CN105784822B - A method of based on chitosan-graphene/gold nanoparticle composite film electrochemical DNA biosensor preparation and application - Google Patents
A method of based on chitosan-graphene/gold nanoparticle composite film electrochemical DNA biosensor preparation and application Download PDFInfo
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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
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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
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- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
Abstract
The invention discloses a kind of ionic liquid carbon paste electrodes (CILE) complex film modified using gold nano grain (AuNPs) and chitosan (CTS)-graphene (GR), as a kind of model electrochemical DNA sensor of platform construction, and it is applied to the measurement of staphylococcus aureus characterizing gene sequence.Ionic liquid 1- hexyl pyridine hexafluorophosphate and graphite powder are mixed to prepare CILE in certain proportion, probe ssDNA sequence hybridizes after being fixed on CTS-GR/AuNPs/CILE by Electrostatic Absorption with target ssDNA sequence, and the hybridization reaction with target sequence is detected as indicator using methylene blue.The experimental results showed that composite material has big specific surface area and good electric conductivity, the load capacity of ssDNA probe can be increased, to effectively improve electrochemical response signal.
Description
Technical field
The present invention relates to a kind of preparation of the electrochemical DNA biosensor of novel nanocomposite materials modified electrode and applications
Method
Background technique
DNA electrochemica biological sensor is one kind using ssDNA as recognition component, is hybridized by DNA sequence dna and carries out molecule
The biosensor of identification, the presence of target ssDNA sequence can be changed into detectable electric signal by it.With traditional label
Technique of gene detection compares, and DNA electrochemica biological sensor has the characteristics that more quick, easy, pollution-free, and avoids
Quantitatively and the problem of leading to false positive cannot be contaminated.Furthermore DNA molecular identification layer is sufficiently stable and is easy to regenerate, this makes
It obtains DNA electrochemica biological sensor to play an important role in field of genetic analysis, is widely used in medicine, food work
The fields such as industry, environmental monitoring, medicament research and development.The key step of electrochemistry ssDNA sensor preparation is as follows: (1) by probe ssDNA
Sequence is fixed on electrode surface；(2) hybridized with target ssDNA sequence；(3) instruction of hybridization reaction；(4) hybridization signal
Electrochemical Detection.Wherein the most key step is the fixation of probe ssDNA and the Electrochemical Detection of hybridization reaction, probe
SsDNA will affect the sensitivity of electrochemical sensor in the fixed amount of electrode surface and the detection method of hybridization reaction.
Carbon paste electrode is to be blended into paste using the graphite powder of electric conductivity and the adhesive of hydrophobicity, is subsequently filled in electricity
Manufactured one kind carbon electrode in pole pipe, it, which has, prepares simple, cheap, selective good, high sensitivity, the applicable model of current potential
The advantages that wide and surface is easily updated is enclosed, ionic liquid at room temperature refers to be made of zwitterion completely under room temperature and neighbouring temperature
Liquid substance, there is electrochemical window mouth width, conductivity high, thermal stability and good chemical stability.Ionic liquid can
To be mixed with Ionic Liquid Modified carbon paste electrode (CILE) as adhesive and graphite powder, the presence of ionic liquid can be effective
Ground changes the performance of traditional carbon paste electrode, can not only increase the stability of carbon paste electrode, but also can increase electrical efficiency.
Nano material has the characteristics that large specific surface area, bio-compatibility be good and good conductivity, is often applied to DNA electrification
The building of Biosensors.Gold nanoparticle has preferable conductive capability, can play the work of conducting wire or conductive channel
With acceleration biomolecule and interelectrode electronics transfer can accelerate the electron transport rate of biphase interface.Decorated by nano-gold electricity
The effective area that electrode can extremely be increased, can be improved DNA fixed amount when being used to prepare DNA sensor.
Graphene (graphene, GR) is with sp2The New Two Dimensional single layer of carbon atom layer crystal body of hydridization connection, it is unique
Two-dimensional structure can be used as the load of biomolecule and nano material.Since it has, electrochemical window mouth width, stability are high, compare
The advantages that surface area is big, electro catalytic activity is high, good conductivity has important application prospect in electrochemical sensor.
Chitosan is a kind of natural polymer for being largely present in nature, with nontoxic, film forming ability is strong, bio-compatible
Property good, high-intensitive, high adhesive ability the advantages that, the presence of a large amount of amino in surface can occur Electrostatic Absorption with DNA molecular and make
With, and be largely used to prepare electrochemical DNA biosensor.
Electrochemical deposition method is, by changing electrochemical conditions, such as to change current potential in containing the solution for wanting depositing ions
Either sedimentation time will depositing ions be uniformly deposited in cathode or anode template.Electrochemical deposition method has the advantage that
Such as: can react under room temperature, reaction temperature is low；It can control the thickness of deposition film by the charge number of monitoring transfer
Degree；It is formed and defect can control；Can in the substrate of various complicated shapes deposition film；It can carry out non-equilibrium phase
Deposition；Driving force can be accurately controlled；Expend the advantages that small.Electrochemical deposition method is according to deposition voltage and current system
Difference, constant current electrodeposition process, constant pressure electrodeposition process, pulse electrodeposition method, cyclic voltammetry etc. can be divided into.
Staphylococcus aureus is widespread in nature, it is a kind of important pathogenic microorganisms, by golden yellow Portugal
The infection of grape coccus can cause a variety of diseases, including food poisoning, pseudomembranous enteritis, scald skin syndrome, Endotoxin Shock synthesis
Disease, purulent inflammation and septicemia etc. can cause to human health and greatly threaten and lose.Staphylococcus aureus at present
Detection method generally comprise traditional method for cultivation of bacteria and biochemical identification, immunological method, fluorescence quantitative PCR method, fluorescence
Hybridization in situ (FISH) and gene chips etc..
This experiment is prepared for Ionic Liquid Modified carbon paste electrode (CILE) first, by the method for electrochemical deposition in CILE
Surface first deposits gold nano grain, is then prepared using drop-coating in its surface modification chitosan-graphene (CTS-GR) film
A kind of electrochemical DNA biosensor of chitosan-graphene/gold nano grain composite material modified electrode, further with methylene
Blue (MB) is indicator to detect its hybridization reaction with target sequence.
Summary of the invention
The object of the present invention is to provide a kind of specific sequence DNA, fast and accurately electrochemical detection method, specific method are
Nanogold particle is modified by electrochemical deposition method on Ionic Liquid Modified carbon paste electrode surface, then using drop-coating at it
Probe ssDNA is fixed on electrode surface, preparation one using Electrostatic Absorption by surface modification chitosan-graphene (CTS-GR) film
The novel electrochemical DNA biosensor of kind is that indicator is anti-with hybridizing for target ssDNA sequence to detect with methylene blue (MB)
It answers.
It is an object of the present invention to provide a kind of preparation methods of electrochemical DNA biosensor: Ionic Liquid Modified
The preparation of carbon paste electrode, using potentiostatic electrodeposition method in CILE Surface Creation nanogold particle, chitosan-graphene composite material
Then probe ssDNA is adsorbed onto the fixation for carrying out probe sequence on the electrode after modification, that is, made by drop coating to modified electrode surface
It obtains based on chitosan-graphene/gold nano grain composite modified electrode electrochemical DNA biosensor, specifically includes following procedure:
(1) graphite powder and ionic liquid n-hexyl pyridine hexafluorophosphate certain proportion mixed grinding uniformly obtain ionic liquid
Body modifies carbon paste, then inserts in vitreous electricity pole pipe Ionic Liquid Modified carbon paste and is compacted, and interpolation copper wire can be obtained as conducting wire
Ionic Liquid Modified carbon paste electrode (CILE).
(2) by gold chloride (HAuCl4) and sodium nitrate (NaNO3) be mixed in a certain proportion and prepare electrolyte, using CILE as work
Make electrode, platinized platinum is auxiliary electrode, and saturated calomel electrode is reference electrode, is received using potentiostatic electrodeposition method on the surface CILE
Rice gold particle (AuNPs) obtains AuNPs/CILE afterwards wash with distilled water, is dried in vacuo spare.
(3) dispersant liquid drop of CTS-GR composite material is applied to the surface AuNPs/CILE, obtains modified electrode after drying
CTS-GR/AuNPs/CILE。
(4) on modified electrode surface, uniform drop coating contains the buffer solution of probe ssDNA sequence, makes respectively after natural drying
It is rinsed 3 times with 0.5% dodecyl sodium sulfate (SDS) solution and secondary distilled water, to remove unadsorbed probe ssDNA sequence
Column, can be obtained the modified electrode (ssDNA/CTS-GR/AuNPs/CILE) for being fixed with probe sequence.
The preparation method, what is stated in step 1 is graphite powder and ionic liquid n-hexyl pyridine hexafluorophosphate
(HPPF6) quality be respectively 0.8g and 1.6g；It is preferred that milling time is 1.5h~3h, the preferably time is 2h；Institute is old in step 1
The glass bore stated is 3mm~5mm, and preferably glass bore is 4mm, the both ends sand paper (80 of glass tube#~1200#) beat
Ground smooth.
The preparation method, that stated in step 2 is HAuCl in mixed electrolytic solution4Concentration be 3.0mmol/L,
NaNO3Concentration be 0.1mol/L, mixed solution ultrasonic time be 0.5h~1h；Nanogold particle sedimentary condition: sedimentation potential
For -0.2V (vs.SCE), sedimentation time 30s；Distilled water is secondary distilled water.
The preparation method, stated in step 3 to be that 1.0mL 1.0mg/mL chitosan (CTS) is added in 1.0mg GR molten
CTS-GR dispersion liquid is obtained in liquid, after ultrasonic disperse, drop coating is 8.0 μ L in the drop coating amount of electrode surface.
The method, what is stated in step 4 is to contain 1.0 × 10 in electrode surface drop coating-6Mol/L probe ssDNA
The drop coating amount of the PBS buffer solution (50.0mmol/L, pH=7.0) of sequence is 10.0 μ L.
Probe sequence: 5'-TGGACG TGG CTT AGC GTATAT T-3'；
It is a further object to provide a kind of application methods of electrochemical DNA biosensor, including with
Lower step:
(1) different modifying electrode is immersed in methylene blue (MB) solution and adsorbs certain time, successively with 0.5% after taking-up
SDS solution and secondary distilled water are sufficiently washed, and to remove the MB of adsorption, study MB using Differential Pulse Voltammetry (DPV)
Electrochemical behavior on different modifying electrode.
(2) by the direct drop coating of PBS buffer solution of the target sequence containing various concentration in ssDNA/CTS-GR/AuNPs/
The surface CILE is rinsed after hybridization with 0.5% SDS solution and secondary distilled water, respectively to remove non-hybridized target sequence.It will
Electrode after hybridization, which immerses in MB solution, to be adsorbed in certain time, and 0.5%SDS solution and secondary distilled water are successively used after taking-up
Sufficiently washing is changed using Differential Pulse Voltammetry (DPV) measurement reduction peak current, obtains complementary ssDNA concentration and current value
Change the standard working curve of size, to study the sensitivity of electrochemical DNA biosensor.
(3) ssDNA/CTS-GR/AuNPs/CILE is hybridized from different ssDNA sequences, the electrode after hybridization is immersed
It adsorbs in certain time in MB solution, is successively sufficiently washed with 0.5%SDS solution and secondary distilled water after taking-up, using showing difference
Pulse voltammetry (DPV) measures and records the response current of MB to study the selectivity of constructed DNA sensor.
The application method, the PBS buffer solution (50.0mmol/L, pH=7.0) for the target ssDNA sequence stated
Drop coating amount is 8.0 μ L.
The application method, the MB solution concentration for absorption stated are 2.0 × 10-5Mol/L, adsorption time are
10min, the concentration of the Tris-HCl buffer solution (pH=7.0) containing MB are 50.0mmol/L, DPV determination condition are as follows: current potential
Increment 0.008V, pulse width 0.05s, pulse period 0.2s.
The application method, the different modifying electrode stated in step 1 is respectively ssDNA/CTS/CILE, ssDNA/
CTS/AuNPs/CILE, ssDNA/CTS-GR/CILE, ssDNA/CTS-GR/AuNPs/CILE, dsDNA/CTS-GR/AuNPs/
CILE。
The application method, the various concentration for the target sequence stated in step 2 are 0,1.0 × 10-13, 1.0 × 10-12, 1.0 × 10-11, 1.0 × 10-10, 1.0 × 10-9, 1.0 × 10-8, 1.0 × 10-7With 1.0 × 10-6mol/L。
The application method, the different ssDNA sequences stated in step 3 are respectively incomplementarity ssDNA sequence, three alkali
Base mispairing ssDNA sequence, single base mismatch ssDNA sequence and complementary target ssDNA sequence.
Complementary target ssDNA sequence: 5'-AATATA CGC TAA GCCACG TCCA-3'；
Single base mismatch ssDNA sequence: 5'-AAGATACGC TAA GCCACG TCC A-3'；
Three base mispairing ssDNA sequences: 5'-AAGATACGC TAC GCCACG TCTA-3'；
Incomplementarity ssDNA sequence: 5'-GCG GTT GAATCG GCGATG GGT G-3'.
The beneficial effects of the present invention are: the present invention provides a kind of specific sequence DNA fast and accurately new side of Electrochemical Detection
Method.Ionic Liquid Modified carbon paste electrode has preparation simply, cheap, and selectivity is good, and electrochemical window mouth width, surface is easy to more
Newly, easy to use, the advantages that electric conductivity is good.It can be with using the method for electrochemistry on Ionic Liquid Modified carbon paste electrode surface
Nanogold particle material is conveniently deposited, then using drop-coating in its surface modification CTS-GR film, finally fixes probe
SsDNA sequence obtains a kind of model electrochemical DNA sensor, can be with rapid and accurate determination specific objective DNA.The present invention selects
Nanogold particle surface drop coating chitosan-graphene composite material, makes full use of nanogold particle and graphene large specific surface area,
The characteristics of the advantages of conducting electricity very well and chitosan can be by direct Electrostatic Absorption fixed dna molecules is prepared a kind of novel
Electrochemical DNA biosensor, the experimental results showed that CTS-GR/AuNPs composite material is a kind of electricity for being preferably used for DNA sensor
Pole material.
Detailed description of the invention
Fig. 1: (A) different modifying electrode is in 1.0mmol/L K3[Fe(CN)6] and 0.5mol/L KCl mixed solution in follow
Ring voltammogram, sweep speed 100mV/s；(B) different modifying electrode is in 10.0mmol/L [Fe (CN)6]3-/4-And 0.1mol/L
Electrochemical AC impedance figure in KCl mixed solution, frequency 104~0.1Hz (a:CILE；B:AuNPs/CILE；C:CTS-
GR/CILE；D:CTS-GR/AuNPs/CILE).
Fig. 2: MB differential pulse voltammetry voltammogram (a:ssDNA/CTS/CILE, the b:ssDNA/CTS/ on different modifying electrode
AuNPs/CILE, c:ssDNA/CTS-GR/CILE, d:ssDNA/CTS-GR/AuNPs/CILE and e:dsDNA/CTS-GR/
AuNPs/CILE)。
Fig. 3: MB in ssDNA/CTS-GR/AuNPs/CILE and the differential pulse voltammetry on electrode after the hybridization of different ssDNA sequences
Voltammogram (a: before hybridization, b: after hybridizing with non-complementary sequence, c: after hybridizing with three base mispairing sequences, d: with single base mismatch
Sequence hybridization after and e: after hybridizing with target sequence).
Fig. 4: ssDNA/CTS-GR/AuNPs/CILE hybridize with the target ssDNA sequence of various concentration after MB show poor arteries and veins
Rushing voltammogram, (the target sequence concentration of a to i is followed successively by 0,1.0 × 10-13, 1.0 × 10-12, 1.0 × 10-11, 1.0 × 10-10,
1.0×10-9, 1.0 × 10-8, 1.0 × 10-7With 1.0 × 10-6Mol/L) illustration: reduction peak current and the logarithm of concentration it is linear
Relation curve.
Specific embodiment
In order to be more clearly understood that technology contents of the invention, spy lifts following embodiment and is described in detail, and purpose is only
It is to be best understood from the protection scope that the contents of the present invention are not intended to limit the present invention.
The preparation method of embodiment 1DNA sensor
(1) 1.6g graphite powder and 0.8g HPPF are accurately weighed with electronic balance6, by load weighted graphite powder and HPPF6It puts
Enter in agate mortar and grind uniformly, obtains Ionic Liquid Modified carbon paste, then slowly insert Ionic Liquid Modified carbon paste a length of
6cm, for internal diameter to be compacted in the vitreous electricity pole pipe of 4mm, piece fine copper wire of interpolation obtains Ionic Liquid Modified carbon paste electricity as conducting wire
Pole (CILE) is polished into mirror surface on clean polishing paper using preceding.
(2) HAuCl of certain mass is accurately weighed with electronic balance4With NaNO3, it is dissolved in 50ml secondary distilled water, ultrasound
It is uniformly dispersed to get 3.0mmol/L HAuCl4With 0.1mol/L NaNO3Mixed solution.Using CILE as working electrode, platinized platinum is
Auxiliary electrode, saturated calomel electrode are reference electrode, deposit nanogold in CILE electrode surface using the method for potentiostatic electrodeposition
Particle, sedimentation potential are -0.2V (vs.SCE), and sedimentation time 30s can obtain one layer of uniform nanogold on the surface CILE
Particle (AuNPs) obtains AuNPs/CILE wash with distilled water after taking-up, be dried in vacuo spare.
(3) 1.0mg GR is accurately weighed with electronic balance to be scattered in 1.0mL 1.0mg/mL chitosan (CTS) solution, surpass
Sound is uniformly dispersed to get CTS-GR dispersion liquid, and 8.0 μ L dispersion liquid of drop coating, is modified after natural drying on AuNPs/CILE
Electrode CTS-GR/AuNPs/CILE.
(4) on the surface CTS-GR/AuNPs/CILE, uniform 10.0 μ L of drop coating contains 1.0 × 10-6Mol/L probe ssDNA sequence
The PBS buffer solution (50.0mmol/L, pH=7.0) of column, probe ssDNA can be by the negative electricity and band of DNA phosphoric acid backbone just
Electrical CTS polycation film Electrostatic Absorption is to be fixed on modified electrode surface.After natural drying respectively using 0.5%
SDS solution and secondary distilled water are rinsed, and to remove unadsorbed probe ssDNA sequence, be can be obtained and are fixed with probe ssDNA sequence
The electrode (ssDNA/CTS-GR/AuNPs/CILE) of column.
The electrochemical Characterization of embodiment 2DNA sensor
Different modifying electrode is prepared according to example 1, is investigated electrode interface using cyclic voltammetry and AC impedence method
Variation, and to electrode surface carry out electrochemical Characterization, as a result as shown in Figure 1.Cyclic voltammetry experiment is in 1.0mmol/L K3[Fe
(CN)6] and 0.5mol/L KCl mixed solution in carry out, potential window be -0.3~0.7V, scanning speed 100mV/s；Electricity
Chemical AC impedance experiment is in 10.0mmol/L K3[Fe(CN)6]/K4[Fe(CN)6] and 0.1mol/L KCl mixed solution in into
Row, frequency range are 0.1~104Hz；Wherein working electrode is respectively (a:CILE；B:AuNPs/CILE；C:CTS-GR/CILE；
D:CTS-GR/AuNPs/CILE), platinized platinum is auxiliary electrode, and saturated calomel electrode is reference electrode.From Figure 1A it can be seen that GR-
CTS and AuNPs synergistic effect is capable of increasing electrode effective area and improves the electric conductivity of electrode, accelerates the speed of electrochemical reaction
Rate is to increase current value.From Figure 1B it can be seen that synergistic effect makes CTS-GR/AuNPs/CILE between AuNPs and CTS-GR
The electric conductivity at interface increases, and impedance value reduces step by step.
The detection of electrochemical behavior of the embodiment 3MB on different modifying electrode
Different modifying electrode is prepared according to example 1, then 2.0 × 10-58min is impregnated in the MB solution of mol/L concentration,
It is successively sufficiently washed with 0.5%SDS solution and secondary distilled water after taking-up, to remove non-hybridized ssDNA sequence, using showing difference
Pulse voltammetry studies electrochemical behavior of the MB on different modifying electrode, as a result as shown in Figure 2.Electrolyte is 50.0mmol/L
Tris-HCl buffer solution, platinized platinum are auxiliary electrode, and saturated calomel electrode is reference electrode, and different modified electrodes is work electricity
Pole (a:ssDNA/CTS/CILE, b:ssDNA/CTS/AuNPs/CILE, c:ssDNA/CTS-GR/CILE, d:ssDNA/CTS-
GR/AuNPs/CILE, e:dsDNA/CTS-GR/AuNPs/CILE), potential window is 0.2~-0.7V.As can be seen from Figure 2
After various nano materials are added into CTS film, the peak current of MB is gradually increased, this is because the addition of decorative material makes electrode
Effective area is gradually increased, and then increases with it the adsorbance of DNA, so that transducer sensitivity be made to improve.Due to probe
SsDNA is that electrode surface is fixed on by Electrostatic Absorption, and MB is easy intercalation in the double-stranded big minor groove of dsDNA after hybridization
In, so that MB is greater than the signal on ssDNA modified electrode in the signal on dsDNA modified electrode.
The detection of 4 sensor selectivity of embodiment
SsDNA/CTS-GR/AuNPs/CILE is hybridized from different ssDNA sequences, then 2.0 × 10-5Mol/L is dense
8min is impregnated in the MB solution of degree, is successively sufficiently washed with 0.5%SDS solution and secondary distilled water after taking-up, using showing poor arteries and veins
It rushes voltammetric determination and records the response current of MB to study the selectivity of constructed DNA sensor, as a result as shown in Figure 3.
Electrolyte is 50.0mmol/L Tris-HCl buffer solution, and platinized platinum is auxiliary electrode, and saturated calomel electrode is reference electrode, point
Not to be added dropwise 1.0 × 10-6Mol/L different ssDNA sequence (b: incomplementarity ssDNA sequence, c: three base mispairing ssDNA sequences,
D: single base mismatch ssDNA sequence, e: target ssDNA) electrode after hybridization is working electrode, potential window is 0.2~-
0.7V.The electrochemical DNA biosensor constructed as can be seen from Figure 3 has good selectivity different sequences, can distinguish
The mismatch of different bases.
The detection of 5 transducer sensitivity of embodiment
On the sensor prepared, 10.0 μ L are contained to the PBS buffer solution of various concentration target ssDNA sequence
(50.0mmol/L, pH=7.0) directly drop coating is on the surface ssDNA/CTS-GR/AuNPs/CILE, after hybridizing at room temperature respectively
It is rinsed with 0.5% SDS solution and secondary distilled water, to remove non-hybridized target sequence, then in 2.0 × 10-5mol/L
8min is impregnated in the MB solution of concentration, is successively sufficiently washed with 0.5% SDS solution and secondary water after taking-up, uses differential pulse voltammetry
Voltammetry detects electric current, as a result as shown in Figure 4.Electrolyte is 50.0mmol/L Tris-HCl buffer solution, and platinized platinum is auxiliary electricity
Pole, saturated calomel electrode are reference electrode, respectively various concentration target ssDNA sequence (the target sequence concentration of a to i is added dropwise
It is followed successively by 0,1.0 × 10-13, 1.0 × 10-12, 1.0 × 10-11, 1.0 × 10-10, 1.0 × 10-9, 1.0 × 10-8, 1.0 × 10-7With
1.0×10-6Mol/L) modified electrode after hybridization reaction is working electrode, and potential window is 0.2~-0.7V.It can from Fig. 4
The reduction peak current value of MB increases with the increase of target ssDNA sequence concentration out, and 1.0 × 10-13~1.0 × 10- 6With the logarithm of target ssDNA sequence concentration in good linear relationship, equation of linear regression in the concentration range of mol/L
For Δ I (μ A)=1.93log [c/ (mol/L)]+26.04 (n=8, γ=0.998) (wherein c is the concentration of target sequence,
mol/L；Δ I is the peak current difference of hybridization front and back MB, μ A), detection is limited to 3.33 × 10-14(3 σ, σ are blank solution to mol/L
Standard deviation).
Claims (1)
1. it is a kind of based on chitosan-graphene/gold nano grain composite modified electrode electrochemical DNA biosensor in golden yellow Portugal
The application of grape coccus characteristic sequence context of detection, it is characterised in that
The preparation method of the electrochemical DNA biosensor includes the following steps:
(1) graphite powder and ionic liquid n-hexyl pyridine hexafluorophosphate are mixed in a certain proportion grinding and uniformly obtain ionic liquid
Modify carbon paste, then will Ionic Liquid Modified carbon paste insert vitreous electricity pole pipe in be compacted, interpolation copper wire as conducting wire, can be obtained from
Sub- liquid decorating carbon paste electrode (CILE)；
(2) gold chloride and sodium nitrate solution are mixed in a certain proportion and prepare electrolyte, CILE is working electrode, and platinized platinum is auxiliary
Electrode, saturated calomel electrode are reference electrode, obtain gold nano grain on the surface CILE using potentiostatic electrodeposition method, use distilled water
AuNPs/CILE is obtained after cleaning, it is spare after vacuum drying；
(3) dispersion liquid of 8.0 μ L CTS-GR composite materials is applied to the surface AuNPs/CILE, obtains modified electrode after drying
CTS-GR/AuNPs/CILE；
(4) buffer solution for containing probe ssDNA sequence in the uniform drop coating of CTS-GR/AuNPs/CILE electrode surface, dries in the air naturally
It is rinsed 3 times using 0.5% dodecyl sodium sulfate (SDS) solution and secondary distilled water respectively after dry, to remove unadsorbed spy
The electrode ssDNA/CTS-GR/AuNPs/CILE for being fixed with probe sequence can be obtained in needle ssDNA sequence；
Step (1) the glass electrode bore is 3mm~5mm, both ends sand paper 80#~1200#It polishes smooth, the grinding
Time be 1.5h~3h, the mass ratio of graphite powder and ionic liquid n-hexyl pyridine hexafluorophosphate is 2:1；
The concentration of gold chloride is 3.0mmol/L in step (2), and the concentration of sodium nitrate is 0.1mol/L, and potentiostatic electrodeposition method is sunk
Product current potential is -0.2V (vs.SCE), sedimentation time 30s；
The dispersion liquid of CTS-GR composite material is obtained in 1.0mL chitosan by 1.0mg GR ultrasonic disperse in step (3)；
Contain 1.0 × 10 in electrode surface drop coating in step (4)-6The PBS buffer solution of the probe ssDNA sequence of mol/L, PBS
The concentration of buffer solution is 50.0mmol/L, and pH 7.0, drop coating amount is 10.0 μ L；The sequence of the probe ssDNA is 5'-TGG
ACG TGG CTTAGC GTATAT T-3'；
It is described application the following steps are included:
(1) different modifying electrode is immersed in methylene blue (MB) solution and adsorbs certain time, 0.5%SDS is successively used after taking-up
Solution and secondary distilled water are sufficiently washed, to remove the MB of adsorption, using Differential Pulse Voltammetry (DPV) research MB not
With the electrochemical behavior on modified electrode；The different modifying electrode is respectively ssDNA/CTS/CILE, ssDNA/CTS/
AuNPs/CILE, ssDNA/CTS-GR/CILE, ssDNA/CTS-GR/AuNPs/CILE, dsDNA/CTS-GR/AuNPs/CILE；
(2) by the direct drop coating of PBS buffer solution of the target sequence containing various concentration in ssDNA/CTS-GR/AuNPs/CILE
Surface is rinsed respectively with 0.5% SDS solution and secondary distilled water after hybridization, to remove non-hybridized target sequence, will be hybridized
Electrode afterwards, which immerses in MB solution, adsorbs certain time, is successively sufficiently washed with 0.5%SDS solution and secondary distilled water after taking-up
It washs, is changed using Differential Pulse Voltammetry (DPV) measurement reduction peak current, obtain complementary ssDNA concentration and current value variation greatly
Small standard working curve, to study the sensitivity of electrochemical DNA biosensor；The various concentration of the target sequence is 0,1.0
×10-13, 1.0 × 10-12, 1.0 × 10-11, 1.0 × 10-10, 1.0 × 10-9, 1.0 × 10-8, 1.0 × 10-7With 1.0 × 10- 6mol/L；
(3) ssDNA/CTS-GR/AuNPs/CILE is hybridized from different ssDNA sequences, it is molten that the electrode after hybridization is immersed MB
Certain time is adsorbed in liquid, is successively sufficiently washed with 0.5%SDS solution and secondary distilled water after taking-up, is lied prostrate using differential pulse voltammetry
Peace method (DPV) measures and records the response current of MB to study the selectivity of constructed DNA sensor；It is described different
SsDNA sequence is respectively incomplementarity ssDNA sequence, three base mispairing ssDNA sequences, single base mismatch ssDNA sequence and complementation
Target ssDNA sequence；
Complementary target ssDNA sequence: 5'-AATATA CGC TAA GCCACG TCCA-3'；
Single base mismatch ssDNA sequence: 5'-AAGATACGC TAA GCCACG TCC A-3'；
Three base mispairing ssDNA sequences: 5'-AAGATACGC TAC GCCACG TCTA-3'；
Incomplementarity ssDNA sequence: 5'-GCG GTT GAATCG GCGATG GGT G-3'.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610240528.5A CN105784822B (en) | 2016-04-19 | 2016-04-19 | A method of based on chitosan-graphene/gold nanoparticle composite film electrochemical DNA biosensor preparation and application |
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| JP2019041626A (en) * | 2017-08-30 | 2019-03-22 | 株式会社東芝 | Sensor, reagent, probe molecule production method, sensor production method, and polymer molecule production method |
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| CN109596697B (en) * | 2018-12-21 | 2021-08-31 | 嘉兴学院 | Electrochemical immunosensor and preparation method and application thereof |
| CN110568036A (en) * | 2019-08-16 | 2019-12-13 | 成都理工大学 | Method for electrochemically detecting mercury ions based on nucleic acid dye |
| CN110672688B (en) * | 2019-08-30 | 2021-11-12 | 佛山市南海区里水镇经济促进局 | Electrochemical biosensor for detecting tetrabromobisphenol A bis (2-hydroxyethyl) ether and preparation method and application thereof |
| CN112588277A (en) * | 2020-11-06 | 2021-04-02 | 兰州城市学院 | Preparation method of solid-phase micro-extraction fiber based on nanogold/chitosan coating |
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