；(h)1×10-6mol/L。
FIG. 2 is a standard curve of the difference of the luminous intensity before and after the addition of enrofloxacin and the logarithm of the concentration of enrofloxacin in example 1;
FIG. 3 is Ti prepared in example 13C2-scanning electron microscopy of AgBr nanocomposites.
Detailed Description
In the following examples, aptamers having a base sequence of 3 '-CCC ATC AGG GGG CTA GGC TAACAC GGT TCG GCT CTC TGA GCC CGG GTT ATT TCA GGG GGA-5' COOH were obtained from Biotechnology engineering (Shanghai) Co., Ltd and supported on Ti3C2The method for modifying the surface of the glassy carbon electrode by the-AgBr nano composite material comprises the following steps: firstly, adding a solution containing KCl, NaCl and MgCl into the aptamer2And EDTA in Tris-HCl buffer solution to prepare aptamer solution with aptamer concentration of 2.5. mu. mol/L, and then transferring 6. mu.L of the aptamer solution and dripping the aptamer solution on the Ti3C2-a glassy carbon electrode surface modified by AgBr nanocomposite. Specifically, the method comprises the following steps: (1) carrying out vortex oscillation for 5-10 min before uncovering the purchased aptamer, and then centrifuging for 20min at 5000 rpm; (2) slowly opening the tube cover, adding appropriate amount of 0.2mol/L KCl, 0.1mol/L NaCl and 5.0mmol/L MgCl according to the mark on the tube2And 1.0mmol/L EDTA in 0.05mol/L Tris-HCl buffer solution; (3) fully and uniformly oscillating, diluting to the concentration of 2.5 mu mol/L, and refrigerating in a refrigerator at 4 ℃ for standby.
The preparation method of the enrofloxacin standard solutions with different concentrations in the following examples comprises the following steps: preparing enrofloxacin solution, and then adding the enrofloxacin solution into the solution containing 0.1mol/L K2S2O8Obtaining a series of enrofloxacin standard solutions with different concentrations in 0.1mol/L PBS buffer solution with the pH of 7.4, wherein the concentrations of enrofloxacin in the enrofloxacin standard solutions in the embodiment are 0 respectively; 1X 10-12mol/L；1×10-11mol/L；1×10-10mol/L；1×10-9mol/L；1×10-8mol/L；1×10- 7mol/L；1×10-6mol/L。
Example 1:
assembling Aptamer/Ti3C2-AgBr/GCE sensor
(1)Ti3C2-AgBr compoundingPreparation of the material:
with Ti3C2As a starting material, AgNO3Is silver source, 1-hexadecyl-3-methylimidazolium bromide is bromine source, and Ti is synthesized by a wet chemical method3C2-AgBr nanocomposite. 87.74mg of silver nitrate is weighed in a centrifugal tube and dissolved in 2mL of ammonia water to obtain reaction liquid; 600 μ LTi was added to a 50mL round bottom flask3C2(5mg/mL) and 8mL of ethylene glycol, performing ultrasonic treatment for 20min, adding 200.10mg of 1-hexadecyl-3-methylimidazolium bromide, stirring for 30min at room temperature, slowly dropwise adding the reaction solution into the round-bottom flask, and performing oil bath for 6h at 90 ℃; centrifuging after oil bath to obtain precipitate, washing with water, and freeze-drying for 12h to obtain Ti3C2-AgBr nanocomposite (of Ti3C2The content of the composite material is 3 percent), and the composite material is stored at normal temperature in a dark place. Taking 20mg of Ti3C2Dispersing the-AgBr nano composite material in 10mL of N, N-Dimethylformamide (DMF) to obtain a dispersion liquid;
(2)Aptamer/Ti3C2preparation of-AgBr/GCE sensor
Firstly, polishing powder (Al) for glassy carbon electrode2O3) And polishing the chamois leather into a mirror surface, then ultrasonically cleaning the chamois leather by using a nitric acid solution, an ethanol solution and ultrapure water in sequence, and drying the chamois leather at room temperature to obtain the pretreated glassy carbon electrode. Transferring 6 mu L of 2mg/mL dispersion liquid prepared in the step (1) by using a liquid transfer gun, and dripping the dispersion liquid on the surface of the pretreated glassy carbon electrode to obtain Ti3C2The AgBr/GCE modified electrode is naturally dried for standby; at Ti3C2Dripping 6 mu L of prepared Aptamer solution on the surface of the-AgBr/GCE modified electrode, and naturally airing for 6h to obtain Aptamer/Ti3C2AgBr/GCE sensor as a sensor element for electrochemiluminescence tests.
(II) based on Aptamer/Ti3C2Method for detecting enrofloxacin by-AgBr/GCE sensor
(1) Drawing of standard curve
The Aptamer/Ti in (A) is added3C2the-AgBr/GCE sensor is used as a sensing element and is immersed in enrofloxacin standard solutions with different concentrationsSoaking for 30min, taking out, rinsing, using as working electrode, Ag/AgCl as reference electrode, platinum electrode as counter electrode to form three-electrode system, and adding water to obtain solution containing 0.1mol/L K2S2O8The 0.1mol/L PBS buffer solution with the pH value of 7.4 is used as electrolyte to measure the luminous intensity, in the electrochemical window range of-1.8 to 0V, the photomultiplier is under the high pressure of 800V, the sweep rate is 0.1V/s, cyclic voltammetry scanning is carried out, a luminous intensity-time curve is recorded, a linear relation between the luminous intensity difference before and after the electrochemical luminescence aptamer sensor is combined with enrofloxacin and the enrofloxacin concentration logarithm value in the enrofloxacin standard solution is established, and the obtained corresponding linear regression equation is as follows: Δ ECL 4251.2612+962.1239lgC (nmol/L) with a detection range of 1 × 10- 12mol/L～1×10-6mol/L, detection limit of 5.92 multiplied by 10-13mol/L(S/N＝3)。
(2) Detection of samples
Taking a water sample of a certain pond, naturally standing for a period of time, centrifugally separating and absorbing an upper layer solution, filtering through a 0.22 mu m filter membrane to collect filtrate, and adding a solution containing 0.1mol/L K2S2O8Adjusting the pH value of the 0.1mol/L PBS buffer solution to 7.4, taking 25mL of the obtained solution for electrochemical luminescence analysis, and calculating the concentration of enrofloxacin in the sample to be detected according to the linear regression equation obtained in the step (1), wherein the results are shown in Table 1.
This example uses a two-dimensional layered material Ti3C2Synthesizing Ti as a base material3C2the-AgBr nano composite material has the appearance shown in figure 3, uniformly distributed points on a sheet, novel material, good conductivity, good stability, good sensor selectivity and good stability.
Comparative example 1:
(1)Aptamer/Ti3C2preparation of/GCE sensor
Remove 2mL of Ti3C2(5mg/mL) is dispersed in 3mL of N, N-Dimethylformamide (DMF), and ultrasonic dispersion is carried out uniformly to obtain 2mg/mL of Ti3C2And (3) dispersing the mixture.
Transfer 6. mu.L of 2mg/mL Ti with a pipette3C2DMF dispersion, drop-coating onto pretreatedThe glassy carbon electrode (pretreatment method same as example 1) was used for surface preparation to obtain Ti3C2The electrode is chemically modified by GCE and naturally dried for later use; at Ti3C2Dripping 6 mu L of 2.5 mu mol/L enrofloxacin Aptamer on the surface of the/GCE chemically modified electrode, and naturally airing for 6h to obtain Aptamer/Ti3C2the/GCE sensor is used as a sensing element for electrochemiluminescence test.
(2) Drawing of standard curve
The Aptamer/Ti prepared in the step (1) is added3C2the/GCE sensor is used as a sensing element and is placed in enrofloxacin standard solutions with different concentrations for soaking for 30min, the enrofloxacin standard solutions are taken out and rinsed to be used as a working electrode, Ag/AgCl is used as a reference electrode, a platinum electrode is used as a counter electrode to form a three-electrode system, and the sensing element is characterized in that the sensing element contains 0.1mol/L K2S2O8The 0.1mol/L PBS buffer solution with the pH value of 7.4 is used as electrolyte to measure the luminous intensity, in the electrochemical window range of-1.8 to 0V, the photomultiplier is under the high pressure of 800V, the sweep speed is 0.1V/s, cyclic voltammetry scanning is carried out, a luminous intensity-time curve is recorded, a linear relation between the luminous intensity difference before and after the electrochemical luminescence aptamer sensor is combined with enrofloxacin and the enrofloxacin concentration logarithm value in the enrofloxacin standard solution is established, and a corresponding linear regression equation is obtained.
(3) Detection of samples
Taking a water sample of a certain pond, naturally standing for a period of time, centrifugally separating and absorbing an upper layer solution, filtering through a 0.22 mu m filter membrane to collect filtrate, and adding a solution containing 0.1mol/L K2S2O8Adjusting the pH value of the 0.1mol/L PBS buffer solution to 7.4, taking 25mL of the obtained solution for electrochemical luminescence analysis, and calculating the concentration of enrofloxacin in the sample to be detected according to the linear regression equation obtained in the step (2), wherein the results are shown in Table 1.
Comparative example 2:
(1) preparation of Aptamer/AgBr/GCE sensor
87.74mg of silver nitrate is weighed in a centrifugal tube and dissolved in 2mL of ammonia water to obtain reaction liquid; adding 8mL of ethylene glycol and 200.10mg of 1-hexadecyl-3-methylimidazolium bromide into a 50mL round-bottom flask, stirring at room temperature for 30min, slowly dropwise adding the reaction solution into the round-bottom flask, and performing oil bath at 90 ℃ for 6 h; after oil bath, the precipitate is obtained by centrifugation, washed by water, frozen and dried for 12 hours, and dispersed in N, N-Dimethylformamide (DMF) to prepare 2mg/mL AgBr dispersion.
Transferring 6 mu L of 2mg/mL AgBr DMF dispersion liquid by using a liquid transfer gun, dripping the DMF dispersion liquid on the surface of a pretreated glassy carbon electrode (the pretreatment method is the same as that in the embodiment 1) to obtain an AgBr/GCE chemically modified electrode, and naturally airing for later use; and dripping 6 mu L of 2.5 mu mol/L enrofloxacin Aptamer on the surface of the AgBr/GCE chemically modified electrode, and naturally airing for 6h to obtain the Aptamer/AgBr/GCE sensor serving as a sensing element for electrochemiluminescence test.
(2) Drawing of standard curve
Taking the Aptamer/AgBr/GCE sensor prepared in the step (1) as a sensing element, soaking the sensor in enrofloxacin standard solutions with different concentrations for 30min, taking out the sensor, leaching the sensor to be used as a working electrode, taking Ag/AgCl as a reference electrode, taking a platinum electrode as a counter electrode to form a three-electrode system, and taking the sensor containing 0.1mol/L K2S2O8The 0.1mol/L PBS buffer solution with the pH value of 7.4 is used as electrolyte to measure the luminous intensity, in the electrochemical window range of-1.8 to 0V, the photomultiplier is under the high pressure of 800V, the sweep speed is 0.1V/s, cyclic voltammetry scanning is carried out, a luminous intensity-time curve is recorded, a linear relation between the luminous intensity difference before and after the electrochemical luminescence aptamer sensor is combined with enrofloxacin and the enrofloxacin concentration logarithm value in the enrofloxacin standard solution is established, and a corresponding linear regression equation is obtained.
(3) Detection of samples
Taking a water sample of a certain pond, naturally standing for a period of time, centrifugally separating and absorbing an upper layer solution, filtering through a 0.22 mu m filter membrane to collect filtrate, and adding a solution containing 0.1mol/L K2S2O8Adjusting the pH value of the 0.1mol/L PBS buffer solution to 7.4, taking 25mL of the obtained solution for electrochemical luminescence analysis, and calculating the concentration of enrofloxacin in the sample to be detected according to the linear regression equation obtained in the step (2), wherein the results are shown in Table 1.
TABLE 1 determination of water samples from a pond
Remarking:ais the average of three determinations
As shown in Table 1, the samples are parallelly measured for 3 times, the recovery rate of the added standard is 96.6-101.6%, the relative standard deviation is less than 5%, and the recovery effect is good. The above experimental results show that Ti is not used3C2Modification of-AgBr nanocomposite with Ti alone3C2Or the sensor element is further assembled after the glassy carbon electrode is modified by AgBr, so that the enrofloxacin can not be detected, and the sensor can be used for detecting the enrofloxacin in the pond water sample.
The above embodiments are only used for illustrating the present invention, and are not meant to be limiting, and those skilled in the relevant art can make various changes without departing from the scope of the present invention, and therefore all technical solutions formed by equivalent substitutions or equivalent modifications belong to the protection scope of the present invention.
Sequence listing
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<120> electrochemiluminescence aptamer sensor for detecting enrofloxacin, preparation method thereof and method for detecting enrofloxacin
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Claims (10)
1. An electrochemiluminescence aptamer sensor for detecting enrofloxacin, which is characterized in that: the electrochemical luminescence aptamer sensor is characterized in that an aptamer containing a 3 '-CCC ATC AGG GGG CTA GGC TAA CAC GGT TCG GCT CTC TGA GCC CGG GTT ATT TCA GGG GGA-5' COOH base sequence is supported on Ti3C2The surface of the glassy carbon electrode modified by the-AgBr nano composite material.
2. The method for preparing an electrochemiluminescence aptamer sensor for detecting enrofloxacin as claimed in claim 1, wherein: the method comprises the following steps:
(1) preparation of Ti by wet chemical method3C2-AgBr nanocomposite, followed by Ti3C2Dispersing the-AgBr nano-composite in DMF to obtain a dispersion liquid;
(2) polishing the glassy carbon electrode, sequentially ultrasonically cleaning the glassy carbon electrode by using a nitric acid solution, an ethanol solution and ultrapure water, and drying the glassy carbon electrode at room temperature to obtain a pretreated glassy carbon electrode for later use; using a liquid-transfering gun to transfer the dispersion liquid prepared in the step (1) to be coated on the surface of the pretreated glassy carbon electrode, and naturally drying to obtain the Ti3C2A glassy carbon electrode modified by an AgBr nano composite material, and then the aptamer is loaded on Ti through electrostatic adsorption3C2And (4) naturally airing the surface of the glassy carbon electrode modified by the AgBr nano composite material to prepare the electrochemical luminescence aptamer sensor.
3. The method for preparing an electrochemiluminescence aptamer sensor for detecting enrofloxacin according to claim 2, wherein the electrochemiluminescence aptamer sensor comprises: ti in the dispersion liquid obtained in the step (1)3C2The concentration of the-AgBr nanocomposite is 2 +/-1 mg/mL; and/or the transfer amount of the dispersion liquid in the step (2) is 6 +/-2 mu L.
4. The method for preparing an electrochemiluminescence aptamer sensor for detecting enrofloxacin according to claim 2, wherein the electrochemiluminescence aptamer sensor comprises: in the step (2), the aptamer is loaded on Ti3C2The method for modifying the surface of the glassy carbon electrode by the-AgBr nano composite material comprises the following steps: firstly, KCl, NaCl and MgCl are added2Adding the aptamer into Tris-HCl buffer solution of EDTA to prepare aptamer solution with aptamer concentration of 0.5-10 mu mol/L, transferring 6 +/-2 mu L of the aptamer solution, and dripping the aptamer solution on the Ti3C2-a glassy carbon electrode surface modified by AgBr nanocomposite.
5. The method for preparing an electrochemiluminescence aptamer sensor for detecting enrofloxacin according to claim 4, wherein the electrochemiluminescence aptamer sensor comprises: the concentration of the aptamer in the aptamer solution is 2.0-5.0 mu mol/L.
6. The method for preparing an electrochemiluminescence aptamer sensor for detecting enrofloxacin according to claim 4, wherein the electrochemiluminescence aptamer sensor comprises: the concentration of the aptamer in the aptamer solution is 2.5 mu mol/L.
7. A method for detecting enrofloxacin, which is characterized by comprising the following steps: the method comprises the following steps:
the electrochemiluminescence aptamer sensor for detecting enrofloxacin as claimed in claim 1 is used as a working electrode, Ag/AgCl is used as a reference electrode, a platinum wire electrode is used as a counter electrode to form a three-electrode system, enrofloxacin in a sample to be detected is quantitatively captured on the surface of the sensor, and detection is carried out through a generated luminescence signal.
8. The method for detecting enrofloxacin as claimed in claim 7, further comprising the steps of:
step 1, containing K2S2O8Preparation of PBS buffer solution of (1):
using a pH of 7.0-8.0 of 0.1mol/LPBS buffer solution is prepared to contain 0.05-0.1 mol/L K2S2O8PBS buffer solution of (4);
step 2, preparing enrofloxacin standard solutions with different concentrations: firstly preparing enrofloxacin solution, and then adding the enrofloxacin solution into the solution containing K in the step 12S2O8Obtaining a series of enrofloxacin standard solutions with different concentrations in the PBS buffer solution, wherein the concentration range of the enrofloxacin standard solution is 1 multiplied by 10-12mol/L～1×10-6mol/L；
Step 3, drawing a standard curve:
placing the electrochemiluminescence aptamer sensor in the enrofloxacin standard solutions with different concentrations prepared in the step 2, soaking for the same time to enable the electrochemiluminescence aptamer sensor to be combined with enrofloxacin, taking out the electrochemiluminescence aptamer sensor, leaching to serve as a working electrode, taking Ag/AgCl as a reference electrode, taking a platinum electrode as a counter electrode to form a three-electrode system, and taking the K-containing standard solution in the step 1 as a K-containing standard solution2S2O8The PBS buffer solution is electrolyte, within an electrochemical window range of-1.8-0V, the photomultiplier tube has a high voltage of 600-800V and a sweep rate of 0.05-0.125V/s, cyclic voltammetry scanning is carried out, a luminous intensity-time curve is recorded, a linear relation between a luminous intensity difference value before and after the combination of an electrochemiluminescence aptamer sensor and enrofloxacin and a logarithm value of enrofloxacin concentration in an enrofloxacin standard solution is established, and a corresponding linear regression equation is obtained;
step 4, detecting enrofloxacin in the sample to be detected
Filtering the sample and then using the K-containing sample obtained in the step 12S2O8Adjusting the pH of the PBS buffer solution to the value containing K2S2O8And (3) the PBS buffer solutions are consistent, then the electrochemical luminescence aptamer sensor is placed into the electrochemical luminescence aptamer sensor to be soaked for the same time, the electrochemical luminescence aptamer sensor is combined with enrofloxacin, then the electrochemical luminescence aptamer sensor is taken out and rinsed to be used as a working electrode, the method in the step 3 is adopted to detect the luminescence intensity, and the concentration of enrofloxacin in the sample is calculated according to a linear regression equation.
9. The method for detecting enrofloxacin based on an electrochemiluminescent aptamer sensor of claim 8, wherein: and the soaking time in the step 3 is 20-40 min.
10. The method for detecting enrofloxacin as claimed in claim 8, wherein:
the pH of the PBS buffer solution in the step 1 is 7.4, and the prepared K-containing solution2S2O8The concentration of the PBS buffer solution is 0.1 mol/L;
the soaking time in the step 3 is 30min, the photomultiplier high pressure is 800V, and the sweeping speed is 0.1V/s.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113533279A (en) * | 2021-07-15 | 2021-10-22 | 河北农业大学 | Method for detecting enrofloxacin by using fluorescent dipeptide nano microspheres/nucleic acid aptamer |
| CN114487058A (en) * | 2021-12-10 | 2022-05-13 | 江苏大学 | Preparation method and application of photoelectrochemical aptamer sensor for detecting fluoroquinolone antibiotics |
| CN114544720A (en) * | 2022-01-12 | 2022-05-27 | 肇庆学院 | Electrochemical sensor for ultra-sensitive detection of enrofloxacin and preparation method thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103760151A (en) * | 2014-02-18 | 2014-04-30 | 周亚民 | Method for detecting quinolone antibiotics by electrochemiluminescence sensor |
| KR20150003013A (en) * | 2013-06-28 | 2015-01-08 | 호서대학교 산학협력단 | Nucleic acid aptamer specifically binding to enrofloxacin or ciprofloxacin |
| CN104830866A (en) * | 2015-05-20 | 2015-08-12 | 江南大学 | ssDNA aptamer for recognizing ofloxacin with specificity and application of ssDNA aptamer |
| CN107764887A (en) * | 2017-10-26 | 2018-03-06 | 天津科技大学 | A kind of 24 site microarray screen-printing electrochemical sensing devices and its application |
| CN110988070A (en) * | 2019-12-20 | 2020-04-10 | 常州大学 | Electrochemical luminescence aptamer sensor and method for detecting chloramphenicol |
| CN111004804A (en) * | 2019-12-12 | 2020-04-14 | 中国海洋大学 | Aptamer and kit for detecting enrofloxacin and ciprofloxacin and application of aptamer and kit |
| CN111257397A (en) * | 2020-02-07 | 2020-06-09 | 烟台大学 | Preparation method of imprinted electroluminescent sensor for detecting enrofloxacin |
-
2020
- 2020-11-03 CN CN202011211366.5A patent/CN112505024A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20150003013A (en) * | 2013-06-28 | 2015-01-08 | 호서대학교 산학협력단 | Nucleic acid aptamer specifically binding to enrofloxacin or ciprofloxacin |
| CN103760151A (en) * | 2014-02-18 | 2014-04-30 | 周亚民 | Method for detecting quinolone antibiotics by electrochemiluminescence sensor |
| CN104830866A (en) * | 2015-05-20 | 2015-08-12 | 江南大学 | ssDNA aptamer for recognizing ofloxacin with specificity and application of ssDNA aptamer |
| CN107764887A (en) * | 2017-10-26 | 2018-03-06 | 天津科技大学 | A kind of 24 site microarray screen-printing electrochemical sensing devices and its application |
| CN111004804A (en) * | 2019-12-12 | 2020-04-14 | 中国海洋大学 | Aptamer and kit for detecting enrofloxacin and ciprofloxacin and application of aptamer and kit |
| CN110988070A (en) * | 2019-12-20 | 2020-04-10 | 常州大学 | Electrochemical luminescence aptamer sensor and method for detecting chloramphenicol |
| CN111257397A (en) * | 2020-02-07 | 2020-06-09 | 烟台大学 | Preparation method of imprinted electroluminescent sensor for detecting enrofloxacin |
Non-Patent Citations (1)
| Title |
|---|
| 中国冶金百科全书总编辑委员会《金属材料卷》编辑委员会编: "《中国冶金百科全书 金属材料》", 31 March 2001 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113533279A (en) * | 2021-07-15 | 2021-10-22 | 河北农业大学 | Method for detecting enrofloxacin by using fluorescent dipeptide nano microspheres/nucleic acid aptamer |
| CN113533279B (en) * | 2021-07-15 | 2022-07-29 | 河北农业大学 | Method for detecting enrofloxacin by using fluorescent dipeptide nano microspheres/nucleic acid aptamer |
| CN114487058A (en) * | 2021-12-10 | 2022-05-13 | 江苏大学 | Preparation method and application of photoelectrochemical aptamer sensor for detecting fluoroquinolone antibiotics |
| CN114544720A (en) * | 2022-01-12 | 2022-05-27 | 肇庆学院 | Electrochemical sensor for ultra-sensitive detection of enrofloxacin and preparation method thereof |
| CN114544720B (en) * | 2022-01-12 | 2023-09-05 | 肇庆学院 | Electrochemical sensor for ultra-sensitively detecting enrofloxacin and preparation method thereof |
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