CN107121469A - A kind of construction method of high stability nucleic acid sensor - Google Patents
A kind of construction method of high stability nucleic acid sensor Download PDFInfo
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- CN107121469A CN107121469A CN201710294507.6A CN201710294507A CN107121469A CN 107121469 A CN107121469 A CN 107121469A CN 201710294507 A CN201710294507 A CN 201710294507A CN 107121469 A CN107121469 A CN 107121469A
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- gold electrode
- dsdna
- nucleic acid
- dna1
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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
Abstract
The invention discloses a kind of construction method of high stability nucleic acid sensor, this method is that gold electrode is immersed in the tetrahydrofuran solution containing 4 [(trimethyl silicon substrate) acetenyl] benzoic acid and tetrabutyl ammonium fluoride, reacted in lucifuge and room temperature environment, obtain the gold electrode of 4 carboxyl phenyl acetenyls modification；DNA1 and DNA2 is subjected to specific reaction, dsDNA is obtained；The gold electrode of 4 carboxyl phenyl acetenyl modification is carried out after amidation process with dsDNA, and gold electrode surfaces are closed using MCH solution, produces that stability is good, the rapid aptamer sensor of electron transmission；And this method is simple, the used time is short, is conducive to promoting the use of.
Description
Technical field
The present invention relates to a kind of construction method of nucleic acid sensor, more particularly to a kind of structure of high stability nucleic acid sensor
Construction method, belongs to biosensor technique field.
Background technology
Electrochemica biological sensor by single stranded DNA, oligonucleotide probe by being fixed on electrode surface, using molten with sample
The complementary dna chain in liquid or oligonucleotide base pairing is solved to be identified.By the electric current for detecting electroactive indicator
Change, or produce the change of electric, interfacial property to monitor hybridisation events due to double-spiral structure formation.In aptamers electrification
In the evolution for learning sensor, the fixation of aptamers has very important effect for the performance of whole sensor.Adaptation
Stability, adhesion and the specificity to target molecule of body sensor both depend on aptamers on electrode i.e. sensor surface
Technique for fixing.Therefore, reduce fit non-specific adsorption, sensing interface is controlled, to ensure the fit stabilization being connected with surface
Property, it is the key for building stabilization of nucleic acids sensor.
In the prior art, relatively common electrode surface construction method has following several：
1) the affine force fixing method of biotin.This method is by biotin-labeled pentylamine, streptavidin element and neutral chain parent
Strong affinity between element is fixed, due to be this method of biomolecule to solution environmental and operating process requirement compared with
Height, and expensive starting materials, are not readily used for daily use.
2) fixation is adsorbed.A kind of simplest mode of fixed adaptation body is absorption method, and this method need not both be tried
Agent is mainly be combined with each other also without special modification is carried out to nucleic acid by electrostatic interaction.This method adhesion is not strong enough,
Nucleic acid is easy for surface desorption of the meeting from motor during hybridization, and causes object joint efficiency low.
3) chemically react fixation.Chemical reaction fixation is the chemical reaction between substrate surface and reactant, typical case
Be that sulfydryl is combined with gold electrode and is fixed on electrode surface, this method is unstable during electro-chemical test and easily takes off
Fall.
The content of the invention
The technical problem existed for nucleic acid sensor of the prior art, the purpose of the present invention is to be to provide a kind of letter
The single, used time is short, and structure stability is good, the method for the rapid aptamer sensor of electron transmission.
, should the invention provides a kind of construction method of high stability nucleic acid sensor in order to realize above-mentioned technical purpose
Method comprises the following steps：
1) gold electrode is immersed in the tetrahydrochysene furan containing 4- [(trimethyl silicon substrate) acetenyl] benzoic acid and tetrabutyl ammonium fluoride
Mutter in solution, reacted in lucifuge and room temperature environment, obtain the gold electrode of 4- carboxyl phenyls acetenyl modification；
2) DNA1 and DNA2 is subjected to specific reaction, obtains dsDNA (double-stranded DNA)；The terminal modified amino of the DNA1 mono-,
Another terminal modified biotin (biotin)；DNA2 mono- terminal modified methylene blue (MB) signaling molecule；The DNA1 and DNA2 is complete
It is complementary；
3) gold electrode of the 4- carboxyl phenyls acetenyl modification is carried out after amidation process with dsDNA, using MCH solution
Gold electrode surfaces are closed, are produced.
It is preferred that scheme, 1) in reaction time be 0.5~1.5h；Most preferably 1h.
It is preferred that scheme, DNA1 and DNA2 are in cushioning liquid, in 35~39 DEG C, react 1~3h, obtain dsDNA.It is optimal
The reaction condition of choosing is to react 2h at 37 DEG C.Cushioning liquid is Tris-HCl solution.
It is preferred that scheme, the process of the amidation process is：The gold electrode immersion that 4- carboxyl phenyls acetenyl is modified
In the mixed aqueous solution containing dsDNA, EDC and NHS, in light protected environment, 3~6h is reacted at room temperature.
The DNA1 and DNA2 of the present invention can be directly by being commercially available, such as raw work bioengineering Shanghai (share) limited public affairs
Department.The DNA1 that the present invention is used is a terminal modified-NH2, another terminal modified biotin；The terminal modified methylene blue MB signals of DNA2 mono-
Molecule.DNA1 obtains dsDNA with DNA2 complete complementaries.-NH2For being condensed with-COOH, dsDNA is set to be fixed to electrode surface.
The molecular probe that the present invention is used, MB is reducing substances, and square wave is measured in PBS, is had in -0.27V or so
The peak of obvious methylene blue；According to the change of the square wave current of Multiple-Scan, it can be determined that interface electron transmission speed and steady
Whether determine.
In the building process of the nucleic acid sensor of the present invention, assembling sequence is crucial, i.e., first generate Au-C keys, then modify
DsDNA, first forms Au-C keys in tetrahydrofuran (THF) hydrophobic environment, Au-C keys is conducive in tetrahydrofuran hydrophobic environment
Generation, then generate in water environment dsDNA and progress-COOH and-NH2Dehydrating condensation, is conducive to improving DNA in water environment
Structural stability and assembling fastness, combination and detection to succeeding target thing are all favourable.If first carrying out DNA hybridization
With carboxyamino dehydrating condensation, then react and to form Au-C keys, then DNA assembling will be in hydrophobic THF hydrophobic environments, and it is stable
Property is subject to influence.
The nucleic acid sensor building mode of the present invention includes step in detail below：
1) first 4- [(trimethyl silicon substrate) acetenyl] benzoic acid solution is mixed with tetrabutyl ammonium fluoride solution, taken a certain amount of
Into type centrifuge tube, tip upside down on electrode, electrode is completely soaked in the solution；Room temperature places 1h in dark, makes 4- [(three
Methylsilyl) acetenyl] the de- trimethyl silane protection group of benzoic acid, by the modification of Au-C keys in gold electrode surfaces, obtain 4- carboxylics
The gold electrode of base phenylene-ethynylene modification；
2) DNA complementary pairing reactions are carried out, DNA1 is mixed with DNA2 and is placed in 37 DEG C, 2h is reacted, obtains dsDNA；DNA1
For a terminal modified-NH2, another terminal modified biotin；The terminal modified methylene blue MB signaling molecules of DNA2 mono-；DNA1 is complete with DNA2
It is complementary；
3) gold electrode of 4- carboxyl phenyls acetenyl modification is rinsed well with tetrahydrofuran THF；By dsDNA solution and EDC
(1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides) and NHS (n-hydroxysuccinimide) solution are mixed, and are taken
In small-sized centrifuge tube, tip upside down on electrode, electrode is completely soaked in the solution, room temperature places 5h in the dark, makes ds-DNA
- NH2Occurs dehydrating condensation with the-COOH of 4- [(trimethyl silicon substrate) acetenyl] benzoic acid, so that dsDNA is connected to electricity
Pole surface；
4) after the dsDNA is connected on electrode, adds MCH solution and closed, on the one hand to electrode surface room point
Closed, on the other hand, remove the dsDNA of non-specific binding.
Technical scheme key is that being used as " bridged bond " by 4- [(trimethyl silicon substrate) acetenyl] benzoic acid incites somebody to action
The dsDNA of the signaling molecule containing methylene blue is keyed to gold electrode surfaces, substantially increases the stability at nucleic acid sensor interface, together
When improve electron transmission, be conducive to obtaining wider detection range and improve sensitivity.4- [(trimethyl silicon substrate) acetenyl] benzene
Formic acid after Deprotection by carbon-gold bond by being closed in gold electrode surfaces, and many experiments surface passes through carbon-gold key stability
It is especially good, interface stability can be greatly improved, while will be introduced between dsDNA and gold electrode with big conjugated system base
Group, can speed up electron transmission, increases electric current, be conducive to obtaining wider detection range and detection sensitivity.
Compared with the prior art, technical scheme advantage is：
1) sensing interface stability of the invention is good, and electro-chemical test will not produce influence to signal；
2) sensing interface velocity of electrons of the invention is very fast, is conducive to high sensitivity to detect；
3) the sensing interface of the invention construction used time is shorter, efficiency high.
4) sensing interface of the invention is used for biological detection, mild condition, and upper terminal modified flexibly can be used for plurality of target
Analyte detection.
Brief description of the drawings
【Fig. 1】For nucleic acid sensor synthetic route；
【Fig. 2】The square wave voltammetry SWV Electrochemical Detection curves of the present invention；
【Fig. 3】For control group square wave voltammetry SWV Electrochemical Detection curves.
Embodiment
Following examples are intended to further illustrate present invention, rather than limit the protection model of the claims in the present invention
Enclose.
Embodiment 1
(1) Au-C keys are fixed on electrode
First by four fourths of 20 μM of 50 μM of 10 μ L 4- [(trimethyl silicon substrate) acetenyl] benzoic acid THF solutions and 100 μ L
Base ammonium fluoride THF solution is mixed, and is taken 55 μ L and in small-sized centrifuge tube, is tipped upside down on electrode, electrode is fully immersed in mixing molten
In liquid.Room temperature places 1h in dark, is allowed to form Au-C keys.
(2) formation of double-stranded DNA (dsDNA)
The DNA1Tris-HCl solution and 10 μM of DNA2Tris-HCl solution of 50 μM of preparation, two kinds of solution are mixed respectively
37 DEG C are placed in, 2h is reacted, because two DNA are complete complementaries, it is possible to obtain dsDNA.
(3) connection of dsDNA and electrode surface
Electrode is rinsed well with tetrahydrofuran THF.By dsDNA solution and 100 μM of EDC (1- (3- dimethylaminos third
Base) -3- ethyl-carbodiimide hydrochlorides) and 100 μM of NHS (n-hydroxysuccinimide) solution mix in equal volume, take small-sized
In centrifuge tube, tip upside down on electrode, electrode is completely soaked in the solution.Room temperature places 5h in dark, makes ds-DNA-NH2
Occurs dehydrating condensation with the-COOH of 4- [(trimethyl silicon substrate) acetenyl] benzoic acid, so that dsDNA is connected to electrode table
Face.Afterwards, 1mM MCH solution is added to be closed.On the one hand electrode surface room point is closed, on the other hand, removed
The dsDNA of non-specific binding.
(4) Electrochemical Detection sensing interface
After the completion of sensing interface is built, then carry out square wave voltammetry SWV Electrochemical Detections.Pass through the big of MB electrochemical signals
Small and continuous several times test signal stability.
The present invention is compared using most Au-S keys of present electrode assembling as control group, and specific experiment is such as
Under：DNA1-NH during this is tested2Change-SH- (CH into2)6Other parts are constant, by DNA1 and DNA2 in the solution containing TCEP
It is middle to stand, with opened disulfide bond, 2h is incubated in 37 DEG C of mixing afterwards, heteroduplex DNA is obtained.Gold electrode is pre-processed,
In the mixed liquor that the gold electrode handled well is immersed in pretreated double-stranded DNA, it is stored at room temperature overnight, redistilled water is used afterwards
With cleaning fluid cleaning；Then electrode is immersed in the solution containing MCH, with enclosed-electrode, cleaned afterwards with redistilled water；Will
Electrode after above-mentioned steps processing is as working electrode, and reference electrode, electrode is connected on chem workstation, is lied prostrate with square wave
The methylene blue signal for the sensor that the detection of peace method is prepared, and continuously scan multiple.
Fig. 2 is the SWV Electrochemical Detection curves of this method.Five curves are continuous scan 5 times successively from top to bottom in Fig. 2
The curve that SWV is obtained, it can be seen that the MB reduction peaks at -0.27 are very high, and peak current is very big, illustrate the combination side of this scheme
Formula is conducive to the electron transmission of MB and electrode surface；And after continuous several times scanning, curve is still relatively stablized, MB current decays
Seldom, illustrate that the combination of this scheme is also very stable.
Fig. 3 is the SWV Electrochemical Detection curves of control group.Three curves are continuous scan 3 times successively from top to bottom in Fig. 3
The curve that SWV is obtained, it can be seen that the MB reduction peaks at -0.27 are very low, peak current very little illustrates the combination side of this scheme
Formula is unfavorable for the electron transmission of MB and electrode surface；And after continuous several times scanning, curve is very unstable, and MB current decays are very
Many, the combination for illustrating this scheme is unstable.
Compare two figures to understand, still very stable after Fig. 2 scheme Multiple-Scan, scanning the influence to interface can ignore not
Meter, and MB electric current is bigger than Fig. 3, illustrates that this interface building mode of the present invention is successful.
Claims (4)
1. a kind of construction method of high stability nucleic acid sensor, it is characterised in that：Comprise the following steps：
1) that gold electrode is immersed in into the tetrahydrofuran containing 4- [(trimethyl silicon substrate) acetenyl] benzoic acid and tetrabutyl ammonium fluoride is molten
In liquid, reacted in lucifuge and room temperature environment, obtain the gold electrode of 4- carboxyl phenyls acetenyl modification；
2) DNA1 and DNA2 is subjected to specific reaction, obtains dsDNA；The terminal modified amino of the DNA1 mono-, it is another terminal modified
biotin；The terminal modified methylene blue signaling molecules of DNA2 mono-；The DNA1 and DNA2 complete complementaries；
3) gold electrode of the 4- carboxyl phenyls acetenyl modification is carried out after amidation process with dsDNA, is closed using MCH solution
Gold electrode surfaces, are produced.
2. the construction method of high stability nucleic acid sensor according to claim 1, it is characterised in that：1) during reaction in
Between be 0.5~1.5h.
3. the construction method of high stability nucleic acid sensor according to claim 1, it is characterised in that：DNA1 and DNA2 exists
In cushioning liquid, in 35~39 DEG C, 1~3h is reacted, dsDNA is obtained.
4. the construction method of the high stability nucleic acid sensor according to any one of claims 1 to 3, it is characterised in that：Institute
The process for stating amidation process is：The gold electrode that 4- carboxyl phenyls acetenyl is modified is immersed in mixed containing dsDNA, EDC and NHS
In Heshui solution, in light protected environment, 3~6h is reacted at room temperature.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103792372A (en) * | 2014-02-20 | 2014-05-14 | 南京大学 | Dinucleotide-labelled ratio electrochemical immunosensor |
| CN104020199A (en) * | 2014-06-18 | 2014-09-03 | 青岛科技大学 | Method for electrochemically determining dopamine on basis of aptamer recognition effect |
| CN104020198A (en) * | 2014-06-18 | 2014-09-03 | 青岛科技大学 | Method for detecting DNA by electrochemical transducer with signal amplification technology |
-
2017
- 2017-04-28 CN CN201710294507.6A patent/CN107121469B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103792372A (en) * | 2014-02-20 | 2014-05-14 | 南京大学 | Dinucleotide-labelled ratio electrochemical immunosensor |
| CN104020199A (en) * | 2014-06-18 | 2014-09-03 | 青岛科技大学 | Method for electrochemically determining dopamine on basis of aptamer recognition effect |
| CN104020198A (en) * | 2014-06-18 | 2014-09-03 | 青岛科技大学 | Method for detecting DNA by electrochemical transducer with signal amplification technology |
Non-Patent Citations (2)
| Title |
|---|
| ALINE UTAKA ET AL.: "Electrophilic alkynylation of ketones using hypervalent iodine", 《CHEMCOMM》 * |
| VENKAT REDDY CHINTAREDDY ET AL.: "Tetrabutylammonium Fluoride (TBAF)-Catalyzed Addition of Substituted Trialkylsilylalkynes to Aldehydes, Ketones, and Trifluoromethyl Ketones", 《J.ORG.CHEM.》 * |
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