CN106399492B - A method of renewable DNA hybridization interface is constructed with BSA isoelectric point confining liquid - Google Patents
A method of renewable DNA hybridization interface is constructed with BSA isoelectric point confining liquid Download PDFInfo
- Publication number
- CN106399492B CN106399492B CN201610804155.XA CN201610804155A CN106399492B CN 106399492 B CN106399492 B CN 106399492B CN 201610804155 A CN201610804155 A CN 201610804155A CN 106399492 B CN106399492 B CN 106399492B
- Authority
- CN
- China
- Prior art keywords
- dna
- optical fiber
- interface
- nucleic acid
- solution
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of methods for constructing renewable DNA hybridization interface with BSA isoelectric point confining liquid.BSA isoelectric point solution (pH 4.6) is closed sensing interface as confining liquid and carries out Seal treatment to DNA optical fiber by the present invention, and propose a kind of method for constructing renewable DNA hybridization interface with BSA isoelectric point confining liquid, it realizes and the high efficiency in sensing interface activated adoption site is closed, inhibit suction-operated of the sensing interface of biological functional to non-specific nucleic acid chain, guarantee hybridization of the target segment to sensing interface simultaneously, to improve the signal-to-noise ratio of sensitive design, device sensitivity is promoted.Method of the invention ensures hybridization of the target nucleic acid chain to the interface DNA on the basis of reducing activated adoption site, and this method have the advantages that it is economical, easy, quick.
Description
Technical field
The invention belongs to test and analyze field, and in particular to a kind of to construct renewable DNA hybridization with BSA isoelectric point confining liquid
The method at interface.
Background technique
In bio-sensing method, (affine) identification between biological elements constitutes the basis of sensing.Traditional biology
Identify affine identification intracorporal to (antigen-antibody) and biology to the Immune discrimination including classics to (such as Avidin and biotin).
Generally, a side of tional identification centering belongs to protein.And the protein (such as antibody) for having bio-identification power is generally configured with
Higher acquisition and preservation cost；In addition, the problem that the protein for being related to living body production is unstable there is also property between batch.This
A little disadvantages limit traditional biological identification material in the application of sensory field to a certain extent.
From last century Mo, the sensing analytical method using functional nucleic acid as new bio recognition component also achieves swift and violent
Development.Functional nucleic acid is the oligonucleotide fragment with specific functions such as affine identification, catalysis, with aptamer
It (aptamer) is representative with DNA enzymatic (DNAzyme) two kinds of nucleic acid materials.For the traditional biologicals identification material such as antibody,
Can artificial synthesized functional nucleic acid have the advantages such as production and transport are at low cost, easy to maintain, property is stablized.Have over one hundred kind at present
Functional nucleic acid is seen in report, and identification object covers biotoxin, metal ion, cell metabolite, high molecular weight protein, full cell
Deng hundreds of substance, application field concentrates on bio-sensing and living cells diagnosis and treatment etc..
Also have with the fast development in functional nucleic acid field, in bio-sensing field and large quantities of identification material is made with functional nucleic acid
The detection scheme of material comes into being.Wherein most scheme classes belong to homogeneous detection --- and there is no solid phase interfaces i.e. in detection, single
By means of being mutually distinguishable between liquid phase DNA molecular and object (complementary strand or target molecule), in conjunction with fluorescent marker or dyeing, receive
The technological means such as colorimetric, the enzymatic reaction of rice material auxiliary realize the quantitative analysis to target.Although homogeneous detection has pair
The advantages that hardware requirement is low, reaction speed is fast, but compared to interfacial structure (such as optical fiber, chip of light waveguide, electrode) is based on
Biosensor, it is immobilized that homogeneous strategy cannot achieve probe, thus be difficult to construct it is stable, highly integrated, renewable (can be repeatedly
Utilize, single testing cost is low) senser element.And the interface type sensor of application function nucleic acid can be subdivided into two classes: 1) straight
It connects DNA is covalently immobilized in sensing interface (being known as the interface DNA afterwards)；2) it relies on homogeneous reaction to be detected, realizes a signal
Target concentration signal is changed into transfer material concentration, recycles interface identification interim signal (referred to as indirect interface) by conversion.
Obviously, compared to the interface DNA, indirect interface is related to multiple signal conversion, not only has higher complexity in operating level, more
It is possible that introducing signal bias, loss, or even fall into oblivion.In conclusion from promoting user's operation experience, reduction testing cost, pushing away
Wide bio-sensing detection application range level considers, it is significant to construct the reproducible interface DNA.
Generally, there can be some activated adoption sites on manually modified bio-sensing interface, such site easily passes through
The effects of hydrophobe, electrostatic and hydrogen bond, adsorbs the specific components in prepare liquid.For the interface DNA, no matter adsorbed target
Segment or non-targeted segment are likely to decrease quality, the impact analysis result of sensing.Therefore, in building DNA sensing interface
When, need to evade nucleic acid molecules to " non-specific adsorption " of sensing interface.
In the sensor-based system based on the interface DNA, there are the basic standards that two are examined seal quality: 1) biography after closing
Sense interface should substantially drop the response signal of non-specific chain compared to without closing or the closed sensing interface of other closed materials
It is low；2) sensing interface after closing should ensure that the high RST response to target hybridization chain.However, being based on DNA circle existing
In the sensor-based system in face, the rare high-quality sealer that can meet above-mentioned two standards simultaneously is seen in report.Using low-quality envelope
It closes, tends not to that non-specific adsorption is effectively reduced, or also destroy the life at the interface DNA while reducing non-specific adsorption
Object activity.
Summary of the invention
It is an object of the present invention to provide a kind of confining liquids.
Confining liquid provided by the invention is the solution obtained after mixing the buffer that BSA and pH is 4.4-4.8.
Concentration of the BSA in the confining liquid is 1-3mg/mL.
In above-mentioned confining liquid, the pH is 4.6.
In above-mentioned confining liquid, concentration of the BSA in the confining liquid is 2mg/mL.
In above-mentioned confining liquid, the buffer is citric acid-sodium citrate buffer solution or acetic acid-sodium acetate buffer solution.Institute
State the citric acid-sodium citrate buffer solution that citric acid-sodium citrate buffer solution is 0.1M；The acetic acid-sodium acetate buffer solution
0.2M acetic acid-sodium acetate buffer solution.
The configuration method of above-mentioned 0.1M citric acid-sodium citrate buffer solution is as follows: by the aqueous citric acid solution of 0.1mol/L
(citric acid C6H8O7·H2O: molecular mass 210.14,0.1mol/L, solution are 21.01 grams per liters) with the citric acid of 0.1mol/L
Sodium water solution (sodium citrate Na3C6H5O7·2H2O: molecular mass 294.12,0.1mol/L solution are 29.41 grams per milliliters) according to
The ratio that volume ratio is 103:97 mixes, and 0.1M citric acid-sodium citrate buffer solution can be obtained.
The configuration method of above-mentioned 0.2M acetic acid-sodium acetate buffer solution is as follows: by the aqueous sodium acetate solution (Na of 0.2M2Ac·
3H2O molecular mass=136.09,0.2mol/L solution is 27.22 grams per liters) be according to volume ratio with the acetic acid aqueous solution of 0.2M
The ratio of 49:51 mixes, and 0.2M acetic acid-sodium acetate buffer solution can be obtained.
In above-mentioned confining liquid, the buffer is specially the citric acid-sodium citrate buffer solution of 0.1M.
It is a further object to provide the new applications of above-mentioned confining liquid.
The present invention provides above-mentioned confining liquids it is any in following (1)-(8) in application:
(1) interface DNA and/or the renewable interface DNA of building are constructed；
(2) the activated adoption site at the interface DNA is closed；
(3) interface DNA is improved to the specific adsorption and/or identification of target nucleic acid chain；
(4) interface DNA is reduced to the non-specific adsorption and/or identification of non-targeted nucleic acid chains；
(5) interface DNA is improved to the signal response of target nucleic acid chain；
(6) interface DNA is reduced to the signal response of non-targeted nucleic acid chains；
(7) promote hybridizing for target nucleic acid chain and the interface DNA；
(8) inhibit hybridizing for non-targeted nucleic acid chains and the interface DNA.
It is a still further object of the present invention to provide a kind of construction methods at interface DNA.
The construction method at the interface DNA provided by the invention includes the following steps:
1) there is the optical fiber of aldehyde radical to connect with surface active the DNA molecular of end modified amino using covalent method, obtain DNA
The optical fiber of modification；
2) optical fiber that the DNA modification is closed with above-mentioned confining liquid, the optical fiber after obtaining Seal treatment is to get miscellaneous to DNA
Interface.
It further include by the optical fiber after the Seal treatment if the DNA molecular is double chain DNA molecule in the above method
The step of double chain DNA molecule denaturation, recovery single stranded DNA interface.DNA molecular in method of the invention is double chain DNA molecule, is needed
Denaturation treatment is carried out, becomes single-stranded, but DNA molecular herein is also possible to single strand dna, if it is single stranded DNA
Molecule, then the step of not needing denaturation treatment.
In the above method, the method for the denaturation is the optical fiber after impregnating the Seal treatment with DNA denaturing liquid, described
DNA denaturing liquid is urea liquid；The solvent of the urea liquid is water, and solute is urea and NaCl, solute and in urea liquid
In concentration are as follows: urea 4M, NaCl 50mM.
In the above method, the closed condition is 37 DEG C, and 100rpm shakes 4 hours.
In the above method, the step 1) includes the following steps:
A1 optical fiber) is impregnated with Piranha solution, obtains optical fiber after the processing of Piranha solution；
A2 optical fiber after the Piranha solution is handled) is impregnated with triethoxysilane solution, is obtained at triethoxysilane
Optical fiber after reason；
A3) triethoxysilane is impregnated treated optical fiber with glutaraldehyde water solution, obtaining glutaraldehyde, treated
Optical fiber；
A4) glutaraldehyde is impregnated treated optical fiber with the solution of the DNA molecular of the end modified amino, obtain
The optical fiber of DNA modification.
In the above method,
The step A2) and the step A3) between further include carrying out the triethoxysilane treated optical fiber
The step of ultrasonic cleaning；
The step A4) after further include that the optical fiber of the DNA modification is successively passed through to glycine solution and cyano boron hydrogen
The step of changing sodium water solution processing.
In the above method,
The Piranha solution is by dense H2SO4With 30% (mass percent concentration) H2O2What aqueous solution obtained after mixing
Solution, wherein dense H2SO4With 30%H2O2The volume ratio of aqueous solution is 3:1；
The triethoxysilane solution is the solution obtained after mixing APTS and dry toluene, wherein the body of APTS
Fraction is 1%；
The volume fraction of glutaraldehyde is 1% in the glutaraldehyde water solution；
The concentration of DNA molecular in the solution of the DNA molecular of the end modified amino is 300nM.
In the above method, the method for the ultrasonic cleaning is that treated that optical fiber is soaked in no water beetle by triethoxysilane
It is ultrasonically treated, the optical fiber after being ultrasonically treated, then the optical fiber after cleaning the ultrasonic treatment with dry toluene, is obtained in benzene
Optical fiber after to cleaning；It finally uses and is dried with nitrogen the optical fiber after the cleaning, and toasted.The ultrasonic treatment when
Between be 3 minutes, the number of ultrasonic treatment is 3 times；The number of the cleaning is 5 times；The condition of the baking is in 200 DEG C of baking ovens
Baking 1 hour.
In the above method, the concentration of the glycine solution is 1M；The concentration of the sodium cyanoborohydride aqueous solution is
20mg/mL。
Final object of the present invention is to provide the new application of the above method.
The present invention provides the above methods it is any in following (B1)-(B6) in application:
(B1) interface DNA is improved to the specific adsorption and/or identification of target nucleic acid chain；
(B2) interface DNA is reduced to the non-specific adsorption and/or identification of non-targeted nucleic acid chains；
(B3) interface DNA is improved to the signal response of target nucleic acid chain；
(B4) interface DNA is reduced to the signal response of non-targeted nucleic acid chains；
(B5) promote hybridizing for target nucleic acid chain and the interface DNA；
(B6) inhibit hybridizing for non-targeted nucleic acid chains and the interface DNA.
The present invention utilizes the characteristics of BSA is to sensing interface large amount of adsorption in isoelectric point buffer, by BSA isoelectric point solution
(pH 4.6) closes sensing interface as confining liquid and carries out Seal treatment to DNA optical fiber, and proposes a kind of BSA isoelectric point
The method that confining liquid constructs the renewable interface DNA is realized and is closed to the high efficiency in sensing interface activated adoption site, inhibits biology
Suction-operated of the sensing interface of functionalization to non-specific nucleic acid chain, while guaranteeing hybridization of the target segment to sensing interface,
To improve the signal-to-noise ratio of sensitive design, device sensitivity is promoted.Method of the invention is on the basis for reducing activated adoption site
On, while ensuring hybridization of the target nucleic acid chain to the interface DNA, and this method have the advantages that it is economical, easy, quick.
Detailed description of the invention
Fig. 1 is the situation of change of ultra-violet absorption spectrum before and after DNA hybridization.
Fig. 2 is the preparation method of the optical fiber of DNA modification of the invention.
Fig. 3 is non-object chain through the signal on different confining liquids treated control group optical fiber.
Fig. 4 is that target hybridization chain and non-targeted chain are generated on the DNA optical fiber handled without confining liquid in the present invention
Signal.
Fig. 5 is that target hybridizes chain and non-targeted the chain institute on the DNA optical fiber handled through BSA isoelectric point confining liquid in the present invention
The signal of generation.
Fig. 6 is continuous two days signal intensity situations of DNA optical fiber of the invention.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Quantitative test in following embodiments, is respectively provided with three repeated experiments, and results are averaged.
Using evanescent wave sensor as detection platform, (evanescent wave optical fiber sensing platform is that Chinese Patent Application No. is to the present invention
200610089497.4 biosensor of full fiber optic evanescent wave), using optical fiber as sensing element, Cy 5.5 is fluorescent marker base
Group, but the present invention is not limited by following implementation, and all improvement made based on thought of the invention or deformation should belong to this
The protection scope of invention.
Ultrasonic cleaner KQ218 in following embodiments is the product of Kunshan Ultrasonic Instruments Co., Ltd..
Piranha solution in following embodiments is by dense H2SO4With 30% (mass percent concentration) H2O2Aqueous solution is mixed
The solution obtained after even, wherein dense H2SO4(98% concentrated sulfuric acid of Beijing Chemical Plant's production) and 30%H2O2The body of aqueous solution
Product is than being 3:1.
Optical fiber (abbreviation optical fiber, 600 μM of core diameters) in following embodiments is Nanjing Chunhui Science and Technology Industrial Co Ltd
Product.
Triethoxysilane (APTS) solution in following embodiments be by APTS (product of Sigma-Aldrich company,
Catalog number is the solution obtained after 440140) mixing with dry toluene (product of Beijing Chemical Plant), wherein the body of APTS
Fraction is 1%.
BSA in following embodiments is the product of Sigma-Aldrich company, catalog number V900933.
The configuration method of 0.1M citric acid-sodium citrate buffer solution in following embodiments is as follows: by the lemon of 0.1mol/L
Lemon aqueous acid (citric acid C6H8O7·H2O: molecular mass 210.14,0.1mol/L, solution are 21.01 grams per liters) with
Sodium citrate aqueous solution (the sodium citrate Na of 0.1mol/L3C6H5O7·2H2O: molecular mass 294.12,0.1mol/L solution are
29.41 grams per milliliters) it is mixed according to the ratio that volume ratio is 103:97,0.1M citric acid-sodium citrate buffer solution can be obtained.
Hybridization solution in following embodiments is the product of Thermo Scientific company, name of product Pierce
BupH Dry Blend Buffers Phosphate Buffered Saline (PBS), catalog number 28372.
The solvent of urea liquid in following embodiments is water, and solute is urea and NaCl, solute and in urea liquid
Concentration are as follows: urea 4M, NaCl 50mM.
Embodiment 1, a kind of method for constructing renewable DNA hybridization interface with BSA isoelectric point confining liquid
One, the preparation of DNA optical fibre interface
1, the preparation of the optical fiber (DNA optical fiber) of DNA modification
In the end modified amino of DNA, make optical fiber surface with aldehyde radical by multi-step activation, by the amino of the end DNA and
The aldehyde radical of optical fiber surface through tentatively modifying connects, and obtains DNA optical fiber.The specific preparation process of DNA optical fiber is as shown in Fig. 2, tool
Steps are as follows for body:
(1) DNA hybridization prepares dsDNA
By single strand dna NH2- AG (final concentration of 300nM) and single strand dna NH2The single stranded DNA of-AG complementation point
Sub- CT-Cy5.5 (final concentration of 300nM) and 50mM NaCl aqueous solution mix, and obtain ssDNA mixed liquor；By ssDNA mixed liquor
It is heated 10 minutes in 90 DEG C of hot baths, is slowly cooled to room temperature afterwards, obtain dsDNA solution (dsDNA concentration is 300nM), and put
Enter and saved overnight in 4 DEG C, until using.Single strand dna NH2The nucleic acid sequence of-AG and single strand dna CT-Cy5.5 such as table
Shown in 1.A20-Cy5.5, CT-Cy5.5 and NH in table 12- AG is closed by Sangon Biotech (Shanghai) Co., Ltd.
At.
Specific nucleic acid title and sequence involved by 1 example of table
By ssDNA mixed liquor obtained above and dsDNA solution Hitachi U-3900 it is ultraviolet/visible spectrophotometer is enterprising
Row absorbance tests (using the glass cuvette of light path 1cm).The change of ssDNA mixed liquor and dsDNA solution ultra-violet absorption spectrum
Change situation as shown in Figure 1, as can be seen from the figure: and ssDNA blended liquid phase ratio (the single stranded DNA solution before hybridization), dsDNA is molten
In liquid (double stranded DNA solutions after hybridization), the ultraviolet peak Dependent Red Shift of feature, peak value at 258nm increase, and meet expection,
Show that two complementary DNA chain sections have hybridized to form duplex structure.
(2) optical fiber (purpose is cleaning optical fiber, and makes optical fiber surface that hydroxylating occur) is impregnated with Piranha solution,
Heat 1 hour in 110 DEG C of baking ovens, be cooled to room temperature after heating, then sufficiently washed with ultrapure water optical fiber to washing lotion neutrality, and
With optical fiber is dried with nitrogen, it is put in 110 DEG C of baking ovens baking at least one hour, obtains Piranha solution treated optical fiber；
(3) with triethoxysilane (APTS) solution, (ATPS can be reacted with optical fiber surface hydroxyl, and expose amino-functional
Group) impregnating Piranha solution, treated optical fiber 2 hours, then it is dipped in 3 minutes (ultrasounds of ultrasonic clean in dry toluene
Clean instrument: ultrasonic cleaner KQ218), total ultrasonic clean 3 times, then cleaned optical fiber 5 times with anhydrous toluene solution, it is rear to use
It is dried with nitrogen, and the optical fiber after drying is put into 200 DEG C of baking ovens and is toasted 1 hour, it is cooling, obtain APTS treated optical fiber.
(4) APTS treated optical fiber is immersed into the glutaraldehyde water solution (amino of aldehyde radical and APTS that volume fraction is 1%
Reaction) in, the reaction overnight in room temperature, the shaking table of 50-100rpm, next day is cleaned 5 times with ultrapure water, obtains glutaraldehyde processing
Optical fiber afterwards.
(5) glutaraldehyde treated optical fiber is immersed prepared in 1mL above-mentioned steps (1) dsDNA solution (aldehyde radical with
The amino of dsDNA molecule reacts) in, it is reacted 2 hours in room temperature, the shaking table of 50-100rpm, obtains the optical fiber of modification dsDNA.
(6) to modification dsDNA optical fiber immerse 20 μ L 1M glycine solutions (aldehyde radical extra with optical fiber surface reacts,
To close aldehyde radical site) in, it is reacted 1 hour in room temperature, the shaking table of 50-100rpm, obtains glycine treated optical fiber.
(7) glycine treated optical fiber is immersed into the sodium cyanoborohydride aqueous solution of 20mg/mL (by above-mentioned amino and aldehyde
The schiff bases construction recovery that base is formed is stable amido bond) in, react 1 hour in room temperature, the shaking table of 50-100rpm, so
Optical fiber is cleaned with ultrapure water afterwards, and is immersed in 50mM NaCl aqueous solution, the optical fiber for being modified with DNA is obtained, is saved in 4 DEG C.
2, the preparation of control group optical fiber
DsDNA solution in (4) of above-mentioned steps 1 is substituted for 50mM NaCl aqueous solution, other steps are constant, obtain
Control group optical fiber.
Two, sealing effect of the detection BSA isoelectric point confining liquid for control group optical fiber
Seal treatment is carried out to sensing interface, to guarantee that non-targeted segment will not generate excessively high interference letter at interface
Number, it is the basis for realizing DNA hybridization.Utilize the control group optical fiber and control group nucleic acid A of 2 preparations of step 120- Cy5.5 is examined
Examine do not close and confining liquid difference to signal magnitude caused on optical fiber.Specific step is as follows:
1, the preparation of prepare liquid
A20-Cy5.5 is dissolved in hybridization solution, A20-Cy5.5 prepare liquid is obtained, the concentration of A20-Cy5.5 is 10nM.
2, the processing of control group optical fiber
According to whether being divided into following each group with the difference of confining liquid closing and confining liquid:
(1) not closed group: using control group optical fiber as the optical fiber of not closed group.
(2) BSA isoelectric point confining liquid group: control group optical fiber is immersed in BSA isoelectric point confining liquid, and (BSA is dissolved in 0.1M lemon
The solution that lemon acid-sodium citrate buffer solution obtains, the concentration of BSA are 2mg/mL, pH 4.6) in, it is small to shake up 4 for concussion in shaking table
When (37 DEG C, rpm=100), obtain the optical fiber of BSA isoelectric point confining liquid group.
(3) control group optical fiber BSA aqueous solution group: is immersed in BSA aqueous solution (concentration of BSA is 2mg/mL, pH 6.75)
In, it shakes and shakes up 4 hours (37 DEG C, rpm=100) in shaking table, obtain the optical fiber of BSA aqueous solution group.
(4) PSS group: by control group optical fiber be immersed in PSS aqueous solution (volume fraction 2% of PSS, PSS (MW500,000,
Alfa Aesar, lot D09Z004)) in aqueous solution, shakes and shake up 4 hours (37 DEG C, rpm=100) in shaking table, obtain PSS
The optical fiber of aqueous solution group.
(5) PAA group: by control group optical fiber be immersed in PAA aqueous solution (volume fraction 2% of PAA, PAA (MW 3,000,
MACKLIN it)) in aqueous solution, shakes and shakes up 4 hours (37 DEG C, rpm=100) in shaking table, obtain the optical fiber of PAA aqueous solution group.
3, fiber laser arrays
Respectively by the optical fiber of the not closed group in step 2, the optical fiber of BSA isoelectric point confining liquid group, BSA aqueous solution group light
Fine, PSS aqueous solution treated optical fiber and the optical fiber of PAA aqueous solution group are packed into the matched flow cell of evanescent wave sensing platform institute
It is interior.During fiber laser arrays, optical fiber is rinsed with PBS buffer solution (solution obtained after hybridization solution is diluted 10 times with water) first
Until baseline is steady；After baseline is steady, peristaltic pump is opened, A20-Cy5.5 prepare liquid is pumped into sample cell (sample introduction 15s, pump respectively
10) speed is；Stop peristaltic pump later, A20-Cy5.5 prepare liquid is made to react 240s in reaction tank；It is again turned on peristaltic pump, is led to
Enter eluent (mass fraction 0.5%SDS, pH 1.9) 60s；It is finally passed through PBS buffer solution, until baseline steadily stops sampling afterwards,
Save data.
Testing result is as shown in Figure 3.By the signal in Fig. 3 as it can be seen that the signal value of BSA isoelectric point confining liquid group is significantly lower than
Other groups, BSA isoelectric point confining liquid of the invention can effectively reduce non-specific chain signal caused by Fibre Optical Sensor interface,
Its net signal is only the 6%~14% of other materials.It can be seen that BSA isoelectric point confining liquid can effectively inhibit sensing interface to non-spy
The non-specific adsorption of anisotropic chain.
Three, hybridization signal of detection BSA isoelectric point confining liquid treated the interface DNA to target segment
Other than guaranteeing non-specific chain to the low degree absorption of sensing interface, it is also necessary to which further verifying target hybridizes chain
The height of the signal caused by sensing interface.Selection is modified with the optical fiber of DNA, using CT-Cy5.5 as target hybrid nucleic acid (CT-
The nucleic acid of Cy5.5 and the optical fiber surface for being modified with DNA are in base complementrity, and sequence is as shown in table 1), with A20- Cy5.5 is control group
Nucleic acid (A20- Cy5.5 and the nucleic acid for the optical fiber surface for being modified with DNA is not complementary, and sequence is as shown in table 1).Specific step is as follows:
1, the preparation of prepare liquid
(1) A20-Cy5.5 prepare liquid
A20-Cy5.5 is dissolved in hybridization solution, A20-Cy5.5 prepare liquid is obtained, the concentration of A20-Cy5.5 is 10nM.
(2) CT-Cy5.5 prepare liquid
CT-Cy5.5 is dissolved in hybridization solution, CT-Cy5.5 prepare liquid is obtained, the concentration of CT-Cy5.5 is 10nM.
2, the processing of optical fiber
According to whether being closed with BSA isoelectric point confining liquid, it is divided into following two groups:
(1) optical fiber for being modified with DNA that (6) in the 1 of step 1 obtain closed group: is immersed in the closing of BSA isoelectric point
In liquid (BSA is dissolved in the solution that 0.1M citric acid-sodium citrate buffer solution obtains, and the concentration of BSA is 2mg/mL, pH 4.6), in
Concussion shakes up 4 hours (37 DEG C, rpm=100) in shaking table, obtains confining liquid treated optical fiber.
By confining liquid, treated that optical fiber is immersed in DNA denaturing liquid (urea liquid), in room temperature, the shaking table of 50-100rpm
Middle reaction 1 hour, obtains the optical fiber of closed group.
(2) not closed group: the optical fiber for being modified with DNA that (6) in the 1 of step 1 obtain is immersed in DNA denaturing liquid,
It is reacted 1 hour in room temperature, the shaking table of 50-100rpm, obtains the optical fiber of not closed group.
3, fiber laser arrays
It is matched that the optical fiber of the closed group in step 2 and the optical fiber of non-closed group are packed into evanescent wave sensing platform institute respectively
In flow cell.During fiber laser arrays, optical fiber is rinsed with PBS buffer solution until baseline is steady first；After baseline is steady, open
A20-Cy5.5 prepare liquid and CT-Cy5.5 prepare liquid are pumped into sample cell (sample introduction 15s, pump speed 10) respectively by peristaltic pump；Later
Stop peristaltic pump, A20-Cy5.5 prepare liquid is made to react 240s in reaction tank；It is again turned on peristaltic pump, is passed through eluent (quality
Score 0.5%SDS, pH 1.9) 60s；It is finally passed through PBS buffer solution, until baseline steadily stops sampling afterwards, saves data.
Target hybridize chain and non-targeted chain on the optical fiber of not closed group generated signal results as shown in figure 4, from figure
In it can be seen that target hybridization chain and the signal value of non-targeted chain are not significantly different.Target hybridization chain and non-targeted chain are sealing
It is as shown in Figure 5 to close generated signal results on the optical fiber of group.As can be seen from the figure: the signal value that target hybridizes chain is obviously high
In the signal value of non-targeted chain, wherein net signal caused by target hybridization chain is 28 of net signal caused by non-specific chain
Times.It can be seen that BSA isoelectric point confining liquid can effectively facilitate hybridizing for target nucleic acid chain and sensing interface, sensing interface is improved to mesh
Mark the specific adsorption of nucleic acid chains.
Four, the detection of the reusable ability at the interface DNA
After detecting use, the optical fiber of the closed group in step 3 is stored at room temperature and is buffered full of 10mM PBS
In the flow cell of liquid, after saving one day, the optical fiber of the closed group after saving one day is used again according to the method in above-mentioned steps three
CT-Cy5.5 prepare liquid is detected.
As a result as shown in Figure 6.As can be seen from Figure 6: being compared with the hybridization signal value before saving, after saving one day
The hybridization signal value that the optical fiber of closed group obtains only has 4% decline, illustrates the method according to the invention building and closed DNA circle
Face has renewable (reusable) ability.
Claims (9)
1. a kind of confining liquid is the solution obtained after mixing the buffer that BSA and pH is 4.4-4.8；
Concentration of the BSA in the confining liquid is 1-3 mg/mL；The buffer is citric acid-sodium citrate buffer solution
Or acetic acid-sodium acetate buffer solution.
2. confining liquid according to claim 1, it is characterised in that:
The pH is 4.6；
Concentration of the BSA in the confining liquid is 2 mg/mL.
The application during 3. confining liquid of any of claims 1 or 2 is any in following (1)-(8):
(1) interface DNA and/or the renewable interface DNA of building are constructed；
(2) the activated adoption site at the interface DNA is closed；
(3) interface DNA is improved to the specific adsorption and/or identification of target nucleic acid chain；
(4) interface DNA is reduced to the non-specific adsorption and/or identification of non-targeted nucleic acid chains；
(5) interface DNA is improved to the signal response of target nucleic acid chain；
(6) interface DNA is reduced to the signal response of non-targeted nucleic acid chains；
(7) promote hybridizing for target nucleic acid chain and the interface DNA；
(8) inhibit hybridizing for non-targeted nucleic acid chains and the interface DNA.
4. a kind of construction method at the interface DNA, includes the following steps:
1) there is the optical fiber of aldehyde radical to connect with surface active the DNA molecular of end modified amino using covalent method, obtain DNA modification
Optical fiber；
2) optical fiber of the DNA modification is closed with confining liquid of any of claims 1 or 2, the optical fiber after obtaining Seal treatment, i.e.,
Obtain the interface DNA.
5. according to the method described in claim 4, it is characterized by:
It further include being denaturalized the double chain DNA molecule of the optical fiber after the Seal treatment if the DNA molecular is double chain DNA molecule,
The step of restoring single stranded DNA interface.
6. according to the method described in claim 5, it is characterized by: the method for the denaturation is to impregnate the envelope with urea liquid
The optical fiber that closes that treated.
7. according to the method any in claim 4-6, it is characterised in that: the step 1) includes the following steps:
A1 optical fiber) is impregnated with Piranha solution, obtains optical fiber after the processing of Piranha solution；
A2 optical fiber after the Piranha solution is handled) is impregnated with triethoxysilane solution, after obtaining triethoxysilane processing
Optical fiber；
A3) triethoxysilane is impregnated treated optical fiber with glutaraldehyde water solution, obtain glutaraldehyde treated optical fiber；
A4) glutaraldehyde is impregnated treated optical fiber with the solution of the DNA molecular of the end modified amino, obtain DNA and repair
The optical fiber of decorations.
8. according to the method described in claim 7, it is characterized by:
The step A2) and the step A3) between further include that the triethoxysilane treated optical fiber is subjected to ultrasound
The step of cleaning；
The step A4) after further include that the optical fiber of the DNA modification is successively passed through into glycine solution and sodium cyanoborohydride
The step of aqueous solution processing.
9. in claim 4-8 any method it is any in following (B1)-(B6) in application:
(B1) interface DNA is improved to the specific adsorption and/or identification of target nucleic acid chain；
(B2) interface DNA is reduced to the non-specific adsorption and/or identification of non-targeted nucleic acid chains；
(B3) interface DNA is improved to the signal response of target nucleic acid chain；
(B4) interface DNA is reduced to the signal response of non-targeted nucleic acid chains；
(B5) promote hybridizing for target nucleic acid chain and the interface DNA；
(B6) inhibit hybridizing for non-targeted nucleic acid chains and the interface DNA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610804155.XA CN106399492B (en) | 2016-09-05 | 2016-09-05 | A method of renewable DNA hybridization interface is constructed with BSA isoelectric point confining liquid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610804155.XA CN106399492B (en) | 2016-09-05 | 2016-09-05 | A method of renewable DNA hybridization interface is constructed with BSA isoelectric point confining liquid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106399492A CN106399492A (en) | 2017-02-15 |
| CN106399492B true CN106399492B (en) | 2019-07-12 |
Family
ID=57998355
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610804155.XA Active CN106399492B (en) | 2016-09-05 | 2016-09-05 | A method of renewable DNA hybridization interface is constructed with BSA isoelectric point confining liquid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106399492B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111044716B (en) * | 2019-12-11 | 2023-11-14 | 迈克生物股份有限公司 | Sealing agent and sealing method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004016135A (en) * | 2002-06-18 | 2004-01-22 | Canon Inc | Liquid composition for dissolving probe |
| WO2010115044A2 (en) * | 2009-04-02 | 2010-10-07 | Fluidigm Corporation | Selective tagging of short nucleic acid fragments and selective protection of target sequences from degradation |
| CN104178568A (en) * | 2014-07-25 | 2014-12-03 | 清华大学 | Method for detecting target substance in to-be-detected sample based on fluorescent sensing analysis of aptamer probe |
| CN105256053A (en) * | 2015-11-13 | 2016-01-20 | 清华大学 | DNA-protein conjugate and application thereof in detecting mercury ion concentration |
-
2016
- 2016-09-05 CN CN201610804155.XA patent/CN106399492B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004016135A (en) * | 2002-06-18 | 2004-01-22 | Canon Inc | Liquid composition for dissolving probe |
| WO2010115044A2 (en) * | 2009-04-02 | 2010-10-07 | Fluidigm Corporation | Selective tagging of short nucleic acid fragments and selective protection of target sequences from degradation |
| CN104178568A (en) * | 2014-07-25 | 2014-12-03 | 清华大学 | Method for detecting target substance in to-be-detected sample based on fluorescent sensing analysis of aptamer probe |
| CN105256053A (en) * | 2015-11-13 | 2016-01-20 | 清华大学 | DNA-protein conjugate and application thereof in detecting mercury ion concentration |
Non-Patent Citations (6)
| Title |
|---|
| A rapid DNA biosensor for the molecular diagnosis of infectious disease;AngLim Chuaa et al.;《Biosensors and Bioelectronics》;20110303;3825-3831 |
| Adsorption and desorption of DNA on bovine serum albumin modified gold nanoparticles;Rao Fu et al.;《Colloids and Surfaces A: Physicochemical and Engineering Aspects》;20140111;326-329 |
| Bovine serum albumin as a means to immobilize DNA on a silver-plated bulk acoustic wave DNA biosensor;Hong Zhang et al.;《Talanta》;19981231;171-178 |
| Impact of surface chemistry and blocking strategies on DNA microarrays;Scott Taylor et al.;《Nucleic Acids Research》;20031231;1-19 |
| Optimization of the signal-to-noise ratio in south-western assays by using lipid-free BSA as blocking reagent;Athanasios G. et al.;《Nucleic Acids Research》;19921231;4365-4366 |
| Polyethylenimine-graft-Poly(ethylene glycol) Copolymers_ Influence of Copolymer Block Structure on DNA Complexation and Biological Activities as Gene Delivery System;Holger Petersen et al.;《Bioconjugate Chem》;20021231;845-854 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106399492A (en) | 2017-02-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ali et al. | Molecular imprinted polymer combined with aptamer (MIP-aptamer) as a hybrid dual recognition element for bio (chemical) sensing applications. Review | |
| CN100410664C (en) | Device of containing Nano structure for analysis or separation, preparation method and application | |
| Zhang et al. | Microchip electrophoresis based aptasensor for multiplexed detection of antibiotics in foods via a stir-bar assisted multi-arm junctions recycling for signal amplification | |
| ES2326637T3 (en) | ELECTROCHEMICAL DETECTION SYSTEM. | |
| CN105821132B (en) | A method of the specific Single stranded DNA concentration of Electrochemical Detection based on exonuclease and nucleic acid probe | |
| US20070031961A1 (en) | Biosensors and methods for their use | |
| CN110736777B (en) | electrochemical-ELISA immunosensor based on rolling circle amplification DNA enzyme and covalent organic framework | |
| CN107254550B (en) | SPR sensor for detecting HIV related gene and preparation and application thereof | |
| CN104004850A (en) | Paper-based micro-fluidic chip enhancement type chemiluminescence gene sensing method | |
| CN110305770B (en) | DNA nanostructure modified micro-fluidic chip for optical biosensing, and preparation and application thereof | |
| CN113406329A (en) | Universal aptamer colloidal gold lateral chromatography test paper for detecting small molecular substances | |
| CN113736853A (en) | Surface-enhanced Raman spectroscopy detection method for gene based on CRISPR/Cas12a protein | |
| US20230111586A1 (en) | Consumable for analyte detection | |
| CN106596662A (en) | Temperature-controllable electrochemical DNA biosensor and preparation method thereof | |
| CN105126793B (en) | A kind of preparation method based on single stranded DNA nucleic acid aptamers modification hybrid quartz capillary integral post | |
| CN106399492B (en) | A method of renewable DNA hybridization interface is constructed with BSA isoelectric point confining liquid | |
| CN102576026A (en) | Method for detecting and quantifying a target substance using a biochip | |
| Wang et al. | Fluorescent/electrochemical dual-signal response biosensing strategy mediated by DNAzyme-ferrocene-triggered click chemistry for simultaneous rapid screening and quantitative detection of Vibrio parahaemolyticus | |
| CN111197110A (en) | RPA-FLD method for detecting feline panleukopenia virus | |
| CN105256053A (en) | DNA-protein conjugate and application thereof in detecting mercury ion concentration | |
| CN114276915A (en) | Chip and kit for detecting novel coronavirus nucleic acid, preparation method and application | |
| CN103014117A (en) | Nanogold-polypeptide biological probe and preparation and application method | |
| CN105295447A (en) | Method for biological functionalization of silicon-based material surface | |
| Guan et al. | Surface modification of cellulose paper for quantum dot-based sensing applications | |
| JP4207528B2 (en) | Method for binding selective binding substances |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |