CN105929001B - The gold electrode and preparation method and application of specific DNA pseudoknot structure modification - Google Patents
The gold electrode and preparation method and application of specific DNA pseudoknot structure modification Download PDFInfo
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- CN105929001B CN105929001B CN201610243290.1A CN201610243290A CN105929001B CN 105929001 B CN105929001 B CN 105929001B CN 201610243290 A CN201610243290 A CN 201610243290A CN 105929001 B CN105929001 B CN 105929001B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
Abstract
The invention belongs to electrochemical sensor technology field, and in particular to a kind of gold electrode and preparation method and application of the modification of specific DNA pseudoknot structure.The gold electrode surfaces of specific DNA pseudoknot structure of the present invention modification have DNA pseudoknot structure of the end with methylenum careuleum Redox labels, the binding sequence containing Nanog of stem ring 1 by the way that the effect of golden mercapto key is self-assembled modified, after the biology sensor is reacted using the gold electrode of the DNA pseudoknot structure probe modifications of functionalization with target protein, cause DNA pseudoknot structure conformational changes, by the electric signal molecule MB with satisfactory electrical conductivity, the Sensitive Detection to transcription factor Nanog is realized, can effectively distinguish target protein and other reference proteins.This method is simple to operate, has compared with high sensitivity and specificity, minimum detection limits are 170pM.
Description
Technical field
The invention belongs to electrochemical sensor technology field, and in particular to a kind of gold of specific DNA pseudoknot structure modification
Electrode and preparation method and application.
Background technology
Cell biology it is most interesting the problem of one of be cells pluripotency, cells pluripotency is adjusted by strict supervision
Section is abnormal to may result in birth defects, can not injury repair even cancer.Nanog is the related important transcription of versatility
One of factor (transcription factors, TFs), is a biomolecule for causing everybody great interest in the recent period, and it sends out
It is to be bred by regulating cell, be dead to wave function, determines cell fate., it is known that Nanog in most of normal adult tissues not
Expression, but height is expressed in embryonic stem cell and tumour cell.Research has been discovered that Nanog in tumour for tumour cell
Stem-like cell phenotype be made that contribution.Elevated Nanog levels generally occur with tumour, cell invasion and transfer are relevant.
Nanog strong high expression is generally predictive of cancer grade malignancy height, the resistance to the action of a drug and badness come-off.Therefore, Nanog pairs is detected
It is significant in tumor evaluation, pharmaceutical intervention detection and clinical prognosis.
Traditional TF detection methods have following several：Concentration Testing (such as：western blots,
Immunohistochemistry or ELISA), binding Activity determination (such as：electrophoresis mobility
Shift assay, ChIP) and transcriptional level detection (such as：qPCR).These methods respectively have advantage, but there is also time-consuming
Easily there are the drawbacks such as false negative in long, complex operation, measurement result.In recent years, electrochemical techniques continue to develop, in life science
Field has played highly important effect.Electrochemical DNA (E-DNA) sensor has that simple to operate, sensitivity is high, cost is cheap
Etc. advantage.At present, researcher have been developed some detection TF E-DNA sensors, they all have suitable sensitivity and
Specificity, potential replacement method is provided to TF detections.But these methods, although having these advantages, they actually should
Some defects are still had in.For example, the E-DNA systems largely reported are signal-off types, signal-off body
Easily there is false positive (such as probe degraded) and relatively small detection range in architecture.On the contrary, signal-on types sensor is then
Background signal can be reduced, is had a clear superiority.Therefore, there is the TF electrochemistry that exploitation signal-on has been put into research in the recent period
Sensor.But these methods still have the deficiencies such as DNA complex designing, joint efficiency be low.
The content of the invention
The present invention needs solve the problems, such as gold electrode and its preparation side for being to provide a kind of specific DNA pseudoknot structure modification
Method and the quantitative detection to Nanog.
In order to reach the purpose to solve the above problems, the present invention uses following mechanism：DNA pseudoknot structures include two stem rings
Structure (stem ring 1 and 2), stem ring 1 forms a part for stem ring 2.DNA pseudoknot structure sequences are：ttttgaccga
ttatgtttag atcagttcat aatcggtctt tttttttttt ttctgatct.The terminal modified Asia of DNA pseudoknot structures 3 '
Electrode surface (Fig. 1) is arrived in first indigo plant Redox labels, 5 ' ends by gold-mercapto key modification.Stem ring 1 devises Nanog binding sequences.
Because DNA pseudoknot structures have the structure of relative stiffness, reduce contact of the redox probe with electrode, signal background will most
Smallization.After Nanog bindings, because Nanog molecules are relatively large, steric effect be present, stem ring 2 will be interrupted, discharge an elasticity
Single-stranded signal chain, so as to strengthen electron transmission efficiency, increase induced-current.
Technical scheme：
A kind of surface of the gold electrode of specific DNA pseudoknot structure modification of the present invention by the effect of gold-mercapto key from
Assembling is modified with DNA pseudoknot structure of the end with methylenum careuleum Redox labels, the binding sequence containing Nanog of stem ring 1, and the DNA is false
Junction structure sequence is：ttttgaccga ttatgtttag atcagttcat aatcggtctt tttttttttt
ttctgatct。
The preparation method of the gold electrode of specific DNA pseudoknot structure modification of the present invention is made up of following steps：(1) will
Gold electrode (3.0mm diameters) is soaked in Piranha solution (H2SO4:30%H2O2=3:1) 15 minutes, to eliminate the material of absorption,
And rinsed with ultra-pure water；(2) polished respectively with 1 μm and 0.3 μm of aluminum oxide, and the ultrasound 10 in ethanol and ultra-pure water successively
Minute, dried up with nitrogen；(3) take Piranha solution to be added drop-wise to gold electrode surfaces to react 5 minutes, it is clean with ultrapure water, blow
It is dry；(4) treated gold electrode is placed in 0.5M H2SO4In, voltammetric scan is carried out in 0~1.6V voltage ranges, sweeping speed is
100mV/s, until reaching stable；(5) after gold electrode ultrapure water and drying, it is immersed in the electrode of the pseudoknot structure containing DNA
Modify in solution, be incubated 1.5h at room temperature, then be immersed in 1h in 1mM mercaptoethanol solution, with ultrapure water, drying, produce
To the gold electrode of DNA pseudoknot structures modification.The electrode modification solution of the above-mentioned pseudoknot structure containing DNA is 10mM Tris-HCl pH
Contain DNA pseudoknot structure of the concentration for 0.2 μM, 140mM NaCl, 1mM MgCl in 7.0 Tris-HCl2, 5mM KCl and 1mM
TCEP。
Electrochemical Detection side of the gold electrode of specific DNA pseudoknot structure modification of the present invention to transcription factor Nanog
Method, using the gold electrode of specific DNA pseudoknot structure modification of the present invention, saturated calomel electrode is reference electricity for working electrode
Pole, platinum electrode are to electrode, are concretely comprised the following steps：
(1) Nanog is taken to be added to 10mM Tris-HCl, 140mM NaCl, 1mM MgCl2, and 5mM KCl, 1.0%
A series of reaction system of concentration containing Nanog in the range of 0~45nM is formed in Tween 20, pH 7.0 buffer solution；By work
It is inserted into as electrode in the reaction system, reacts 1h at 37 DEG C；
(2) working electrode handled through step (1) is positioned in the buffer solution of 10mM Tris-HCl, PH 7.0, utilizes three
Electrode system, detected with square wave voltammetry (SWV), and in the range of 5~25nM, obtain the linear pass of electric current and Nanog concentration
It is curve, its linear coefficient is 0.99；
(3) treat test sample using calibration curve method obtained by step (2) and carry out analysis detection, analyze Nanog content.
Beneficial effects of the present invention are as follows：
(1) gold electrode of specific DNA pseudoknot structure detection transcription factor of the present invention, its cost of manufacture is cheap, work
Skill is simple, operation is simple；
(2) electrochemical sensor prepared by this method can successfully detect transcription factor Nanog, and sensitivity, special
Property it is high, there is repeatability well；
(3) because Nanog in itself can be as a kind of detection target, for cells pluripotency analysis, drug resistance of tumor
And Prognosis, technical support is provided to the clinical detection of disease, is with a wide range of applications.
Brief description of the drawings
Fig. 1 is the method schematic of the present invention.
Fig. 2 is the variation diagram of electrochemical signals in the case of 0~45nM of various concentrations Nanog are present.Wherein embedded figure is egg
White linear relationship chart of the concentration in the range of 5~25nM between electrochemical signals changing value.
Fig. 3 be modified with the gold electrodes of DNA pseudoknot structure substrates respectively with blank, 25nM hemoglobins, BSA, NF- κ B,
Nanog reacts 1h electrochemical signals.
Embodiment
Embodiment one：Utilize the gold electrode detection various concentrations Nanog of specific DNA pseudoknot structure modification.
Step 1, gold electrode (3.0mm diameters) is soaked in Piranha solution (H2SO4:30%H2O2=3:1) 15 minutes,
To eliminate the material of absorption, and rinsed with ultra-pure water.Then, polished respectively with 1 μm of 0.3 μm of and aluminum oxide, and successively
Ultrasound 10 minutes, are dried up with nitrogen in ethanol and ultra-pure water.Then take Piranha solution to be added drop-wise to gold electrode surfaces and react 5 points
Clock, drying clean with ultrapure water.
Step 2, treated gold electrode is placed in 0.5M H2SO4In, progress volt-ampere is swept in 0~1.6V voltage ranges
Retouch, it is 100mV/s to sweep speed, until reaching stable；After gold electrode ultrapure water and drying, pseudoknot structure containing DNA is immersed in
Electrode modification solution in, be incubated 1.5h at room temperature, then be immersed in 1h in 1mM mercaptoethanol solution, with ultrapure water, blow
It is dry, that is, obtain the gold electrode of DNA pseudoknot structures modification.
Step 3, Nanog is taken to be added to 10mM Tris-HCl, 140mM NaCl, 1mM MgCl2,and 5mM KCl,
Formed in 1.0%Tween 20, pH 7.0 buffer solution a series of concentration containing Nanog in the range of 0~45nM (0nM, 5nM,
10nM, 15nM, 20nM, 25nM, 45nM) reaction system；Working electrode is inserted into the reaction system, in 37 DEG C of reactions
1h；The working electrode of probe modification is positioned in the buffer solution of 10mM Tris-HCl, PH 7.0, using three-electrode system, with side
Ripple voltammetry (SWV) detects redox electric signal.
As shown in Fig. 2 measure the peak current of each solution as Nanog concentration raises and raise, this be due to Nanog and
After DNA pseudoknot structures binding site combines, because Nanog molecules are relatively large, steric effect be present after binding, stem will be opened
Ring 2, release MB molecules to electrode surface, strengthen electric signal.Nanog concentration is linearly related in 5-25nM and SWV peak responses, most
Small detection range is 170pM, illustrates the detection method of the present invention and has higher sensitivity.
Embodiment two：The special Journal of Sex Research of biology sensor.
For checking present invention detection Nanog specificity, the present invention has used different albumen, such as human hemoglobin, ox blood
Pure albumen (BSA) and transcription factor NF-KB, handled according to the operating process of embodiment one to verify the spy of the biology sensor
The opposite sex.As shown in figure 3, as no Nanog or when being substituted with other non-specific proteins, then almost there is no peak value, only when adding
When entering Nanog, obvious peak-to-peak signal is detected.It is experimentally confirmed that the method has specificity well, this biology sensor detection tool
There is high selectivity.
Claims (4)
- A kind of 1. gold electrode of specific DNA pseudoknot structure modification, it is characterised in that the work that the surface of the electrode passes through gold-mercapto key There is DNA false knot of the end with methylenum careuleum Redox labels, the binding sequences of nanog containing transcription factor of stem ring 1 with self-assembled modified Structure, the DNA pseudoknot structure sequences are：ttttgaccga ttatgtttag atcagttcat aatcggtctt tttttttttt ttctgatct。
- A kind of 2. preparation method of the gold electrode of specific DNA pseudoknot structure modification according to claim 1, it is characterised in that This method concretely comprises the following steps：(1) diameter 3.0mm gold electrode is soaked in H2SO4:30%H2O2=3:15 minutes in 1 Piranha solution, to eliminate The material of absorption, and rinsed with ultra-pure water；(2) polished respectively with 1 μm and 0.3 μm of aluminum oxide, and ultrasound 10 minutes in ethanol and ultra-pure water successively, use nitrogen Drying；(3) take Piranha solution to be added drop-wise to gold electrode surfaces to react 5 minutes, drying clean with ultrapure water；(4) treated gold electrode is placed in 0.5M H2SO4In, voltammetric scan is carried out in 0~1.6V voltage ranges, sweeping speed is 100mV/s, until reaching stable；(5) after gold electrode ultrapure water and drying, it is immersed in the electrode modification solution of the pseudoknot structure containing DNA, at room temperature 1.5h is incubated, then is immersed in 1h in 1mM mercaptoethanol solution, with ultrapure water, drying, that is, obtains the modification of DNA pseudoknot structures Gold electrode, the electrode modification solution of the above-mentioned pseudoknot structure containing DNA is to contain in 10mM Tris-HCl pH 7.0 Tris-HCl There are the DNA pseudoknot structures, 140mM NaCl, 1mM MgCl that concentration is 0.2 μM2, 5mM KCl and 1mM TCEP.
- 3. a kind of gold electrode of specific DNA pseudoknot structure modification is to transcription factor Nanog electrochemical detection method, its feature Be the gold electrode for using the specific DNA pseudoknot structure described in claim 1 to modify for working electrode, saturated calomel electrode for ginseng Than electrode, platinum electrode is to be to electrode, specific detecting step：(1) Nanog is taken to be added to 10mM Tris-HCl, 140mM NaCl, 1mM MgCl2, and 5mM KCl, 1.0%Tween A series of reaction system of concentration containing Nanog in the range of 0~45nM is formed in 20, pH 7.0 buffer solution, by working electrode It is inserted into the reaction system, reacts 1h at 37 DEG C；(2) working electrode handled through step (1) is positioned in the buffer solution of 10mM Tris-HCl, PH 7.0, utilizes three electrodes System, detected with square wave voltammetry, and in the range of 5~25nM, obtain the linear relationship curve of electric current and Nanog concentration, its Linear coefficient is 0.99；(3) treat test sample using calibration curve method obtained by step (2) and carry out analysis detection, analyze Nanog content.
- 4. the gold electrode that specific DNA pseudoknot structure described in claim 1 is modified is in cells pluripotency analysis or drug resistance of tumor Application in detection.
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| CN110470712B (en) * | 2018-05-09 | 2021-10-15 | 南京大学 | miRNA detection device based on DNA nanoflower generation in channel |
| CN110514504A (en) * | 2018-05-21 | 2019-11-29 | 南京大学 | A kind of building of novel cell trace model and the research of property and application |
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