KR20150144644A - Biosensor for DNA Detection and method for manufacturing the same - Google Patents
Biosensor for DNA Detection and method for manufacturing the same Download PDFInfo
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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
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
Abstract
The present invention relates to a biosensor for DNA detection and a method for producing the same, and more particularly, to a biosensor for detecting DNA prepared by forming gold nanoparticles coated with polythionine on a conductive transparent substrate electrode, And it has an excellent detection sensitivity and stability in detecting a single strand DNA.
Description
The present invention relates to a biosensor for DNA detection and a method for producing the same, and more particularly, to a biosensor for DNA detection prepared by forming gold nanoparticles coated with polythionine on a conductive transparent substrate electrode and a method for manufacturing the same.
In order to diagnose diseases early, it is becoming increasingly important to detect highly sensitive disease indicator substances present in biological samples. Recently, a number of techniques have been developed for immuno-PCR, bio-barcode assay, and liposome-PCR.
For example, a conventional detection method related to DNA detection is based on a label technique of detecting fluorescent light by bonding a fluorescent substance to DNA molecules.
Such fluorescent-based detection methods require complex sample preparation procedures, complex optical systems, and specialized and complex analyzes, and they have disadvantages economically disadvantageous.
Therefore, research on label-free technique that can measure without labeling is actively under way.
An electrochemical biosensor can be used as one of the label-free techniques. Generally, biosensors are used when a substance related to an organism is used as a component of a sensor or to be analyzed. Electrochemical biosensors combine the analytical capabilities of electrochemical methods with the specificity of the biological recognition process.
An enzyme, an antigen, an antibody, and a biochemical substance, which are bio-specific reagents, are immobilized or contained on the electrode surface, thereby converting a biological recognition phenomenon into a quantitative current or potential change.
For example, selective binding of a biomolecule such as an antibody to a specific target substance can be utilized. Most commonly reported is an immunosensor based on the immune response of an antigen-antibody. Antibodies have remarkable selectivity due to the stereospecificity of the binding site to the antigen, and bind with a very large binding equilibrium constant to the antigen. In the immunoassay sensor, the antibody is immobilized on the surface of the electrode and the labeling of the target substance itself or the second antibody is carried out as a labeling substance. The result of the specific binding to the target substance is measured by an electric current To be converted.
In another embodiment, a DNA hybridization biosensor is used which uses the cognitive function of a nucleic acid.
This is a sensor based on DNA base pairing. It consists of a single-stranded DNA with a specific sequence on the surface of the electrode and hybridization with a single-stranded DNA in a sample having a sequence capable of complementary binding to a fixed single- It can be said that electric signal is converted by
This can be applied to a wide variety of clinical applications from genetic diagnosis to criminal investigation since it is possible to expect selective specificity for DNA sequence and easy and quick analysis.
As a conventional technology related to such a biosensor for DNA detection, Japanese Unexamined Patent Application Publication No. 10-2012-0093630 (Aug. 23, 2012) discloses a DNA sensor obtained by forming a probe DNA in an organic thin film transistor, No. 10-1293666 (Jul. 31, 2013) discloses a method of sequentially and repeatedly stacking a DNA layer and a protein layer selectively on a metal nanodot formed on a substrate to form a three-dimensional protein multi-layer nanostructure And a technique relating to a biosensor using the same.
However, various methods for manufacturing a biosensor for DNA detection have been proposed including the above-described conventional techniques. However, by using a simple and miniaturized electrode by an electrochemical measurement method, preparation process and measurement for DNA detection A DNA detection biosensor which is more stable and reproducible in a solution containing an impurity while simplifying the process and the presence or absence of a clinically meaningful specific gene using the DNA detection biosensor is simpler and easier than conventional methods such as PCR There is a continuing need for research on electrochemical analysis methods that can be quickly confirmed.
The present invention relates to a DNA preparation method and a DNA preparation method which are simple and easy to prepare for DNA detection but are more stable and reproducible in a solution containing an impurity and can confirm the presence or absence of a specific gene more easily and quickly than conventional methods such as PCR An electrode for a biosensor and a method of manufacturing the same.
The present invention also provides a DNA sensing device which is more stable and reproducible in an aqueous solution, using the electrode thus prepared.
In addition, the present invention provides a method for analyzing DNA with stability and reproducibility by a faster and more economical method using the DNA sensing apparatus.
According to an aspect of the present invention, there is provided a method of fabricating a transparent conductive electrode, comprising the steps of: a) forming and dispersing gold nanoparticles on a conductive transparent electrode; b) forming gold nanoparticles coated with polythionine through polymerization of thionin on the surface of the transparent electrode on which the gold nanoparticles are formed; And c) connecting the probe DNA to the polythionine. The present invention also provides a method for manufacturing an electrode for a biosensor.
In one embodiment, the conductive transparent electrode may be any one selected from ITO, IZO, ATO, FTO,
In one embodiment, the gold nanoparticles can be formed by electrochemically reducing a gold precursor, or by reducing it in the presence of a polyol method or a reducing agent.
In one embodiment, the polythionine may be formed through an electrochemical polymerization reaction in an aqueous solution containing thionin.
In one embodiment, the step c) may include immersing the electrode formed with the polythionine-coated gold nanoparticles obtained in step b) in an aqueous solution containing the probe DNA precursor.
In one embodiment, the probe DNA in step c) may be a single-stranded DNA complementary to the specific DNA to be measured.
In addition, the present invention provides an electrode for a biosensor manufactured by the above production method.
In addition, the present invention provides a biosensor comprising a solution reservoir for storing a solution containing a specific DNA to be measured, a working electrode including the electrode for the biosensor of the present invention, a counter electrode corresponding to the working electrode, And a DNA detection device.
The present invention also provides a method for manufacturing a semiconductor device, which comprises a conductive transparent electrode on which gold nanoparticles are formed and dispersed, wherein the gold nanoparticles are coated with polythionine, and the polythionine is a single strand probe DNA And an electrode for a biosensor is connected by covalent bonding. In this case, the conductive transparent electrode is ITO, and the polythionine may be formed by an electrochemical polymerization method, and the electrode for biosensor may be applied to a device for detecting DNA.
In addition, the present invention provides a method for analyzing DNA complementary to the single strand probe DNA through an electrochemical method using the apparatus for detecting DNA.
According to the present invention, there is provided a biosensor electrode and a method for manufacturing the same, which can simplify and simplify the preparation process and the measurement process for DNA detection and the presence or absence of a specific gene in comparison with existing methods such as the conventional PCR technique .
In addition, the present invention can produce a device for DNA detection capable of measuring more stably and reproducibly on a solution containing an impurity by using the electrode, and the apparatus for DNA detection can detect a single strand DNA Can be detected with excellent sensitivity and selectivity.
FIG. 1 is a diagram illustrating a method for fabricating an electrode for a biosensor according to an embodiment of the present invention, and a process of detecting DNA by the method. Referring to FIG.
FIG. 2A is a graph showing a cyclic voltammetric (CV) curve in which gold nanoparticles are deposited on an indium tin oxide thin film according to an embodiment of the present invention, and FIG. 2B is a scanning electron microscope (C) shows a cyclic voltammetric (CV) curve after electrochemical coupling of thionin to an electrode deposited with gold nanoparticles on an indium tin oxide thin film. FIG. D) is a scanning electron microscope (SEM) image of an indium tin oxide electrode coated with gold nanoparticles with polythionine.
3 (A) is a graph showing the relationship between the voltage applied to the ITO electrode, the electrode on which the gold nanoparticles are deposited on the ITO electrode, the circulation voltage of the ITO electrode on which the gold nanoparticles coated with the polythionine are deposited, (CV) curve, and (B) is an illustration of these electrochemical impedance spectroscopy (EIS) spectra.
FIG. 4A is a cross-sectional view illustrating an ITO electrode on which gold nanoparticles coated with polythionine are deposited in an electrode according to an embodiment of the present invention, electrodes to which probe DNA is bound, complementary DNA to the probe DNA, FIG. 4 (B) is a graph showing changes in current density due to hybridization of the probe DNA and the target DNA, and FIG. 4 (C) is a graph showing the change in current density according to the single- Fig.
Hereinafter, the present invention will be described in detail. But may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
The present invention is capable of various modifications and various forms, and specific embodiments will be illustrated in the drawings and described in detail in the text. It is to be understood, however, that the invention is not intended to be limited to the particular forms disclosed, but on the contrary, is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. The terminology used is used only to describe a specific embodiment and is not intended to limit the invention.
Also, the singular expressions include plural expressions unless the context clearly dictates otherwise. In the present application, the terms "comprise", "having", and the like are intended to specify the presence of stated features, integers, steps, operations, elements, components, or combinations thereof, , Steps, operations, components, parts, or combinations thereof, as a matter of principle.
FIG. 1 shows a method of manufacturing an electrode for a biosensor according to the present invention and a process of detecting DNA using the same.
More specifically, the present invention provides a method of forming a conductive transparent electrode, comprising the steps of: a) forming and dispersing gold nanoparticles on a conductive transparent electrode; b) forming gold nanoparticles coated with polythionine through polymerization of thionin on the surface of the transparent electrode on which the gold nanoparticles are formed; And c) connecting the probe DNA to the polythionin.
In the present invention, the conductive transparent electrode can be formed and dispersed in gold nanoparticles. In the case of a conductive material, the conductive transparent electrode can be used without limitation, and preferably, ITO, IZO, antimony tin oxide Metal oxide such as ATO: antimony tin oxide, fluorine-containing tin oxide (FTO), (SnO2), or (ZnO), and semiconductor materials. More preferably, ITO, or fluorine-containing tin oxide (FTO) may be used.
For example, the fluorine tin oxide (FTO) has an advantage of high safety against high temperature, low resistivity and high transmittance. In the case of indium tin oxide (ITO), the resistivity is low and the etching property required for patterning This is an excellent advantage.
Meanwhile, the conductive transparent electrode used in the present invention is used as an electrochemical sensor in the present invention, but its application is not limited thereto, and it can be used in various display devices, biosensors and the like which can be used as a transparent electrode.
Therefore, the electrode formed with the gold nanoparticles, the polythionine, and the probe DNA formed on the conductive transparent electrode according to the present invention can be used not only for an electrochemical sensor but also for various organic semiconductor devices and biosensors .
Meanwhile, the reduction reaction for forming the gold nanoparticles in the present invention is not limited thereto, and any kind of gold nanoparticles may be used as long as the gold nanoparticles can be formed on the conductive transparent electrode. Preferably, Electrochemically reducing it, or by reducing it in the presence of a polyol method or a reducing agent.
Generally, metal nanoparticles can be obtained by reduction of a metal precursor to form metal nanoparticles, and more preferably, gold nanoparticles can be formed by electrochemical reduction.
The gold precursor used for the production of gold nanoparticles in the present invention is not limited as long as it contains monovalent (Au (I)) or trivalent (Au (III)) ions as gold ions, Preferably, chloroauric acid (HAuCl 4 ) can be used, and in the case of preparing gold nanoparticles in an aqueous solution containing the gold ion, an aqueous solution of HAuCl 4 can be used.
In the present invention, the electrochemical reduction may be performed by applying a voltage lower than a specific value to reduce gold ions in the gold precursor aqueous solution to gold nanoparticles and then reducing the gold nanoparticles for a predetermined time.
Meanwhile, in the present invention, the voltage applied for forming the gold nanoparticles means a value relative to the Ag / AgCl reference electrode. Therefore, the voltage applied for the formation of gold nanoparticles in the present invention should be considered to be a value relative to the Ag / AgCl reference electrode, unless otherwise specified.
In this case, a preferable voltage is a range in which the gold ion can be reduced to gold nanoparticles, and a voltage of -0.45 V or less, preferably -0.5 V or less, more preferably -0.55 V or less You can add
The gold nanoparticles can be prepared by applying the above voltage between 0.2 V and 1.3 V for 1 to 50 cycles.
In addition, a reducing agent may be additionally used in the production of the gold nanoparticles by the electrochemical reduction reaction. Examples of the reducing agent include citric acid, ascorbic acid, sodium borohydride, hydroxymethyl, phosphonium chloride, The reducing agent may be used by mixing two or more reducing agents if necessary. Preferably, hydroxylamine and citric acid can be used, and citric acid not only acts as a reducing agent but also sticks to the surface of gold nanoparticles to prevent the nanoparticles from becoming entangled with each other.
In the present invention, the reduction reaction may be performed in an electrolyte-containing state. The electrolyte which can be used here is not limited to the type that can be used in the electrochemical oxidation-reduction reaction, and preferred examples thereof include tetraalkylammonium salts and alkylimidazolium salts. As their conjugate salts, tetrafluoroborate, hexafluoro Phosphate and the like can be used.
In the present invention, the concentration of the chloroauric acid aqueous solution may be in the range of 0.01 mM to 10 mM, preferably 0.05 mM to 2 mM.
Also, the electrochemical reduction condition may be performed by applying a voltage of -0.5 V or lower for 5 to 600 seconds.
Also, in the present invention, the electrochemical reduction may be performed by a three-electrode system including a conductive transparent electrode as a working electrode and a counter electrode corresponding to the working electrode and a reference electrode.
The counter electrode may be a platinum electrode, and the reference electrode may be a commonly used Ag / AgCl electrode.
The following Reaction Schemes (1) to (2) show the reduction reaction of aqueous chloroauric acid solution on the surface of the conductive transparent electrode as the working electrode according to one embodiment of the present invention.
In the chloroauric acid aqueous solution, trivalent gold ions can be reduced to monovalent gold ions by the reaction formula (1), and monovalent gold ions are then reduced to gold nanoparticles by the reaction formula (2) and formed on the electrode surface.
The diameter of the gold nanoparticles formed by the reduction reaction may be in the range of 30 to 5000 nm, preferably 50 to 1000 nm. Generally, the longer the time of the reduction reaction, the larger the diameter. However, as described above, when the reduction reaction is continued after the metal oxide is reduced, the particles may be desorbed or the uniformity of the size of the particles formed on the surface may be different There should be no further excess reduction reaction.
In the present invention, the step of forming gold nanoparticles coated with polythionine through the polymerization of thionon on the surface of the transparent electrode on which the gold nanoparticles are formed is performed by electrochemical polymerization in an aqueous solution containing thionin, . ≪ / RTI >
Since the polythionine contains an amino group in the polymer, the gold atom and the nitrogen atom of the amino group can be coordinately coordinated to increase the binding force between the gold nanoparticle and the polythionine.
The polythionine may be obtained by polymerizing a thionine monomer, and preferably a thin film may be formed through electrochemical polymerization of a thionine (C14H13N3O2S) monomer on the surface of the electrode.
Illustratively, the electrochemical polymerization may be carried out by dissolving the ionic monomer in the electrolyte solution on the surface of the surface-treated electrode and then performing a potential cycling, a potential step or a galvanostatic method, Or the like can be used to form a thin film, and a current-voltage scanning method can be preferably used.
Specifically, the conductive polymer electrode is prepared by dissolving the thionin monomer as an electrolyte solution in a phosphate buffer solution having a pH of 6.0 to 7.0, applying a constant potential of 1.5 to 2.0 V for 300 to 500 seconds And then repeatedly performing electrochemical polymerization at a constant electrode voltage interval of 100 mV / sec. More preferably, in the phosphate buffer solution of pH 6.5 containing thionin, the electrode is subjected to pretreatment in which an equipotential of 1.8 V is applied for 400 seconds , It is preferable to form a thin film by electrochemically polymerizing a conductive polymer on the surface of the electrode repeatedly and cyclically changing the electrode voltage in the range of -0.4 to 2V. The thickness of the thin film can be controlled by the number of repetitions of the electrochemical polymerization method, so that the thickness of the thin film is not limited thereto, and the electrode voltage range can be changed according to the experimental conditions in consideration of the overall sensitivity of the sensor.
In the present invention, the step of connecting the probe DNA to the polythionine may include the step of immersing the electrode on which the polythionine-coated gold nanoparticles are formed in an aqueous solution containing the probe DNA precursor.
In this case, the probe DNA is single-stranded DNA corresponding complementarily to a specific target DNA to be measured, and can hybridize with a single-stranded DNA of a target DNA to be detected and bind to the double-stranded DNA.
For example, the probe DNA usable in the present invention may be a single-stranded DNA completely complementary to the single-stranded DNA of the target DNA, and may also be a single-stranded DNA that partially matches.
That is, the probe DNA may be designed so as to have a sequence completely complementary to the single-stranded DNA of the target DNA or to have a sequence complementary to the target DNA with a slight difference in nucleotide sequence from the target DNA can do.
In the present invention, when the electrode on which the polythionine-coated gold nanoparticles are formed is immersed in an aqueous solution containing the probe DNA precursor, the nitrogen atom in the amine group in the polythionine is shared with the phosphorus atom of the phosphate group (PO 4 -) of the probe DNA Through the binding, the probe DNA can be immobilized on the polythionine.
In order to remove unreacted probe DNA from the finally obtained electrode for a biosensor containing the probe DNA, it can be washed with a phosphate buffer solution or the like.
The present invention can also provide an electrode for a biosensor obtained by the above production method.
The present invention also provides a conductive transparent electrode comprising gold nanoparticles dispersed on the surface of the electrode, wherein the gold nanoparticles are coated with polythionine, and the polythionin is a single strand probe DNA And an electrode for a biosensor is connected by covalent bonding.
Wherein the conductive transparent electrode is ITO, and the polythionine may be formed by an electrochemical polymerization method as described above.
According to the present invention, it is possible to provide an electrode for a biosensor based on electrochemistry, which can more easily and quickly confirm the presence or absence of a clinically meaningful specific gene through the electrode for a biosensor as compared with a conventional PCR technique.
The present invention also provides an apparatus for detecting DNA comprising the above-described electrode for a biosensor, and is also capable of detecting DNA complementary to the single-stranded probe DNA through an electrochemical method using the DNA- Provides a method for analyzing DNA.
More specifically, the DNA sensing apparatus of the present invention includes a solution reservoir for storing a solution containing a specific DNA to be measured, a working electrode including the electrode for the biosensor, a counter corresponding to the working electrode, An electrode, and a reference electrode. The sensing device may further include a voltage source or a current source for supplying a voltage or a current to the working electrode and the counter electrode, and a voltage meter or a current meter for measuring a voltage value or a current value transmitted from the working electrode .
In this case, the voltage source or the current source and the cyclic voltammeter (CV) may be used as the voltage meter or the current meter.
Wherein the working electrode is an electrode in which gold nanoparticles are coated with polythionine and probe DNA is connected to the polythionine, a counter electrode corresponding to the working electrode is a platinum plate or a platinum wire electrode, A possible Ag / AgCl electrode can be used.
Also, in the present invention, the solution stored in the solution reservoir optionally includes an electron transfer mediator, so that the electrical signal in the working electrode can finally be quantified according to the redox of the electron transfer mediator.
The target DNA can be detected and analyzed by an electrochemical method if the electrical signal changed according to the presence or the content of the target DNA in the DNA detection device containing the probe DNA is measured.
Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. It will be apparent to those skilled in the art, however, that these examples are provided to further illustrate the present invention, and the scope of the present invention is not limited thereto.
(Example)
(Na 2 HPO 4 ) used in the following Examples and sodium dihydrogenphosphate (NaH 2 PO 4 ), N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) HAuCl 4 and thionine acetate (C 12 H 9 N 3 S · C 2 H 4 O 2 ) were purchased from Sigma Aldrich.
Phosphate buffer solution (PBS) was prepared by mixing 0.1 M Na 2 HPO 4 and 0.1 MNaH 2 PO 4 .
The DNA samples were those of Cosmo Genetech as the species having the nucleotide sequence shown below.
pDNA: 5'-PO4-AGT TAA AAG CAG CCC TGG TGA CCA GGC GCC CAA TAC GAC C-3 ';
cDNA: 5'-GGT CGT ATT GGG CGC CTG GTC ACC AGG GCT GCT TTT AAC T-3 ';
1-base mismatch DNA (1bmDNA): 5'-GGT CGT ATT GGG CGC CTG GCC ACC AGG GCT GCT TTT AAC T-3 ';
3-base mismatch DNA (3bmDNA): 5'-GGT CGT ATT GGG CGC CTG ACT ACC AGG GCT GCT TTT AAC T-3 ';
A non-complementary DNA (ncDNA): 5'-AGT TAA AAG CAG CCC TGG TGA CCA GGC GCC CAA TAC GAC C-3 'CHI430A electrochemical workstation (CH instruments, Inc. USA) In order to constitute a three-electrode system including the inventive sensor, an indium oxide (ITO) electrode set to have an area of 0.32 cm < 2 > using an O-ring as a working electrode was used and a platinum wire was used as a counter electrode, An Ag / AgCl electrode (aq. Saturated KCl) was used as a reference electrode.
Differential pulse voltammogram (DPVs) is in the range of -0.3 ~ +0.3 V pulse amplitude is 100 mV · s - 1 with the pulse width was tested with a period of 100ms pulse is had to 2ms.
Electrochemical impedance meter (ESI: Electrochemical impedance spectroscopy, IM6ex , Zahner-Elektrik GmbH & Co. KG) 5 mM K3 by the [Fe (CN) 6] 10 5 under a concentration of - 0.1 Hz of the frequency range and amplitude alternating current of 5mV (E0) of [Fe (CN) 6] < 3 >).
Example 1 Formation of indium tin oxide (ITO) electrode comprising gold nanoparticles coated with polythionine
Step 1: Deposition gold nanoparticles on indium tin oxide electrode
An aqueous solution containing 0.1 mM gold ions is prepared by dissolving a chloroauric acid (HAuCl4) precursor containing gold ions in a phosphate buffer solution (PBS).
The indium tin oxide (ITO) substrate having a size of 20 mm x 30 mm is sonicated in a solution of ethanol and Triton X-100 solution, washed and dried using nitrogen gas.
In order to electrochemically form gold nanoparticles, Ag / AgCl electrode is used as a reference electrode, a platinum wire electrode is used as a counter electrode, and the cleaned indium tin oxide (ITO) electrode is used as a working electrode.
The electrode was immersed in a Teflon cell containing an aqueous solution containing gold ions, and gold nanoparticles were formed on the indium tin oxide (ITO) electrode with a potential of 0.2 V to 1.3 V for 15 cycles or more.
FIG. 2 (A) is a graph showing a cyclic voltammetric (CV) curve of an electrode in which gold nanoparticles are deposited on an indium tin oxide (ITO) thin film by an electrochemical method using an aqueous solution containing gold ions. The results of 1 time, 2 times, 5 times, 10 times and 15 times out of the total 15 cycles are shown.
The results of this first round show a typical current loop of metal deposition due to nucleation, which indicates that over-potential is required for deposition of the ITO phase of gold particles.
As the number of cycles increases from 2 to 15 cycles, it can be seen that the reduction current increases from -0.93 V to 1.0 V, which indicates that gold nanoparticles are formed continuously on the electrode.
On the other hand, the figure on the inside of the figure shows the case where only indium tin oxide (ITO) electrode of 0.1 M aqueous sulfuric acid solution (H 2 SO 4 ) is measured and the cyclic voltammetric curve (CV) in which gold nanoparticles are deposited. As a result, it can be seen that the electrode having the gold nanoparticles deposited forms a gold oxide at 1.0 V, and the total surface area of the gold nanoparticles is the total charge (~ 400 mCcm -2 ) , Which is about 0.21 cm2, which is about 65.6% of the total ITO area.
FIG. 2B is a scanning electron microscope (SEM) image of the surface of an indium tin oxide (ITO) electrode on which gold nanoparticles are deposited. The gold nanoparticles thus deposited are uniformly distributed, It can be seen that it has a size between about 50 nm and 120 nm.
Step 2: Manufacture of indium tin oxide coated with polythionine on gold nanoparticles
An indium tin oxide (ITO) electrode having the gold nanoparticles deposited thereon is immersed in a phosphate buffer solution (pH 7) containing 0.1 mM thionin. Thionine is polymerized in the indium oxide tin electrode containing the gold nanoparticles through electropolymerization at a potential of 0.6 V to +1.2 V for 15 cycles or more so that the gold nanoparticles are coated with the polythionine .
FIG. 2C is a graph showing a cyclic voltammetric curve (CV) of an indium tin oxide electrode coated with gold nanoparticles by electrochemical polymerization of polythionine. The thionine cation-radical oxidation-reduction peak at 0.1 V (I) and 0.2 V (I ') in the above polythionine and the desorption of the hydrogen atom of the primary amine (-NH 2) group at 1.0 V (II) Can be seen. The polythionine can form a bond between the surface of the gold nanoparticle and the atom of Au-N, and the binding force between the gold nanoparticle and the polythionine can be improved.
The continuous formation of the polythionine film on the surface of the gold nanoparticles can also be seen from the redox peak at +0.17 (III) and -0.05 V (III ').
FIG. 2 (D) is a scanning electron microscope (SEM) image of an indium tin oxide electrode coated with polythionine on gold nanoparticles. The size of the gold nanoparticles coated with polythionine is 150-280 nm, It can be seen that the nanoparticles are larger than the case where only the nanoparticles are deposited.
3 (A) shows an ITO electrode in an aqueous solution containing 5 mM Fe (CN) 6 3- / 4- , an electrode in which gold nanoparticles were deposited on an ITO electrode, and gold nanoparticles coated with a polythionine were deposited depicts a cyclic voltammetry (CV) curve of the ITO electrode, (B) are gold nanoparticles is deposited on the ITO electrode, ITO electrode to Fe (CN) 6 aqueous solution containing a 3/4 of 5mM of Figure 3 (Electrochemical Impedance Spectroscopy) spectrum of an ITO electrode deposited with gold nanoparticles coated with polythionine.
Table 1 below shows the results of ΔE p (V), R ct (kΩ), and k app (cms -1 ) a as a result according to FIG. More specifically, in the case of the electrode having the gold nanoparticles formed, the peak-to-peak separation (ΔE p (V)) is smaller than that of the electrode made of only ITO, When the gold nanoparticles are included, the charge transfer resistance is the smallest, indicating that the redox conditions are optimal.
Rct : change transfer resistance
k app : the apparent standard heterogeneous rate constant
Example 2: Immobilization of probe DNA and detection of target DNA
An electrode comprising the gold nanoparticles coated with the polythionine is immersed in an aqueous probe DNA solution. The probe DNA is single stranded DNA and can be complementarily bound to a specific DNA to be measured, and the kind thereof is as described above. 10 mM EDC as an activator was mixed with 1 ml of a phosphate buffer solution (PBS) containing the probe DNA (1 uM), and the mixture was stained at 50 ° C for 6 hours to form an ITO electrode having gold nanoparticles coated with polythionine Of the polythionine and the probe DNA are immobilized to form a covalent bond.
The electrode (pDNA / PTH / GNPs / ITO electrode) to which the probe DNA is immobilized on the surface of the polythionine can be immobilized through covalent bonding of the nitrogen atom of the polythionine to the phosphorus atom of the probe DNA.
As a subsequent step, the pDNA / PTH / GNPs / ITO electrode is washed with a phosphate buffer solution to remove unbound pDNA.
On the other hand, for the detection of single-stranded target DNA, the prepared pDNA / PTH / GNPs / ITO electrode can be used as a single-strand target DNA capable of complementarily binding to the probe DNA or a sample such as mismatched DNA (cDNA, 1bmDNA, 3bmDNA and nDNA), pDNA / PTH / GNPs / ITO electrodes are immersed at 50 ° C for 2 hours in a 1 mL phosphate buffer solution. Then, washing is carried out to remove unbound DNA.
FIG. 4A is a graph showing the relationship between the voltage of an ITO electrode on which gold nanoparticles coated with a polythionine are deposited, an electrode to which probe DNA is bound, and an electrode in which the complementary DNA is hybridized with the probe DNA The current density by DPV (differential pulse voltammograms) is schematized. The graph shows that the current density was reduced to 10.8 uA / cm 2 as the probe DNA was immobilized by covalent bonding, which was 20.5 uA / cm 2 on the electrode coated with polythionine (PTH / GNPs / ITO electrode) have. In addition, when the complementary DNA is hybridized with the probe DNA, it can be detected that the target DNA can be detected because it has a lower current density value.
FIG. 4 (B) is a graph showing a trend of decreasing the current density due to hybridization of the probe DNA and the target DNA. FIG. 4 (b) is a graph showing the amount of change in the current density caused by different hybridization depending on the type of the target DNA.
When the target DNA is cDNA, the amount of change in the current density hybridized with the probe DNA is about 3.5 uA / cm < 2 > , And about 1.25 uA / cm < 2 > for the target DNA of 1 bmDNA , 3bm DNA is about 1.1 uA / cm < 2 > , and ncDNA has about 0.36 uA / cm 2. As a result , it can be confirmed that the current density value is lower as the base sequence complementary to the target DNA is different.
From the above results, it can be seen that the electrode for a biosensor according to the present invention can selectively detect a target DNA of a specific sequence.
FIG. 4 (C) is a graph showing the detection range of single-stranded DNA as a target DNA, and shows a change amount of the current density between 1 uM and 1 fM of the concentration of cDNA. As a result, it can be seen that the variation of the current density has a linear relationship with the oxidation peak current density of the polythionine, and the detection limit of the sensor is about 0.93 fM. As a result, It is possible to detect cDNA at a low concentration and is excellent in detection limit and is suitable as a biosensor for DNA detection.
aPANI-NF = polyaniline nanofiber, bPICA = indole-5-carboxylic acid, cMWCNTs = multiwall carbon nanotubes, dGG1PAMAM = first generation G1 dendrimer on graphene sheet, eGO = graphene oxide
Therefore, the gold nanoparticles prepared according to the present invention are coated with polythionine, and the electrode for the biosensor to which the probe DNA is connected has high detection sensitivity and low detection limit. In addition, it has an advantage of high stability and reproducibility, so it plays a sufficient role as a biosensor for detecting a single strand of DNA.
Meanwhile, in order to evaluate the stability of the electrode for biosensor for DNA detection prepared by the present invention, the electrode was immersed in hot water at 80 ° C for 10 minutes, and then the sensor was cooled in an ice bath. The current density of the sensor was measured and compared with the initial current density. In this case, the current density was reduced by 18% from the initial value, and the decrease value was estimated to be due to desorption of the probe DNA. On the other hand, when the sensor was stored at 4 ° C for 2 weeks, the signal showed 91% of the initial value, indicating that the thermal stability and the long-term storage stability were excellent.
Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific descriptions are only for the preferred embodiments and that the scope of the present invention is not limited thereto Will be blind. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (12)
b) forming gold nanoparticles coated with polythionine through polymerization of thionin on the surface of the transparent electrode on which the gold nanoparticles are formed; And
and c) connecting the probe DNA to the polythionine.
Wherein the conductive transparent electrode is any one selected from the group consisting of ITO, IZO, ATO, FTO, SnO 2, and ZnO.
Wherein the gold nanoparticles are formed by electrochemically reducing a gold precursor or by reducing the gold precursor in the presence of a polyol method or a reducing agent.
Wherein the polythionine is formed through an electrochemical polymerization reaction in an aqueous solution containing thionin.
Wherein the step c) includes a step of immersing the electrode formed with the polythionine-coated gold nanoparticles obtained in the step b) in an aqueous solution containing the probe DNA precursor.
Wherein the probe DNA in step c) is single-stranded DNA complementary to the specific DNA to be measured.
Wherein the conductive transparent electrode is ITO, and the polythionin is formed by an electrochemical polymerization method.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20180129206A (en) * | 2017-05-25 | 2018-12-05 | 가천대학교 산학협력단 | Nano-biosensor with interdigitated electrode for enhanced sensing TNF-alpha by deposition of nanoparticle |
| KR20190048775A (en) * | 2017-10-31 | 2019-05-09 | 전자부품연구원 | Electrode for electrochemical sensor and method for manufacturing the same |
| US10481158B2 (en) | 2015-06-01 | 2019-11-19 | California Institute Of Technology | Compositions and methods for screening T cells with antigens for specific populations |
| CN112505115A (en) * | 2020-12-17 | 2021-03-16 | 东北农业大学 | Preparation and detection method of three-dimensional photosensitive electrode for detecting phospholipids in crude oil |
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| KR20120093630A (en) | 2011-02-15 | 2012-08-23 | 명지대학교 산학협력단 | Dna sensor having organic thin film transistor and method for fabricating the same |
| KR101293666B1 (en) | 2011-03-07 | 2013-08-13 | 고려대학교 산학협력단 | A Three-Dimensional Nanostructured Array of Protein Nanoparticles |
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| KR20120093630A (en) | 2011-02-15 | 2012-08-23 | 명지대학교 산학협력단 | Dna sensor having organic thin film transistor and method for fabricating the same |
| KR101293666B1 (en) | 2011-03-07 | 2013-08-13 | 고려대학교 산학협력단 | A Three-Dimensional Nanostructured Array of Protein Nanoparticles |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US10481158B2 (en) | 2015-06-01 | 2019-11-19 | California Institute Of Technology | Compositions and methods for screening T cells with antigens for specific populations |
| KR20180129206A (en) * | 2017-05-25 | 2018-12-05 | 가천대학교 산학협력단 | Nano-biosensor with interdigitated electrode for enhanced sensing TNF-alpha by deposition of nanoparticle |
| KR20190048775A (en) * | 2017-10-31 | 2019-05-09 | 전자부품연구원 | Electrode for electrochemical sensor and method for manufacturing the same |
| CN112505115A (en) * | 2020-12-17 | 2021-03-16 | 东北农业大学 | Preparation and detection method of three-dimensional photosensitive electrode for detecting phospholipids in crude oil |
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