CN109355356B - DNA biosensor and method for applying same to DNA determination - Google Patents
DNA biosensor and method for applying same to DNA determination Download PDFInfo
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- CN109355356B CN109355356B CN201811239868.1A CN201811239868A CN109355356B CN 109355356 B CN109355356 B CN 109355356B CN 201811239868 A CN201811239868 A CN 201811239868A CN 109355356 B CN109355356 B CN 109355356B
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- 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
Abstract
The invention relates to a DNA biosensor and a method for determining DNA by using the same. The key point of the invention is that the substrate PDMS is a flexible material to determine the flexibility of the electrode, the silver nanowires have lower resistance to determine the good conductivity of the electrode, the gold nanoparticles on the silver nanowires are closely arranged to provide a larger surface area for fixing the sulfhydryl single-stranded DNA on gold, and the good conductivity and the higher active surface area are the guarantee for preparing the high-sensitivity electrochemical sensor. The invention adopts the method of dripping to prepare the silver nanowire conducting layer which is evenly distributed, and then adopts the method of electrochemical deposition to deposit the nano-gold particles on the silver nanowire, and has the greatest advantages of simplicity, easy implementation, no need of synthesizing nano-gold, solving the problem that the nano-gold is difficult to prepare, and simultaneously solving the problem that the active surface area of the gold in the electrochemical detection is small.
Description
Technical Field
The invention relates to the technical field of biosensor preparation and electrochemical analysis, in particular to a preparation and determination method of a novel electrode for DNA determination.
Background
DNA (deoxyribonic acid) deoxyribonucleic acid, which is an important substance for regulating biogenetic. The specific nucleotide sequence in the DNA fragment determines the genetic characteristics of the gene, and DNA variation may cause great harm to organisms. Therefore, it is necessary to establish a simple, rapid, sensitive and highly specific DNA detection method. At present, common DNA detection technologies include gene chip technology, DNA sequencing technology, fluorescence spectroscopy, surface enhanced raman scattering, electrochemical detection, and the like. The gene chip technology, the DNA sequencing technology, the fluorescence spectrum and the surface enhanced Raman scattering need complicated and expensive instruments, have high requirements on the operation of personnel, take time for detection, are large and are inconvenient for field detection. Most of DNA detection electrodes constructed in electrochemistry are hard materials, the size and the shape are not easy to control, and the wide application of the electrodes is limited. The composite metal of the nano material is less applied, so that the detection sensitivity is not high.
Disclosure of Invention
The invention aims to provide a novel silver nanowire/nanogold flexible composite electrode (AuNPs/AgNWs/PDMS electrode) with flexibility and high sensitivity, a DNA biosensor prepared by applying the electrode and an analysis method for determining DNA by applying the electrode.
In order to solve the above technical problems, the present invention provides a DNA biosensor. The DNA biosensor is characterized in that sulfhydryl DNA is fixed on an AuNPs/AgNWs/PDMS electrode.
The preparation method of the AuNPs/AgNWs/PDMS electrode comprises the following steps:
using PDMS as a flexible substrate, modifying a hydrophilic surface layer on the surface of the PDMS by using a mixed solution of 2% polyvinyl alcohol (PVA) and 5% glycerol (Gly), uniformly coating a silver nanowire conducting layer on the hydrophilic modification layer, and using KAuCl4And H2SO4The mixed solution is used as electrolyte, an Ag/AgCl electrode is used as a reference electrode, a platinum wire is used as a counter electrode, a silver nanowire conducting layer is used as a working electrode, and gold is electrochemically deposited on the conducting layer by adopting a chronoamperometry method to prepare the AuNPs/AgNWs/PDMS electrode.
Preferably, the KAuCl is4And H2SO4In mixed electrolytes, KAuCl4The concentration is 0.01-0.05 mol/L, H2SO4The concentration is 0.1-0.5 mol/L.
Preferably, the potential of the electrochemically deposited gold is-0.2V, and the deposition time is 400-1600 s.
The preparation method of the DNA biosensor comprises the following steps:
(1) immersing the AuNPs/AgNWs/PDMS electrode into PBS solution containing a sulfhydryl single-stranded DNA (SH-ssDNA) probe for full reaction, then putting the electrode into blank PBS, stirring at constant temperature to remove non-specifically adsorbed DNA molecules, then immersing into Mercaptoethanol (MCH), further eliminating the non-specifically adsorbed DNA molecules and obtaining a relatively compact DNA monomolecular layer, then washing with the PBS solution and secondary distilled water, and drying with nitrogen;
(2) soaking a DNA probe electrode modified by Mercaptoethanol (MCH) into PBS (phosphate buffer solution) of target DNA with different concentrations, performing thermostatic water bath and stirring to obtain a double-stranded DNA (dsDNA) modified electrode, repeatedly washing secondary water and the PBS to remove non-hybridized target DNA adsorbed on the surface, and drying by nitrogen for later use.
Preferably, the thiol single-stranded DNA concentration is 10-7～10-8Double-stranded DNA concentration of 10-7～10-9。
It is another object of the present invention to provide a method for measuring DNA using the above DNA biosensor, wherein the measurement of cyclic voltammetric signal of ssDNA or dsDNA modified electrode is performed in 1mM Methylene Blue (MB) in PBS (0.1M KCl). The ssDNA or dsDNA modified electrode was soaked in 1mM Methylene Blue (MB) solution for 20min to ensure sufficient electrostatic binding of the Methylene Blue (MB) and DNA strands. Then, washing was repeated with double distilled water, PBS (0.1M, pH 7.4) solution to remove the molecules adsorbed on the electrode surface. Then, the cyclic voltammetric signal in PBS (0.1M, pH 7.4) solution was recorded. Preferably, the cyclic voltammetry experiment parameters are that the potential range is-0.6V; the sweep rate was 100 mV/s.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides the flexible electrode with good conductivity and high detection sensitivity. The electrode has simple preparation process and is easy to realize batch production. The key point of the invention is that the substrate PDMS is a flexible material to determine the flexibility of the electrode, and the silver nanowires have lower resistance to determine the good conductivity of the electrode. The gold nanoparticles on the silver nanowire are closely arranged, so that a large surface area is provided for fixing the sulfhydryl single-stranded DNA on gold. The good conductivity and the higher active surface area are the guarantee for preparing the high-sensitivity electrochemical sensor. The invention adopts the method of drop coating to prepare the silver nanowire conducting layer which is evenly distributed, and then adopts the method of electrochemical deposition to deposit the nano-gold particles on the silver nanowire, thereby having the greatest advantages of simplicity, easy implementation, no need of nano-gold synthesis and solving the problem that the nano-gold is difficult to prepare. Meanwhile, the problem of small gold active surface area in electrochemical detection is solved.
Drawings
The invention is further described with reference to the following drawings and detailed description.
FIG. 1 is a process flow diagram of the present invention;
FIG. 2 is an SEM image of a flexible silver nanowire/nanogold composite electrode prepared by the invention;
FIG. 3 is a diagram for characterizing electrochemical properties of a flexible silver nanowire/nanogold composite electrode in sulfuric acid;
FIG. 4 shows the impedance comparison results of the silver nanowire electrode and the silver nanowire/nanogold composite electrode according to the invention;
FIG. 5 shows comparison results of cyclic voltammetry between a silver nanowire electrode and a silver nanowire/nanogold composite electrode according to the invention.
Detailed Description
The technical solutions of the present invention are further described below with reference to the drawings and the specific embodiments, but the present invention is not limited to the embodiments in any way. The experimental methods described in the examples are all conventional methods unless otherwise specified; unless otherwise indicated, the experimental reagents and materials were commercially available.
As shown in FIG. 1, the process flow diagram of the present invention is shown. The method comprises the steps of taking Polydimethylsiloxane (PDMS) as a flexible substrate, modifying a hydrophilic layer on the surface of the PDMS by using a mixed solution of polyvinyl alcohol (PVA) and glycerol (Gly), uniformly coating a silver nanowire conducting layer on the hydrophilic layer, and depositing gold nanoparticles on the conducting layer by using an electrochemical deposition method. Preparing a novel silver nanowire/nanogold electrode, fixing a DNA probe with sulfydryl on nanogold to construct a DNA biosensor, and quickly detecting the DNA concentration by using Methylene Blue (MB) as an indicator.
Example 1
Modifying the surface of PDMS with a mixed solution of PVA (2%) and Gly (5%) for 3 times to obtain a hydrophilic surface layer, uniformly coating a silver nanowire conductive layer on the hydrophilic surface layer, and using KAuCl4(0.01mol/L) and H2SO4The (0.5mol/L) mixed solution is used as electrolyte, an Ag/AgCl electrode is used as a reference electrode, a platinum wire is used as a counter electrode, and a silver nanowire conducting layer is used as a working electrode. Performing nano gold deposition by using a timing current method, fixing the deposition voltage to be-0.2V and adopting the deposition time of 400sAnd (4) accumulating. And (3) depositing a layer of gold flower-shaped nanogold on the conducting layer through electrochemical deposition to prepare the silver nanowire/nanogold composite electrode.
Immersing an electrode into a bath containing 10-7And (3) reacting in PBS (phosphate buffer solution) of a thiol single-stranded DNA (SH-ssDNA) probe for 12 hours at room temperature in a dark place. The electrode was then placed in blank PBS and stirred at 37 ℃ for 1h to remove non-specifically adsorbed DNA molecules. Then immersed in 1mM MCH for 1h to further eliminate non-specifically adsorbed DNA molecules and obtain a relatively dense DNA monolayer, which was then washed clean with PBS solution and double distilled water and dried under nitrogen for further use.
Immersing the MCH-modified DNA probe electrode into the solution with the concentration of 10-7In PBS solution of the target DNA, stirring for 1.5h in thermostatic water bath at 37 ℃ to obtain a double-stranded DNA (dsDNA) modified electrode. The secondary water and PBS solution are repeatedly washed to remove the non-hybridized target DNA adsorbed on the surface, and dried by nitrogen gas for standby.
(3) The determination of cyclic voltammetric signals was performed on ssDNA or dsDNA modified electrodes in 1mM MB in PBS (0.1M KCl). The ssDNA or dsDNA modified electrode was soaked in 1mM methylene blue solution for 20min to ensure sufficient electrostatic binding of the methylene blue to the DNA strands. Then, the electrode surface was washed repeatedly with redistilled water, PBS (0.1M, pH 7.4) to remove adsorbed molecules, and the cyclic voltammetry signal in the deoxygenated PBS (0.1M, pH 7.4) solution was recorded. CV experimental parameters are potential-0.6V; the sweep rate was 100 mV/s.
Example 2
Modifying the surface of PDMS with a mixed solution of PVA (2%) and Gly (5%) for 3 times to obtain a hydrophilic surface layer, uniformly coating a silver nanowire conductive layer on the hydrophilic surface layer, and using KAuCl4(0.01mol/L) and H2SO4The (0.5mol/L) mixed solution is used as electrolyte, an Ag/AgCl electrode is used as a reference electrode, a platinum wire is used as a counter electrode, and a silver nanowire conducting layer is used as a working electrode. And (3) performing nano-gold deposition by adopting a chronoamperometry, fixing the deposition voltage to be-0.2V and adopting the deposition time of 800 s. And (3) depositing a layer of gold flower-shaped nanogold on the conducting layer through electrochemical deposition to prepare the silver nanowire/nanogold composite electrode.
Immersing the electrodeInto contains 10-7And (3) reacting in PBS (phosphate buffer solution) of a thiol single-stranded DNA (SH-ssDNA) probe for 12 hours at room temperature in a dark place. The electrode was then placed in blank PBS and stirred at 37 ℃ for 1h to remove non-specifically adsorbed DNA molecules. Then immersed in 1mM MCH for 1h to further eliminate non-specifically adsorbed DNA molecules and obtain a relatively dense DNA monolayer, which was then washed clean with PBS solution and double distilled water and dried under nitrogen for further use.
Immersing the MCH-modified DNA probe electrode into the solution with the concentration of 10-8In PBS solution of the target DNA, stirring for 1.5h in thermostatic water bath at 37 ℃ to obtain a double-stranded DNA (dsDNA) modified electrode. The secondary water and PBS solution are repeatedly washed to remove the non-hybridized target DNA adsorbed on the surface, and dried by nitrogen gas for standby.
(3) The determination of cyclic voltammetric signals was performed on ssDNA or dsDNA modified electrodes in 1mM MB in PBS (0.1M KCl). The ssDNA or dsDNA modified electrode was soaked in 1mM methylene blue solution for 20min to ensure sufficient electrostatic binding of the methylene blue to the DNA strands. Then, the electrode surface was washed repeatedly with redistilled water, PBS (0.1M, pH 7.4) to remove adsorbed molecules, and the cyclic voltammetry signal in the deoxygenated PBS (0.1M, pH 7.4) solution was recorded. CV experimental parameters are potential-0.6V; the sweep rate was 100 mV/s.
Example 3
Modifying the surface of PDMS with a mixed solution of PVA (2%) and Gly (5%) for 3 times to obtain a hydrophilic surface layer, uniformly coating a silver nanowire conductive layer on the hydrophilic surface layer, and using KAuCl4(0.01mol/L) and H2SO4The (0.5mol/L) mixed solution is used as electrolyte, an Ag/AgCl electrode is used as a reference electrode, a platinum wire is used as a counter electrode, and a silver nanowire conducting layer is used as a working electrode. And (3) performing nano-gold deposition by adopting a timing current method, fixing the deposition voltage to be-0.2V and adopting the deposition time of 1600 s. And (3) depositing a layer of gold flower-shaped nanogold on the conducting layer through electrochemical deposition to prepare the silver nanowire/nanogold composite electrode.
Immersing an electrode into a bath containing 10-8And reacting for 12 hours in PBS solution of a thiol single-stranded DNA (SH-ssDNA) probe at room temperature in the dark. Then, the electrode was placed in blank PBS and stirred at a constant temperature of 37 ℃ for 1 hour to removeNon-specifically adsorbed DNA molecules. Then immersed in 1mM MCH for 1h to further eliminate non-specifically adsorbed DNA molecules and obtain a relatively dense DNA monolayer, which was then washed clean with PBS solution and double distilled water and dried under nitrogen for further use.
Immersing the MCH-modified DNA probe electrode into the solution with the concentration of 10-9In PBS solution of the target DNA, stirring for 1.5h in thermostatic water bath at 37 ℃ to obtain a double-stranded DNA (dsDNA) modified electrode. The secondary water and PBS solution are repeatedly washed to remove the non-hybridized target DNA adsorbed on the surface, and dried by nitrogen gas for standby.
(3) The determination of cyclic voltammetric signals was performed on ssDNA or dsDNA modified electrodes in 1mM MB in PBS (0.1M KCl). The ssDNA or dsDNA modified electrode was soaked in 1mM methylene blue solution for 20min to ensure sufficient electrostatic binding of the methylene blue to the DNA strands. Then, the electrode surface was washed repeatedly with redistilled water, PBS (0.1M, pH 7.4) to remove adsorbed molecules, and the cyclic voltammetry signal in the deoxygenated PBS (0.1M, pH 7.4) solution was recorded. CV experimental parameters are potential-0.6V; the sweep rate was 100 mV/s.
Example 4
Modifying the surface of PDMS with a mixed solution of PVA (2%) and Gly (5%) for 3 times to obtain a hydrophilic surface layer, uniformly coating a silver nanowire conductive layer on the hydrophilic surface layer, and using KAuCl4(0.01mol/L) and H2SO4The (0.5mol/L) mixed solution is used as electrolyte, an Ag/AgCl electrode is used as a reference electrode, a platinum wire is used as a counter electrode, and a silver nanowire conducting layer is used as a working electrode. And (3) performing nano-gold deposition by adopting a chronoamperometry, fixing the deposition voltage to be-0.2V and adopting the deposition time of 800 s. And (3) depositing a layer of gold flower-shaped nanogold on the conducting layer through electrochemical deposition to prepare the silver nanowire/nanogold composite electrode.
Immersing an electrode into a bath containing 10-7And (3) reacting in PBS (phosphate buffer solution) of a thiol single-stranded DNA (SH-ssDNA) probe for 12 hours at room temperature in a dark place. The electrode was then placed in blank PBS and stirred at 37 ℃ for 1h to remove non-specifically adsorbed DNA molecules. Then immersed in 1mM MCH for 1h to further eliminate non-specifically adsorbed DNA molecules and obtain a relatively dense DNA monomolecular layer, followed by PBS solution and redistilled waterAnd (5) washing, and drying with nitrogen for later use.
The DNA probe electrode modified with MCH was immersed at a concentration of 10-9In PBS solution of the target DNA, stirring for 1.5h in thermostatic water bath at 37 ℃ to obtain a double-stranded DNA (dsDNA) modified electrode. The secondary water and PBS solution are repeatedly washed to remove the non-hybridized target DNA adsorbed on the surface, and dried by nitrogen gas for standby.
(3) The determination of cyclic voltammetric signals was performed on ssDNA or dsDNA modified electrodes in 1mM MB in PBS (0.1M KCl). The ssDNA or dsDNA modified electrode was soaked in 1mM methylene blue solution for 20min to ensure sufficient electrostatic binding of the methylene blue to the DNA strands. Then, the electrode surface was washed repeatedly with redistilled water, PBS (0.1M, pH 7.4) to remove adsorbed molecules, and the cyclic voltammetry signal in the deoxygenated PBS (0.1M, pH 7.4) solution was recorded. CV experimental parameters are potential-0.6V; the sweep rate was 100 mV/s.
As shown in fig. 2, SEM topographic structure image of the electrode. The nano gold is deposited on the silver nano wire uniformly and is in close lap joint.
As shown in fig. 3, the electrochemical behavior of the gold (a)800s, (b)1200s, and (c)1600s deposited composite electrode in sulfuric acid shows that the composite electrode active surface area of the gold (a) 1600s deposited is the largest.
The following experiments all used a deposited gold 1600s composite electrode.
FIG. 4 shows silver nanowire/nanogold composite electrode (a) and dsDNA-modified composite electrode (b). Through impedance analysis, after the dsDNA is modified by the composite electrode, the small semicircle of the high-frequency area is increased, and the slope of the low-frequency area is reduced. It is known that DNA is immobilized on a composite electrode.
FIG. 5 is a cyclic voltammogram of a silver nanowire/nanogold composite electrode (a), a composite electrode modified with ssDNA (b), and a composite electrode modified with dsDNA (c) immersed in 1mM MB PBS solution in PBS (0.1M, pH 7.4). From the figure, it is seen that the peak current (c) > (b) at-0.25V and (a) have no peak, indicating that the present electrode can detect DNA.
The above description is only exemplary of the present invention, and all equivalent changes and modifications made within the scope of the present invention should be covered by the protection scope of the present invention.
Claims (4)
1. A DNA biosensor is characterized in that sulfhydryl DNA is fixed on an AuNPs/AgNWs/PDMS electrode;
the preparation method of the AuNPs/AgNWs/PDMS electrode comprises the following steps:
using PDMS as a flexible substrate, modifying a hydrophilic surface layer on the surface of the PDMS by using a mixed solution of PVA and Gly, uniformly coating a silver nanowire conducting layer on the hydrophilic modification layer, and using KAuCl4And H2SO4The mixed solution is used as electrolyte, an Ag/AgCl electrode is used as a reference electrode, a platinum wire is used as a counter electrode, a silver nanowire conducting layer is used as a working electrode, and gold is electrochemically deposited on the conducting layer by adopting a chronoamperometry method to prepare an AuNPs/AgNWs/PDMS electrode;
the preparation method of the DNA biosensor comprises the following steps:
(1) immersing the AuNPs/AgNWs/PDMS electrode into a PBS solution containing a sulfydryl single-stranded DNA probe for full reaction, then putting the electrode into blank PBS, stirring at constant temperature to remove DNA molecules which are non-specifically adsorbed, then immersing into mercaptoethanol, further eliminating the non-specifically adsorbed DNA molecules and obtaining a relatively compact DNA monomolecular layer, then washing with the PBS solution and secondary distilled water, and drying with nitrogen;
(2) soaking a DNA probe electrode modified by mercaptoethanol into PBS (phosphate buffer solution) solutions of target DNAs with different concentrations, carrying out thermostatic water bath and stirring to obtain a double-stranded DNA modified electrode, repeatedly washing secondary water and the PBS solution to remove non-hybridized target DNAs adsorbed on the surface, and drying the target DNA with nitrogen for later use;
wherein the concentration of the sulfhydryl single-stranded DNA is 10-7～10-8Double-stranded DNA concentration of 10-7～10-9。
2. A method for measuring DNA using the DNA biosensor of claim 1, wherein the single-stranded DNA or double-stranded DNA modified electrode is soaked in a 1mM methylene blue solution, washed repeatedly with double distilled water, a PBS solution to remove molecules adsorbed on the surface of the electrode, and then a cyclic voltammetric signal in the PBS solution is recorded; the cyclic voltammetry is carried out under the conditions that the potential range is-0.6V and the sweep rate is 100 mV/s.
3. The DNA biosensor as set forth in claim 1, wherein KAuCl is added to the electrolyte for preparing AuNPs/AgNWs/PDMS electrodes4The concentration is 0.01-0.05 mol/L, H2SO4The concentration is 0.1-0.5 mol/L.
4. The DNA biosensor as claimed in claim 1, wherein the deposition potential of the electrochemically deposited gold is-0.2V and the deposition time is 400-1600 s when preparing AuNPs/AgNWs/PDMS electrode.
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| CN102072931A (en) * | 2010-12-09 | 2011-05-25 | 华东师范大学 | Method for preparing biosensor based on silicon nanowires and application of biosensor to detecting DNA |
| CN104630869A (en) * | 2015-01-22 | 2015-05-20 | 江南大学 | DNA sensor for detecting staphylococcus aureus as well as preparation method and application of DNA sensor |
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| CN102072931A (en) * | 2010-12-09 | 2011-05-25 | 华东师范大学 | Method for preparing biosensor based on silicon nanowires and application of biosensor to detecting DNA |
| CN104630869A (en) * | 2015-01-22 | 2015-05-20 | 江南大学 | DNA sensor for detecting staphylococcus aureus as well as preparation method and application of DNA sensor |
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