Preparation method of electrochemiluminescence sensor for detecting cadmium and ampicillin in aquatic products

Abstract

The present invention discloses a method for preparing and using an electrochemiluminescent aptamer sensor for simultaneously detecting cadmium ions and ampicillin in aquatic products, which is characterized by utilizing the high conductivity of bismuth sulfide and gold complexes, the high specific surface area of ​​the willow-leaf flower shape, and two bioluminescent probes of Au@Luminol and CdS QDs combined with aptamers to identify cadmium ions and ampicillin at different potentials, thereby constructing an electrochemiluminescent biosensor for simultaneous dual detection. The sensor of the present invention can determine whether the detected aquatic products contain target ions based on the changes in the electrochemiluminescence peaks of cadmium ions and ampicillin in the detection results. The advantages of the present invention are: the sensor is low in cost and the detection operation is simple; it can simultaneously perform qualitative and quantitative determinations on cadmium ions and ampicillin; it has high sensitivity and low detection limit, and provides ideas for the detection of other ions and small molecules.

Description

Technical Field

[0001] The invention relates to an electrochemiluminescent aptamer sensor and a preparation method and a use method of the sensor, belonging to the technical field of electrochemiluminescent aptamer sensors. Background Art

[0002] Aquatic food is an important source of animal food in my country and a large part of the food consumed in our daily life. It is rich in various amino acids, easy for the human body to absorb, and has a higher edible value than general meat. It is also the best food for patients with hyperlipidemia and hypertension. However, with the advancement of industrial society, the quality of aquatic products has been seriously affected, resulting in aquatic products being contaminated by heavy metals, excessive drug residues, and excessive antibiotic content. Among them, aquatic products contaminated by heavy metals and antibiotics cause the greatest harm to the body after people accidentally eat them. For aquatic animals, they are often contaminated by toxic metals and antibiotics, including: exogenous, endogenous, etc., and exogenous pollution, namely, pollution caused by feed, water quality, air, and drugs. Ultimately, these toxic and harmful metals and antibiotics that endanger health enter the human body through the food chain, endangering the nervous and blood systems, gastrointestinal tract, cardiovascular, renal system, and immune system. Therefore, it is particularly important to develop a convenient, efficient, fast and practical analysis method for heavy metal and antibiotic pollution in aquatic products. This work has important theoretical and practical significance for protecting the ecological environment, improving food quality, and protecting human safety.

[0003] Detection methods for heavy metals and antibiotics: Currently, the main detection methods for heavy metals include gas chromatography, high performance liquid chromatography, atomic absorption spectroscopy, spectrophotometry, atomic emission spectroscopy, inductively coupled plasma mass spectrometry, colorimetry, etc. Although these traditional heavy metal detection methods are relatively mature, have high sensitivity and accuracy, low detection limits, and wide detection ranges, the detection of antibiotics mainly includes liquid chromatography, enzyme immunoassay, capillary electrophoresis, etc., but these instruments are expensive, time-consuming, sample processing is complicated, inconvenient to carry, and cannot be continuously monitored and measured on-site, which greatly limits their wide application. Therefore, seeking a simple, rapid, and sensitive technology for simultaneous detection of heavy metal ions and antibiotics is still a goal that people are constantly pursuing. In order to effectively ensure food safety and human life safety, it is necessary to establish an efficient, sensitive, and practical method for rapid detection of heavy metals and antibiotics.

[0004] The purpose of the invention is to provide a method for preparing an electrochemiluminescent aptamer sensor that can overcome the above problems and has simple preparation, flexible operation, high sensitivity, high accuracy, good selectivity and stability. The constructed electrochemiluminescent aptamer sensor does not require the catalytic action of an enzyme, directly binds to the target substance through the aptamer, and can detect two substances at the same time. The method can realize the specific detection of heavy metal cadmium ions and ampicillin in aquatic products. Summary of the invention

[0005] The technical solution is: a method for preparing an aptamer sensor for detecting heavy metals of cadmium and ampicillin, characterized in that: the sensitive interface composition of the aptamer sensor includes bismuth sulfide and gold (Bi 2 S 3 @Au) composites, gold and luminol (Au@Luminol) composites, and cadmium sulfide (CdS QDs) were used to immobilize the aptamers of heavy metal cadmium and ampicillin.

[0006] The method for preparing a dual-detection aptamer sensor is characterized by: cleaning of the glassy carbon electrode (d = 3 mm), construction of the sensitive interface of the aptamer sensor and process characterization (preparation of Bi 2 S 3 @Au, Au@Luminol, CdSQDs, using nanoflower-like Bi 2 S 3 @Au is used to immobilize the aptamer, Au@Luminol and CdS QDs are connected to the aptamer that recognizes the target substance to form a bioluminescent probe, and the electrode is modified together), the working curve of the aptamer sensor is established, the performance of the adapter sensor is tested, and the aptamer sensor is tested on actual samples.

[0007] The method for preparing a simultaneous dual-detection aptamer sensor is characterized in that: the optimization of experimental conditions mainly includes the aptamer concentration, the pH of the test base solution, the base solution concentration, and the incubation time of the aptamer and the target substance. The working curve of the prepared aptamer sensor is: y=7574.57-1051.10lgx(R 2 =0.998), y=5608.49-916.00lgx(R 2 =0.996), and the performance test of the aptamer sensor included the determination of specific selectivity, reproducibility, stability, and the recovery rate of the aptamer sensor for aquatic products.

[0008] Electrochemiluminescence detection of target ions: The electrochemiluminescence detection method is used to detect aquatic products using a three-electrode system under optimal experimental conditions. According to the analysis of the electrochemiluminescence intensity value in the test results, it can be known that the peak value of the electrochemiluminescence intensity is inversely proportional to the concentration of cadmium metal ions and ampicillin, and the concentration information of the target substance can be obtained based on this.

[0009] The preparation principle of the present invention is: using the high affinity characteristics of nucleic acid aptamers and the principle of base complementarity, the aptamer ends are cross-linked to the solid phase carrier as capture molecules to capture the target substance in the sample to be tested. At the same time, the aptamer is connected to the corresponding luminescent indicator as a signal molecule for detecting the target substance. When the signal molecule combines with the corresponding sample to be tested, a signal is generated, thereby achieving the purpose of detection. The present invention uses willow-shaped nanoflower Bi 2 S 3 @Au, Au@Luminol and CdS QDs were used to modify the glassy carbon electrode. 2 S 3 @Au has high specific surface area and conductivity, which not only provides more active sites for the aptamer, but also increases the electron transport capacity and stability of the electrode surface. 2 S 3 The high specific surface area of ​​Au@Luminol provides a good sensor interface for the fixation of aptamers that identify target substances, maintains the effective fixation amount of cadmium heavy metal and ampicillin aptamers, and thus increases the detection accuracy of the sensor. At the same time, Au@Luminol and CdS QDs are used as two luminescent substances to display electrochemiluminescence signal values ​​at different potentials to achieve the detection of two target substances, cadmium ions and ampicillin. The aptamer sensor prepared by the above steps is simple and sensitive, has good stability and specificity, good reproducibility, and the recovery rate meets the requirements.

[0010] To achieve the above objectives, the following technical solutions are adopted: a method for preparing a dual-detection aptamer sensor, characterized in that: (1) before preparing the aptamer sensor, the bare glassy carbon electrode is cleaned, activated and tested for performance, and the electrode is tested under cyclic voltammetry until it meets the requirements; (2) the surface of the cleaned glassy carbon electrode is drop-coated with a uniformly dispersed nanoflower Bi 2 S 3 @Au, and then fix the aptamer DNA1 chain on Bi 2 S 3 @Au, and use hexamercaptohexanol (MCH) to block the unbound sites, then drop-coat the constructed bioluminescent probe Au@Luminol and CdS QDs to complement the base pairing of the aptamer DNA 1 chain, and finally add two target substances; after the preparation of the aptamer sensor is completed, it is stored in a 4°C refrigerator for future use.

[0011] To achieve the above objectives, the following technical solutions are adopted: a method for preparing a dual-detection aptamer sensor, characterized in that: (1) the prepared aptamer sensor is detected by electrochemiluminescence in a working base solution to obtain the relationship between the peak value and the two target substances; (2) a series of cadmium ion and ampicillin concentration standard solutions are prepared and electrochemiluminescence is used to measure the concentration, and the working curve, detection range and detection limit of the prepared aptamer sensor are further obtained; (3) a series of common heavy metal and antibiotic solutions are prepared to detect the anti-interference property of the prepared aptamer sensor; (4) the stability of the aptamer sensor is verified multiple times by electrochemiluminescence; and (5) the actual aquatic products are analyzed to obtain the recovery rate of the aptamer sensor.

[0012] The preparation process of the aptamer sensor is as follows: 6 μL of nanoflower Bi 2 S 3 @Au, dry the electrode at room temperature to obtain Bi 2 S 3 @Au / GCE. Then in Bi 2 S 3 6 μL of 200 nM aptamer DNA 1 chain was added to the Au / GCE electrode and reacted with Bi 2 S 3 @Au is combined through the chemical bond Au-S, and then MCH is added to block the non-specific binding sites. The electrode is allowed to dry naturally at room temperature, and finally 6 μL of Au@Luminol and CdS QDs bioluminescent probes are added. The electrode surface is allowed to dry, and the aptamer sensor is prepared and stored at 4 °C for later use. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Characterization of substrate materials for electrochemiluminescence sensors.

[0014] Figure 2 Characterization of luminescent materials for electrochemiluminescence sensors.

[0015] Figure 3 Electrochemical characterization of the electrochemiluminescent sensor.

[0016] Figure 4 Aptamer concentration optimization for electrochemiluminescence sensors.

[0017] Figure 5 Optimization of incubation time for electrochemiluminescence sensors.

[0018] Figure 6 Optimization of pH of electrochemiluminescence sensor base solution.

[0019] Figure 7Optimization of electrochemiluminescence sensor base solution concentration.

[0020] Figure 8 Standard curves of different concentrations of heavy metal cadmium and ampicillin measured by electrochemiluminescence sensor.

[0021] Fig. 9 Study on the specificity of electrochemiluminescence sensors.

[0022] Fig.10 Reproducibility study of electrochemiluminescence sensor.

[0023] Fig.11 Study on the stability of electrochemiluminescence sensor. DETAILED DESCRIPTION

[0024] Example:

[0025] (1)Bi 2 S 3 Preparation of Au: 0.5 g Bi(NO 3 ) 3 ·5H 2 O and 20 mL of ethylene glycol were uniformly mixed. Then 0.4 g of thiourea was added to the solution, and then the uniform mixture was transferred to an oil bath and kept at 160 ° C for 20 hours. The product was then collected by several centrifugation cycles using ethanol and ultrapure water and dried under vacuum conditions at 60 ° C for 12 hours. The product Bi 2 S 3 Re-disperse, add the prepared Au NPs solution, and stir for 2 hours to make the Au NPs particles in Bi 2 S 3 Surface growth, followed by centrifugation and washing, and finally Bi 2 S 3 @Au redisperse the spare parts;

[0026] (2) Preparation of Au@Luminol: To prepare Au@Luminol, 4 mL of 0.01 M luminol and 0.6 mL of 0.1 M NaBH 4 Cool with ice and then add 20 mL of 2.5 × 10 -4 M HAuCl 4 After stirring in an ice bath for 10 min, the solution color changed to purple-red, indicating the formation of Au NPs, and finally the product was stirred at room temperature for 6 h to promote the growth of colloidal Au@Luminol;

[0027] (3) Preparation of CdS QDs: L-cysteine ​​(0.363 g), CdCl 2 .5H 2 O(0.275g) and Na 2S.9H 2 O (0.762 g) was dissolved in 15.0 mL, 45.0 mL, and 45.0 mL of ultrapure water to prepare the corresponding solutions. Next, the newly prepared L-cysteine ​​solution was injected into CdCl under stirring. 2 solution, and then the resulting mixture was injected into Na 2 S solution, refluxed continuously at 75 °C for 6 hours, centrifuged at 12000 rpm for 15 minutes to separate the obtained CdS quantum dots, and washed with anhydrous ethanol and ultrapure water respectively;

[0028] (4) Preparation of Au@Luminol-DNA2 and CdS QDs-DNA3 probes: 10 μL of 10 μM aptamer DNA2 solution was added to the prepared 1 mL Au@Luminol solution, and the mixed solution was stirred at room temperature for 6 h to obtain an Au@Luminol probe with normal DNA2 function. 40 mM EDC and 10 mM NHS solution were added to 1 mL CdS QDs solution to activate the carboxyl groups on the surface of CdS QDs. Then 10 μL of aptamer DNA3 (1 μM) was dropped into the solution. The above mixture was fully reacted at 37°C for 2 h. The generated CdS QDs-DNA3 was centrifuged at 10,000 rpm for 10 minutes to remove unbound DNA3, and washed twice with anhydrous ethanol and ultrapure water.

[0029] (5) Cleaning of glassy carbon electrode: Before modification of glassy carbon electrode, use 0.3 μm Al 2 O 3 Polish the suede to a mirror surface, wash with deionized water to remove surface dirt, and then move it to an ultrasonic water bath for cleaning, 5 minutes each time, repeat 2 times, ultrasonically clean with anhydrous ethanol and deionized water, and dry in a nitrogen environment;

[0030] (6) Test of glassy carbon electrode: In the presence of 0.1M KCl and 5mM [Fe(CN) 6 ] 3- / 4- Run a cyclic voltammetry curve in a 0.01M PBS solution to test the performance of the glassy carbon electrode, with a scanning speed of 50 mV / s and a scanning potential of -0.1 to 0.6 V. When the redox peak potential difference in the cyclic voltammetry curve is below 100 mV, the glassy carbon electrode can be further used, otherwise return to step (5) to continue processing the glassy carbon electrode until it meets the requirements;

[0031] (7) Add 6 μL of Bi nanoflowers onto the glassy carbon electrode (GCE). 2 S 3 @Au, dry the electrode at room temperature to obtain Bi 2 S 3@Au / GCE, then in Bi 2 S 3 6 μL of 200 nM aptamer DNA 1 chain was added to the Au / GCE electrode and incubated with Bi 2 S 3 @Au is combined through the chemical bond Au-S, and then MCH is added to block the non-specific binding sites. The electrode is allowed to dry naturally at room temperature, and finally 6 μL of Au@Luminol and CdS QDs bioluminescent probes are added. The electrode surface is allowed to dry, and the aptamer sensor is prepared and stored at 4 °C for later use.

[0032] (8) A three-electrode system was used to optimize the experimental factors such as the test base solution pH, base solution concentration, aptamer concentration and incubation time by electrochemiluminescence. The test base solution pH ranged from 6.5 to 9.0, the test base solution concentration ranged from 4 nM to 24 nM, the aptamer concentration ranged from 50 nM to 400 nM, and the incubation time ranged from 20 to 70 min.

[0033] (9) Under the optimal test conditions, the concentration gradient quantitative analysis test was performed using the electrochemiluminescence method. The aptamer concentration was 200 nM, the test base solution pH was 7.5, and the target substance concentration range for the test was 1 pg / L to 1 mg / L. The regression equations were y=7574.57-1051.10lgx(R 2 =0.998), y=5608.49-916.00lgx(R 2 =0.996);

[0034] (10) Add interfering ions of copper, iron, silver, zinc, nickel, mercury, cobalt, magnesium, and lead, as well as antibiotics such as tetracycline, streptomycin, oxytetracycline, chloramphenicol, erythromycin, and kanamycin to the test system, and test it by electrochemiluminescence to detect its anti-interference ability; select the prepared electrode and repeat the measurement six times to detect its stability;

[0035] (11) After grinding, filtering, and centrifuging the aquatic products, cadmium heavy metal and ampicillin solution were added to conduct actual sample testing and analysis.

[0036] This type of electrochemiluminescent aptamer sensor is not only low-cost, simple to operate, short in detection time, strong in anti-interference ability, good in stability, and strong in specificity, but it can also simultaneously detect heavy metal cadmium and ampicillin in aquatic products, which is in line with the development and internationalization requirements of rapid detection technology for heavy metal cadmium and ampicillin in aquatic products in my country.

Claims

1. An electrochemiluminescent biosensor for simultaneous detection of cadmium ions and ampicillin in aquatic products, characterized in that: Bismuth sulfide and gold complex, gold and luminol complex, and cadmium sulfide quantum dots were introduced to construct aptamer sensors for the simultaneous detection of cadmium ions and ampicillin in aquatic products. The preparation steps of bismuth sulfide and gold complex Bi2S3@Au, gold and luminol complex Au@Luminol, and cadmium sulfide quantum dots CdS QDs are as follows: Preparation of Bi2S3@Au: 0.5 g Bi(NO3)3·5H2O and 20 mL ethylene glycol were uniformly mixed, and then 0.4 g thiourea was added to the solution. Subsequently, the uniform mixture was transferred to an oil bath and kept at 160°C for 20 h. The product was then collected by several centrifugation cycles using ethanol and ultrapure water and dried at 60°C under vacuum conditions for 12 h. The product Bi2S3 was then redispersed and the prepared Au NPs solution was added. The mixture was stirred for 2 h to allow Au NPs particles to grow on the surface of Bi2S3. The mixture was then centrifuged and washed. Finally, Bi2S3@Au was redispersed for later use. Preparation of Au@Luminols: 4 mL of 0.01 M luminol and 0.6 mL of 0.1 M NaBH4 were cooled with ice, and then 20 mL of 2.5×10 -4 M HAuCl4 was stirred in an ice bath for 10 min, and the solution color changed to purple-red, indicating the formation of Au NPs. Finally, the product was stirred at room temperature for 6 h to synthesize colloidal Au@Luminol; Preparation of CdS QDs: 0.363 g of L-cysteine ​​was dissolved in 15.0 mL of ultrapure water, 0.275 g of CdCl2·5H2O was dissolved in 45.0 mL of ultrapure water, and 0.762 g of Na2S·9H2O was dissolved in 45.0 mL of ultrapure water to prepare the corresponding solutions. Next, the newly prepared L-cysteine ​​solution was injected into the CdCl2 solution under stirring, and then the resulting mixture was injected into the Na2S solution. The mixture was refluxed continuously at 75°C for 6 hours, and the obtained CdS quantum dots were separated by centrifugation at 12000 rpm for 15 min, and washed with anhydrous ethanol and ultrapure water, respectively.

2. The electrochemiluminescent biosensor for simultaneous detection of cadmium ions and ampicillin in aquatic products according to claim 1, characterized in that: The glassy carbon electrode was modified using willow-shaped nanoflowers Bi2S3@Au, Au@Luminol and CdS QDs. The specific steps were as follows: 6 μL of nanoflowers Bi2S3@Au was dripped onto the glassy carbon electrode GCE, and the electrode was dried at room temperature to obtain Bi2S3@Au / GCE. Then 6 μL of 200 nM aptamer DNA1 chain was dripped onto the Bi2S3@Au / GCE electrode, and it was combined with Bi2S3@Au through the chemical bond Au-S bond at room temperature. MCH was then dripped to block the nonspecific binding sites. The electrode was allowed to dry naturally at room temperature. Finally, 6 μL of Au@Luminol and CdS QDs bioluminescent probes were dripped, and the electrode surface was allowed to dry. The aptamer sensor was prepared and stored at 4°C for later use.

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