CN115963159A - Pollutant action mode discrimination method based on electrochemical DNA logic switch - Google Patents
Pollutant action mode discrimination method based on electrochemical DNA logic switch Download PDFInfo
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Abstract
The invention discloses a pollutant action mode discrimination method based on an electrochemical DNA logic switch, which belongs to the technical field of electrochemical analysis and comprises the following steps: designing a logic switch DNA; (2) activating an electrode; (3) activation of DNA; (4) preparing an electrochemical DNA logic switch; (5) judging the action mode of the organic pollutants: the change value S of the electrochemical signal according to each electrochemical DNA logic switch E And judging the action mode of the organic pollutants and the DNA. The method can be used for analyzing the action modes of the DNA and the organic pollutants, and realizes the quick, accurate, high-sensitivity and identification analysis of the action modes of the DNA and the pollutants according to the structural specificities of different DNAs and the electrochemical signal change of each detection unit.
Description
Technical Field
The invention belongs to the technical field of electrochemical analysis, and particularly relates to a pollutant action mode discrimination method based on an electrochemical DNA logic switch.
Background
The pollutants in the environment are various, such as heavy metals, polycyclic aromatic hydrocarbons, pesticides, antibiotics and the like can exist in the environment for a long time, not only can damage the environmental ecosystem and break the ecosystem balance, but also can enter the human body through a food chain to generate acute/chronic toxicity or generate teratogenesis, carcinogenesis and mutagenesis effects under the action of DNA (deoxyribonucleic acid), and cause diseases, so that the human body health is harmed.
Research shows that organic pollutants can react with DNA and influence the DNA replication process after entering human bodies, wherein the action modes mainly comprise: 1) Organic pollutants are directly inserted into the middle of the DNA base layer and generate pi-pi action and the like with the base through a benzene ring; 2) Organic pollutants directly generate hydrogen bond action with the basic group of the DNA; 3) Organic pollutants generate active free radicals under the action of in vivo enzymes, and the active free radicals react with DNA active sites to cause untwistable damage. Therefore, the method for researching the action mode of the DNA and the organic pollutant micromolecules and establishing the DNA and organic pollutant micromolecule analysis method has important significance for disclosing the pathogenic mechanism of the pollutant and has important values for researching the DNA toxicity of the pollutant, evaluating the eating risk and the occupational exposure risk of the pollutant and the like.
At present, the method for researching the action modes of pollutants and DNA mostly adopts spectroscopy (such as ultraviolet-visible spectroscopy, fluorescence spectroscopy), an electrochemical DNA sensing method and the like, and although the method can research the action modes of DNA and pollutants, the method has some defects, such as large sample requirement and low sensitivity of spectroscopy, and the electrochemical method is difficult to independently identify different action modes and only can reveal or partially reveal the action of DNA and pollutants.
Therefore, the research method for researching the action mode of the DNA and the organic pollutant, which improves the sensitivity of the method, reduces the use amount of the sample and improves the identification performance of the action mode, is one of important directions for expanding the technical field of DNA biosensing, and has important significance for pollutant toxicological analysis, exposure risk evaluation, environmental sample analysis and the like.
Disclosure of Invention
The invention discloses a pollutant action mode distinguishing method based on an electrochemical DNA logic switch, which can be used for analyzing the action modes of DNA and organic pollutants, and can realize quick, accurate, high-sensitivity and identification analysis of the action modes of the DNA and the pollutants according to the structural specificities of different DNAs and the electrochemical signal change of each detection unit.
In order to achieve the purpose, the invention adopts the following technical scheme:
the method for distinguishing the action mode of the pollutants based on the electrochemical DNA logic switch comprises the following steps:
(1) Designing logic switch DNA
Designing different DNAs according to the DNA base complementary principle and molecular structure characteristics;
(2) Electrode activation
Carrying out acid solution activation and/or electrochemical activation on the electrode to obtain an activated electrode;
(3) Activation of DNA
Dissolving different DNAs respectively, and adding an activating agent for incubation to obtain different activated DNA solutions;
(4) Preparation of electrochemical DNA logic switch
Respectively dripping different activated DNA solutions on the surfaces of the activated electrodes, and carrying out drip washing and blow drying after incubation to obtain different electrochemical DNA logic switches; carrying out electrochemical measurement on different electrochemical DNA logic switches, and recording an electrochemical signal S1;
(5) Organic contaminant mode of action discrimination
Dripping organic pollutant solution to be detected on the surfaces of different electrochemical DNA logic switches, carrying out rinsing and blow-drying after incubation, carrying out electrochemical determination, and respectively recording electrochemical signals S2;
calculating S E = S2-S1| depending on the electrochemical signal variation S of the respective electrochemical DNA logic switch E And judging the action mode of the organic pollutants and the DNA.
Further, the different DNAs are hairpin DNAs;
different GC or AT base pair ratios of double-stranded stem parts of the DNA, base pairs of double-stranded stem parts, different numbers of loop bases and/or different base ratio of loops.
The hairpin DNA sequence is short, the price is low, the preparation is convenient, the thermal stability is strong, the hairpin DNA sequence has the structures of single-stranded DNA and double-stranded DNA, and has the characteristics of two structures, so that the detection cost is reduced, and the detection sensitivity is high.
According to the comparison of the change values of the electrochemical signals of the DNA with different GC or AT base pair ratios of the double-stranded stem part, whether the change values are related to the insertion of a GC or AT base layer can be judged;
according to the comparison of the change values of the electrochemical signals of the DNA with different base pairs of the double-stranded stem part, whether the change values are related to the insertion of the base layer can be judged;
according to the comparison of the change values of the electrochemical signals of the DNA with different loop base numbers, whether the change values are related to the base action can be judged;
according to the comparison of the change values of the electrochemical signals of the DNA with different base ratios of the loop, whether the change values are related to the action of the specific type of the base can be judged.
Further, the different DNAs include DNA with a double-stranded stem part GC base pair ratio of > 80% and DNA with a double-stranded stem part AT base pair ratio of > 80%.
Preferably, the different DNAs include DNA1, DNA2, DNA3, DNA4; the loop base number of DNA1 and DNA2 is 5-25, and the loop base number of DNA3 and DNA4 is 10-60;
preferably, the number of base pairs of the double-stranded stem portion in DNA1, DNA2 is 5 to 20, and the number of base pairs of the double-stranded stem portion in DNA3, DNA4 is 3 to 15.
Furthermore, the 3 '-end and/or the 5' -end of the hairpin DNA are modified with active functional groups, and the active functional groups comprise one or more of amino, carboxyl and sulfydryl.
Further, the first complementary base pairs of different DNA double strand stem portions are all GC base pairs.
Further, the electrode is a screen printing gold electrode;
the acid is one or more of sulfuric acid, nitric acid and hydrochloric acid.
Further, the activator is tris (2-carboxyethyl) phosphine hydrochloride, and the working concentration is 500-5000 mu M.
Further, after different DNAs are dissolved, adding an activating agent for incubation for 30min;
dripping different activated DNA solutions on the surface of the activated electrode and incubating for 1-10h;
the solution of the organic pollutants to be detected is dripped on the surfaces of different electrochemical DNA logic switches to be incubated for 10-100min.
Preferably, 5-500 μ L of organic pollutant solution to be measured with the concentration of 1-500 μm is dripped on the surface of the electrochemical DNA logic switch.
Preferably, in the preparation process of the electrochemical DNA logic switch, different electrochemical signals of the electrochemical DNA logic switch are the same by regulating and controlling parameters such as DNA concentration, incubation time, 6-mercaptohexan-1-ol incubation time and the like.
Further, the electrochemical measurement method is an electrochemical impedance method,
the electrochemical active substance is one or more of ruthenium pyridine, ruthenium hexamine, potassium ferricyanide and potassium ferrocyanide.
The invention provides an organic pollutant action mode discrimination method based on an electrochemical DNA logic switch, establishes, perfects and develops an organic pollutant and DNA action mode discrimination technology, has important significance for disclosing a pollutant pathogenesis, and has important values for researching pollutant DNA toxicity, evaluating the edible risk and occupational exposure risk of pollutants and the like. Meanwhile, the method has the characteristics of quick response, high sensitivity, low cost, strong stability, strong anti-interference performance, strong applicability and the like, and has good reference value for developing a novel DNA biosensing detection technology.
The method obviously improves the sensitivity of the DNA logic switch, reduces the discrimination difficulty and improves the discrimination efficiency by introducing hairpin DNA, provides a new analysis idea for the research of the action mode of organic pollutants and DNA, and has important significance.
Drawings
FIG. 1 is a graph showing the effect of activating a screen-printed electrode in example 1;
FIG. 2 shows the results of the electrochemical impedance measurement of the electrochemical DNA logic switch DAE1 of example 1;
FIG. 3 shows the measurement results of sample A to be tested in example 1.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
1. Designing logic switch DNA
The nucleotide sequence of the DNA1 is shown as SEQ ID NO.1:5 'TTTTTTTGGGCGCGCGGAGGGCTGGAGGAGGAGGAGCGCCC-3' HO- (CH) 2 ) 6 -S-S-(CH 2 ) 6 The group is modified on the 1 st T base at the 5' end and is used for linking and activating a silk-screen printing gold electrode interface, the number of the bases of the stem part is 6 pairs, and the number of the bases of the loop part is 20;
the nucleotide sequence of the DNA2 is shown as SEQ ID NO.2:5 'TTTTTTTTTTTTTTTTAAAGGGGAGGGG TGGCTGGAGGAGGATTTAAG-3', HO- (CH) 2 ) 6 -S-S-(CH 2 ) 6 The group is modified on the 1 st T base at the 5' end and is used for linking and activating a screen printing gold electrode interface, the number of the base in the stem part is 6 pairs, and the number of the base in the loop part is 22;
the nucleotide sequence of the DNA3 is shown as SEQ ID NO.3:5 'TTTTTTTGGGCGCGCGGAGGGGTGGCTGGGTGGAGGAGGAGCGCCC-3' HO- (CH) 2 ) 6 -S-S-(CH 2 ) 6 The group is modified on the 1 st T base at the 5' end and is used for linking and activating a screen printing gold electrode interface, the base number of stem part is 6 pairs, and the base number of loop part is 28;
the nucleotide sequence of the DNA4 is shown as SEQ ID NO.4:5' HO- (CH) TTTTTTTTTTTTATATACATTTTATC TATTTTATTTTTTATTTTATATATATAAG-3 2 ) 6 -S-S-(CH 2 ) 6 The group is modified on the 1 st T base at the 5' end and used for linking and activating a silk-screen printing gold electrode interface, the base number of stem part is 8 pairs, and the base number of loop part is 24.
2. Preparation of activated screen printing gold electrode
Soaking screen printing gold electrode in sulfuric acid-H 2 O 2 The mixed solution (V/V = 7) is placed in an electrochemical detection cell containing 0.5M sulfuric acid solution for 5min, an electrode is activated by sampling electrochemical voltammetry, the oxidation-reduction potential range is-0.1 to +1.2V, the number of activation cycles is 20, and after the activation cycles are finished, the electrode is taken out, and the ultra-pure solution is prepared by the following stepsWater cleaning, nitrogen drying and standby.
The electrode activation results are shown in FIG. 1. As can be seen from the figure, the method is adopted to activate the screen printing gold electrode, the activated electrode has a higher electrochemical current value, and the oxidation reduction peak potential difference of the electrode is smaller, so that the electrochemical performance of the screen printing gold electrode can be remarkably improved by adopting the method.
Preparation of DNA solution
Centrifuging each designed DNA (synthetic product purchased) sample vial for 3-5min, then gently opening the lid, and taking the newly configured Tris-NaClO 4 (20mM, pH 7.5) solution, transferred to a DNA sample vial, sealed, treated with a vortex shaker for 5min, and mixed well to obtain a 50. Mu.M DNA solution for use.
4. Preparation of activated DNA solution
With Tris-NaClO 4 (20mM, pH 7.5) 4mM Tris (2-carboxyethyl) phosphine hydrochloride solution, 10. Mu.L of the solution was transferred to a centrifugal tube, 2. Mu.L of the prepared DNA solution was added thereto, and Tris-NaClO was finally added 4 (20mM, pH 7.5) to 50. Mu.L, and left standing for 30min to obtain an activated DNA solution at a concentration of 2. Mu.M for use.
5. Preparation of electrochemical DNA logic switch
Taking 10 μ L of prepared 2 μ M activated DNA1 solution, dripping on the interface of an activated screen printing gold electrode, incubating for 6h, taking out, and adding Tris-NaClO 4 (20mM, pH 7.5) buffer, then placed in Tris-NaClO containing 2mM 6-mercaptohexan-1-ol 4 (20mM, pH 7.5) for 25min, and taking out the solution, adding Tris-NaClO 4 (20mM, pH 7.5) and then the buffer solution is rinsed and dried by nitrogen to obtain the electrochemical DNA logic switch DAE1 for later use.
A50 mM KCl solution was prepared and 4mM K was prepared from the solution 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]5ml of the solution is added into an electrochemical detection cell. And (3) measuring the electrochemical impedance S1 by taking the electrochemical DNA logic switch as a working electrode, a platinum wire as a counter electrode and silver/silver chloride as a reference electrode. FIG. 2 is a representation of electrochemical DNA logic switches by electrochemical impedance method. As can be seen from the figure, the activated silk after modification of DNAElectrochemical impedance of screen printed gold electrode interface>1000, which shows that the gold electrode interface prepared by the method has higher activity and can successfully modify the DNA film.
Furthermore, an activated DNA2 solution, an activated DNA3 solution and an activated DNA4 solution are respectively used for replacing the activated DNA1 solution to prepare the electrochemical DNA logic switch, and the DNA concentration, the incubation time and the 6-mercaptohexane-1-alcohol incubation time are regulated and controlled in the preparation process so as to achieve the same assembly effect (electrochemical impedance signal S1) as the electrochemical DNA logic switch DAE 1. The obtained electrochemical DNA logic switches are DAE2, DAE3 and DAE4 respectively.
6. Contaminant action mode discrimination
Dripping 100 μ L of 1 μ M sample A (2, 3-naphthylamine) to be tested on the surfaces of electrochemical DNA logic switches DAE1, DAE3 and DAE4, incubating for 30min, and incubating with Tris-NaClO 4 (20mM, pH 7.5) buffer rinsing, nitrogen blow-drying, and electrochemical impedance measurement: a50 mM KCl solution was prepared and 4mM K was prepared from the solution 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]5ml of the solution is added into an electrochemical detection cell. And (3) performing electrochemical impedance measurement by using the DNA modified electrode as a working electrode, a platinum wire as a counter electrode and silver/silver chloride as a reference electrode.
Measuring electrochemical impedance S2, and calculating electrochemical signal change value S of each electrochemical DNA logic switch E = S2-S1|, i.e. S E 1、S E 1、S E 3、S E 4, judging the action mode of the organic pollutants and the DNA according to the change value. The discrimination method is shown in Table 1.
TABLE 1
The result of the measurement of sample A to be tested (2, 3-naphthylamine) is shown in FIG. 3. As can be seen from FIG. 3, the samples A to be tested were used differentlyThe electrochemical DNA logic switches DAE1, DAE2, DAE3 and DAE4 are determined to present different detection signals, and the detection signal relationship is S E 1>S E 2>S E 3，S E 4, it can be judged that the sample A to be tested can act on DNA in such a manner that the G-C base layer is inserted. The result shows that the method can be used for researching the action mode of organic molecules and DNA.
Dripping 100 μ L of 5 μ M sample chlorobenzene on the surface of electrochemical DNA logic switches DAE1, DAE3, DAE4, incubating for 30min, and incubating with Tris-NaClO 4 (20mM, pH 7.5) buffer rinse, nitrogen blow dry, electrochemical impedance measurements were performed: preparing 50mM KCl solution, and preparing 4mM K from the solution 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]5ml of the solution is added into an electrochemical detection cell. And (3) performing electrochemical impedance measurement by using the DNA modified electrode as a working electrode, a platinum wire as a counter electrode and silver/silver chloride as a reference electrode.
The chlorobenzene sample to be detected presents similar detection signals by using different electrochemical DNA logic switches DAE1, DAE2, DAE3 and DAE4 for determination, S E 1(47)，S E 2(38)，S E 3(45)，S E 4 (53), all are less than<100, the chlorobenzene sample to be detected has no obvious effect on DNA basically.
The chlorocyclohexane of the sample to be tested presents similar detection signals by using different electrochemical DNA logic switches DAE1, DAE2, DAE3 and DAE4 for determination, S E 1(52)，S E 2(57)，S E 3(49)，S E 4 (44) are all less than<100, so that the chlorocyclohexane of the sample to be detected has no obvious effect on DNA basically.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to the above-described embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (9)
1. The method for distinguishing the action mode of the pollutants based on the electrochemical DNA logic switch is characterized by comprising the following steps of:
(1) Designing logic switch DNA
Designing different DNAs according to the DNA base complementary principle and molecular structure characteristics;
(2) Electrode activation
Carrying out acid solution activation and/or electrochemical activation on the electrode to obtain an activated electrode;
(3) Activation of DNA
Dissolving different DNAs respectively, and adding an activating agent for incubation to obtain different activated DNA solutions;
(4) Preparation of electrochemical DNA logic switch
Respectively dripping different activated DNA solutions on the surfaces of the activated electrodes, and carrying out drip washing and blow drying after incubation to obtain different electrochemical DNA logic switches; carrying out electrochemical measurement on different electrochemical DNA logic switches, and recording an electrochemical signal S1;
(5) Organic contaminant mode of action discrimination
Dripping organic pollutant solution to be detected on the surfaces of different electrochemical DNA logic switches, carrying out rinsing and blow-drying after incubation, carrying out electrochemical determination, and respectively recording electrochemical signals S2;
calculating S E = S2-S1|, the value S is varied in accordance with the electrochemical signal of each electrochemical DNA logic switch E And judging the action mode of the organic pollutants and the DNA.
2. The method for discriminating the action mode of a contaminant based on electrochemical DNA logic switch as claimed in claim 1,
the different DNAs are all hairpinDNA;
the different DNA double-stranded stem parts have different GC or AT base pair occupation ratios, double-stranded stem part base pairs and/or loop base numbers.
3. The method for discriminating the action mode of a contaminant based on electrochemical DNA logic switch as claimed in claim 2,
the different DNAs include DNA with a double-stranded stem part GC base pair ratio of > 80% and DNA with a double-stranded stem part AT base pair ratio of > 80%.
4. The method for discriminating the action mode of a contaminant based on electrochemical DNA logic switch as claimed in claim 2,
the 3 '-end and/or the 5' -end of the hairpinDNA are modified with active functional groups, and the active functional groups comprise one or more of amino, carboxyl and sulfydryl.
5. The method of claim 1, wherein the method of discriminating the mode of action of contaminants based on electrochemical DNA logic switch,
the first complementary base pairs of the different DNA double stranded stem parts are all GC base pairs.
6. The method of claim 1, wherein the method of discriminating the mode of action of contaminants based on electrochemical DNA logic switch,
the electrode is a screen printing gold electrode;
the acid is one or more of sulfuric acid, nitric acid and hydrochloric acid.
7. The method of claim 1, wherein the method of discriminating the mode of action of contaminants based on electrochemical DNA logic switch,
the activating agent is tris (2-carboxyethyl) phosphine hydrochloride, and the working concentration is 500-5000 mu M.
8. The method for discriminating the action mode of a contaminant based on electrochemical DNA logic switch as claimed in claim 1,
adding an activating agent to incubate for 30min after different DNAs are dissolved;
dripping different activated DNA solutions on the surface of the activated electrode and incubating for 1-10h;
the solution of the organic pollutants to be detected is dripped on the surfaces of different electrochemical DNA logic switches to be incubated for 10-100min.
9. The method for discriminating the action mode of a contaminant based on electrochemical DNA logic switch as claimed in claim 1,
the electrochemical measuring method is an electrochemical impedance method,
the electrochemical active substance is one or more of ruthenium pyridine, ruthenium hexamine, potassium ferricyanide and potassium ferrocyanide.
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| CN102375021A (en) * | 2010-08-25 | 2012-03-14 | 中国科学院大连化学物理研究所 | Electrochemical method employing DNA as probe to detect environmental pollutant |
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| CN115166131A (en) * | 2022-07-28 | 2022-10-11 | 华中科技大学 | Method for detecting in-vitro DNA adduct catalyzed by drug metabolizing enzyme through single-molecule manipulation |
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| CN102375021A (en) * | 2010-08-25 | 2012-03-14 | 中国科学院大连化学物理研究所 | Electrochemical method employing DNA as probe to detect environmental pollutant |
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| CN115166131A (en) * | 2022-07-28 | 2022-10-11 | 华中科技大学 | Method for detecting in-vitro DNA adduct catalyzed by drug metabolizing enzyme through single-molecule manipulation |
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