CN107121469B - 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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- CN107121469B CN107121469B CN201710294507.6A CN201710294507A CN107121469B CN 107121469 B CN107121469 B CN 107121469B CN 201710294507 A CN201710294507 A CN 201710294507A CN 107121469 B CN107121469 B CN 107121469B
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- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
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Abstract
The invention discloses a kind of construction methods of high stability nucleic acid sensor, this method is to be immersed in gold electrode in the tetrahydrofuran solution containing 4- [(trimethyl silicon substrate) acetenyl] benzoic acid and tetrabutyl ammonium fluoride, be protected from light and room temperature environment in react, obtain 4- carboxyl phenyl acetenyl modification gold electrode；DNA1 and DNA2 is subjected to specific reaction, obtains dsDNA；After the gold electrode and dsDNA of the 4- carboxyl phenyl acetenyl modification carry out amidation process, the electron transmission rapid aptamer sensor good to get stability using MCH solution closing gold electrode surfaces；And this method is simple, the used time is short, is conducive to promote the use of.
Description
Technical field
The present invention relates to a kind of construction method of nucleic acid sensor, in particular to a kind of structure of high stability nucleic acid sensor
Construction method belongs to biosensor technique field.
Background technique
Electrochemica biological sensor is by being fixed on electrode surface for single stranded DNA, oligonucleotide probe, using molten with sample
Complementary dna chain or oligonucleotide base pairing in liquid are solved to be identified.By the electric current for detecting electroactive indicator
Variation, or electric, interfacial property variation is generated to monitor hybridisation events due to double-spiral structure formation.In aptamers electrification
In the development process for learning sensor, the fixation of aptamers has very important effect the performance of entire sensor.Adaptation
The stability of body sensor, binding force and aptamers are both depended on electrode i.e. sensor surface to the specificity of target molecule
Technique for fixing.Therefore, reduce the non-specific adsorption of aptamer, control sensing interface, to guarantee stabilization that aptamer is connect with surface
Property, it is the key that 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 does not both need to try
Agent does not need to carry out nucleic acid special modification yet, is mainly be combined with each other by electrostatic interaction.This method binding force is not strong enough,
Nucleic acid is easy for during hybridization can be from the surface desorption of motor, and causes object joint efficiency low.
3) fixation is chemically reacted.Chemical reaction fixation is the chemical reaction between substrate surface and reactant, typical
Be that sulfydryl is fixed on electrode surface in conjunction with gold electrode, this method is unstable and easy to be de- during electro-chemical test
It falls.
Summary of the invention
For nucleic acid sensor in the prior art, the purpose of the invention is to provide a kind of letters
List, used time are short, and building stability is good, the method for the rapid aptamer sensor of electron transmission.
In order to achieve the above technical purposes, the present invention provides a kind of construction methods of high stability nucleic acid sensor, should
Method the following steps are included:
1) gold electrode is immersed in the tetrahydro furan containing 4- [(trimethyl silicon substrate) acetenyl] benzoic acid and tetrabutyl ammonium fluoride
Mutter in solution, be protected from light and room temperature environment in react, obtain the gold electrode of 4- carboxyl phenyl 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) after the gold electrode and dsDNA of the 4- carboxyl phenyl acetenyl modification carry out amidation process, using MCH solution
Close gold electrode surfaces to get.
Preferred scheme, 1) reaction time in is 0.5~1.5h；Most preferably 1h.
Preferred scheme, DNA1 and DNA2 are in buffer solution, in 35~39 DEG C, react 1~3h, obtain dsDNA.It is optimal
The reaction condition of choosing is in 37 DEG C of reaction 2h.Buffer solution is Tris-HCl solution.
Preferred scheme, the process of the amidation process are as follows: impregnate the gold electrode of 4- carboxyl phenyl acetenyl modification
In the mixed aqueous solution containing dsDNA, EDC and NHS, in light protected environment, 3~6h is reacted at room temperature.
DNA1 and DNA2 of the 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 uses is a terminal modified-NH2, another terminal modified biotin；The terminal modified methylene blue MB signal of DNA2 mono-
Molecule.DNA1 and DNA2 complete complementary obtain dsDNA.-NH2For being condensed with-COOH, dsDNA is made to be fixed to electrode surface.
The molecular probe that the present invention uses, MB are reducing substances, and square wave is measured in PBS, are had in -0.27V or so
The peak of apparent methylene blue；According to the variation of the square wave current of Multiple-Scan, it can be determined that interface electron transmission speed and steady
Whether fixed.
In the building process of nucleic acid sensor of the invention, assembling sequence is key, i.e., first generates Au-C key, then modify
DsDNA first forms Au-C key in the hydrophobic environment of tetrahydrofuran (THF), is conducive to Au-C key in tetrahydrofuran hydrophobic environment
Generation, then dsDNA and progress-COOH and-NH are generated in water environment2Dehydrating condensation is conducive to improve DNA in water environment
Structural stability and assembling fastness, combination and detection to succeeding target object are all advantageous.If first carrying out the hybridization of DNA
It with carboxyamino dehydrating condensation, then reacts and to form Au-C key, then the assembling of DNA will stablize in hydrophobic THF hydrophobic environment
Property is subject to influence.
Nucleic acid sensor building mode of the invention comprising the following specific steps
1) first 4- [(trimethyl silicon substrate) acetenyl] benzoic acid solution is mixed with tetrabutyl ammonium fluoride solution, is taken a certain amount of
Into type centrifuge tube, back-off on the electrode, be completely soaked electrode in the solution；It is placed at room temperature for 1h in dark, makes 4- [(three
Methylsilyl) acetenyl] the de- trimethyl silane protecting group of benzoic acid, it is modified by Au-C key in gold electrode surfaces, obtains 4- carboxylic
The gold electrode of base phenylene-ethynylene modification；
2) reaction of DNA complementary pairing is 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 molecule of DNA2 mono-；DNA1 is complete with DNA2
It is complementary；
3) gold electrode of 4- carboxyl phenyl acetenyl modification is rinsed well with tetrahydrofuran THF；By dsDNA solution and EDC
The mixing of (1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride) and NHS (n-hydroxysuccinimide) solution, takes
In small-sized centrifuge tube, back-off on the electrode, is completely soaked electrode in the solution, is placed at room temperature for 5h in the dark, make ds-DNA
- NH2Dehydrating condensation occurs 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, MCH solution is added and is closed, on the one hand to electrode surface vacancy point
It is closed, on the other hand, removes the dsDNA of non-specific binding.
Technical solution of the present invention key is to be used as " bridged bond " will by 4- [(trimethyl silicon substrate) acetenyl] benzoic acid
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, help to obtain wider detection range and improve sensitivity.4- [(trimethyl silicon substrate) acetenyl] benzene
For formic acid by being incorporated in gold electrode surfaces by carbon-Jin Jian after Deprotection, many experiments surface passes through carbon-gold key stability
It is especially good, interface stability can be greatly improved, while will introduce between dsDNA and gold electrode with big conjugated system base
Group, can speed up electron transmission, increases electric current, help to obtain wider detection range and detection sensitivity.
Compared with the prior art, technical solution of the present invention advantage is:
1) sensing interface stability of the invention is good, and electro-chemical test will not have an impact signal；
2) sensing interface velocity of electrons of the invention is very fast, is conducive to high sensitivity detection；
3) the sensing interface of the invention construction used time is shorter, high-efficient.
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.
Detailed description of the invention
[Fig. 1] is nucleic acid sensor synthetic route；
Square wave voltammetry SWV Electrochemical Detection curve [Fig. 2] of the invention；
[Fig. 3] is control group square wave voltammetry SWV Electrochemical Detection curve.
Specific embodiment
Following embodiment is intended to further illustrate the content of present invention, rather than limits the protection model of the claims in the present invention
It encloses.
Embodiment 1
(1) Au-C key is fixed on the electrode
First by four fourths of 50 μM of 10 μ L 4- [(trimethyl silicon substrate) acetenyl] 20 μM of benzoic acid THF solution and 100 μ L
The mixing of base ammonium fluoride THF solution, takes in 55 μ L and small-sized centrifuge tube, and back-off on the electrode, makes electrode be fully immersed in mixing molten
In liquid.It is placed at room temperature for 1h in dark, is allowed to form Au-C key.
(2) formation of double-stranded DNA (dsDNA)
50 μM of DNA1Tris-HCl solution and 10 μM of DNA2Tris-HCl solution are prepared respectively, and two kinds of solution are mixed
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 hydrochloride) and 100 μM of NHS (n-hydroxysuccinimide) solution mix in equal volume, take small-sized
In centrifuge tube, back-off on the electrode, is completely soaked electrode in the solution.It is placed at room temperature for 5h in dark, makes-the NH of ds-DNA2
Dehydrating condensation occurs with-the COOH of 4- [(trimethyl silicon substrate) acetenyl] benzoic acid, so that dsDNA is connected to electrode table
Face.Later, 1mM MCH solution is added to be closed.On the one hand electrode surface vacancy point is closed, on the other hand, is removed
The dsDNA of non-specific binding.
(4) Electrochemical Detection sensing interface
After the completion of sensing interface building, then carry out square wave voltammetry SWV Electrochemical Detection.Pass through the big of MB electrochemical signals
The stability of small and continuous several times test signal.
The present invention is compared using most Au-S keys of present electrode assembling as control group, and specific experiment is such as
Under: by this test in DNA1-NH2Change-SH- (CH into2)6Other parts are constant, by DNA1 and DNA2 in the solution containing TCEP
Middle standing is incubated for 2h in 37 DEG C of mixing later, is obtained heteroduplex DNA with opened disulfide bond.Gold electrode is pre-processed,
The gold electrode handled well is immersed in the mixed liquor of pretreated double-stranded DNA, is stored at room temperature overnight, uses secondary distilled water later
It is cleaned with cleaning solution；Then electrode is immersed in the solution containing MCH, with enclosed-electrode, is cleaned later with secondary distilled water；It will
Above-mentioned steps treated electrode is lied prostrate with square wave as working electrode and reference electrode, be connected on chem workstation to electrode
The methylene blue signal for the sensor that the detection of peace method prepares, and continuous scanning is multiple.
Fig. 2 is the SWV Electrochemical Detection curve of this method.Five curves are successively continuous scanning 5 times from top to bottom in Fig. 2
The curve that SWV is obtained, it can be seen that the MB reduction peak at -0.27 is very high, and peak current is very big, illustrates 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 more stable, MB current decay
Seldom, illustrate that the combination of this scheme is also very stable.
Fig. 3 is the SWV Electrochemical Detection curve of control group.Three curves are successively continuous scanning 3 times from top to bottom in Fig. 3
The curve that SWV is obtained, it can be seen that the MB reduction peak at -0.27 is very low, and 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 decay is very
It is more, illustrate that the combination of this scheme is unstable.
Two figures are compareed it is found that still very stable after the scheme Multiple-Scan of Fig. 2, scanning the influence to interface can ignore not
Meter, and electric current ratio Fig. 3's of MB is bigger, illustrates that this interface building mode of the invention is successful.
Claims (4)
1. a kind of construction method of high stability nucleic acid sensor, it is characterised in that: the following steps are included:
1) that gold electrode is immersed in the tetrahydrofuran containing 4- [(trimethyl silicon substrate) acetenyl] benzoic acid and tetrabutyl ammonium fluoride is molten
In liquid, be protected from light and room temperature environment in react, obtain 4- carboxyl phenyl acetenyl modification gold electrode；
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 molecule of DNA2 mono-；The DNA1 and DNA2 complete complementary；
3) it after the gold electrode and dsDNA of the 4- carboxyl phenyl acetenyl modification carry out amidation process, is closed using MCH solution
Gold electrode surfaces to get.
2. the construction method of high stability nucleic acid sensor according to claim 1, it is characterised in that: when reaction in 1)
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 exist
In buffer solution, in 35~39 DEG C, 1~3h is reacted, dsDNA is obtained.
4. the construction method of described in any item high stability nucleic acid sensors according to claim 1~3, it is characterised in that: institute
State the process of amidation process are as follows: be immersed in the gold electrode that 4- carboxyl phenyl acetenyl is modified mixed containing dsDNA, EDC and NHS
In Heshui solution, in light protected environment, 3~6h is reacted at room temperature.
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| 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 |
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- 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 |
|---|
| Electrophilic alkynylation of ketones using hypervalent iodine;Aline Utaka et al.;《ChemComm》;20140214;第50卷;3810-3813 |
| Tetrabutylammonium Fluoride (TBAF)-Catalyzed Addition of Substituted Trialkylsilylalkynes to Aldehydes, Ketones, and Trifluoromethyl Ketones;Venkat Reddy Chintareddy et al.;《J.Org.Chem.》;20110425;第76卷;4482-4488 |
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