CN114460019A - Aptamer-based colorimetric sensor for neurogenic shellfish toxin and detection method - Google Patents

Aptamer-based colorimetric sensor for neurogenic shellfish toxin and detection method Download PDF

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CN114460019A
CN114460019A CN202210103613.2A CN202210103613A CN114460019A CN 114460019 A CN114460019 A CN 114460019A CN 202210103613 A CN202210103613 A CN 202210103613A CN 114460019 A CN114460019 A CN 114460019A
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王平
孔留兵
王心怡
马驰宇
孙先佑
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Zhejiang University ZJU
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Abstract

The invention discloses an aptamer-based colorimetric sensor for neurogenic shellfish toxin and a detection method, wherein the sensor comprises the following substances: the reagent kit comprises a 96-hole polystyrene microporous plate, a leucine-modified nano-gold solution, an aptamer diluent, a toxin sample diluent and a TMB solution. 10. mu.L of the aptamer dilution with a concentration of 0.3. mu.M was added to a 96-well plate, and 20. mu.L of the toxin sample dilutions with different concentrations were added and reacted for 30 minutes. And adding 100 mu L of the nano gold solution modified by the leucine into the solution after the reaction, and reacting for 5 minutes. Finally, 5. mu.L of TMB solution was added and reacted for 5 minutes. Putting the reacted 96-well plate into an enzyme-labeling instrument, reading absorbances at 530nm and 610nm, and calculating the ratio of the absorbances, namely A610nm/A530nm. The invention utilizes the aptamer to detect the specificity of the neurogenic shellfish toxin and realizes the quantitative detection of the neurogenic shellfish toxin by TMB induced nanogold coagulation. The detection method provided by the invention is low in cost, convenient and fast to operate and short in detection time.

Description

Aptamer-based colorimetric sensor for neurogenic shellfish toxin and detection method
Technical Field
The invention relates to a shellfish neurotoxin detection and analysis technology, in particular to a shellfish neurotoxin detection and analysis method based on an aptamer colorimetric sensor.
Background
The Nerve Shellfish Poison (NSP) is a fat-soluble cyclic polyester poison with stable chemical property, the main component of the NSP is short gymnodinia toxin (Brevetoxin), wherein Brevetoxin-2 is the main existing form. The NSP poisoning symptoms are mainly divided into gastrointestinal tract symptoms and nervous system symptoms, wherein the gastrointestinal tract poisoning symptoms comprise nausea, vomiting, diarrhea and abdominal colic, and the nervous system poisoning symptoms comprise mouth, lip, tongue far-end paresthesia, mouth and tooth unclogness and dizziness headache. When NSP poisoning is severe, respiratory distress and paralysis of limbs can develop. At present, the detection methods of marine product neurotoxin at home and abroad mainly comprise a mouse biological Method (MBA), high performance liquid chromatography-mass spectrometry (HPLC-MS), enzyme-linked immunosorbent assay (ELISA) and the like. The MBA method has strong specialty and large individual difference; although HPLC-MS has the advantage of high sensitivity and accuracy, its equipment is bulky and expensive; enzyme-linked immunosorbent assay (ELISA) and other enzyme-linked immunosorbent assay (ELISA) technologies have the defects of high price, high assay cost and the like, and the assay kit is monopolized abroad. Aptamers are artificially synthesized single-stranded DNA or RNA sequences, as they can fold into secondary and tertiary structures, allowing the aptamers to specifically bind to a target substance. Aptamers have a high affinity for target molecules with dissociation constants ranging from nanomolar to picomolar levels. Compared with the antibody, the antibody has higher stability and can be subjected to subsequent amplification through a polymerase chain reaction, so that the cost is greatly saved. The method for detecting and analyzing the neurotoxin in the marine aquatic products based on the aptamer colorimetric sensor has the advantages of low cost, simplicity and convenience in operation and the like, is convenient for establishing a platform for detecting and analyzing the neurotoxin in the marine aquatic products, and is expected to become a new tool in the field of detection and analysis of marine toxins and be popularized.
Disclosure of Invention
The invention aims to provide a shellfish neurotoxin detection and analysis method based on an aptamer colorimetric sensor aiming at the defects of the prior art.
The purpose of the invention is realized by the following technical scheme: in one aspect, the invention provides an aptamer-based colorimetric sensor for neurogenic shellfish toxin, which comprises a 96-hole polystyrene microporous plate, a complex acid modified nanogold solution, an aptamer BT10 diluent, a neurogenic shellfish toxin sample diluent and a dimethylbenzidine TMB solution. Adding 96-hole polystyrene microporous plates into the aptamer BT10 diluent and the toxin sample diluent in a volume ratio of 1:2, and mixing and incubating at room temperature to combine the two; adding a leucine modified nano-gold solution, wherein the volume ratio of the leucine modified nano-gold solution to the toxin sample diluent is 5:1, and the aptamer BT10 which is not combined with the toxin is combined with the nano-gold so as to protect the nano-gold from coagulation; adding a dimethylbenzidine TMB solution, wherein the volume ratio of the dimethylbenzidine TMB solution to the toxin sample diluent is 1:4, inducing nano gold to perform coagulation, and the color and the absorbance are different due to different coagulation degrees, so as to construct a colorimetric sensor for detecting the concentration of the neurogenic shellfish toxin;
the nano gold solution modified by the leucine is as follows: weighing the leucine powder and the potassium hydroxide powder in a mass ratio of 6:17, putting the leucine powder and the potassium hydroxide powder into a clean beaker, adding ultrapure water, and fully stirring and dissolving; the solution in the beaker was heated to boiling and then 2mL of a 1% by mass chloroauric acid solution was added rapidly. After continuing heating for 20 minutes, stopping heating and cooling to room temperature; dialyzing the solution with a dialysis bag overnight to obtain a nano gold solution modified by the leucine; the nano-gold modified by the leucine reduces the adsorption of the neurogenic shellfish toxin on the surface of the nano-gold;
the aptamer BT10 diluent is: centrifuging the aptamer BT10 powder at 4000rpm for 5 minutes, and then adding phosphate buffer solution with the pH of 7.0 to dilute the aptamer BT10 into stock solution with the concentration of 50 mu M; then carrying out aptamer secondary structure treatment, specifically heating the centrifuge tube filled with the stock solution in 90 ℃ water bath for 5 minutes, then rapidly placing the centrifuge tube in a 4 ℃ environment for cooling for 5 minutes, and finally placing the centrifuge tube at room temperature for 10 minutes to finish secondary structure treatment; the aptamer stock can be diluted to different concentrations by using phosphate buffer at pH 7.0;
the toxin sample dilutions were: the Brevetoxin-2 solution of the nervoshellfish toxin is diluted into different concentrations by pure water.
Further, the sequence of the aptamer BT10 is: 5'-GGCCACCAAACCACACCGTCGCAACCGCGAGAACCGAAGTAGTGATCATGTCCCTGCGTG-3' are provided.
On the other hand, the invention provides a method for detecting the neurogenic shellfish toxin based on the colorimetric sensor, which comprises the following steps:
(1) respectively adding 10 mu L of aptamer BT10 diluent with the concentration of 0.3 mu M into a 96-hole polystyrene micropore plate, correspondingly adding 20 mu L of toxin sample diluent with different concentrations, and reacting for 30 minutes;
(2) adding 100 mu L of the nano gold solution modified by the leucine into the solution after the reaction, and reacting for 5 minutes; finally adding 5 mu L of TMB solution, and reacting for 5 minutes;
(3) putting the well-reacted 96-hole polystyrene microporous plate into an enzyme-labeling instrument, reading absorbances at 530nm and 610nm, and calculating the ratio of the absorbances, namely A610nm/A530nmAnd obtaining the concentration of the neurogenic shellfish poison.
The invention has the beneficial effects that: the invention utilizes the aptamer with a specific sequence to specifically detect the neurogenic shellfish toxin, and realizes the quantitative detection of the neurogenic shellfish toxin Brevetoxin-2 by adding TMB to induce the coagulation of the nano-gold modified by the glycine. The detection method provided by the invention is low in cost and convenient and fast to operate, and the detection time is less than 1 hour.
Drawings
FIG. 1 is a schematic diagram of the method of the present invention;
FIG. 2 is a representation diagram of the absorption spectrum of the complex amino acid modified nano-gold solution;
FIG. 3 is a graph of the response of sodium chloride-induced nanogold coagulation to aptamers in accordance with the invention;
FIG. 4 is a graph of TMB-induced nanogold coagulation versus aptamer response in accordance with the invention;
FIG. 5 is a graph of aptamer concentration optimization according to the invention;
FIG. 6 is a graph showing the results of detecting different concentrations of Brevetoxin-2 in accordance with the present invention;
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments.
The invention provides an aptamer-based colorimetric sensor for neurogenic shellfish toxin, which comprises the following substances: the reagent kit comprises a 96-hole polystyrene microporous plate, a leucine-modified nano-gold solution, an aptamer diluent, a toxin sample diluent and a TMB solution.
Specifically, the complex acid modified nano gold solution is as follows: 0.006g of powder of the leucine and 0.017g of powder of the potassium hydroxide were weighed into a clean beaker, and 300mL of ultrapure water was added to the beaker and sufficiently stirred to dissolve. The solution in the beaker was heated to boiling with an alcohol burner and then 2mL of a 1% strength by mass chloroauric acid solution was added rapidly. Heating was continued until the volume of the solution was reduced to 150mL, heating was stopped and cooling to room temperature was carried out. Dialyzing the solution with a dialysis bag of 8000-14000D overnight to obtain the nano-gold solution modified by the leucine.
Specifically, the aptamer diluent is: the aptamer (SEQ ID NO: 5'-GGCCACCAAACCACACCGTCGCAACCGCGAGAACCGAAGTAGTGATCATGTCCCTGCGTG-3') powder synthesized by Shanghai Biometrics Ltd was centrifuged at 1200rpm for 5 minutes, and then diluted to a 50. mu.M concentration by adding phosphate buffer at pH 7.0. And then carrying out aptamer secondary structure treatment, specifically heating the centrifuge tube filled with the stock solution in a water bath at 90 ℃ for 5 minutes, then rapidly placing the centrifuge tube in a 4 ℃ environment for cooling for 5 minutes, and finally placing the centrifuge tube at room temperature for 10 minutes to finish secondary structure treatment. The aptamer stock can be diluted to different concentrations by using phosphate buffer at pH 7.0.
Specifically, the toxin sample diluent is: the Brevetoxin-2 solution of the nervoshellfish toxin is diluted into different concentrations by pure water.
The principle of the method for detecting the neurogenic shellfish toxin based on the aptamer colorimetric sensor is shown in figure 1, and specifically comprises the following steps:
and adding 10 mu L of aptamer diluent with the concentration of 0.3 mu M into a 96-hole polystyrene micropore plate, adding 20 mu L of toxin sample diluent with different concentrations, and reacting for 30 minutes to ensure that the aptamer and the toxin are fully combined. Adding 100 μ L of tyrosine to the post-reaction solutionThe nano-gold solution reacts for 5 minutes, the aptamer which is not combined with the toxin can be wound on the surface of the nano-gold to play a role in protecting the nano-gold, but the aptamer which is combined with the toxin cannot. And finally, adding 5 mu L of TMB solution, reacting for 5 minutes, wherein the TMB can induce the coagulation of the nano-gold by positively charging the TMB, and the coagulation degree is related to the amount of the aptamer on the surface of the nano-gold, namely the concentration of the toxin. Putting the well-reacted 96-hole polystyrene microporous plate into an enzyme-labeling instrument, reading absorbances at 530nm and 610nm, and calculating the ratio of the absorbances, namely A610nm/A530nmThe ratio reflects the coagulation degree of the nano gold.
Example 1:
preparing the nano gold modified by the leucine: 0.006g of powder of the leucine and 0.017g of powder of the potassium hydroxide were weighed into a clean beaker, and 300mL of ultrapure water was added to the beaker and sufficiently stirred to dissolve. The solution in the beaker was heated to boiling with an alcohol burner and then 2mL of a 1% strength by mass chloroauric acid solution was added rapidly. Heating was continued until the volume of the solution was reduced to 150mL, heating was stopped and cooling to room temperature was carried out. Dialyzing the solution with a dialysis bag of 8000-14000D overnight to obtain the nano-gold solution modified by the leucine. The prepared nano-gold solution is characterized by scanning absorption spectrum by an enzyme-labeling instrument, and as shown in figure 2, a characteristic absorption peak can be found at 520nm, which indicates that the nano-gold is successfully prepared.
Example 2:
the test group was prepared by adding 10. mu.L of 0.3. mu.M aptamer diluent to the test group, and 10. mu.L of a pH 7.0 phosphate buffer (i.e., 0. mu.M) to a 96-well polystyrene microplate to the control group, adding 100. mu.L of a complex acid-modified nanogold solution, and reacting for 5 minutes. The nanogold coagulation induced by adding 5 μ L of 10% sodium chloride solution or 5 μ L of TMB solution to the experimental group and the control group, as shown in fig. 3 and 4, was found to be insensitive to low concentration aptamers and not effectively distinguished using the conventional sodium chloride-induced nanogold coagulation. The TMB induced nano gold coagulation is sensitive to low-concentration aptamers and can be well distinguished.
Example 3:
in order to study the optimal aptamer concentration to be added to achieve the highest sensitivity, the response of the TMB-induced nanogold coagulation principle to the aptamer concentration was first explored. To each well, 100. mu.L of the nanogold solution was added, followed by 10. mu.L of aptamer dilutions (concentrations of 0.1. mu.M, 0.2. mu.M, 0.3. mu.M, 0.4. mu.M, 0.5. mu.M, 1. mu.M, 2. mu.M, respectively) at different concentrations to react for 5 minutes, followed by 5. mu.L of TMB solution to induce nanogold coagulation, and the results are shown in FIG. 5. It can be found that when the aptamer concentration is 0.3 μ M, the discrimination is larger as the aptamer concentration continues to decrease. Considering that the principle of the sensor is the binding of aptamers to the toxin, such that the amount of nanogold surface aptamers is reduced, 0.3 μ M aptamer concentration was chosen as the optimal concentration.
Example 4:
under the reaction conditions and the optimized conditions, 10 muL of aptamer diluent with the concentration of 0.3 muM is added into a 96-hole polystyrene micropore plate, 20 muL of toxin sample diluent with different concentrations (the concentration range is 0-5ppm) is added, and the reaction is carried out for 30 minutes, so that the aptamer and the toxin are fully combined. And adding 100 mu L of the complex acid modified nano gold solution into the solution after the reaction, and reacting for 5 minutes. Finally, 5. mu.L of TMB solution was added and reacted for 5 minutes. Putting the well-reacted 96-hole polystyrene microporous plate into an enzyme-labeling instrument, reading absorbances at 530nm and 610nm, and calculating the ratio of the absorbances, namely A610nm/A530nm. The relationship between the absorbance ratio and the toxin concentration is shown in FIG. 6. The sensor showed good linearity over the 0-1ppm concentration range. Linear relation of A610nm/A530nm0.2267X +0.7011, where X represents toxin concentration with a linearity of 0.9949.
It should be noted that the summary and the detailed description of the invention are intended to demonstrate the practical application of the technical solutions provided by the present invention, and should not be construed as limiting the scope of the present invention. Any modification and variation of the present invention within the spirit of the present invention and the scope of the claims will fall within the scope of the present invention.

Claims (3)

1. The colorimetric sensor for the neurogenic shellfish toxin based on the aptamer is characterized by comprising a 96-hole polystyrene micropore plate, a complex acid modified nano gold solution, an aptamer BT10 diluent, a neurogenic shellfish toxin sample diluent and a dimethyl benzidine TMB solution. Adding 96-hole polystyrene microporous plates into the diluted solution of the aptamer BT10 and the diluted solution of the toxin sample at a volume ratio of 1:2, and mixing and incubating at room temperature to combine the two solutions; adding a leucine modified nano-gold solution, wherein the volume ratio of the leucine modified nano-gold solution to the toxin sample diluent is 5:1, and the aptamer BT10 which is not combined with the toxin is combined with the nano-gold so as to protect the nano-gold from coagulation; adding a dimethylbenzidine TMB solution, wherein the volume ratio of the dimethylbenzidine TMB solution to the toxin sample diluent is 1:4, inducing nano gold to perform coagulation, and the color and the absorbance are different due to different coagulation degrees, so as to construct a colorimetric sensor for detecting the concentration of the neurogenic shellfish toxin;
the nano gold solution modified by the leucine is as follows: weighing the leucine powder and the potassium hydroxide powder in a mass ratio of 6:17, putting the leucine powder and the potassium hydroxide powder into a clean beaker, adding ultrapure water, and fully stirring and dissolving; the solution in the beaker was heated to boiling and then 2mL of a 1% by mass chloroauric acid solution was added rapidly. After continuing heating for 20 minutes, stopping heating and cooling to room temperature; dialyzing the solution with a dialysis bag overnight to obtain a nano gold solution modified by the leucine; the nano-gold modified by the leucine reduces the adsorption of the neurogenic shellfish toxin on the surface of the nano-gold;
the aptamer BT10 diluent is: centrifuging the aptamer BT10 powder at 4000rpm for 5 minutes, and then adding phosphate buffer solution with the pH of 7.0 to dilute the aptamer BT10 into stock solution with the concentration of 50 mu M; then carrying out aptamer secondary structure treatment, specifically heating the centrifuge tube filled with the stock solution in 90 ℃ water bath for 5 minutes, then rapidly placing the centrifuge tube in a 4 ℃ environment for cooling for 5 minutes, and finally placing the centrifuge tube at room temperature for 10 minutes to finish secondary structure treatment; the aptamer stock can be diluted to different concentrations by using phosphate buffer at pH 7.0;
the toxin sample dilutions were: the Brevetoxin-2 solution of the nervoshellfish toxin is diluted into different concentrations by pure water.
2. The aptamer-based colorimetric sensor for neurogenin according to claim 1, wherein the sequence of the aptamer BT10 is: 5'-GGCCACCAAACCACACCGTCGCAACCGCGAGAACCGAAGTAGTGATCATGTCCCTGCGTG-3' are provided.
3. A method for detecting a neurotoxic shellfish toxin based on the colorimetric sensor of claim 1, which comprises the following steps:
(1) respectively adding 10 mu L of aptamer BT10 diluent with the concentration of 0.3 mu M into a 96-hole polystyrene micropore plate, correspondingly adding 20 mu L of toxin sample diluent with different concentrations, and reacting for 30 minutes;
(2) adding 100 mu L of the nano gold solution modified by the leucine into the solution after the reaction, and reacting for 5 minutes; finally adding 5 mu L of TMB solution, and reacting for 5 minutes;
(3) putting the well-reacted 96-hole polystyrene microporous plate into an enzyme-labeling instrument, reading absorbances at 530nm and 610nm, and calculating the ratio of the absorbances, namely A610nm/A530nmAnd obtaining the concentration of the neurogenic shellfish poison.
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