CN112147200A - Electrochemical luminescence aptamer sensor for detecting kanamycin and preparation method thereof - Google Patents
Electrochemical luminescence aptamer sensor for detecting kanamycin and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of electrochemical luminescence detection, and particularly relates to an electrochemical luminescence aptamer sensor for detecting kanamycin and a preparation method thereof. The aptamer is loaded on the surface of a glassy carbon electrode modified by a composite material Au @ HKUST-1/PTC-Cys, the Au-S bond between the Au @ HKUST-1 and the PTC-Cys is combined and modified on the surface of the glassy carbon electrode, so that the sensitivity and stability of electrochemical luminescence are remarkably improved, the aptamer is loaded to obtain an electrochemical luminescence aptamer sensor, a target molecule kanamycin can be specifically identified, and the selectivity of kanamycin detection is improved. The detection range of the electrochemical luminescence aptamer sensor is 1.0 multiplied by 10‑13～1.0×10‑8M, minimum detection limit of 4.2 × 10‑14And M. The preparation method of the modified electrode is simple and rightThe kanamycin detection has high sensitivity, good selectivity and wide linear range.
Description
Technical Field
The invention belongs to the field of electrochemical luminescence detection, and particularly relates to an electrochemical luminescence aptamer sensor for detecting kanamycin and a preparation method thereof.
Background
Food safety issues have severely affected people's lives. The monitoring of food safety is very important. Kanamycin (KAN), an aminoglycoside antibiotic, has potential toxicity, and excessive use of KAN can cause serious side effects to human and animal bodies, including ototoxicity, nephrotoxicity and anaphylactic shock, and can affect the health of consumers. In order to improve the awareness of the harm of antibiotic residues and effectively protect human health, the European Union has clearly specified that the Maximum Residual Limit (MRL) of kanamycin in milk is 150 mug kg-1Improper use of kanamycin can lead to residues in meat, dairy products and other animal derived foods that can enter the human body through the biological circulatory system, which can be a health hazard to the human body and can be a public health hazard. Therefore, it is important to enhance the detection of kanamycin residues.
In the prior art, common methods for detecting kanamycin mainly comprise a high performance liquid chromatography, an enzyme-linked immunosorbent assay, a liquid chromatography, a gas chromatography, an electrochemical method and the like, but the methods generally have the defects of complex experimental operation, long time consumption and the like, and are difficult to popularize in the market for field detection.
The existing aptamer sensor has high detection sensitivity, but has high detection cost, long detection time and larger result error. Therefore, it is necessary to establish a kanamycin detection technology which has low cost, high sensitivity and high selectivity and can be used for rapid detection on the food safety site.
Disclosure of Invention
In view of the defects in the prior kanamycin detection pointed out in the background art, the invention develops an electrochemiluminescence aptamer sensor for detecting kanamycin, so as to improve the kanamycin detection efficiency, the sensitivity and the selectivity and make the kanamycin detection more practical.
The first purpose of the invention is to provide an electrochemiluminescence aptamer sensor for detecting kanamycin, which has the advantages of high sensitivity, good reproducibility, good selectivity and wide linear range.
The technical purpose of the invention is realized by the following technical scheme:
the invention provides an electrochemiluminescence aptamer sensor for detecting kanamycin, which is prepared by loading an aptamer on the surface of a glassy carbon electrode modified by a composite material Au @ HKUST-1/PTC-Cys. The sensor disclosed by the invention fully utilizes the Au-S bond combination effect between the PTC-Cys and the Au @ HKUST-1 to jointly modify the surface of the glassy carbon electrode, so that the sensitivity and stability of electrochemical luminescence are obviously improved, and then an Aptamer is loaded to further obtain an electrochemical luminescence Aptamer sensor (Aptamer/Au @ HKUST-1/PTC-Cys/GCE sensor for short), so that a target molecule kanamycin can be specifically identified, and the selectivity of kanamycin detection is improved.
Wherein the aptamer is an aptamer comprising a base sequence of 5'-AGATGGGGGTTGAGGCTAAGCCGA-3'.
The PTC-Cys is independently modified on the surface of the electrode, the electrochemical luminescence response intensity is not high and unstable, when Au @ HKUST-1 is loaded, the composite material has more stable performance through the combination effect of Au-S bonds, and the composite material on the electrode cannot fall off when the electrode is soaked in a potassium persulfate solution, so that the stability of the sensor can be improved.
The preparation method of the composite material Au @ HKUST-1/PTC-Cys modified glassy carbon electrode comprises the following steps:
s1, preparing PTC-Cys: 0.1g of perylenetetracarboxylic dianhydride (PTCDA) is dissolved in 20mL of an aqueous solution containing 0.04g of NaOH, a 1M HCl solution is slowly added to the solution until a bright red precipitate is formed, the precipitate is washed several times with deionized water until the pH value of the suspension is about 7, and the washed Precipitate (PTCA) is dried for later use.
5mg of PTCA prepared above was weighed accurately, dispersed in 10mL of deionized water, ultrasonically dispersed uniformly, stirred in an ice-water bath for 30min, and 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added, stirring was continued for 2h to activate the carboxyl group, then 100mg of cysteine was added, stirring was continued overnight, and finally, the product PTC-Cys was collected by centrifugation. Dispersing the PTC-Cys in DMF to uniformly disperse the PTC-Cys to obtain a DMF dispersion liquid of the PTC-Cys;
among these, 1-ethyl- (3-dimethylaminopropyl) carbodiimides hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (m/m ═ 1:1-4: 1).
The concentration of the resulting DMF dispersion of PTC-Cys was 1 mg/mL.
S2. preparation of Au @ HKUST-1:
Cu2and (3) synthesis of O: 0.2g polyvinylpyrrolidone (PVP (K30)) was added to 100mL of 0.01M copper nitrate trihydrate (Cu (NO)3)2·3H2O), then 25mL of 1.5M sodium hydroxide (NaOH) solution was added to produce a blue precipitate immediately, stirred, 25mL of 0.1M ascorbic acid was added, and after 15min, Cu was added2Generating an O precipitate, and dispersing the O precipitate in ethanol;
Au@Cu2o heterostructure: 10mL of 3mg/mL Cu prepared as described above was removed2Oalcoholic solution, 10mL of 3 mM chloroauric acid (HAuCl) was added4) Standing, centrifuging to collect the product (8000rpm, 5min), and washing to obtain Au @ Cu2O-heterostructure dispersed in ethanol.
5mL of 0.16M trimesic acid (H)3BTC), 5mL of N, N-Dimethylformamide (DMF) and 10mL of 10mg/mL Au @ Cu prepared as described above2Mixing the ethanol solution of the O heterojunction, stirring the mixture overnight, performing centrifugal separation, washing, and drying to obtain a green crystal Au @ HKUST-1; dispersing Au @ HKUST-1 in DMF, and performing ultrasonic treatment to uniformly disperse the Au @ HKUST-1 in the DMF to obtain a DMF dispersion liquid of Au @ HKUST-1;
the concentration of the DMF dispersion of Au @ HKUST-1 was 1 mg/mL.
S3, polishing the glassy carbon electrode, sequentially carrying out ultrasonic cleaning by using a nitric acid solution, an ethanol solution and ultrapure water, and blow-drying at room temperature to obtain a pretreated glassy carbon electrode for later use; and sequentially transferring the DMF dispersion liquid of the PTC-Cys in the step S1 and the DMF dispersion liquid of the Au @ HKUST-1 in the step S2 by using a micro-injector, dripping the DMF dispersion liquid on the surface of the pretreated glassy carbon electrode, and naturally airing to obtain the glassy carbon electrode modified by the Au @ HKUST-1/PTC-Cys composite material.
Wherein the volume ratio of the composite modification of the PTC-Cys and the Au @ HKUST-1 is as follows: 4:1-4:5.
The second purpose of the invention is to provide a preparation method of an electrochemiluminescence aptamer sensor for detecting kanamycin.
The technical purpose of the invention is realized by the following technical scheme:
according to the preparation method of the electrochemical luminescence aptamer sensor for detecting kanamycin, the aptamer is loaded on the surface of the glassy carbon electrode modified by the Au @ HKUST-1/PTC-Cys composite material through the gold-sulfur bond combination effect, and the glassy carbon electrode is naturally dried to prepare the electrochemical luminescence aptamer sensor.
The specific method for loading the aptamer on the surface of the glassy carbon electrode modified by the Au @ HKUST-1/PTC-Cys composite material comprises the following steps: firstly, KCl, NaCl and MgCl are added2And adding an aptamer into a Tris-HCl buffer solution of ethylene diamine tetraacetic acid to prepare an aptamer solution with the aptamer concentration of 2-10 mu M, and then transferring and dripping the aptamer solution on the surface of the glassy carbon electrode modified by the Au @ HKUST-1/PTC-Cys nano composite material.
Further, the concentration of the aptamer in the aptamer solution was 8. mu.M.
The third purpose of the invention is to provide an application method of an electrochemiluminescence aptamer sensor for detecting kanamycin.
The technical purpose of the invention is realized by the following technical scheme:
the specific method of the electrochemiluminescence aptamer sensor for detecting kanamycin provided by the invention is characterized in that the electrochemiluminescence aptamer sensor 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, kanamycin in a sample is quantitatively captured on the surface of the sensor, and kanamycin detection is realized through a generated luminescence signal.
Further, the method comprises the following specific steps:
A1. containing K2S2O8Preparation of PBS buffer solution of (1): 0.05M K was made up with 0.1M PBS buffer at pH 7.42S2O8PBS buffer solution of (4);
A2. preparation of kanamycin standard solutions of different concentrations: first, 1X 10 of the mixture was prepared-4Dissolving the kanamycin solution of M in ultrapure water, and then diluting with water to obtain a plurality of kanamycin standard solutions with different concentrations, wherein the concentration range of the kanamycin standard solution is 1.0 multiplied by 10-13～1.0×10-8M；
A3. Drawing a standard curve: placing the electrochemiluminescence aptamer sensor in kanamycin standard solutions with different concentrations prepared in the step A2 to be soaked for the same time, enabling the electrochemiluminescence aptamer sensor to be combined with kanamycin, taking out and leaching the kanamycin 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 kanamycin standard solution in the step A1 as a K-containing system2S2O8The PBS buffer solution is electrolyte, in the range of an electrochemical window of-1.7-0V, the photomultiplier has high voltage of 800V and the sweep rate of 0.1V/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 electrochemical luminescence aptamer sensor is combined with kanamycin and a kanamycin concentration logarithm value in a kanamycin standard solution is established, and a corresponding linear regression equation is obtained;
A4. detection of kanamycin in the samples: the sample was pretreated and then subjected to K-containing step A12S2O8And (3) adjusting the pH value by using the PBS buffer solution, then putting the prepared kanamycin with different concentrations into an electrochemiluminescence aptamer sensor to be soaked for the same time, enabling the electrochemiluminescence aptamer sensor to be combined with the kanamycin, then taking out and leaching the kanamycin to be used as a working electrode, detecting the luminescence intensity by adopting the method of the step A3, and calculating the concentration of the kanamycin in the sample according to a linear regression equation.
Further, the soaking time in the step A3 is 25 min.
In conclusion, the invention has the following beneficial effects:
the invention designs an electrochemiluminescence aptamer sensor based on a copper-based metal organic framework Au @ HKUST-1 composite material of a Per derivative PTC-Cys and doped gold nanoparticles, wherein the two materials are connectedThe invention fully utilizes the advantages of the aptamer and the electrochemiluminescence sensor, successfully realizes the sensitive detection of kanamycin through the enhancement effect of kanamycin on the ECL signal intensity of the system, and the sensing platform can specifically identify a detection object kanamycin and has high selectivity. The detection range of the invention is 1.0 multiplied by 10-13～1.0×10- 8M, minimum detection limit of 4.2 × 10-14And M. The kanamycin detection method is simple to operate, good in selectivity, low in detection cost and high in sensitivity. The invention has important significance for popularizing the practical application of the aptamer sensor in the aspect of food safety.
Drawings
FIG. 1 is a schematic flow chart of the preparation of an electrochemiluminescent aptamer sensor according to the invention and the detection of kanamycin.
FIG. 2 is a graph of ECL response of the electrochemiluminescent aptamer sensor constructed in example 1 after binding with different concentrations of kanamycin, which are, from left to right: (a) 1.0X 10-13M；(b)1.0×10-12M；(c)1.0×10-11M；(d)1.0×10-10M；(e)1.0×10-9M；(f)1.0×10-8M。
FIG. 3 is a standard curve of the difference in luminescence intensity between before and after addition of kanamycin and the logarithmic value of kanamycin concentration in example 1;
FIG. 4 is a scanning electron micrograph of the Au @ HKUST-1/PTC-Cys composite prepared in example 1;
FIG. 5 is a graph of the electrochemiluminescence stability characterization of the sensor Aptamer/Au @ HKUST-1/PTC-Cys/GCE prepared in example 1 for 15 consecutive cyclic scans;
FIG. 6 is a preferred characterization of the ratio of EDC to NHS in amidation reaction of example 3 at 4:1 (m/m); in the figure, curve a represents EDC: NHS ═ 1:1(m/m), and curve b represents EDC: NHS ═ 4:1 (m/m);
FIG. 7 is an optimized characterization plot of aptamer concentration in example 4.
Detailed Description
The present invention is further described below with reference to examples, but is not limited thereto.
In the following examples, aptamers containing 5'-AGATGGGGGTTGAGGCTAAGCCGA-3' base sequences were purchased from Biotechnology engineering (Shanghai) Inc., and the methods for loading the aptamers on the surface of a glassy carbon electrode modified with Au @ HKUST-1/PTC-Cys composite material were as follows: firstly, KCl, NaCl and MgCl are added2And adding the aptamer into a Tris-HCl buffer solution of ethylene diamine tetraacetic acid to prepare an aptamer solution, and then transferring the aptamer solution and dropwise coating the aptamer solution on the surface of the glassy carbon electrode modified by the Au @ HKUST-1/PTC-Cys composite material. Specifically, the method comprises the following steps:
A1. before opening the cover of the purchased aptamer, carrying out vortex oscillation for 10min, and then centrifuging at 4000rpm for 20 min;
A2. slowly open the tube cap, add 1020. mu.l of a solution containing 0.2mol/L KCl, 0.1mol/L NaCl, 5.0mmol/L MgCl, as labeled on the tube2And 1.0mmol/L EDTA in 0.05M Tris-HCl buffer solution;
A3. fully and uniformly oscillating, diluting to a concentration of 10 mu M, and refrigerating in a refrigerator at 4 ℃ for standby.
The preparation method of kanamycin standard solutions with different concentrations in the following examples is as follows: preparing kanamycin solution, and then diluting with ultrapure water to obtain a series of kanamycin standard solutions with different concentrations, wherein the concentration of kanamycin in the kanamycin standard solution in the embodiment is (a)1.0 multiplied by 10 respectively-13M；(b)1.0×10-12M；(c)1.0×10-11M；(d)1.0×10-10M；(e)1.0×10-9M；(f)1.0×10-8M。
Example 1
(I) electrochemical luminescence aptamer sensor for detecting kanamycin by assembly
(1) Preparation of PTC-Cys and Au @ HKUST-1 materials:
s1, preparing a precursor PTCA, namely dissolving 0.1g of PTCDA in 20mL of aqueous solution containing 0.04g of NaOH, slowly adding 1M HCl solution into the solution until bright red precipitates are generated, washing the precipitates for several times by deionized water until the pH value of the suspension is equal to 7, and drying for later use. Accurately weighing 5mg of the prepared PTCA, dispersing the PTCA in 10mL of deionized water, uniformly dispersing the PTCA by ultrasonic, stirring the mixture in an ice-water bath for 30min, adding 40mg of EDC and 10mg of NHS (m/m is 4:1), continuously stirring the mixture for 2h to activate carboxyl, then adding 100mg of cysteine, stirring the mixture overnight, and finally, collecting a product, namely PTC-Cys by centrifugation; and (3) dispersing the PTC-Cys in DMF (dimethyl formamide) uniformly to obtain a DMF dispersion liquid of the PTC-Cys with the concentration of 1 mg/mL.
S2, preparing Au @ HKUST-1
Cu2And (3) synthesis of O: 0.2g polyvinylpyrrolidone (PVP (K30)) was added to 100mL of 0.01M copper nitrate trihydrate (Cu (NO)3)2·3H2O), then 25mL of 1.5M sodium hydroxide (NaOH) solution is added, a blue precipitate is formed immediately, stirred, 0.4403g of ascorbic acid are added, after 15min Cu2Generating an O precipitate, and dispersing the O precipitate in ethanol;
Au@Cu2o heterostructure: 10mL of 3mg/mL Cu prepared as described above was removed2Oalcoholic solution, 10mL of 3 mM chloroauric acid (HAuCl) was added4) Standing, centrifuging to collect the product (8000rpm, 5min), and washing to obtain Au @ Cu2O-heterostructure dispersed in ethanol.
5mL 0.16M H3BTC, 5mL DMF and 10mL10 mg/mL Au @ Cu prepared as described above2Mixing ethanol solutions of the O heterojunction, stirring the mixture overnight, performing centrifugal separation, and washing to obtain Au @ HKUST-1; dispersing Au @ HKUST-1 in DMF, and performing ultrasonic treatment to uniformly disperse the Au @ HKUST-1 to obtain 1mg/mL of DMF dispersion liquid of Au @ HKUST-1.
(2) Preparation of electrochemiluminescence aptamer sensor for detecting kanamycin
Firstly, polishing powder (Al) for glassy carbon electrode2O3) And polishing the chamois leather into a mirror surface, then ultrasonically cleaning for 3min by using a nitric acid solution, an ethanol solution and ultrapure water in sequence, and drying at room temperature to obtain the pretreated glassy carbon electrode. Sequentially transferring 4 mu L of 1mg/mL DMF dispersion liquid of PTC-Cys prepared in the step (1) by using a micro-injector, naturally airing, then dropwise coating 3 mu L of 1mg/mL DMF dispersion liquid of Au @ HKUST-1, naturally airing to obtain an Au @ HKUST-1/PTC-Cys/GCE modified electrode, and naturally airing for later use; in Au @ HKUST-1/PTC-CysAnd 5 mu L of prepared Tris-HCl buffer solution containing the Aptamer is dripped on the surface of the GCE modified electrode, and the mixture is naturally dried for 10h to obtain the Aptamer/Au @ HKSUT-1/PTC-Cys/GCE sensor which is used as a sensing element for electrochemical luminescence test.
Wherein the concentration of the aptamer in the aptamer solution is 8 mu M.
(II) application method of electrochemiluminescence aptamer sensor for detecting kanamycin
A1. Containing K2S2O8Preparation of PBS buffer solution of (1):
0.05M K was made up with 0.1M PBS buffer at pH 7.42S2O8PBS buffer solution of (4);
A2. preparation of kanamycin standard solutions of different concentrations: first, 1X 10 of the mixture was prepared-4Dissolving the kanamycin solution of M in ultrapure water, and then diluting with ultrapure water to obtain a series of kanamycin standard solutions with different concentrations, wherein the concentration range of the kanamycin standard solution is 1.0 multiplied by 10-13～1.0×10-8M; in this example, the concentration of kanamycin in the kanamycin standard solution was (a) 1.0X 10-13M；(b)1.0×10-12M；(c)1.0×10-11M；(d)1.0×10-10M；(e)1.0×10-9M；(f)1.0×10-8M。
A3. Drawing a standard curve: taking an electrochemiluminescence aptamer sensor for detecting kanamycin in A1 as a sensing element, soaking the electrochemiluminescence aptamer sensor in kanamycin standard solutions with different concentrations for 25min, taking out the electrochemiluminescence aptamer sensor, leaching the electrochemiluminescence aptamer sensor 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 electrochemiluminescence aptamer sensor containing 0.05M K2S2O8Measuring the luminous intensity by using 0.1M PBS buffer solution with the pH of 7.4 as electrolyte, performing cyclic voltammetry scanning on the electrolyte at the high voltage of 800V and the scanning speed of 0.1V/s of a photomultiplier tube within the range of an electrochemical window of-1.7-0V, recording a luminous intensity-time curve, establishing a linear relation between the luminous intensity difference before and after the electrochemical luminescence aptamer sensor is combined with kanamycin and the kanamycin concentration logarithm value in a kanamycin standard solution, and obtaining a corresponding linear regression equation as follows; Δ ECL 9277.5598+2109.2115lgC (nM), detection range is 1.0X 10-13～1.0×10-8M, detection limit of 4.2 × 10-14M。
A4. And (3) detection of the sample: briefly, 10mL milk samples were diluted with 10mL PBS solution (pH 7.4, 0.01. mu.M). Followed by centrifugation at 12,000rpm for about 20 minutes and standing at room temperature for 20 minutes. The supernatant of the milk was extracted, filtered through a 0.22mm microporous membrane, and then KAN standard solution was added to the five-fold diluted milk to prepare kanamycin sample solutions of different concentrations for analytical determination. And adding a solution containing 0.05M K2S2O8The pH of the test sample was adjusted to 7.4 with 0.1M PBS buffer solution, 25mL of the obtained solution was used for electrochemiluminescence analysis, and the concentration of kanamycin in the test sample was calculated according to the linear regression equation obtained in step A1, and the results are shown in Table 1.
In the embodiment, PTC-Cys is used as a substrate material, and the composite material is obtained by dripping Au @ HKUST-1 for the second time, the shape is shown in figure 4, the PTC-Cys and the HKUST-1 can be stably combined through Au-S bonds, the material combination is novel, the electrochemical luminescence intensity of an individual material can be greatly improved, the sensitivity is good, the stability is good, the selectivity of a sensor is good, and the stability is good.
Example 2
Example 1 (one) Assembly of an electrochemiluminescent aptamer sensor for detecting kanamycin
In the preparation process of the PTC-Cys material in the step (1), the ratio of EDC to NHS is changed to 1:1(m/m) in amidation reaction of S1, and the other steps are the same as in example 1.
Example 3
An electrochemiluminescence aptamer sensor for detecting kanamycin is assembled in example 1, and in the step 2, the concentration of an aptamer in an aptamer solution is 2-10 mu M.
Comparative example 1
(1) Preparation of Aptamer/Au @ HKUST-1/GCE sensor
Transferring 3 mu L of 1mg/mL DMF dispersion of Au @ HKUST-1 by using a liquid transfer gun, dripping the DMF dispersion on the surface of a pretreated glassy carbon electrode (the pretreatment method is the same as that in example 1) to obtain an Au @ HKUST-1/GCE chemically modified electrode, and naturally airing for later use; 5 muL of 8 muM Aptamer is dripped on the surface of the Au @ HKUST-1/GCE chemically modified electrode, and the sensor is naturally aired for 10h to obtain the Aptamer/Au @ HKUST-1/GCE sensor which is used as a sensing element for electrochemical luminescence test.
(2) Drawing of standard curve
Taking the Aptamer/Au @ HKUST-1/GCE sensor prepared in the step (1) as a sensing element, soaking the sensing element in kanamycin standard solutions with different concentrations for 25min, taking out the sensing element, leaching the sensing element 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 sensing element containing 0.05M K2S2O8The 0.1M PBS buffer solution with the pH value of 7.4 is used as electrolyte to measure the luminous intensity, the high voltage of a photomultiplier tube is 800V within the electrochemical window range of-1.7-0V, 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 kanamycin and the kanamycin concentration logarithm value in a kanamycin standard solution is established, and a corresponding linear regression equation is obtained.
(3) Detection of samples
A4. And (3) detection of the sample: briefly, 10mL milk samples were diluted with 10mL PBS solution (pH 7.4, 0.01. mu.M). Followed by centrifugation at 12,000rpm for about 20 minutes and standing at room temperature for 20 minutes. Extracting the supernatant of milk, filtering with 0.22mm microporous membrane, adding KAN standard solution to diluted milk five times to prepare kanamycin sample solution with different concentration for analysis, and adding solution containing 0.05M K2S2O8The pH of the sample was adjusted to 7.4 with 0.1M PBS buffer solution, 25mL of the obtained solution was used for electrochemiluminescence analysis, and the concentration of kanamycin in the sample to be assayed was calculated according to the linear regression equation obtained in step (2), and the results are shown in Table 1.
Comparative example 2
(1) Preparation of Aptamer/PTC-Cys/GCE sensor
Transferring 4 mu L of 1mg/mL DMF dispersion solution of PTC-Cys by using a micro-injector, dripping the dispersion solution on the surface of a pretreated glassy carbon electrode (the pretreatment method is the same as that in example 1) to obtain a PTC-Cys/GCE chemically modified electrode, and naturally airing for later use; and (3) dripping 5 mu L of 8 mu M on the surface of the PTC-Cys/GCE chemically modified electrode, and naturally airing for 10h to obtain the Aptamer/PTC-Cys/GCE sensor serving as a sensing element for electrochemical luminescence test.
(2) Drawing of standard curve
Taking the Aptamer/PTC-Cys/GCE sensor prepared in the step (1) as a sensing element, soaking the sensing element in kanamycin standard solutions with different concentrations for 25min, taking out the sensing element, leaching the sensing element 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 sensing element containing 0.05M K2S2O8The 0.1M PBS buffer solution with the pH value of 7.4 is used as electrolyte to measure the luminous intensity, the high voltage of a photomultiplier tube is 800V within the electrochemical window range of-1.7-0V, 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 kanamycin and the kanamycin concentration logarithm value in a kanamycin standard solution is established, and a corresponding linear regression equation is obtained.
(3) Detection of samples
Briefly, 10mL milk samples were diluted with 10mL PBS solution (pH 7.4, 0.01. mu.M). Followed by centrifugation at 12,000rpm for about 20 minutes and standing at room temperature for 20 minutes. Extracting the supernatant of milk, filtering with 0.22mm microporous membrane, adding KAN standard solution to diluted milk five times to prepare kanamycin sample solution with different concentration for analysis, and adding solution containing 0.05M K2S2O8The pH of the sample was adjusted to 7.4 with 0.1M PBS buffer solution, 25mL of the obtained solution was used for electrochemiluminescence analysis, and the concentration of kanamycin in the sample to be assayed was calculated according to the linear regression equation obtained in step (2), and the results are shown in Table 1.
TABLE 1 measurement results of certain milk samples
Wherein: actual quantityaIs 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 between 96% and 103%, the relative standard deviation is less than 5%, and the recovery effect is good. The experimental results show that kanamycin cannot be detected by further assembling a sensing element after a glassy carbon electrode is modified by Au @ HKUST-1 or PTC-Cys alone without modification by an Au @ HKUST-1/PTC-Cys composite material, so that the sensor disclosed by the invention can be used for detecting kanamycin in milk.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. The electrochemical luminescence aptamer sensor is characterized in that the electrochemical luminescence aptamer sensor is prepared by loading an aptamer on the surface of a glassy carbon electrode modified by a composite material Au @ HKUST-1/PTC-Cys.
2. The electrochemiluminescent aptamer sensor according to claim 1, wherein the aptamer is an aptamer comprising 5'-AGATGGGGGTTGAGGCTAAGCCGA-3' base sequence.
3. The electrochemical luminescence aptamer sensor according to claim 1, wherein the composite material Au @ HKUST-1/PTC-Cys modified glassy carbon electrode is prepared by the following method:
s1, preparing PTC-Cys: dissolving perylene tetracarboxylic dianhydride (PTCDA) in an aqueous solution of sodium hydroxide, adding HCl to obtain a red precipitate, washing the red precipitate with deionized water to remove excessive reactants, dispersing to obtain a PTCA solution, sequentially adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the PTCA solution, adding L-cysteine, continuously stirring, centrifuging, and drying to obtain a pink powdery product PTC-Cys; dispersing the PTC-Cys in DMF to uniformly disperse the PTC-Cys to obtain a DMF dispersion liquid of the PTC-Cys;
s2. preparation of Au @ HKUST-1: benzene tricarboxylic acid (H)3BTC), N-Dimethylformamide (DMF) and Au @ Cu2Mixing ethanol solutions of the O heterojunction, stirring the mixture overnight, performing centrifugal separation and washing to obtain Au @ HKUST-1, dispersing the Au @ HKUST-1 in N, N-dimethylformamide, and performing ultrasonic treatment to uniformly disperse the Au @ HKUST-1 to obtain a DMF (dimethyl formamide) dispersion liquid of the Au @ HKUST-1;
s3, polishing the glassy carbon electrode, sequentially carrying out ultrasonic cleaning by using a nitric acid solution, an ethanol solution and ultrapure water, and blow-drying at room temperature to obtain a pretreated glassy carbon electrode for later use; and sequentially transferring the DMF dispersion liquid of the PTC-Cys in the step S1 and the DMF dispersion liquid of the Au @ HKUST-1 in the step S2, dropwise coating the DMF dispersion liquid on the surface of the pretreated glassy carbon electrode, and naturally airing to obtain the glassy carbon electrode modified by the Au @ HKUST-1/PTC-Cys composite material.
4. The electrochemiluminescent aptamer sensor according to claim 3, wherein the molar ratio of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to N-hydroxysuccinimide (NHS) in step S1 is 1:1-4: 1; the concentration of the resulting DMF dispersion of PTC-Cys was 1 mg/mL.
5. The electrochemiluminescent aptamer sensor of claim 3, wherein the concentration of the DMF dispersion of Au @ HKUST-1 obtained in the step S2 is 1 mg/mL.
6. The electrochemiluminescent aptamer sensor according to claim 3, wherein the volume ratio of the complex modification of PTC-Cys and Au @ HKUST-1 in the step S3 is: 4:1-4:5.
7. The method for preparing the electrochemical luminescence aptamer sensor according to any one of claims 1 to 6, wherein the method is characterized in that an aptamer is loaded on the surface of a glassy carbon electrode modified by an Au @ HKUST-1/PTC-Cys composite material through Au-S bond combination, and the glassy carbon electrode is naturally dried to prepare the electrochemical luminescence aptamer sensor.
8. The method for preparing the electrochemical luminescence aptamer sensor according to claim 7, wherein the specific method for loading the aptamer on the surface of the glassy carbon electrode modified by the Au @ HKUST-1/PTC-Cys nanocomposite material comprises the following steps: firstly, KCl, NaCl and MgCl are added2And adding an aptamer into a Tris-HCl buffer solution of ethylene diamine tetraacetic acid to prepare an aptamer solution with the aptamer concentration of 2-10 mu M, and then transferring and dripping the aptamer solution on the surface of the glassy carbon electrode modified by the Au @ HKUST-1/PTC-Cys composite material.
9. An electrochemiluminescence aptamer sensor according to any of claims 1 to 6 for detecting kanamycin, wherein the application method is as follows: an electrochemiluminescence aptamer sensor 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, kanamycin in a sample is quantitatively captured on the surface of the sensor, and kanamycin detection is realized through a generated luminescence signal.
10. The use of an electrochemiluminescence aptamer sensor according to claim 9, wherein the specific steps of the application method are as follows:
A1. containing K2S2O8Preparation of PBS buffer solution of (1): 0.05M K was made up with 0.1M PBS buffer at pH 7.42S2O8PBS buffer solution of (4);
A2. preparation of kanamycin standard solutions of different concentrations: first, 1X 10 of the mixture was prepared-4M Carnacina moldDissolving the kanamycin standard solution with ultrapure water, and diluting with ultrapure water to obtain kanamycin standard solutions with different concentrations, wherein the concentration range of the kanamycin standard solution is 1.0 multiplied by 10-13～1.0×10-8M；
A3. Drawing a standard curve: placing the electrochemiluminescence aptamer sensor in kanamycin standard solutions with different concentrations prepared in the step A2 to be soaked for the same time, enabling the electrochemiluminescence aptamer sensor to be combined with kanamycin, taking out and leaching the kanamycin 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 kanamycin standard solution in the step A1 as a K-containing system2S2O8The PBS buffer solution is electrolyte, in the range of an electrochemical window of-1.7-0V, the photomultiplier has high voltage of 800V and the sweep rate of 0.1V/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 electrochemical luminescence aptamer sensor is combined with kanamycin and a kanamycin concentration logarithm value in a kanamycin standard solution is established, and a corresponding linear regression equation is obtained;
A4. detection of kanamycin in the samples: the sample was pretreated and then subjected to K-containing step A12S2O8And (3) adjusting the pH value by using the PBS buffer solution, then putting the PBS buffer solution into an electrochemiluminescence aptamer sensor to be soaked for the same time, enabling the electrochemiluminescence aptamer sensor to be combined with kanamycin, then taking out and leaching the electrochemiluminescence aptamer sensor to be used as a working electrode, detecting the luminescence intensity by adopting the method in the step A3, and calculating the concentration of kanamycin in the sample according to a linear regression equation.
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