CN115950937B - 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
- Publication number
- CN115950937B CN115950937B CN202310054338.4A CN202310054338A CN115950937B CN 115950937 B CN115950937 B CN 115950937B CN 202310054338 A CN202310054338 A CN 202310054338A CN 115950937 B CN115950937 B CN 115950937B
- Authority
- CN
- China
- Prior art keywords
- dna
- electrode
- small molecule
- binding constant
- electrochemical
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 230000027455 binding Effects 0.000 title claims abstract description 44
- 238000005516 engineering process Methods 0.000 title claims abstract description 12
- 238000004364 calculation method Methods 0.000 title abstract description 5
- 238000000034 method Methods 0.000 claims abstract description 31
- 150000003384 small molecules Chemical class 0.000 claims abstract description 30
- 239000003344 environmental pollutant Substances 0.000 claims abstract description 29
- 231100000719 pollutant Toxicity 0.000 claims abstract description 28
- 230000008859 change Effects 0.000 claims abstract description 16
- 238000001514 detection method Methods 0.000 claims abstract description 13
- 239000000243 solution Substances 0.000 claims description 38
- 239000002585 base Substances 0.000 claims description 32
- 238000011534 incubation Methods 0.000 claims description 13
- 230000004913 activation Effects 0.000 claims description 12
- 239000000356 contaminant Substances 0.000 claims description 10
- 238000004140 cleaning Methods 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 238000004132 cross linking Methods 0.000 claims description 4
- PBVAJRFEEOIAGW-UHFFFAOYSA-N 3-[bis(2-carboxyethyl)phosphanyl]propanoic acid;hydrochloride Chemical compound Cl.OC(=O)CCP(CCC(O)=O)CCC(O)=O PBVAJRFEEOIAGW-UHFFFAOYSA-N 0.000 claims description 3
- 229910021607 Silver chloride Inorganic materials 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 3
- 229910052709 silver Inorganic materials 0.000 claims description 3
- 239000004332 silver Substances 0.000 claims description 3
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 claims description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 2
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 2
- 239000007983 Tris buffer Substances 0.000 claims description 2
- 239000003513 alkali Substances 0.000 claims description 2
- -1 amino, carboxyl Chemical group 0.000 claims description 2
- 238000004082 amperometric method Methods 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 150000001718 carbodiimides Chemical class 0.000 claims description 2
- 239000003431 cross linking reagent Substances 0.000 claims description 2
- 239000008151 electrolyte solution Substances 0.000 claims description 2
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 claims description 2
- 125000000524 functional group Chemical group 0.000 claims description 2
- 238000007306 functionalization reaction Methods 0.000 claims description 2
- 229910021397 glassy carbon Inorganic materials 0.000 claims description 2
- 238000000691 measurement method Methods 0.000 claims description 2
- 238000004313 potentiometry Methods 0.000 claims description 2
- 239000012266 salt solution Substances 0.000 claims description 2
- 238000007650 screen-printing Methods 0.000 claims description 2
- 238000002791 soaking Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 2
- GVJXGCIPWAVXJP-UHFFFAOYSA-N 2,5-dioxo-1-oxoniopyrrolidine-3-sulfonate Chemical compound ON1C(=O)CC(S(O)(=O)=O)C1=O GVJXGCIPWAVXJP-UHFFFAOYSA-N 0.000 claims 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims 1
- 229910052799 carbon Inorganic materials 0.000 claims 1
- 230000008901 benefit Effects 0.000 abstract description 4
- 230000009471 action Effects 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 abstract description 2
- 238000000840 electrochemical analysis Methods 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 61
- RUFPHBVGCFYCNW-UHFFFAOYSA-N 1-naphthylamine Chemical compound C1=CC=C2C(N)=CC=CC2=C1 RUFPHBVGCFYCNW-UHFFFAOYSA-N 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 150000002343 gold Chemical class 0.000 description 5
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 5
- 229910052737 gold Inorganic materials 0.000 description 5
- 239000010931 gold Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000002593 electrical impedance tomography Methods 0.000 description 4
- 238000005498 polishing Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 102000053602 DNA Human genes 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 231100000025 genetic toxicology Toxicity 0.000 description 3
- 230000001738 genotoxic effect Effects 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- JBIJLHTVPXGSAM-UHFFFAOYSA-N 2-naphthylamine Chemical compound C1=CC=CC2=CC(N)=CC=C21 JBIJLHTVPXGSAM-UHFFFAOYSA-N 0.000 description 2
- 241000282994 Cervidae Species 0.000 description 2
- 238000007702 DNA assembly Methods 0.000 description 2
- 102100033072 DNA replication ATP-dependent helicase DNA2 Human genes 0.000 description 2
- 101000927313 Homo sapiens DNA replication ATP-dependent helicase DNA2 Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000000861 blow drying Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000004744 fabric Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000002386 leaching Methods 0.000 description 2
- 230000009149 molecular binding Effects 0.000 description 2
- 239000002002 slurry Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- CCMKPCBRNXKTKV-UHFFFAOYSA-N 1-hydroxy-5-sulfanylidenepyrrolidin-2-one Chemical compound ON1C(=O)CCC1=S CCMKPCBRNXKTKV-UHFFFAOYSA-N 0.000 description 1
- 229910018072 Al 2 O 3 Inorganic materials 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 231100000693 bioaccumulation Toxicity 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000002484 cyclic voltammetry Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000000835 electrochemical detection Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 238000002847 impedance measurement Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000003993 organochlorine pesticide Substances 0.000 description 1
- 150000003071 polychlorinated biphenyls Chemical class 0.000 description 1
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 235000011008 sodium phosphates Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 231100000378 teratogenic Toxicity 0.000 description 1
- 230000003390 teratogenic effect Effects 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 238000004506 ultrasonic cleaning Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A20/00—Water conservation; Efficient water supply; Efficient water use
- Y02A20/20—Controlling water pollution; Waste water treatment
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a small molecule-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; (5) determining the binding constant. The invention can change the electrochemical performance of the DNA film layer by the action of the small molecules and the DNA modified by the interface, thereby realizing the analysis of the binding capacity and the binding constant. The method has the advantages of simplicity in 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 small molecule-DNA binding constant calculation method based on an electrochemical technology.
Background
Contaminants in the environment, particularly organic, toxic contaminants such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls, organochlorine pesticides, organic dyes, etc., are bioaccumulative and are extremely harmful to the ecosystem and human health. Research shows that pollutant enters human body through direct contact, respiration, diet and other ways, and may accumulate in human body to produce toxic effect, and may be converted into intermediate and derivative with high bioactivity and high toxicity through activation, etc. the active intermediate may interact with biomacromolecule to alter the structure and function of the macromolecule and damage the body, so that the product has powerful carcinogenic, teratogenic and mutagenic functions. In addition, some environmental pollutant molecules can directly act with DNA and generate genotoxicity, so that the research on the binding capacity of small pollutant molecules and DNA can provide important reference basis for distinguishing the genotoxicity of the pollutants, revealing the toxicity of the pollutants, evaluating the ecological risk and the like.
The binding constant K is a direct and key indicator reflecting the binding performance of small molecules to DNA. However, the method 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 molecule pollutants and in-vivo DNA effect is difficult to obtain and determine. In addition, the binding performance and binding constant of the existing small molecules with DNA are often analyzed by adopting a fluorescence method, and the binding constant of the molecules with weak fluorescence or no fluorescence property is difficult to determine.
Therefore, the research of the specific recognition DNA film is developed, and an accurate, reliable and sensitive small molecule-DNA binding constant K measuring method is established on the basis, so that the existing small molecule-DNA binding constant K measuring method can be enriched, perfected and supplemented, and has important significance.
Disclosure of Invention
The invention discloses a method for calculating a small molecule-DNA binding constant based on an electrochemical technology, wherein the electrochemical performance of a DNA film layer can be changed by the action of the small molecule and an interface modified DNA, so that the binding capacity and the binding constant analysis are realized.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the method for calculating the small molecule-DNA binding constant based on the electrochemical technology comprises the following steps:
(1) Preparing a DNA modified electrode:
preparing the end functionalized DNA into a solution, coating the solution on an electrode interface, and cleaning and drying after cross-linking incubation to obtain a DNA modified electrode;
(2) Measuring electrochemical signals:
determining the electrochemical signal S1 of the DNA modified electrode;
preparing small pollutant molecules into solutions with different concentrations, and soaking the DNA modified electrode in the small pollutant molecule solution for incubation; after incubation, cleaning and drying the DNA modified electrode, and measuring an electrochemical signal S2;
calculating electrochemical signal change values Sw= |S 2-S1|ofthe DNA modified electrode before and after incubating the pollutant small molecule solution with each concentration;
(3) Determining the detection concentration of small molecules of pollutants:
according to the maximum value S of electrochemical signal variation value C Determining the electrochemical signal change value as S CK Concentration C of contaminant small molecule solution of (C) k To determine the detection concentration of the binding constant of the contaminant small molecule to the DNA; s is S 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 of the step (2), and calculating electrochemical signal change values; drawing a standard curve according to the combination constant and the electrochemical signal change value;
(5) Determination of binding constant:
the concentration is configured as C k Is a small molecule of the pollutant to be detected; and (3) measuring an electrochemical signal according to the method of the step (2), calculating an electrochemical signal change value, and calculating a binding constant by combining a standard curve.
Further, in the step (1),
the site of end functionalization is at the 5 'or 3' end of the DNA;
functional groups include amino, carboxyl, aldehyde, sulfhydryl, and the like.
Further, in the step (1),
the DNA is hairpin DNA, wherein the base pair number of the double-stranded stem part is not less than 5.
Preferably, the base pair number of the double stranded stem portion of the hairpin DNA is 5-11.
Further preferably, the base pair number of the double stranded stem portion of the hairpin DNA is 5-8.
Preferably, the number of bases of the loop portion of the hairpin DNA is 18-26.
Further preferably, the number of bases of the loop portion of the hairpin DNA is 18-22.
Further, in the step (1),
the electrode is an activated gold electrode, a glassy carbon electrode, a screen printing electrode or the like;
the activation treatment includes acid solution activation, alkali solution activation, salt solution activation and/or electrochemical activation.
Further preferably, the acid may be sulfuric acid, the base may be sodium hydroxide, the salt may be sodium phosphate, sodium dihydrogen phosphate, or the like.
Further, in the step (1),
the cross-linking agent used in cross-linking incubation is tri (2-carboxyethyl) phosphine hydrochloride, dicyclohexylcarbodiimide, carbodiimide or N-hydroxy thiosuccinimide, etc.;
the incubation time is 10-120min.
Further preferably, the incubation time is 30-60min.
Further, in the step (2),
measuring an electrochemical signal by adopting a three-electrode measuring system;
the method comprises the steps that a DNA modified electrode is used as a working electrode before and after incubating a pollutant small molecule solution, a platinum wire electrode is used as a counter electrode, and a silver/silver chloride electrode is used as a reference electrode;
the measurement method includes a amperometric method, an potentiometric method or a resistive method.
Further, in the step (2),
the electrolyte solution is PB, tris, acetate or carbonate buffer solution, etc., the concentration is 1-200mM, and the pH is 6.0-8.0;
the indicator substance used for measuring the electric 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 optical 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-chain structure under the condition of fewer pairing bases, thereby reducing the number of bases and lowering the cost;
5) The electrochemical hairpin DNA film adopted by the invention has the advantage of small sample quantity.
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, not only provides an important reference basis for distinguishing the genotoxicity of pollutants, revealing the toxicity of the pollutants, evaluating the ecological risk and the like, but also solves the difficult 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 simplicity in 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 the electrochemical impedance measurement of an activated gold electrode of example 1;
FIG. 2 is a diagram showing the electrochemical impedance measurement of the DNA modified electrode of example 1;
FIG. 3 shows the binding constant discriminant curve of example 1;
FIG. 4 is a graph showing electrical impedance measurements of different DNA modified electrodes of example 2.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
1. Activation of gold electrodes
Taking 1 μm gamma-Al 2 O 3 Adding distilled water into the powder, stirring into slurry, coating on distilled water-wetted deer skin polishing cloth, polishing gold electrode on the deer skin polishing cloth in a circular manner for 5min, and respectively using ethanol and ultrapure water for ultrasonic cleaning;
according to the above operation method, gamma-Al of 0.3 μm and 0.05 μm are used in sequence 2 O 3 Polishing the gold electrode by slurry for 5min, and respectively ultrasonically cleaning in ethanol and ultrapure water;
the polished gold is electrically chargedH at 1M 2 SO 4 Scanning the solution for 20 times by cyclic voltammetry (-0.6V to +0.8V), taking out, washing with ultrapure water, and drying with nitrogen to obtain an activated gold electrode for later use.
The electrochemical impedance measurement is carried out on the prepared activated gold electrode, and the result is that the resistance of the electrode interface is very small and is only 26.5 Ω cm as shown in figure 1 2 The method can effectively remove impurities at the electrode interface and remarkably improve the electrochemical performance of the electrode interface.
Preparation of DNA modified electrode
Thiol-modified solid DNA1 (5'-ttttttttttggccgttcttgctaggtgggtgtcggcc-3', SEQ ID NO.1, HO- (CH) 2 ) 6 -S-S-(CH 2 ) 6 Modification of the group at the 5' -terminal 1 st T base, 5 pairs of base numbers in the stem part and 18 base numbers in the loop part) with an appropriate amount of Tris-HClO 4 The solution (20 mM, pH=7.5) was dissolved, vortexed for 5min, incubated in hot water at 85℃for 5min, and naturally cooled to room temperature to give a 200. Mu.M stock solution of DNA for further use.
Preparation of Tris-HClO containing 1mM EDTA, 500mM NaCl, 2mM Tris (2-carboxyethyl) phosphine hydrochloride 4 (20 mM, pH=7.5), the DNA stock solution prepared above was diluted to 1. Mu.M, and incubated at room temperature for 30min to give a DNA assembly solution for use.
Dripping the DNA assembly liquid on the interface of the activated gold electrode, incubating for 6 hours, and then using Tris-HClO 4 (20 mM, pH=7.5) solution was purged and dried with nitrogen, then in Tris-HClO containing 1mM 6-mercaptohex-1-ol 4 Incubation in (20 mM, pH=7.5) solution for 1h, finally with Tris-HClO 4 (20 mM, pH=7.5) solution was purged and dried with nitrogen to obtain a DNA modified electrode for use.
Electrochemical impedance measurement is carried out on the prepared DNA modified electrode, the result is shown in figure 2, and after DNA is assembled at the interface of the activated bare electrode, 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 interface of the gold electrode to prepare the DNA recognition film.
3. Electrochemical impedance detection and determination of contaminant small molecule detection concentration
Preparation of Tris-HClO containing 100mM KCl 4 (20 mM, pH=7.5) solution, with which 4mM K was prepared 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]The solution was then added to the electrochemical detection cell by taking 5ml of the solution. The DNA modified electrode prepared in the above way is used as a working electrode, a platinum wire electrode is used as a counter electrode, saturated KCl solution silver/silver chloride is used as a reference electrode, a three-electrode measuring system is constructed, and the electrochemical impedance S1 of the working electrode is measured; the working electrode was removed using Tris-HClO 4 (20 mM, pH=7.5) solution leaching, N 2 Blow-drying;
using Tris-HClO 4 (20 mm, ph=7.5) solution contaminant small molecules were made into solutions of different concentrations (10, 50, 100, 200, 400, 500 nM), and DNA modified electrodes were immersed in each concentration of contaminant small molecule solution and incubated for 30min; tris-HClO for DNA modified electrode after incubation 4 (20 mM, pH=7.5) solution leaching, N 2 Blow-drying, and measuring electrochemical impedance S2 again as a working electrode;
calculating electrochemical impedance change values Sw= |S 2-S1|ofthe DNA modified electrode before and after incubating the pollutant small molecule solution with each concentration, wherein the largest electrochemical impedance change value is S C The corresponding concentration of the small molecular solution of the pollutant is shown in table 1, and the detection concentration of the small molecular solution of the subsequent pollutant is determined.
TABLE 1
4. Constructing a standard curve:
selecting different pollutant micromolecules 1-naphthylamine, 2-naphthylamine and 1, 5-naphthylamine with known binding constants, respectively preparing into solutions with the concentration of 200nM, measuring electrochemical impedance according to the method in the step 3, and calculating electrochemical impedance change values; the standard curve is drawn according to the combination constant and the electrochemical impedance change value, and the result is shown in figure 3, and the correlation between the electrochemical impedance signal and the combination constant is higher, which indicates that the method can be used for analyzing the small molecular combination constant.
5. Determination of binding constant:
concentration of configurationIs C k (S CK /S C >0.70 Small molecules of the contaminant to be detected; and (3) measuring electrochemical impedance according to the method of the step (3), calculating an electrochemical impedance change value, and calculating a 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
Using hairpin DNA1 (5'-ttttttttttggccgttcttgctaggtgggtgtcggcc-3', SEQ ID NO.1, HO- (CH) 2 ) 6 -S-S-(CH 2 ) 6 The group is modified at the 5' -end at the 1 st T base, the number of bases of the stem part is 5 pairs, the number of bases of the loop part is 18), single-stranded DNA2 (5'-ttttttttttattcaatttgaggcgggtgggtggg-3', SEQ ID NO.2, HO- (CH) 2 ) 6 -S-S-(CH 2 ) 6 Group modification at the 5' end 1 st T base), double-stranded DNA (3+4) (DNA 3:5'-ttttttttttagcggaacggcgggt-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'-tcgccttgccgccca-3', SEQ ID NO. 4), instead of the DNA of example 1, the preparation of DNA modified electrodes and the detection were carried out, the results are shown in FIG. 4, and the electrochemical impedance detection results for 500nM 1, 5-naphthylamine are: s is S hairpinDNA >S DNA-(3+4) >S DNA The hairpin DNA is more favorable for researching the binding property of small molecules and DNA, and the hairpin structure is obviously superior to that of single-stranded DNA and double-stranded DNA by further comparative analysis.
Example 3 comparison of DNA Properties
Using hairpin DNA1 (5'-ttttttttttggccgttcttgctaggtgggtgtcggcc-3', SEQ ID NO.1, HO- (CH) 2 ) 6 -S-S-(CH 2 ) 6 The group is modified at the 5' -end at 1 st T base, the number of bases of the stem part is 5 pairs, the number of bases of the loop part is 18), the hairpin DNA 5 (5'-ttttttttttcggggccgttcttgctaggtgggtgtcggccccg-3', SEQ ID NO.5, HO- (CH) 2 ) 6 -S-S-(CH 2 ) 6 The group is modified at the 5' end with 1 st T base, the number of bases of the stem part is 8 pairs, and the number of bases of the loop part is 18、hairpin DNA 6(5’-ttttttttttgaacggggccgttcttgctaggtgggtgtcggccccgttc-3’，SEQ ID NO.6，HO-(CH 2 ) 6 -S-S-(CH 2 ) 6 The group is modified at the 5' -end at 1 st T base, the number of bases in the stem part is 11 pairs, the number of bases in the loop part is 18), the hairpin DNA 7 (5'-ttttttttttggccgttcttatttgctaggtgggtgtcggcc-3', SEQ ID NO.7, HO- (CH) 2 ) 6 -S-S-(CH 2 ) 6 The group is modified at the 5' -end at 1 st T base, the number of bases in the stem part is 5 pairs, the number of bases in the loop part is 22), hairpin DNA 8 (5'-ttttttttttggccgttcttatttgctagtagttgggatgtcggcc-3', SEQ ID NO.8, HO- (CH) 2 ) 6 -S-S-(CH 2 ) 6 The group was modified at 1 st T base at the 5' end, the number of bases in the stem portion was 5 pairs, and the number of bases in the loop portion was 26), instead of the DNA in example 1, the preparation and detection of the DNA modified electrode were performed, and the results of electrochemical impedance detection on 500nM 1, 5-naphthylamine are shown in Table 2, and the structure of the hairpin DNA was significantly superior to that of hairpin DNA2, hairpin DNA3, hairpin DNA4, hairpin DNA 5, and the hairpin DNA was the best.
TABLE 2
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer 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 embodiments described above 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:
(1) Preparing a DNA modified electrode:
preparing the end functionalized DNA into a solution, coating the solution on an electrode interface, and cleaning and drying after cross-linking incubation to obtain a DNA modified electrode;
(2) Measuring electrochemical signals:
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, cleaning and drying the DNA modified electrode, and measuring an electrochemical signal S2;
calculating electrochemical signal change values Sw= |S 2-S1|ofthe DNA modified electrode before and after incubating the pollutant small molecule solution with each concentration;
(3) Determining the detection concentration of small molecules of pollutants:
according to the maximum value S of electrochemical signal variation value C Determining the electrochemical signal change value as S CK Concentration C of contaminant small molecule solution of (C) k To determine the detection concentration of the binding constant of the contaminant small molecule to the DNA; s is S CK /S C＞ 0.70；
(4) Constructing a standard curve:
selecting different pollutant small molecules with known binding constants, and respectively configuring the different pollutant small molecules into the concentration of C k Measuring electrochemical signals according to the method of the step (2), and calculating electrochemical signal change values; drawing a standard curve according to the combination constant and the electrochemical signal change value;
(5) Determination of binding constant:
the concentration is configured as C k Is a small molecule of the pollutant to be detected; and (3) measuring an electrochemical signal according to the method of the step (2), calculating an electrochemical signal change value, and calculating a binding constant by combining a standard curve.
2. The method for calculating a small molecule-DNA binding constant according to claim 1, wherein,
in the step (1), the step of (a),
the site of end functionalization is at the 5 'or 3' end of the DNA;
the functional groups include amino, carboxyl, aldehyde, sulfhydryl.
3. The method for calculating a small molecule-DNA binding constant according to claim 1, wherein,
in the step (1), the step of (a),
the DNA is hairpin DNA, wherein the base pair number of the double-stranded stem part is not less than 5.
4. The method for calculating a small molecule-DNA binding constant according to claim 1, wherein,
in the step (1), the step of (a),
the electrode is an activated gold electrode, a glassy carbon electrode, a carbon electrode or a screen printing electrode;
the activation treatment includes acid solution activation, alkali solution activation, salt solution activation and/or electrochemical activation.
5. The method for calculating a small molecule-DNA binding constant according to claim 1, wherein,
in the step (1), the step of (a),
the cross-linking agent used in the cross-linking incubation is tri (2-carboxyethyl) phosphine hydrochloride, dicyclohexylcarbodiimide, carbodiimide or N-hydroxysulfosuccinimide;
the incubation time is 10-120min.
6. The method for calculating a small molecule-DNA binding constant according to claim 1, wherein,
in the step (2), the step of (C),
measuring an electrochemical signal by adopting a three-electrode measuring system;
the method comprises the steps that a DNA modified electrode is used as a working electrode before and after incubating a pollutant small molecule solution, a platinum wire electrode is used as a counter electrode, and a silver/silver chloride electrode is used as a reference electrode;
the measurement method includes a amperometric method, an potentiometric method or a resistive method.
7. The method for calculating a small molecule-DNA binding constant according to claim 6, wherein,
in the step (2), the step of (C),
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 used for measuring the electric signal is K 3 [Fe(CN) 6 ]And K 4 [Fe(CN) 6 ]。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310054338.4A CN115950937B (en) | 2023-02-03 | 2023-02-03 | Small molecule-DNA binding constant calculation method based on electrochemical technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310054338.4A CN115950937B (en) | 2023-02-03 | 2023-02-03 | Small molecule-DNA binding constant calculation method based on electrochemical technology |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115950937A CN115950937A (en) | 2023-04-11 |
| CN115950937B true CN115950937B (en) | 2023-11-10 |
Family
ID=87282573
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310054338.4A Active CN115950937B (en) | 2023-02-03 | 2023-02-03 | Small molecule-DNA binding constant calculation method based on electrochemical technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115950937B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2023
- 2023-02-03 CN CN202310054338.4A patent/CN115950937B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| 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.2013,第48卷第238-243页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115950937A (en) | 2023-04-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Goud et al. | Disposable and portable electrochemical aptasensor for label free detection of aflatoxin B1 in alcoholic beverages | |
| Wang et al. | Enhanced catalytic and dopamine sensing properties of electrochemically reduced conducting polymer nanocomposite doped with pure graphene oxide | |
| Li et al. | Supportless electrochemical sensor based on molecularly imprinted polymer modified nanoporous microrod for determination of dopamine at trace level | |
| Xie et al. | Electrochemical sensor for 2, 4-dichlorophenoxy acetic acid using molecularly imprinted polypyrrole membrane as recognition element | |
| Yang et al. | Electrochemical sensor based on molecularly imprinted polymer film via sol–gel technology and multi-walled carbon nanotubes-chitosan functional layer for sensitive determination of quinoxaline-2-carboxylic acid | |
| Wen et al. | One-step fabrication of poly (o-aminophenol)/multi-walled carbon nanotubes composite film modified electrode and its application for levofloxacin determination in pharmaceuticals | |
| CN110618185B (en) | Ratiometric electrochemical detection method of ochratoxin A | |
| Pereira et al. | Electrochemical sensing of lactate by using an electrode modified with molecularly imprinted polymers, reduced graphene oxide and gold nanoparticles | |
| Mohammad-Razdari et al. | Detection of sulfadimethoxine in meat samples using a novel electrochemical biosensor as a rapid analysis method | |
| Ziyatdinova et al. | Complex electrochemical and impedimetric evaluation of DNA damage by using DNA biosensor based on a carbon screen-printed electrode | |
| Galandova et al. | Disposable electrochemical biosensor with multiwalled carbon nanotubes—Chitosan composite layer for the detection of deep DNA damage | |
| CN109072284A (en) | System for Electrochemical Detection relevant molecule | |
| Chiorcea-Paquim et al. | Nanostructured material–based electrochemical sensing of oxidative DNA damage biomarkers 8-oxoguanine and 8-oxodeoxyguanosine: A comprehensive review | |
| Tao et al. | Fiber based organic electrochemical transistor integrated with molecularly imprinted membrane for uric acid detection | |
| Simionca et al. | Siloxane‐Crown Ether Polyamide Based Electrode for Electrochemical Determination of Lead (II) in Aqueous Solution | |
| CN105806924A (en) | 8-OHdG sensor as well as preparation method and application thereof | |
| Asadi et al. | Graphene-based electrochemical biosensor for impedimetric detection of miRNAs as potential cancer biomarkers | |
| CN105067598A (en) | Chloramphenicol molecular imprinting electrochemical luminescence sensor and chloramphenicol detection method thereof | |
| Geto et al. | Electrochemical sensor for caffeine in coffee and beverages using a naphthalene sulfonic acid polymer film–based modified electrode | |
| CN103901020A (en) | Method for detecting DNA (Deoxyribose Nucleic Acid) transmethylase by using electrochemical luminescence biosensor and application of biosensor | |
| CN105866211B (en) | A kind of preparation method and application of ampicillin molecular engram sensor | |
| Ferancová et al. | Association interaction and voltammetric determination of 1-aminopyrene and 1-hydroxypyrene at cyclodextrin and DNA based electrochemical sensors | |
| Díaz et al. | Molecularly Imprinted Polypyrrole for the selective detection of Dopamine and Serotonin | |
| CN110044987A (en) | The method of the preparation method and its Electrochemical Detection troponin of the covalent organic frame modified electrode of ferrocenyl | |
| CN115950937B (en) | Small molecule-DNA binding constant calculation method based on electrochemical technology |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |