Preparation method of co-reactant-free anti-pollution electrochemical luminescence sensor for detecting cadmium in grains

Abstract

The invention discloses a preparation and use method of a co-reactant-free anti-pollution electrochemical luminescence sensor for detecting cadmium ions in grains, which is characterized in that the oxygen evolution reaction activity of porphyrin, the anti-pollution capability and conductivity of poly (3, 4-ethyldioxythiophene)-polystyrene sulfonate chitosan hydrogel and the luminescence stability of luminol are utilized to detect the cadmium ions in the grains. According to the recognition effect of cadmium ions on aptamers and the signal amplification strategy of a DNA walker, a non-co-reactant anti-pollution electrochemical luminescence sensor for detecting the cadmium ions in the grains in an on-off mode is constructed. The sensor disclosed by the invention can be used for judging whether the detected grain contains target ions or not according to the change of the electrochemical luminescence peak value of the cadmium ions in a detection result. The method has the advantages that the sensor is low in cost, simple in detection operation, free of co-reactants and high in pollution resistance; the cadmium heavy metal can be qualitatively and quantitatively measured; the method has high sensitivity and low detection limit, and provides a thought for detection of other ions and small molecular substances.

Description

Technical Field

[0001] The present invention relates to a co-reactant-free anti-fouling electrochemiluminescence sensor, a preparation method and a usage method of such a sensor, and belongs to the technical field of electrochemiluminescence aptamer sensors. Background Art

[0002] China's grain production is still in the stage of scattered sowing and harvesting, with a certain risk of heavy metal pollution. According to incomplete statistics of the National Environmental Protection Agency, the amount of grain polluted by heavy metals across the country reaches 1,200 tons annually, causing direct economic losses of more than 20 billion yuan. Food crops have the strongest ability to enrich heavy metal cadmium, resulting in the most serious cadmium pollution in grains. The cadmium content in grains is low but the harm is serious; combined with the risk of heavy metal cadmium pollution, it is urgent to classify and store grains by type. Therefore, establishing a rapid and highly sensitive heavy metal cadmium monitoring method has become a strategic requirement for ensuring national food security.

[0003] Detection methods for heavy metals: Currently, the main detection methods for heavy metals include gas chromatography, high-performance liquid chromatography, atomic absorption spectrometry, spectrophotometry, atomic emission spectrometry, inductively coupled plasma mass spectrometry, colorimetry, etc. Although these traditional heavy metal detection methods are relatively mature in application, have high sensitivity and accuracy, low detection limits, and wide detection ranges, the instrument prices are expensive, the time consumption is long, the sample treatment is complex, they are not convenient to carry, and they cannot be continuously monitored and measured on-site, etc., which greatly limits their wide application. Therefore, seeking a simple, rapid, and sensitive heavy metal ion detection technology is still the goal that people constantly pursue. In order to effectively ensure food safety and human life safety, it is necessary to establish an efficient, sensitive, and practical rapid heavy metal detection method.

[0004] The object of the invention is to provide a preparation method of an electrochemiluminescence aptamer sensor that can overcome the above problems, is simple to prepare, flexible to operate, has high sensitivity, high accuracy, good selectivity and stability, and the constructed co-reactant-free anti-fouling electrochemiluminescence sensor does not require the catalytic action of an enzyme, directly binds to the target substance cadmium ion through an aptamer, and the DNA walker amplifies the signal, and can specifically detect heavy metal cadmium ions in grains. Summary of the Invention

[0005] The technical solution is: A preparation method of a co-reactant-free anti-fouling electrochemiluminescence sensor for detecting cadmium heavy metals, characterized in that: the sensing surface composition of the co-reactant-free anti-fouling electrochemiluminescence sensor includes a composite material of porphyrin and luminol, and a composite material of poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate and chitosan hydrogel, and further immobilizes an aptamer for heavy metal cadmium.

[0006] The preparation method of a coreactant-free anti-fouling electrochemiluminescence sensor for detecting cadmium heavy metal is characterized by: cleaning the glassy carbon electrode (d = 3 mm), constructing and characterizing the sensing interface of the coreactant-free anti-fouling electrochemiluminescence sensor (preparing porphyrin and luminol, poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate and chitosan hydrogel, and using the fixed catalytic action of porphyrin and luminol, poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate and chitosan hydrogel to modify the electrode jointly), establishing the working curve of the coreactant-free anti-fouling electrochemiluminescence sensor, detecting the performance of the coreactant-free anti-fouling electrochemiluminescence sensor, and detecting the actual sample with the coreactant-free anti-fouling electrochemiluminescence sensor.

[0007] The preparation method of a coreactant-free anti-fouling electrochemiluminescence sensor for detecting cadmium heavy metal is characterized by: optimizing the experimental conditions, mainly including the aptamer concentration, the pH of the test bottom solution, and the incubation time of the aptamer and the target substance; the working curve of the prepared coreactant-free anti-fouling electrochemiluminescence sensor is: y = 1525.187 + 1182.821gC Cd 2+ (R 2 = 0.995); the performance detection of the coreactant-free anti-fouling electrochemiluminescence sensor includes the determination of specific selectivity, reproducibility, stability, and the recovery rate of the electrochemical sensor sample.

[0008] Electrochemiluminescence detection of target ions: Using the electrochemiluminescence detection method to detect grain samples under the optimal experimental conditions with a three-electrode system, and analyzing according to the electrochemiluminescence intensity value in the detection result, it can be known that the electrochemiluminescence intensity peak is proportional to the concentration of the metal ion, and the concentration information of the heavy metal ion can be obtained accordingly.

[0009] The preparation principle of the present invention is: Nucleic acid aptamers are single-stranded DNA or RNA nucleic acid molecules with specific recognition functions, which can bind to target substances quickly, with high affinity and high specificity. Therefore, the detection of heavy metals by aptamers has also developed. Cross-link the end of the aptamer to a solid-phase carrier as a capture molecule to capture the target substance in the specimen to be tested, and the 5 ，The end is labeled with corresponding indicators or catalytic substances, such as fluorescein, biotin, radioisotopes, or nanogold and silver nanoparticles, etc., which are transformed into detection molecules. When the detection molecules bind to the corresponding specimen to be detected, signals will be generated, thus achieving the detection purpose. In the present invention, porphyrin, luminol, poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate, and chitosan hydrogel are used to modify the glassy carbon electrode. Due to the high conductivity of poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate and the electrical neutrality and hydrophilicity of chitosan hydrogel, the electron transfer ability and anti-fouling ability of the electrode surface are increased. Through the high specific surface area of porphyrin and luminol, the electrode surface has a better ability to orient and immobilize the arm chain / aptamer chain, thus providing a good sensor interface for the immobilization of the arm chain / aptamer chain, maintaining the effective immobilization amount of the arm chain / aptamer chain, and further increasing the detection accuracy of the sensor. The reagentless anti-fouling electrochemical sensor prepared by the above steps is simple, sensitive, has good stability and specificity, good reproducibility, and the recovery rate meets the requirements.

[0010] To achieve the above object, the following technical solutions are adopted: A method for preparing a reagentless anti-fouling electrochemiluminescence sensor for detecting cadmium heavy metal, characterized in that: (1) Before preparing the electrochemiluminescence sensor, the bare glassy carbon electrode is cleaned, activated, and its performance is tested. If the peak potential difference in the cyclic voltammogram curve is below 100 mV and the oxidation peak and reduction peak are symmetric, then the said glassy carbon electrode can be used; otherwise, it needs to be returned to the cleaning step until it meets the requirements. (2) A uniformly dispersed solution of poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate and chitosan hydrogel is drop-coated on the surface of the cleaned glassy carbon electrode, then porphyrin and luminol are modified, and then the arm chain / aptamer chain is fixed on the porphyrin and luminol. After that, cDNA chain is drop-coated, mercaptohexanol is added to prevent specific adsorption, the target substance is added, and finally exonuclease is added for cleavage. After the preparation of the aptasensor is completed, it is stored in a refrigerator at 4 °C for standby.

[0011] To achieve the above object, the following technical solutions are adopted: A method for preparing a reagentless anti-fouling electrochemiluminescence sensor for detecting cadmium heavy metal, characterized in that: (1) The prepared aptasensor is detected by electrochemiluminescence method in the working base solution to obtain the relationship between the peak value and the target substance; (2) A series of standard solutions of cadmium ion concentrations are prepared and measured by electrochemistry to further obtain the working curve, detection range, and detection limit of the prepared aptasensor; (3) A series of common heavy metal solutions are prepared to detect the selectivity of the prepared aptasensor; (4) The stability of the above aptasensor is verified by electrochemiluminescence method multiple times; (5) The actual grain samples are analyzed to obtain the recovery rate of the aptasensor.

[0012] The present invention uses a simple cross-linking method to synthesize poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate and chitosan hydrogel to promote the electron transfer rate on the electrode surface, increase the response current on the electrode, improve the microenvironment of the electrode surface, enhance the anti-fouling ability of the electrode, and prepare an aptamer sensor with strong response signal and high sensitivity; porphyrin and luminol are synthesized by the self-assembly method. Porphyrin can carry out an oxygen evolution reaction to generate reactive oxygen species, enhance the electrochemiluminescence signal, enhance the change range of the peak value, and thus increase the detection accuracy of the aptamer sensor. In addition, the sensor preparation method is simple, has high stability and specificity, and can be used for the detection of heavy metal cadmium in grain samples, laying a foundation for the development of a portable device for rapid detection of heavy metal pollution in grain.

[0013] The preparation process of the co-reactant-free anti-fouling electrochemiluminescence sensor is as follows: Drop 5 μL of the hydrogel solution on a glassy carbon electrode (GCE), and place it at room temperature for half an hour to obtain hydrogel / GCE. Then, drop 5 μL of 2 μM swing arm chain / aptamer chain on the hydrogel / GCE electrode, and then drop 5 μL of cDNA chain. Place it at room temperature for half an hour. After the electrode is naturally dried, drop 5 μL of mercaptoethanol (MCH), place it at room temperature for half an hour, wait for the electrode to dry naturally, then drop 5 μL of the treated target solution, place it at room temperature for half an hour, wait for the electrode to dry naturally, then drop 5 μL of 2 u exonuclease solution, place it at room temperature for half an hour, wait for the electrode to dry naturally, and the aptamer sensor is prepared and stored at 4 °C for later use. Description of the Drawings

[0014] Figure 1 Material characterization of the hydrogel.

[0015] Figure 2 Luminescent material characterization of the co-reactant-free anti-fouling electrochemiluminescence sensor.

[0016] Figure 3 Electrochemical characterization of the co-reactant-free anti-fouling electrochemiluminescence sensor. (a: hydrogel / naked electrode; b: porphyrin and luminol / hydrogel / naked electrode; c: cDNA chain + swing arm chain and aptamer chain / porphyrin and luminol / hydrogel / naked electrode; d: MCH / cDNA chain / swing arm chain and aptamer chain / porphyrin and luminol / hydrogel / naked electrode; e: Cd 2+ / MCH / cDNA chain + swing arm chain and aptamer chain / porphyrin and luminol / hydrogel / naked electrode; f: ExoⅢ exonuclease / Cd 2+ / MCH / cDNA chain + swing arm chain and aptamer chain / porphyrin and luminol / hydrogel / naked electrode).

[0017] Figure 4 Optimization of the aptamer concentration of the co-reactant-free anti-fouling electrochemiluminescence sensor.

[0018] Figure 5 Optimization of the incubation time of a coreactant-free anti-fouling electrochemiluminescence sensor.

[0019] Figure 6 Optimization of the bottom solution pH of a coreactant-free anti-fouling electrochemiluminescence sensor.

[0020] Figure 7 Standard curve of a coreactant-free anti-fouling electrochemiluminescence sensor for measuring different concentrations of heavy metal cadmium.

[0021] Figure 8 Specificity study of a coreactant-free anti-fouling electrochemiluminescence sensor.

[0022] Figure 9 Reproducibility study of an electrochemiluminescence sensor.

[0023] Figure 10 Stability study of a coreactant-free anti-fouling electrochemiluminescence sensor. Detailed implementation mode

[0024] Example: (1) Preparation of tetrahydroxyphenylporphyrin: Under a nitrogen atmosphere, 1.5275 g of p-hydroxybenzaldehyde was dissolved in 125 mL of pre-dried propionic acid solvent placed in a 250 mL three-necked flask by vigorous stirring. Subsequently, the reaction system was heated to the reflux temperature of propionic acid (i.e., 145 °C), and under this condition, a solution formed by dissolving 870 μL of freshly distilled pyrrole in 15 mL of propionic acid was slowly added. The reflux state was maintained for another 2 hours to promote complete reaction. After the reaction was completed, the propionic acid solvent was removed by distillation technology, and the residue was cooled to room temperature. Next, the above residue was thoroughly mixed with 100 mL of ethanol and allowed to stand overnight to promote the precipitation of the product. Subsequently, the mixture was treated by crystallization, the precipitate was collected by filtration, and chloroform (CHCl 3 ) was used as the washing solvent until the washing liquid became clear, thus obtaining a preliminary purple solid product. For further purification, the above crude product was dried and then dissolved in acetone to form a saturated solution. Subsequently, column chromatography was used for separation, with 200 - 300 mesh silica gel as the stationary phase and acetone as the mobile phase. During the collection process, special attention was paid to and the second purple chromatographic band was collected, which is the main enrichment area of the target product. The collected product was recrystallized, using a mixed solvent of acetone - ether as the eluent, filtered again and thoroughly dried, and finally a high-purity purple tetrahydroxyphenylporphyrin product was obtained.

[0025] (2)Preparation of porphyrin and luminol complex: Weigh 2 mg of tetrahydroxyphenyl porphyrin (THPP) and dissolve it in 4 mL of ultrapure water. Sonicate for 5 minutes until dissolved. Then weigh 1.77 mg of luminol and add it to the above solution. Sonicate for 5 hours to obtain the self-assembled material, porphyrin and luminol complex. Immediately place the prepared material in a refrigerator at 4 °C in the dark for later use.

[0026] (3)Preparation of poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate and chitosan citral hydrogel complex: Weigh 0.2 g of chitosan and dissolve it in 10 mL of acetic acid (1 wt%). Then add 0.1 mL of poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate (PEDOT:PSS) solution and homogenize. After homogenization, add 5 mL of citral solution (1 wt%). Stir the mixture at room temperature for 6 hours for complete reaction.

[0027] (4)Cleaning of glassy carbon electrode: Before modifying the glassy carbon electrode, polish it on suede with 0.3 μm of Al 2 O 3 until it becomes mirror-like. After polishing, wash the surface dirt with deionized water, then transfer it to an ultrasonic water bath for cleaning, 5 min each time, repeat 2 times, clean with absolute ethanol and deionized water by ultrasound, and dry it under a nitrogen atmosphere.

[0028] (5)Testing of glassy carbon electrode: Run cyclic voltammetry curves in a 0.01 M PBS solution containing 0.1 M KCl and 5 mM [Fe(CN) 6 3- / 4- to test the performance of the glassy carbon electrode. The scanning speed is 50 mV / s, and the scanning potential is -0.1 to 0.6 V. When the potential difference between the oxidation and reduction peak in the cyclic voltammetry curve is below 100 mV, the glassy carbon electrode can be further used; otherwise, return to step (5) to continue treating the glassy carbon electrode until it meets the requirements.

[0029] (6)Preparation of grain samples: First, crush the sample. Weigh 0.2 g of the sample and add 10 mL of nitric acid and 0.5 mL of perchloric acid. React at 120 °C for 1 h until white smoke appears and the digestion solution is colorless and transparent or slightly yellow. Take out the digestion tube, cool it, and make the volume up to 10 mL with water, then mix well for later use.

[0030] ​(7) 5 μL of the hydrogel solution was dropped onto a glassy carbon electrode (GCE) and left at room temperature for half an hour to obtain hydrogel / GCE. Then, 5 μL of 2 μM arm chain / aptamer chain was dropped onto the hydrogel / GCE electrode, followed by 5 μL of cDNA chain. After leaving it at room temperature for half an hour, when the electrode was naturally dried, 5 μL of mercaptoethanol (MCH) was dropped onto it and left at room temperature for half an hour. After the electrode was naturally dried, 5 μL of the treated target solution was dropped onto it and left at room temperature for half an hour. After the electrode was naturally dried, 5 μL of 2 u exonuclease solution was dropped onto it and left at room temperature for half an hour. After the electrode was naturally dried, the aptamer sensor was prepared and stored at 4 °C for later use.

[0031] (8) A three-electrode system was adopted, and electrochemiluminescence was used to optimize experimental factors such as the pH of the test bottom solution, aptamer concentration, and incubation time. The range of the pH of the test bottom solution was 4.0 - 9.0, the range of the aptamer concentration was 0.5 μM - 3.0 μM, the range of the target incubation time was 20 - 120 min, and the range of the enzyme incubation time was 10 - 60 min.

[0032] (9) Under the optimal test conditions, concentration gradient quantitative analysis tests were carried out using electrochemiluminescence. The aptamer concentration was 2 μM, the pH value of the test bottom solution was 8.0, and the concentration range of cadmium used for testing was 1 - 10 10 ng / L. The lowest detection limits of cadmium were 0.51 ng / L respectively, and the regression equations were I ECL = 1525.187 + 1182.821lgC Cd 2+ , R 2 = 0.996, as shown in Figure 7 .

[0033] (10) Interference ions of copper, iron, zinc, magnesium, and lead with a concentration of 50 μg / L were respectively added to the test system, and electrochemiluminescence was used for testing to detect its anti-interference ability; one prepared electrode was selected and repeatedly measured six times when the cadmium concentration was 10 6 ng / L to detect its stability.

[0034] (11) After filtering, ultrasonicating, and centrifuging the grain samples, cadmium heavy metal solution was added for actual sample detection and analysis.

[0035] This co-reactant-free anti-fouling electrochemiluminescence sensor can detect cadmium heavy metals in grains. The sensor has a low cost, simple operation, short detection time, strong anti-interference ability, good stability, high specificity, and high detection sensitivity, meeting the development of rapid detection technology for cadmium heavy metals in grains in China and international requirements.

Claims

1. A co-reactant-free electrochemiluminescent biosensor for detecting cadmium ions in food samples, characterized in that: Porphyrin and luminol complex (THPP-Lum) was introduced as an electrochemiluminescent group without co-reactants. The preparation steps of THPP-Lum were as follows: 2 mg of tetrahydroxyphenylporphyrin (THPP) was weighed and dissolved in 4 mL of ultrapure water, and ultrasonically dissolved for 5 minutes. Then 1.77 mg of luminol (Luminol) was weighed and added to the above solution. Ultrasonic treatment was performed for 5 hours to obtain the self-assembled material porphyrin and luminol complex (THPP-Lum). The prepared material was then placed in a 4°C refrigerator away from light for later use.

2. An anti-fouling electrochemiluminescent biosensor for detecting cadmium ions in food samples, characterized in that: Poly(3,4-ethyldioxythiophene)-polystyrene sulfonate (PEDOT:PSS) and chitosan citral hydrogel composite (PEDDOT:PSS-CS) were introduced as antifouling sensing interface. The preparation steps of PEDDOT:PSS-CS were as follows: 0.2 g chitosan (CS) was weighed and dissolved in 10 mL acetic acid (1 wt%), and then 0.1 mL poly(3,4-ethyldioxythiophene)-polystyrene sulfonate (PEDOT:PSS) solution was added and homogenized. After homogenization, 5 mL citral solution (1 wt%) was added and the mixture was stirred at room temperature for 6 hours for complete reaction.

3. Used to prepare the co-reactant-free anti-fouling electrochemiluminescent biosensor interface for cadmium ion detection according to claims 1 and 2, characterized in that: The hydrophilic and electrically neutral PEDDOT:PSS-CS hydrogel and THPP-Lum were used to modify the glassy carbon electrode. The specific steps were as follows: 5 μL of PEDDOT:PSS-CS solution was added to the glassy carbon electrode (GCE) and allowed to stand at room temperature for half an hour to obtain PEDDOT:PSS-CS / GCE, and then 5 μL of THPP-Lum was added to the PEDDOT:PSS-CS / GCE electrode. Then, 5 μL of 2 μM swing arm chain / aptamer chain was added to the THPP-Lum / PEDDOT:PSS-CS / GCE electrode, and then 5 μL of cDNA chain was added, and allowed to stand at room temperature for half an hour. After the electrode was naturally dried, 5 μL of mercaptoethanol (MCH) was added, and allowed to stand at room temperature for half an hour. After the electrode was naturally dried, 5 μL of the treated target solution was added, and allowed to stand at room temperature for half an hour. After the electrode was naturally dried, 5 μL of 2 u exonuclease solution was placed at room temperature for half an hour, and the electrode was naturally dried. The aptamer sensor was prepared and stored at 4°C for later use. Cadmium ion detection was performed under the conditions of base solution pH 8, aptamer concentration 2 μM, cadmium ion incubation time 60 minutes, exonuclease concentration 2 u, and exonuclease incubation time 40 minutes. The standard curve of cadmium ion was obtained and actual sample detection was performed.

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