CN115950937A - Small molecule-DNA binding constant calculation method based on electrochemical technology - Google Patents
Small molecule-DNA binding constant calculation method based on electrochemical technology Download PDFInfo
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Abstract
The invention discloses a micromolecule-DNA binding constant calculation method based on an electrochemical technology, which belongs to the technical field of electrochemical analysis and comprises the following steps: (1) preparing a DNA modified electrode; (2) measuring an electrochemical signal; (3) determining the detection concentration of the small molecules of the pollutants; (4) constructing a standard curve; and (5) measuring the binding constant. The invention can change the electrochemical performance of the DNA film layer through the action of the micromolecules and the interface modified DNA, thereby realizing the analysis of the binding capacity and the binding constant. The method has the advantages of simple operation, rapidness, high sensitivity, low cost, strong anti-interference performance and the like, and has important significance and better application value for perfecting the existing binding constant K determination technology.
Description
Technical Field
The invention belongs to the technical field of electrochemical analysis, and particularly relates to a micromolecule-DNA binding constant calculation method based on an electrochemical technology.
Background
Pollutants in the environment, particularly organic and toxic pollutants, such as polycyclic aromatic hydrocarbon, polychlorinated biphenyl, organochlorine pesticide, organic dye and the like, have bioaccumulation and great harm to an ecosystem and human health. Research shows that pollutants enter a human body through various ways such as direct contact, respiration, diet and the like, can be accumulated in the human body to generate toxic action, can be converted into intermediates/derivatives with higher biological activity and stronger toxicity through activation action (human metabolism action, enzyme degradation action) and the like, and the active intermediates can interact with biological macromolecules (protein, lipid, DNA and the like) to cause the change of the structure and the function of the macromolecules so as to cause damage to the organism, so that the pollutants also have stronger carcinogenic, teratogenic and mutagenic 'tri-inducing' effects. In addition, some environmental pollutant molecules can directly react with DNA to generate genotoxicity, so that the research on the binding capacity of the pollutant micromolecules and the DNA can provide important reference basis for judging the genotoxicity of the pollutants, disclosing the toxicology of the pollutants, evaluating ecological risks and the like.
The binding constant K is a direct and key index reflecting the binding performance of the small molecule and the DNA. But is limited by factors such as DNA sequence difference, three-dimensional structure difference, in vivo and in vitro environment difference and the like, so that the binding performance of the small molecular pollutants and the in vivo DNA effect is difficult to obtain and measure really. In addition, the binding performance and binding constant of the conventional small molecules to DNA are often analyzed by fluorescence, and it is difficult to measure the binding constant of molecules with weak fluorescence or no fluorescence.
Therefore, the method for establishing the accurate, reliable and sensitive micromolecule-DNA binding constant K determination method on the basis of developing the research of the specific recognition DNA membrane has important significance for enriching and perfecting the existing micromolecule-DNA binding constant K determination method.
Disclosure of Invention
The invention discloses a micromolecule-DNA binding constant calculation method based on an electrochemical technology, which can change the electrochemical performance of a DNA film layer through the action of micromolecules and interface modified DNA, thereby realizing the analysis of binding capacity and binding constant.
In order to achieve the purpose, the invention adopts the following technical scheme:
the small molecule-DNA binding constant calculation method based on the electrochemical technology comprises the following steps:
(1) Preparing a DNA modified electrode:
preparing a solution of the end functionalized DNA, coating the solution on an electrode interface, and cleaning and drying after cross-linking incubation to obtain a DNA modified electrode;
(2) Measuring an electrochemical signal:
measuring an electrochemical signal S1 of the DNA modified electrode;
preparing solution with different concentrations by taking small molecules of the pollutants, and soaking the DNA modified electrode in the solution of the small molecules of the pollutants for incubation; after incubation, washing and drying the DNA modified electrode, and measuring an electrochemical signal S2;
calculating the electrochemical signal change value Sw = | S2-S1| of the DNA modified electrode before and after incubation of the pollutant small-molecule solution with each concentration;
(3) Determining the detection concentration of the small molecules of the pollutants:
maximum value S of variation value according to electrochemical signal C Determining the electrochemical signal variation value as S CK Concentration of contaminant small molecule solution C k Detecting concentration for determining the binding constant of the pollutant micromolecules and the DNA; s. the CK /S C＞ 0.70；
(4) Constructing a standard curve:
selecting different pollutant small molecules with known binding constants, respectively preparing the different pollutant small molecules into solutions with the concentration of C, measuring electrochemical signals according to the method in the step (2), and calculating the change value of the electrochemical signals; drawing a standard curve according to the binding constant and the electrochemical signal change value;
(5) Determination of binding constant:
prepared at a concentration of C k The pollutant micromolecules to be detected; and (3) measuring the electrochemical signal according to the method in the step (2), calculating the change value of the electrochemical signal, and calculating the binding constant by combining with the standard curve.
Further, in the step (1),
the site of end functionalization is at the 5 'end or 3' end of the DNA;
the functional group includes amino, carboxyl, aldehyde group, sulfhydryl group, etc.
Further, in the step (1),
the DNA is hairpin DNA in which the number of base pairs in a double-stranded stem portion is not less than 5.
Preferably, the base pair number of the hairpin DNA double-stranded stem portion is 5 to 11.
Further preferably, the base pair number of the hairpin DNA double-stranded stem portion is 5 to 8.
Preferably, the number of bases in the loop portion of hairpin DNA is 18 to 26.
Further preferably, the number of bases in the hairpin DNA loop portion is 18 to 22.
Further, in the step (1),
the electrode is a gold electrode, a glassy carbon electrode, a carbon electrode or a screen printing electrode and the like which are subjected to activation treatment;
the activation treatment includes acid solution activation, alkali solution activation, salt solution activation, and/or electrochemical activation.
Further preferably, the acid is selected from sulfuric acid, the base is selected from sodium hydroxide, and the salt is selected from sodium phosphate, sodium dihydrogen phosphate, etc.
Further, in the step (1),
the cross-linking agent used for cross-linking incubation is tris (2-carboxyethyl) phosphine hydrochloride, dicyclohexylcarbodiimide, carbodiimides or N-hydroxy thiosuccinimide;
the incubation time is 10-120min.
Further preferably, the incubation time is 30-60min.
Further, in the step (2),
measuring electrochemical signals by using a three-electrode measuring system;
taking a DNA modified electrode before and after incubation of the pollutant micromolecule solution as a working electrode, a platinum wire electrode as a counter electrode, and a silver/silver chloride electrode as a reference electrode;
the measurement method includes a current method, a potential method or a resistance method.
Further, in the step (2),
the electrolyte solution is PB, tris, acetate or carbonate buffer solution with the concentration of 1-200mM and the pH of 6.0-8.0;
the indicator substance for measuring the electrical signal is K 3 [Fe(CN) 6 ]And K 4 [Fe(CN) 6 ]。
The invention has the beneficial effects that:
1) The electrochemical technology adopted by the invention has the advantages of simple operation, rapid detection and high reliability;
2) The electrochemical technology adopted by the invention can be suitable for detecting weak or non-fluorescent optically active molecules;
3) The electrochemical technology adopted by the invention has higher anti-interference performance;
4) The hairpin DNA adopted by the invention can form a double-stranded structure under the condition of less base pairs, thereby reducing the number of the base and lowering the cost;
5) The electrochemical hairpin DNA membrane used in the invention has the advantage of small sample size.
The invention provides a method for determining a small molecule-DNA binding constant based on interface immobilized DNA, which establishes, perfects and develops a calculation method of the small molecule binding constant K, provides an important reference basis for judging the genotoxicity of pollutants, revealing the toxicology of the pollutants, evaluating ecological risks and the like, and solves the problem that the small molecule binding constant K with weak fluorescence or no fluorescence activity is difficult to measure. The method has the advantages of simple operation, rapidness, high sensitivity, low cost, strong anti-interference performance and the like, and has important significance and better application value for perfecting the existing binding constant K determination technology.
Drawings
FIG. 1 is a graph showing electrochemical impedance measurement of an activated gold electrode in example 1;
FIG. 2 is a graph showing electrochemical impedance measurement of the DNA modified electrode in example 1;
FIG. 3 is a graph of the binding constant discrimination criteria of example 1;
FIG. 4 is a graph showing the measurement of electrical impedance of various DNA-modified electrodes in example 2.
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. Activation of gold electrodes
Taking 1 μm of gamma-Al 2 O 3 Adding distilled water into the powder, stirring into slurry, smearing on the deerskin polishing cloth wetted by distilled water, polishing the deerskin polishing cloth with gold electrode in a circle manner for 5min, and then respectively ultrasonically cleaning with ethanol and ultrapure water;
according to the above operation method, 0.3 μm and 0.05 μm of gamma-Al are sequentially used 2 O 3 Polishing the gold electrode with slurry for 5min, and ultrasonically cleaning in ethanol and ultrapure water respectively;
putting the polished gold electrode at H of 1M 2 SO 4 Scanning the solution for 20 times by cyclic voltammetry (-0.6V- + 0.8V), taking out, washing with ultrapure water, and drying with nitrogen to obtain the activated gold electrode for later use.
Electrochemical impedance measurement was performed on the prepared activated gold electrode, and as a result, as shown in FIG. 1, the resistance of the electrode interface was very small, only 26.5. Omega. Cm 2 The method can effectively remove impurities on the electrode interface and obviously improve the electrochemical performance of the electrode interface.
Preparation of DNA modified electrode
Thiol-modified solid DNA1 (5 2 ) 6 -S-S-(CH 2 ) 6 Modifying the 1 st T base at the 5' end with 5 pairs of stem part bases and 18 loop part bases) and adding proper amount of Tris-HClO 4 (20mM, pH = 7.5), vortex shaking for 5min, incubating in hot water at 85 ℃ for 5min, and naturally cooling to room temperature to obtain 200. Mu.M DNA stock solution for use.
Tris-HClO containing 1mM EDTA, 500mM NaCl, 2mM Tris (2-carboxyethyl) phosphine hydrochloride was prepared 4 (20mM, pH = 7.5) solution, the prepared DNA stock solution was diluted to 1. Mu.M, and incubated at room temperature for 30min to obtain a DNA assembly solution for use.
Droplet application of DNA assembly to activated gold electrodesPolar boundary, incubation for 6h with Tris-HClO 4 (20mM, pH = 7.5), washed with nitrogen gas, and then dried in Tris-HClO containing 1mM 6-mercaptohex-1-ol 4 (20mM, pH = 7.5) for 1h, and finally Tris-HClO 4 (20mM, pH = 7.5) and then the solution was washed and blown dry with nitrogen gas to obtain a DNA-modified electrode.
Electrochemical impedance measurement is carried out on the prepared DNA modified electrode, and the result is shown in figure 2, after the DNA is assembled by activating the bare electrode interface, the resistance of the electrode interface is obviously increased to 1070.4 omega cm 2 The method of the invention can successfully assemble DNA to the gold electrode interface to prepare the DNA recognition membrane.
3. Electrochemical impedance detection and determination of pollutant small molecule detection concentration
Tris-HClO containing 100mM KCl is prepared 4 (20mM, pH = 7.5) solution, and 4mM K was prepared in the solution 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]The solution was then added to 5ml of the solution in an electrochemical cell. Constructing a three-electrode measuring system by taking the prepared DNA modified electrode as a working electrode, a platinum wire electrode as a counter electrode and saturated KCl solution silver/silver chloride as a reference electrode, and measuring the electrochemical impedance S1 of the working electrode; taking out the working electrode, and using Tris-HClO 4 (20mM, pH = 7.5) solution elution, N 2 Drying;
using Tris-HClO 4 (20mM, pH = 7.5) solutions the pollutant small molecules are prepared into solutions with different concentrations (10, 50, 100, 200, 400 and 500 nM), and the DNA modification electrode is soaked in the pollutant small molecule solution with each concentration for incubation for 30min; tris-HClO for DNA modified electrode after incubation 4 (20mM, pH = 7.5) solution elution, N 2 Drying, and measuring the electrochemical impedance S2 as a working electrode again;
calculating the electrochemical impedance change value Sw = | S2-S1| of the DNA modified electrode before and after incubation of the pollutant small molecule solution with each concentration, wherein the maximum electrochemical impedance change value is S C And the concentration of the corresponding pollutant small molecule solution is shown in table 1, and the subsequent pollutant small molecule detection concentration is determined.
TABLE 1
4. Constructing a standard curve:
selecting different pollutant micromolecules with known binding constants, namely 1-naphthylamine, 2-naphthylamine and 1, 5-naphthylamine, respectively preparing the different pollutant micromolecules into solutions with the concentration of 200nM, measuring electrochemical impedance according to the method in the step 3, and calculating the change value of the electrochemical impedance; a standard curve is drawn according to the binding constant and the electrochemical impedance change value, and the result is shown in FIG. 3, the electrochemical impedance signal has high correlation with the binding constant, which indicates that the method can be used for small molecule binding constant analysis.
5. Determination of binding constant:
configured concentration of C k (S CK /S C >0.70 A small molecule of the contaminant to be detected; and (3) measuring the electrochemical impedance according to the method in the step (3), calculating the change value of the electrochemical impedance, and calculating the binding constant by combining a standard curve.
According to the above operation method, 2, 3-naphthylamine and 1, 8-naphthylamine are used as research objects, and the calculated binding constants are respectively 2.35 × 10 6 ，1.66×10 7 。
Example 2 comparison of DNA Properties
By using hairpin DNA1 (5 2 ) 6 -S-S-(CH 2 ) 6 The group is modified on the 1 st T base at the 5' end, the number of the stem part bases is 5 pairs, the number of the loop part bases is 18), single-stranded DNA2 (5 2 ) 6 -S-S-(CH 2 ) 6 Group modified at the 1 st T base at the 5' end), double-stranded DNA (3 + 4) (DNA 3:5 'ttttttttttagacggaacggcggg-3', SEQ ID NO.3, HO- (CH) 2 ) 6 -S-S-(CH 2 ) 6 The group is modified at the 1 st T base at the 5' end; DNA4:5 'tcgccttgccca-3', SEQ ID NO. 4) instead of the DNA in example 1, the preparation and detection of the DNA-modified electrode were performed, and the results are shown in FIG. 4, and the electrochemical impedance detection results for 500nM 1, 5-naphthylamine are: s hairpinDNA >S DNA-(3+4) >S DNA The method proves that hairpin DNA is more beneficial to researching the binding property of small molecules and DNA, and the hairpin structure is obviously superior to single-strand DNA and double-strand DNA through further comparative analysis.
Example 3DNA Performance comparison
By using hairpin DNA1 (5 2 ) 6 -S-S-(CH 2 ) 6 The group is modified at the 1 st T base at the 5' end, the number of the stem part bases is 5 pairs, the number of the loop part bases is 18), hairpin DNA 5 (5- 2 ) 6 -S-S-(CH 2 ) 6 The group is modified on the 1 st T base at the 5' end, the number of the stem part bases is 8 pairs, the number of the loop part bases is 18), hairpin DNA 6 (5 2 ) 6 -S-S-(CH 2 ) 6 The group is modified on the 1 st T base at the 5' end, the number of the stem part bases is 11 pairs, the number of the loop part bases is 18), hairpin DNA 7 (5 2 ) 6 -S-S-(CH 2 ) 6 The group is modified on the 1 st T base at the 5' end, the number of the stem part bases is 5 pairs, the number of the loop part bases is 22), hairpin DNA 8 (5 2 ) 6 -S-S-(CH 2 ) 6 The group is modified on the 1 st T base at the 5' end, the number of the stem part bases is 5 pairs, and the number of the loop part bases is 26), DNA modified electrodes are prepared and detected instead of the DNA in the example 1, the electrochemical impedance detection result of 500nM 1, 5-naphthylamine is shown in Table 2, and the detection effect of the hairpin DNA structure is obviously better than that of the hairpin DNA2, the hairpin DNA3, the hairpin DNA4, the hairpin DNA 5 and the hairpin DNA is the best.
TABLE 2
In the present specification, the embodiments 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 (7)
1. The method for calculating the small molecule-DNA binding constant based on the electrochemical technology is characterized by comprising the following steps of:
(1) Preparing a DNA modified electrode:
preparing a solution of the end functionalized DNA, coating the solution on an electrode interface, and cleaning and drying after cross-linking incubation to obtain a DNA modified electrode;
(2) Measuring an electrochemical signal:
determining the electrochemical signal S1 of the DNA-modified electrode;
preparing pollutant micromolecules into solutions with different concentrations, and soaking the DNA modified electrode in the pollutant micromolecule solution for incubation; after incubation, washing and drying the DNA modified electrode, and measuring an electrochemical signal S2;
calculating the electrochemical signal change value Sw = | S2-S1| of the DNA modified electrode before and after incubation of the pollutant small molecule solution with each concentration;
(3) Determining the detection concentration of the small molecules of the pollutants:
maximum value S of variation value according to electrochemical signal C Determining the electrochemical signal variation value as S CK Concentration C of the contaminant small molecule solution k Detecting concentration for determining the binding constant of the pollutant micromolecules and the DNA; s CK /S C＞ 0.70；
(4) Constructing a standard curve:
selection knotDifferent pollutant small molecules with known synthetic constant are respectively configured to have the concentration of C k Measuring an electrochemical signal according to the method in the step (2), and calculating a change value of the electrochemical signal; drawing a standard curve according to the binding constant and the electrochemical signal change value;
(5) Determination of binding constant:
configured concentration of C k The pollutant micromolecules to be detected; and (3) measuring the electrochemical signal according to the method in the step (2), calculating the change value of the electrochemical signal, and calculating the binding constant by combining with the standard curve.
2. The method for calculating small molecule-DNA binding constant based on electrochemical technique according to claim 1,
in the step (1), the step (c),
the site of end functionalization is at the 5 'end or 3' end of the DNA;
the functional group comprises amino, carboxyl, aldehyde group and sulfhydryl.
3. The method for calculating small molecule-DNA binding constant based on electrochemical technique according to claim 1,
in the step (1), the step (c),
the DNA is hairpin DNA, wherein the base number of the double-stranded stem part is not less than 5.
4. The method for calculating small molecule-DNA binding constant based on electrochemical technique according to claim 1,
in the step (1), the step (c),
the electrode is a gold electrode, a glassy carbon electrode, a carbon electrode or a screen printing electrode which is subjected to activation treatment;
the activation treatment includes acid solution activation, alkaline solution activation, salt solution activation, and/or electrochemical activation.
5. The method for calculating small molecule-DNA binding constant based on electrochemical technique according to claim 1,
in the step (1), the step (c),
the cross-linking agent used for cross-linking incubation is tris (2-carboxyethyl) phosphine hydrochloride, dicyclohexylcarbodiimide, carbodiimides or N-hydroxy thiosuccinimide;
the incubation time is 10-120min.
6. The method for calculating small molecule-DNA binding constant based on electrochemical technique according to claim 1,
in the step (2),
measuring electrochemical signals by using a three-electrode measuring system;
taking a DNA modified electrode before and after incubation of a pollutant micromolecule solution as a working electrode, a platinum wire electrode as a counter electrode, and a silver/silver chloride electrode as a reference electrode;
the measurement method includes a current method, a potential method or a resistance method.
7. The method for calculating small molecule-DNA binding constant based on electrochemical technique according to claim 6,
in the step (2),
the electrolyte solution is PB, tris, acetate or carbonate buffer solution, the concentration is 1-200mM, and the pH value is 6.0-8.0;
the indicator substance for measuring the electrical signal is K 3 [Fe(CN) 6 ]And K 4 [Fe(CN) 6 ]。
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Citations (4)
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|---|---|---|---|---|
| US5593834A (en) * | 1993-06-17 | 1997-01-14 | The Research Foundation Of State University Of New York | Method of preparing DNA sequences with known ligand binding characteristics |
| CN102692435A (en) * | 2012-04-17 | 2012-09-26 | 北京师范大学 | Method for detecting 1,8-diaminonaphthalene based on electrochemical DNA biosensor |
| CN103898093A (en) * | 2014-03-13 | 2014-07-02 | 中央民族大学 | Application of magnolia liliflora pigment A for stabilizing four-chain unit DNA (deoxyribonucleic acid) |
| CN103983555A (en) * | 2014-05-28 | 2014-08-13 | 国家纳米科学中心 | Method for detecting interaction of biomolecules |
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|---|---|---|---|---|
| US5593834A (en) * | 1993-06-17 | 1997-01-14 | The Research Foundation Of State University Of New York | Method of preparing DNA sequences with known ligand binding characteristics |
| CN102692435A (en) * | 2012-04-17 | 2012-09-26 | 北京师范大学 | Method for detecting 1,8-diaminonaphthalene based on electrochemical DNA biosensor |
| CN103898093A (en) * | 2014-03-13 | 2014-07-02 | 中央民族大学 | Application of magnolia liliflora pigment A for stabilizing four-chain unit DNA (deoxyribonucleic acid) |
| CN103983555A (en) * | 2014-05-28 | 2014-08-13 | 国家纳米科学中心 | Method for detecting interaction of biomolecules |
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| GANG LIANG 等: "Electrochemical detection of the amino-substituted naphthalene compounds based on intercalative interaction with hairpin DNA Ćby electrochemical impedance spectroscopy", BIOSENSORS AND BIOELECTRONICS, vol. 48, pages 238 - 243 * |
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