CN116858909A - MC-LR detection method based on ortho-position connection electrochemiluminescence analysis - Google Patents

MC-LR detection method based on ortho-position connection electrochemiluminescence analysis Download PDF

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CN116858909A
CN116858909A CN202310351701.9A CN202310351701A CN116858909A CN 116858909 A CN116858909 A CN 116858909A CN 202310351701 A CN202310351701 A CN 202310351701A CN 116858909 A CN116858909 A CN 116858909A
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aptamer
dna1
dna2
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高红方
王登弘
丁怡琳
平平
马钰杰
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Wuxi University
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Abstract

The application discloses a detection MC-LR method based on ortho-position connection electrochemiluminescence analysis, which comprises the following steps of S1, preparing Ru (bpy) 3 2+ Mixing AuNPs and Nafion uniformly, and modifying on the surface of a glassy carbon electrode to form Ru (bpy) 3 2+ An electrochemiluminescence platform of/AuNPs/Nafion/GCE; s2, capturing DNA is self-assembled and fixed on the surface of the electrochemiluminescence platform through gold-sulfur bonds, and an electrochemiluminescence aptamer sensor is obtained; s3, immersing the prepared electrochemiluminescence aptamer sensor into a detection solution of MC-LR to be detected, and incubating; s4, taking the incubated electrochemiluminescence aptamer sensor as a working electrode, immersing the working electrode into PBS buffer solution containing tripropylamine, and adopting electrochemiluminescenceMeasuring the MC-LR content in the solution to be measured by a light instrument; the detection solution comprises MC-LR aptamer, DNA1 and DNA2. The application is based on the design that four DNA are cooperated to form an ortho-position connection compound, three quenching groups are introduced into one ortho-position connection compound, and the sensitivity and the specificity of the method are improved; the method is one-step detection, has simple steps and quick analysis, and can be used for measuring the MC-LR content.

Description

MC-LR detection method based on ortho-position connection electrochemiluminescence analysis
Technical Field
The application belongs to the field of analytical chemistry, and particularly relates to an MC-LR detection method based on an ortho-position connection electrochemiluminescence analysis method.
Background
Along with the rapid development of industrial and agricultural production, the water resource eutrophication phenomenon is increasingly serious, the blue algae bloom phenomenon happens sometimes, algae in water grow in a large amount due to the explosion of the blue algae bloom and release a large amount of blue algae toxins after death, the toxins not only can influence the balance of an ecological system and pollute the water environment, but also can cause acute poisoning and even death of people and animals, and seriously threaten the health of people. The blue algae toxins are various in variety, different in content and different in toxicity, wherein microcystins- (Leucine-arginine) (Microcystin- (leucoine-Argine), MC-LR (Microcystin-LR) are the most studied blue algae toxins at present due to strong toxicity and great harm. In view of this, the World Health Organization (WHO) in 2004 revised "drinking Water quality guidelines" and in 2006 in China "sanitary standards for Drinking Water", GB 5749-2006, both specify that the MC-LR content in drinking water should not exceed 1. Mu.g/L (1 nM). Therefore, the determination of the cyanobacteria toxin in the water body has important significance for early warning of cyanobacteria bloom and safety guarantee of drinking water.
At present, common MC-LR detection methods mainly comprise a mouse biological method, a liquid chromatography method, an immunoassay method and the like (Xiaoying. Research and application of an electrochemiluminescence peptide biosensing method. Shaanxi university, 2013), and the detection methods have the problems of long detection time, low sensitivity, overlarge equipment, difficult operation, need of professional technicians and the like. Therefore, development of a method for realizing MC-LR detection in water body with high sensitivity, high selection, simplicity and rapidness is needed.
Disclosure of Invention
The application aims to provide a method for simply, rapidly and highly sensitively detecting MC-LR in a water body based on an ortho-position connection electrochemiluminescence analysis technology.
In order to achieve the above purpose, the present application adopts the following technical scheme:
an MC-LR detection method based on ortho-junction electrochemiluminescence analysis comprises the following steps:
s1, ru (bpy) 3 2+ Mixing AuNPs and Nafion uniformly, and modifying on the surface of a glassy carbon electrode to form Ru (bpy) 3 2+ An electrochemiluminescence platform of/AuNPs/Nafion/GCE;
s2, self-assembling and fixing the capture DNA on the surface of the electrochemiluminescence platform in the step S1 through gold-sulfur bonds to obtain an electrochemiluminescence aptamer sensor;
s3, immersing the prepared electrochemiluminescence aptamer sensor into a detection solution of MC-LR to be detected, and incubating;
s4, immersing the incubated electrochemiluminescence aptamer sensor serving as a working electrode into PBS buffer solution containing tripropylamine, recording the electrochemiluminescence intensity by using an electrochemiluminescence instrument, and determining the MC-LR content in the solution to be detected according to a standard curve established by the electrochemiluminescence intensity and the known MC-LR content;
the detection solution comprises MC-LR aptamer, DNA1 and DNA2;
the MC-LR aptamer is a DNA single-chain aptamer;
the capture DNA is of a single-chain structure, one end of the capture DNA is modified with sulfhydryl, and a base sequence far away from one end of the modified sulfhydryl is complementary with one end of the MC-LR aptamer; ferrocene is modified at both ends of the DNA1, the base sequence at one end of the DNA1 is complementary with one end of the capture DNA close to the modified sulfhydryl, and the base sequence in the middle of the DNA1 is complementary with the middle of the MC-LR aptamer; one end of the DNA2 is modified with ferrocene, a base sequence close to one end of the ferrocene is complementary with the other end of the DNA1, and a base sequence far away from one end of the DNA2 is complementary with the other end of the MC-LR aptamer.
The application is to capture DNA and electrochemiluminescence platform Ru (bpy) 3 2+ The electrochemical luminescence aptamer sensor formed by self-assembly of AuNPs/Nafion/GCE through gold-sulfur bonds is immersed in a detection solution containing aptamer, DNA1 and DNA2;when MC-LR is not contained in the detection solution, the capture DNA, the aptamer, the DNA1 and the DNA2 are subjected to ortho hybridization, so that ferrocene marked on the DNA1 and the DNA2 is close to the surface of an electrode, and the electrochemical luminescence is reduced; when MC-LR exists, the MC-LR is combined with the aptamer, so that the formation of an ortho-position connection complex is destroyed, ferrocene on DNA1 and DNA2 is far away from the surface of the electrode, and the electrochemiluminescence signal is enhanced.
Further, the capture DNA and DNA1 contain 9-21 complementary base pairs.
Preferably, the capture DNA and DNA1 contain 15 complementary base pairs.
Further, the capture DNA and MC-LR aptamer contain 15-27 complementary base pairs at one end.
Preferably, the capture DNA and MC-LR aptamer contain 21 complementary base pairs at one end.
Further, the middle of DNA1 and the middle of MC-LR aptamer contain 10-18 complementary base pairs.
Preferably, the DNA1 middle and aptamer middle contain 14 complementary base pairs.
Further, the DNA1 and DNA2 contain 9-21 complementary base pairs.
Preferably, the DNA1 and DNA2 contain 15 complementary base pairs.
Further, the other end of the DNA2 and MC-LR aptamer contains 15-27 complementary base pairs.
Preferably, the other end of the DNA2 and MC-LR aptamer contains 21 complementary base pairs.
Preferably, the capture DNA sequence is shown as SEQ ID NO. 1, the DNA1 sequence is shown as SEQ ID NO. 2, the DNA2 sequence is shown as SEQ ID NO. 3, and the MC-LR aptamer sequence is shown as SEQ ID NO. 4.
Further, the detection solution in step S3 contains 1-10nmol/L aptamer, 1-10nmol/L DNA1 and 1-10nmol/L DNA2.
Further, the solvent of the detection solution is PBS buffer solution, the pH is 7.4, and the buffer solution concentration is 75-150mmol/L.
Preferably, the concentration of PBS buffer is 100mmol/L.
Further, the step S2 capture DNA is self-assembled and fixed on the surface of the electrochemiluminescence platform through gold-sulfur bonds, and the method comprises the following steps: ru (bpy) 3 2+ And (3) immersing the AuNPs/Nafion/GCE electrochemiluminescence platform into a capture DNA solution with the mass concentration of 5-20 mu mol/L, incubating for 1-5h, immersing into mercaptoethanol for sealing after incubation, and obtaining the electrochemiluminescence aptamer sensor.
Further, the mercaptoethanol species is present in an amount ranging from 0.5 to 2mmol/L and the blocking reaction time is from 20 to 50 minutes.
The purpose of the addition of mercaptoethanol after self-assembly is to carry out blocking in order to prevent non-specific adsorption of the components after incubation.
Furthermore, 5-20mmol/L PB buffer is used for sealing after incubation of Ru (bpy) before immersion in mercaptoethanol 3 2+ the/AuNPs/Nafion/GCE electrochemiluminescence platform was rinsed to remove DNA1 not assembled on the surface of the electrochemiluminescence platform.
Further, the PB buffer had a pH of 7.4.
Still further, the preparation of the electrochemiluminescence aptamer sensor is performed at room temperature.
Further, the incubation is performed in the step S3, and the reaction condition is that the incubation is performed for more than 30 minutes at the temperature of 25-37 ℃.
Further, the incubation is performed in the step S3, and the reaction condition is that the incubation is performed for 30-60min at 25-37 ℃.
Preferably, the incubation is performed as described in step S3, with reaction conditions of 25-37℃for 45min.
Further, the electrochemical luminescence aptamer sensor in step S4 needs to be rinsed with a PB buffer solution with a pH of 7.2,5-20mmol/L before being immersed in tripropylamine solution.
Further, the concentration of the tripropylamine solution in step S4 is 25-75mmol/L.
Further, the tripropylamine solution is prepared by taking tripropylamine as a solute and taking 75-150mmol/L PBS as a solvent, wherein the pH of the PBS is 7.4.
Further, in step S4, the electrochemiluminescence intensity is recorded by using an electrochemiluminescence apparatus, wherein the reference electrode is Ag/AgCl, and the counter electrode is a platinum wire.
Further, in step S4, the electrochemiluminescence intensity is recorded by using an electrochemiluminescence apparatus, and the conditions are that cyclic voltammetry scanning is performed within a potential range of 0-1.45V, and the scanning speed is high: 100mV/s, negative high pressure: -500V, the electrochemiluminescence intensity was recorded.
The application provides application of the capture DNA, the MC-LR aptamer, the DNA1 and the DNA2 in determination of MC-LR content.
Compared with the prior art, the application has the beneficial effects that:
according to the MC-LR detection method based on ortho-position connection electrochemiluminescence analysis, the capture DNA, the DNA1, the DNA2 and the MC-LR are competitively combined with an aptamer to destroy the formation of an ortho-position connection complex of the capture DNA, the DNA1 and the DNA2 with the aptamer, and the quencher ferrocene is far away from the surface of an electrode, so that an electrochemiluminescence signal is enhanced.
The application is based on the design that four DNA are cooperated to form an ortho-position connection compound, three quenching groups are introduced into one ortho-position connection compound, so that the sensitivity of the method is improved; meanwhile, DNA1 mediates capture of DNA, aptamer and DNA2 to cooperatively generate ortho hybridization, so that the specificity of the method is improved, and the aptamer and MC-LR react in a homogeneous solution, so that the recognition efficiency is improved; the method is one-step detection, and has simple steps and rapid analysis.
Drawings
FIG. 1 is a schematic diagram of the MC-LR detection method based on ortho-ligation electrochemiluminescence analysis.
FIG. 2 is a graph of electrochemiluminescence properties of the electrochemiluminescence aptamer sensor of example 3 after incubation with varying concentrations of MC-LR.
FIG. 3 is a graph of the electrochemiluminescence properties of the electrochemiluminescence aptamer sensors of comparative example 1, comparative example 2, and example 3 after incubation with 0.05ng/mL MC-LR, respectively.
FIG. 4 is a graph of electrochemiluminescence intensity as a function of time for comparative example 1 and comparative example 3 electrochemiluminescence aptamer sensors analyzing 0.05ng/mL MC-LR;
in the figure, symbol a represents the measurement result of the method of comparative example 3, and symbol b represents the measurement result of the method of comparative example 1.
Detailed Description
The application is further illustrated in the following drawings and specific examples, which are not intended to limit the application in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present application are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The term noun is interpreted as follows:
Ru(bpy) 3 2+ represents tris (2, 2' -bipyridine) ruthenium dichloride;
AuNPs represent gold nanoparticles prepared by citrate reduction of chloroauric acid (preparation references: turkevicthJ, stevenson P C, hillier J, nucleation and Growth Process in the Synthesis of Colloidal gold. Discuss. Faraday Soc.1951, 11:55-75.);
nafion represents perfluoro (4-methyl-3, 6-dioxa-7-octene) sulfonyl fluoride, a 5% low-fat alcohol and water mixture;
wherein, a mixture of tris (2, 2' -bipyridine) ruthenium dichloride, chloroauric acid, perfluoro (4-methyl-3, 6-dioxa-7-octene) sulfonyl fluoride, 5% low-fatty alcohol and water was purchased from Sigma-Aldrich company;
GCE represents a glassy carbon electrode, available from GaoshiRui company.
Example 1
An MC-LR detection method based on ortho-junction electrochemiluminescence analysis comprises the following steps:
s1, ru (bpy) 3 2+ Mixing AuNPs and Nafion uniformly, and modifying on the surface of a glassy Carbon Electrode (CEC) to form Ru (bpy) 3 2+ An electrochemiluminescence platform of/AuNPs/Nafion/GCE;
s2, self-assembling and fixing the capture DNA on the surface of the electrochemiluminescence platform in the step S1 through gold-sulfur bonds to obtain an electrochemiluminescence aptamer sensor;
s3, immersing the prepared electrochemiluminescence aptamer sensor into a detection solution of MC-LR to be detected, and incubating;
s4, immersing the incubated electrochemiluminescence aptamer sensor serving as a working electrode into PBS buffer solution containing tripropylamine, recording the electrochemiluminescence intensity by using an electrochemiluminescence instrument, and determining the MC-LR content in the solution to be detected according to a standard curve established by the electrochemiluminescence intensity and the known MC-LR content;
the detection solution comprises MC-LR aptamer, DNA1 and DNA2; the MC-LR aptamer is a DNA single-stranded aptamer or an RNA single-stranded aptamer; the capture DNA is of a single-chain structure, one end of the capture DNA is modified with sulfhydryl, and a base sequence far away from one end of the modified sulfhydryl is complementary with one end of the MC-LR aptamer; ferrocene is modified at both ends of the DNA1, the base sequence at one end of the DNA1 is complementary with one end of the capture DNA close to the modified sulfhydryl, and the base sequence in the middle of the DNA1 is complementary with the middle of the MC-LR aptamer; one end of the DNA2 is modified with ferrocene, a base sequence close to one end of the ferrocene is complementary with the other end of the DNA1, and a base sequence far away from one end of the DNA2 is complementary with the other end of the MC-LR aptamer.
FIG. 1 is a schematic diagram of detection of MC-LR based on ortho-ligation electrochemiluminescence analysis according to the present application, wherein capture DNA and an electrochemiluminescence platform Ru (bpy) are used 3 2+ The electrochemical luminescence aptamer sensor formed by the self-assembly of the AuNPs/Nafion/GCE through gold-sulfur bonds is immersed in MC-LR aptamer, DNA1 and DNA2 and added with detection solution with unknown MC-LR content, when MC-LR is not present, DNA1 mediates capturing DNA, aptamer and DNA2 to cooperatively generate ortho hybridization, so that ferrocene marked on the DNA1 and the DNA2 is close to the surface of an electrode, and electrochemical luminescence is reduced; when MC-LR exists, the MC-LR is combined with the aptamer, so that the formation of an ortho-position connection complex is destroyed, ferrocene on DNA1 and DNA2 is far away from the surface of the electrode, and the electrochemiluminescence signal is enhanced.
Example 2
An MC-LR detection method based on ortho-junction electrochemiluminescence analysis comprises the following steps:
s1, ru (bpy) 3 2+ Mixing AuNPs and Nafion uniformly, and modifying on the surface of a glassy carbon electrode to form Ru (bpy) 3 2+ An electrochemiluminescence platform of/AuNPs/Nafion/GCE; the detailed steps are as follows:
0.5% Nafion, auNPs and 1mmol/L Ru (bpy) 3 2+ Uniformly mixing the solutions in a volume ratio of 1:2:1, performing ultrasonic treatment for 30 minutes, then dripping 10 mu L of the mixed solution on the surface of a 2mm glassy carbon electrode, and airing at room temperature to obtain Ru (bpy) 3 2+ /AuNPs/Nafion/GCE。
S2, capturing DNA (deoxyribonucleic acid) is self-assembled and fixed on the surface of an electrochemiluminescence platform through gold-sulfur bonds, and an electrochemiluminescence aptamer sensor is obtained; the detailed steps are as follows:
the electrochemiluminescence platform was immersed in a capture DNA solution with an amount concentration of 5. Mu. Mol/L of the substance at room temperature. Incubating for 1h, enabling the DNA to be self-assembled on the surface of the electrochemiluminescence platform through gold sulfide bonds, and flushing a PB counter electrode with the concentration of 5mmol/L and the pH value of 7.4 to remove capture DNA which is not assembled on the surface of the electrochemiluminescence platform; then immersing the sensor into mercaptoethanol with the mass concentration of 0.5mmol/L, and sealing for 20min to obtain the electrochemiluminescence aptamer sensor.
The capture DNA is of a single-chain structure, one end of the capture DNA is modified with sulfhydryl, and the other end of the capture DNA is complementary with a base sequence at one end of the MC-LR aptamer. The structure of the modified sulfhydryl is as follows: 5' -HS (CH) 2 ) 6 GCATGGTATTTTTCGCCCTACCCCGGGACCCAAAAA-3' and the sequence is shown as SEQ ID NO. 1.
S3, immersing the prepared electrochemiluminescence aptamer sensor into a detection solution containing MC-LR, and incubating; the incubation conditions were 25℃for 30min. The detailed steps are as follows:
taking PBS buffer solution as a solvent of a detection solution, wherein the concentration of the buffer solution is 75mmol/L, and the pH of the PBS buffer solution is 7.4; MC-LR aptamer, DNA1 and DNA2 were added to the buffer solution, respectively, so that the detection solution contained 1nmol/L MC-LR aptamer, 1nmol/L DNA1 and 1nmol/L DNA2, and incubated at 25℃for 30min.
The detection solution comprises MC-LR aptamer, DNA1 and DNA2; the MC-LR aptamer is a DNA single-chain aptamer; the capture DNA is of a single-chain structure, one end of the capture DNA is modified with sulfhydryl, and a base sequence far away from one end of the modified sulfhydryl is complementary with one end of the MC-LR aptamer; ferrocene is modified at both ends of the DNA1, the base sequence at one end of the DNA1 is complementary with one end of the capture DNA close to the modified sulfhydryl, and the base sequence in the middle of the DNA1 is complementary with the middle of the MC-LR aptamer; one end of the DNA2 is modified with ferrocene, a base sequence close to one end of the ferrocene is complementary with the other end of the DNA1, and a base sequence far away from one end of the DNA2 is complementary with the other end of the MC-LR aptamer.
The structure of the DNA1 containing ferrocene is as follows:
the sequence of 5'-Fc-CGTACCATAAAAAGCTACGCTCCATACCTTATGGTCGAATAAGT-Fc-3' is shown as SEQ ID NO. 2.
The structure of the ferrocene modified at one end of the DNA2 is as follows:
the sequence of 5'-Fc-ACTTATTCGACCATACCCCAGGAACAAAGGGAGAAC-3' is shown as SEQ ID NO. 3. The MC-LR aptamer sequence is shown as SEQ ID NO. 4, and specifically comprises the following components: 5'-TTT TTG GGT CCC GGG GTA GGG ATG GGAGGT ATG GAG GGG TCC TTG TTT CCC TCT TG-3'.
S4, after incubation is completed, immersing an electrochemiluminescence aptamer sensor serving as a working electrode into PBS buffer solution containing tripropylamine, recording the electrochemiluminescence intensity by using an electrochemiluminescence instrument, and determining the content of MC-LR according to a standard curve established by the electrochemiluminescence intensity and the known content of MC-LR; the detailed steps are as follows:
the electrochemical luminescence aptamer sensor after reaction is used as a working electrode, PB buffer solution with the buffer solution concentration of 7.4 and the concentration of 5mmol/L is used for flushing the working electrode, then the working electrode is immersed into tripropylamine solution with the mass concentration of 25mmol/L, tripropylamine solution is prepared by taking tripropylamine as a solute and PBS with the concentration of 75mmol/L as a solvent, and the pH of the PBS is 7.4. In the electrochemiluminescence reaction, a reference electrode is Ag/AgCl, a counter electrode is a platinum wire, and cyclic voltammetry scanning is carried out within the potential range of 0-1.45V, and the scanning speed is high: 100mV/s, negative high pressure: -500V, recording the electrochemiluminescence intensity, respectively analyzing and detecting the electrochemiluminescence properties of the solution with the concentration of 0, 0.05 and 0.5ng/mLMC-LR according to the electrochemiluminescence intensity at 1.22V, establishing a standard curve, and measuring the content of the unknown concentration MC-LR according to the steps after establishing the standard curve.
Example 3
An MC-LR detection method based on ortho-junction electrochemiluminescence analysis comprises the following steps:
s1, ru (bpy) 3 2+ Mixing AuNPs and Nafion uniformly, and modifying on the surface of a glassy carbon electrode to form Ru (bpy) 3 2+ An electrochemiluminescence platform of/AuNPs/Nafion/GCE; the specific procedure is as in example 2S 1.
S2, capturing DNA (deoxyribonucleic acid) is self-assembled and fixed on the surface of an electrochemiluminescence platform through gold-sulfur bonds, and an electrochemiluminescence aptamer sensor is obtained; the detailed steps are as follows:
the electrochemiluminescence platform was immersed in a capture DNA solution with an amount concentration of 10. Mu. Mol/L of the substance at room temperature. Incubating for 3h, enabling the DNA to be self-assembled on the surface of the electrochemiluminescence platform through gold sulfide bonds, and flushing the surface of the electrochemiluminescence platform through a PB counter electrode with the concentration of 10mmol/L and the pH value of 7.4 to remove capture DNA which is not assembled on the surface of the electrochemiluminescence platform; then immersing the sensor into mercaptoethanol with the mass concentration of 1mmol/L, and sealing for 30min to obtain the electrochemiluminescence aptamer sensor.
The capture DNA is of a single-chain structure, one end of the capture DNA is modified with sulfhydryl, and the other end of the capture DNA is complementary with a base sequence at one end of the MC-LR aptamer. The structure of the modified sulfhydryl is as follows: 5' -HS (CH) 2 ) 6 GCATGGTATTTTTCGCCCTACCCCGGGACCCAAAAA-3' and the sequence is shown as SEQ ID NO. 1.
S3, immersing the prepared electrochemiluminescence aptamer sensor into a detection solution containing MC-LR, and incubating; the incubation conditions were 37℃for 45min. The detailed steps are as follows:
taking PBS buffer solution as a solvent of a detection solution, wherein the concentration of the buffer solution is 100mmol/L, and the pH of the PBS buffer solution is 7.4; MC-LR aptamer, DNA1 and DNA2 were added to the buffer solution, respectively, so that the detection solution contained 5nmol/L MC-LR aptamer, 5nmol/L DNA1 and 5nmol/L DNA2, and incubated at 37℃for 60min.
The detection solution comprises MC-LR aptamer, DNA1 and DNA2;
the MC-LR aptamer is a DNA single-chain aptamer;
the capture DNA is of a single-chain structure, one end of the capture DNA is modified with sulfhydryl, and a base sequence far away from one end of the modified sulfhydryl is complementary with one end of the MC-LR aptamer; ferrocene is modified at both ends of the DNA1, the base sequence at one end of the DNA1 is complementary with one end of the capture DNA close to the modified sulfhydryl, and the base sequence in the middle of the DNA1 is complementary with the middle of the MC-LR aptamer; one end of the DNA2 is modified with ferrocene, a base sequence close to one end of the ferrocene is complementary with the other end of the DNA1, and a base sequence far away from one end of the DNA2 is complementary with the other end of the MC-LR aptamer.
The structure of the DNA1 containing ferrocene is as follows:
the sequence of 5'-Fc-CGTACCATAAAAAGCTACGCTCCATACCTTATGGTCGAATAAGT-Fc-3' is shown as SEQ ID NO. 2.
The structure of the ferrocene modified at one end of the DNA2 is as follows:
the sequence of 5'-Fc-ACTTATTCGACCATACCCCAGGAACAAAGGGAGAAC-3' is shown as SEQ ID NO. 3. The MC-LR aptamer sequence structure is as follows: 5'-TTT TTG GGT CCC GGG GTA GGG ATG GGA GGT ATG GAG GGG TCC TTG TTT CCC TCT TG-3' is shown as SEQ ID NO. 4.
S4, after incubation is completed, immersing an electrochemiluminescence aptamer sensor serving as a working electrode into PBS buffer solution containing tripropylamine, recording the electrochemiluminescence intensity by using an electrochemiluminescence instrument, and determining the content of MC-LR according to a standard curve established by the electrochemiluminescence intensity and the known content of MC-LR; the detailed steps are as follows:
the electrochemical luminescence aptamer sensor after reaction is used as a working electrode, the working electrode is washed by PB buffer solution with the pH value of 7.4 and 10mmol/L, then the working electrode is immersed into tripropylamine solution with the mass concentration of 50mmol/L, the tripropylamine solution is prepared by using tripropylamine as a solute and PBS with the pH value of 100mmol/L as a solvent, and the pH value of the PBS is 7.4. In the electrochemiluminescence reaction, a reference electrode is Ag/AgCl, a counter electrode is a platinum wire, and cyclic voltammetry scanning is carried out within the potential range of 0-1.45V, and the scanning speed is high: 100mV/s, negative high pressure: -500V, recording the electrochemiluminescence intensity, establishing a standard curve from the electrochemiluminescence intensity at 1.22V and the known different concentrations of MC-LR; and respectively analyzing the electrochemiluminescence properties of the detection solutions with the concentrations of 0, 0.05 and 0.5ng/mLMC-LR, establishing a standard curve, and measuring the content of the MC-LR with unknown concentration according to the steps after establishing the standard curve.
Example 4
An MC-LR detection method based on ortho-junction electrochemiluminescence analysis comprises the following steps:
s1, ru (bpy) 3 2+ Mixing AuNPs and Nafion uniformly, and modifying on the surface of a glassy carbon electrode to form Ru (bpy) 3 2+ An electrochemiluminescence platform of/AuNPs/Nafion/GCE; the specific procedure is as in example 2S 1.
S2, capturing DNA (deoxyribonucleic acid) is self-assembled and fixed on the surface of an electrochemiluminescence platform through gold-sulfur bonds, and an electrochemiluminescence aptamer sensor is obtained; the detailed steps are as follows:
the electrochemiluminescence platform was immersed in a capture DNA solution with an amount concentration of 20. Mu. Mol/L of the substance at room temperature. Incubating for 5h, enabling the DNA to be self-assembled on the surface of the electrochemiluminescence platform through gold sulfide bonds, and flushing the surface of the electrochemiluminescence platform through a PB counter electrode with 20mmol/L and pH of 7.4 to remove capture DNA which is not assembled on the surface of the electrochemiluminescence platform; then immersing the sensor into mercaptoethanol with the mass concentration of 2mmol/L, and sealing for 50min to obtain the electrochemiluminescence aptamer sensor.
The structure of the capture DNA was the same as in example 3.
S3, immersing the prepared electrochemiluminescence aptamer sensor into a detection solution containing MC-LR, and incubating; the incubation conditions were 37℃for 60min. The detailed steps are as follows:
taking PBS buffer solution as a solvent of a detection solution, wherein the concentration of the buffer solution is 150mmol/L, and the pH of the PBS buffer solution is 7.4; MC-LR aptamer, DNA1 and DNA2 were added to the buffer solution, respectively, so that the detection solution contained 10nmol/L MC-LR aptamer, 10nmol/L DNA1 and 10nmol/L DNA2, and incubated at 37℃for 60min.
The detection solution comprises MC-LR aptamer, DNA1 and DNA2;
the MC-LR aptamer is a DNA single-chain aptamer;
the capture DNA is of a single-chain structure, one end of the capture DNA is modified with sulfhydryl, and a base sequence far away from one end of the modified sulfhydryl is complementary with one end of the MC-LR aptamer; ferrocene is modified at both ends of the DNA1, the base sequence at one end of the DNA1 is complementary with one end of the capture DNA close to the modified sulfhydryl, and the base sequence in the middle of the DNA1 is complementary with the middle of the MC-LR aptamer; one end of the DNA2 is modified with ferrocene, a base sequence close to one end of the ferrocene is complementary with the other end of the DNA1, and a base sequence far away from one end of the DNA2 is complementary with the other end of the MC-LR aptamer.
The structures of the capture DNA, MC-LR aptamer, DNA1 and DNA2 are the same as in example 3
S4, after incubation is completed, immersing an electrochemiluminescence aptamer sensor serving as a working electrode into PBS buffer solution containing tripropylamine, recording the electrochemiluminescence intensity by using an electrochemiluminescence instrument, and determining the content of MC-LR according to a standard curve established by the electrochemiluminescence intensity and the known content of MC-LR; the detailed steps are as follows:
the electrochemical luminescence aptamer sensor after reaction is used as a working electrode, PB buffer solution with the buffer solution concentration of 20mmol/L and the pH value of 7.4 is used for flushing the working electrode, then the working electrode is immersed into tripropylamine solution with the mass concentration of 75mmol/L, tripropylamine solution is prepared by taking tripropylamine as a solute and PBS with the concentration of 150mmol/L as a solvent, and the pH value of the PBS is 7.4. In the electrochemiluminescence reaction, a reference electrode is Ag/AgCl, a counter electrode is a platinum wire, and cyclic voltammetry scanning is carried out within the potential range of 0-1.45V, and the scanning speed is high: 100mV/s, negative high pressure: -500V, recording the electrochemiluminescence intensity, analyzing the electrochemiluminescence properties of the detection solution according to the electrochemiluminescence intensity at 1.22V and the known electrochemiluminescence properties of the detection solution with the concentration of 0, 0.05 and 0.5 ng/lmc-LR, respectively, establishing a standard curve, and measuring the content of the unknown concentration of MC-LR according to the steps after establishing the standard curve.
Comparative example 1
The procedure is as in example 3, except that no DNA1 is present, i.e.the ortholinked complex is formed from three DNA strands, the sequence of the capture DNA being kept unchanged (same as in example 3), the number of bases complementary to the capture DNA and the aptamer being unchanged, the number of base pairs complementary to DNA2 and the capture DNA being 15, the number of base pairs complementary to DNA2 and the aptamer being 36 (same as in example 3), the sequence being: fc-CGT ACC ATA AAA AGC TAC GCT CCA TAC CTC CCC AGG AAC AAA GGG AGA AC has a sequence shown in SEQ ID NO. 5, and one end of the Fc-CGT ACC ATA AAA AGC TAC GCT CCA TAC CTC CCC AGG AAC AAA GGG AGA AC is modified with ferrocene.
Comparative example 2
The procedure was as in example 3, except that only one ferrocene was modified in DNA1.
Comparative example 3
The procedure was as in example 3, except that DNA1 did not modify ferrocene. The electrochemiluminescence property of the concentration of 0.05ng/mLMC-LR in the detection solution is analyzed.
Experimental example 1 Gibbs free energy
1. Experimental method
According to the method of example 3, capture DNA, DNA1 and DNA2 were designed and analyzed for gibbs free energy of hybridization of capture DNA, DNA1, DNA2, MC-LR aptamers, capture DNA and capture DNA, DNA1 and DNA1, DNA2 and DNA2, MC-LR aptamers and MC-LR aptamers, capture DNA and DNA1, capture DNA and MC-LR aptamers, DNA1 and DNA2, DNA2 and MC-LR aptamers using RNA structure software.
2. Experimental results
Table 1 shows the free energy of Gibbs for hybridization of different DNAs to each other. As can be seen from Table 1, the Gibbs free energies of the capture DNA, DNA1, DNA2, MC-LR aptamers were-0.8, -3.2, -1, and-8.8 eV, respectively; the gibbs free energies of hybridization of capture DNA and capture DNA, DNA1 and DNA1, DNA2 and DNA2, MC-LR aptamer and MC-LR aptamer were-9.8, -12.8, -7.4 and-24.6 eV, respectively; the gibbs free energies of the hybridization of the capture DNA and DNA1, the capture DNA and MC-LR aptamer, the DNA1 and DNA2, and the DNA2 and MC-LR aptamer are-11.1, -34.7, -17.5, -19.9, -18.2eV, respectively, which are greater than the gibbs free energies of the other hybridization, thus, hybridization is easier, i.e., it is demonstrated that the capture DNA, DNA1, and DNA2 of the present protocol design is feasible.
TABLE 1 Gibbs free energy of hybridization of different DNAs to each other
The "initial" in the table indicates: capturing the initial gibbs free energy of DNA, DNA1, DNA2 and MC-LR aptamers; "/" means: not detected.
Experimental example 2
1. Experimental method
According to the method of example 3, the electrochemiluminescence properties of MC-LR with concentrations of 0, 0.05 and 0.5ng/mL in the detection solution were analyzed respectively; and the electrochemiluminescence property of the detection solution was analyzed according to the method of comparative example 1-2, wherein the concentration of MC-LR was 0.05 ng/mL.
2. Experimental results
FIG. 2 is a graph showing the electrochemiluminescence properties of the electrochemiluminescence aptamer sensor of example 3 for analysis of MC-LR at 0, 0.05, and 0.5ng/mL, respectively. As can be seen from FIG. 2, when the concentration of MC-LR is 0ng/mL, the electrochemiluminescence intensity is 2064+ -82; when the concentration of MC-LR is 0.05ng/mL, the electrochemiluminescence intensity is increased to 6070+/-233; when the concentration of MC-LR was 0.5ng/mL, the electrochemiluminescence intensity was further increased to 10719.+ -.190. The design scheme is proved to have extremely high electrochemiluminescence intensity response and higher sensitivity when the concentration of MC-LR is changed.
FIG. 3 is a graph of electrochemiluminescence properties of comparative example 1, comparative example 2 and example 3 electrochemiluminescence aptamer sensors analyzed at 0.05ng/mL MC-LR, respectively. As shown in FIG. 3, when the concentration of MC-LR is 0.05ng/mL, the electrochemiluminescence intensity in comparative example 1 is 3070.+ -.140, which indicates that the distance of the ortho-position junction complex containing one quencher on the electrode surface when the target is present causes a smaller electrochemiluminescence intensity; in contrast, the increase in electrochemiluminescence intensity of comparative example 2 was 4103.+ -. 198, which is 33.6% greater than example 1, indicating that the recognition of the aptamer and target, when present, disrupts the formation of the ortho-linked complex containing both quenchers, greatly enhancing the electrochemiluminescence signal; correspondingly, in example 3, the electrochemical luminescence intensity was increased by 6070+ -233, which was 97.7% higher than in example 1. Indicating that recognition of the target and aptamer, when present, disrupts the formation of ortho-linked complexes containing three quenchers, greatly enhancing the electrochemiluminescent signal, indicating that our method has high sensitivity.
Experimental example 3
1. Experimental method
According to the methods of comparative example 3 and comparative example 1, the change of the electrochemiluminescence intensity with the tending time in the detection solution at a concentration of 0.05ng/mL MC-LR was analyzed, and since comparative example 3 and example 3 were identical in structure, only DNA1 did not modify ferrocene, which was different from comparative example 1 only in DNA sequence, to verify the DNA structure specificity identical to that of example 3.
2. Experimental results
FIG. 4 is a graph of electrochemiluminescence intensity as a function of time for comparative example 1 (curve b) and comparative example 3 (curve a) electrochemiluminescence aptamer sensor analysis of 0.05ng/mL MC-LR. As can be seen from the graph of fig. 4a, as the tending time increases from 10min to 40min, the electrochemiluminescence intensity gradually increases, continuing to increase the tending time to 50min and 60min, with the electrochemiluminescence intensity being substantially unchanged; however, the electrochemiluminescence intensity in the b curve increases with increasing incubation time from 10min to 50min, while remaining unchanged. It is shown that in the orthotopic ligation complex formed by four DNA, DNA1 mediates capture of DNA, aptamer and DNA2 to cooperatively generate orthotopic hybridization, thereby improving recognition between DNA, accelerating reaction speed and further improving the specificity of the method.
The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present application should be made in the equivalent manner, and the embodiments are included in the protection scope of the present application.

Claims (10)

1. An MC-LR detection method based on ortho-junction electrochemiluminescence analysis is characterized by comprising the following steps:
s1, ru (bpy) 3 2+ Mixing AuNPs and Nafion uniformly, and modifying on the surface of a glassy carbon electrode to form Ru (bpy) 3 2+ An electrochemiluminescence platform of/AuNPs/Nafion/GCE;
s2, self-assembling and fixing the capture DNA on the surface of the electrochemiluminescence platform in the step S1 through gold-sulfur bonds to obtain an electrochemiluminescence aptamer sensor;
s3, immersing the prepared electrochemiluminescence aptamer sensor into a detection solution of MC-LR to be detected, and incubating;
s4, immersing the incubated electrochemiluminescence aptamer sensor serving as a working electrode into PBS buffer solution containing tripropylamine, recording the electrochemiluminescence intensity by using an electrochemiluminescence instrument, and determining the MC-LR content in the solution to be detected according to a standard curve established by the electrochemiluminescence intensity and the known MC-LR content;
the detection solution comprises MC-LR aptamer, DNA1 and DNA2;
the MC-LR aptamer is a DNA single-chain aptamer;
the capture DNA is of a single-chain structure, one end of the capture DNA is modified with sulfhydryl, and a base sequence far away from one end of the modified sulfhydryl is complementary with one end of the MC-LR aptamer; ferrocene is modified at both ends of the DNA1, the base sequence at one end of the DNA1 is complementary with one end of the capture DNA close to the modified sulfhydryl, and the base sequence in the middle of the DNA1 is complementary with the middle of the MC-LR aptamer; one end of the DNA2 is modified with ferrocene, a base sequence close to one end of the ferrocene is complementary with the other end of the DNA1, and a base sequence far away from one end of the DNA2 is complementary with the other end of the MC-LR aptamer.
2. The method of claim 1, wherein the capture DNA and DNA1 comprise 9-21 complementary base pairs, one end of the capture DNA and MC-LR aptamer comprises 15-27 complementary base pairs, the middle of DNA1 and the middle of the MC-LR aptamer comprises 10-18 complementary base pairs, DNA1 and DNA2 comprise 9-21 complementary base pairs, and the other end of the DNA2 and MC-LR aptamer comprises 15-27 complementary base pairs.
3. The method according to claim 2, wherein the capture DNA sequence is shown in SEQ ID NO. 1, the DNA1 sequence is shown in SEQ ID NO. 2, the DNA2 sequence is shown in SEQ ID NO. 3, and the MC-LR aptamer sequence is shown in SEQ ID NO. 4.
4. The method according to claim 1, wherein the detection solution in step S3 comprises 1-10nmol/L MC-LR aptamer, 1-10nmol/L DNA1 and 1-10nmol/L DNA2.
5. The method according to claim 1, wherein the capturing DNA in step S2 is self-assembled and immobilized on the surface of the electrochemiluminescence platform by gold-sulfur bond, and the method comprises the steps of: ru (bpy) 3 2+ And (3) immersing the AuNPs/Nafion/GCE electrochemiluminescence platform into a capture DNA solution with the mass concentration of 5-20 mu mol/L, incubating for 1-5h, and immersing into mercaptoethanol for sealing after tending to obtain the electrochemiluminescence aptamer sensor.
6. The method according to claim 5, wherein the mercaptoethanol is present in an amount of 0.5 to 2mmol/L and the blocking reaction time is 20 to 50min.
7. The method of claim 1, wherein the incubation in step S3 is performed at a temperature of 25-37 ℃ for 30-60min.
8. The method according to claim 1, wherein the electrochemical luminescence aptamer sensor of step S4 is further rinsed with a PB buffer having a pH of 7.2,5-20mmol/L before being immersed in tripropylamine solution.
9. The method according to claim 8, wherein the tripropylamine solution in step S4 has a mass concentration of 25-75mmol/L.
10. Use of the capture DNA, MC-LR aptamers, DNA1 and DNA2 of any one of claims 1-9 in the determination of MC-LR content.
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