KR20160025122A - Biosensor using bimetallic nanodendrite coated by gold and silver having surface-enhanced Raman scattering activity and method for preparing the same - Google Patents

Abstract

The present invention relates to a target DNA detecting biosensor using bimetal nanodendrite coated with gold and silver having surface-enhanced Raman scattering activity, and to a manufacturing method thereof. According to the present invention, the biosensor to detect the target DNA is manufactured using bimetal nanodendrite coated with gold and silver having surface-enhanced Raman scattering activity, thereby having excellent sensitivity and stability of a Raman signal; and when the biosensor is used in target DNA detection, having excellent target DNA detecting effect with a simple and economical method.

Description

TECHNICAL FIELD The present invention relates to a biosensor using bimetallic nanodendrite coated with gold and silver having surface enhanced Raman scattering activity and a method for preparing the biosensor using bimetallic nanodendrite coated gold and silver having a surface enhanced Raman scattering activity.

The present invention relates to a biosensor for detecting target DNA using gold and silver coated bimetallic nanodendrite having surface enhanced Raman scattering activity and a method for producing the same.

Detection of specific DNA sequences is used in important applications in biological studies or medical diagnostics. For example, since 1997 when H5N1 infection has been identified in human cells, the virus has been considered one of the deadliest viruses to threaten the human body. Thus, probes of specific DNA sequences are important in disease diagnosis, food and drug industry monitoring, gene therapy and medical research. However, since DNA exists at a very low concentration, a more sensitive detection technique is required for DNA analysis. DNA amplification and fluorescence detection techniques using PCR have been widely used for general DNA detection. Among these, Raman spectroscopy has the advantage that the biological and structural properties of compounds can be identified in trace amounts. When light passes through a type medium, some amount deviates from its natural direction, which is known as Raman scattering. In addition, some of the scattered light differs in frequency from the original stimulated light due to the absorption of light and excitation of the electrons to high energy levels. The wavelength of the Raman emission spectrum indicates the chemical composition and structural characteristics of the light absorbing molecules in the sample, and the intensity of light scattering differs depending on the concentration of molecules in the sample. However, the possibility of Raman interactions between stimulated light in the sample and individual molecules is very low, limiting the applicability of Raman analysis due to the low sensitivity.

Surface enhanced Raman spectroscopy (SERS) using silver nanoparticles showed the potential to overcome low sensitivity, a problem that existed in Raman spectroscopy. In addition, The Raman signals of the molecules were very high in the case of silver, gold, copper and the like. However, gold exhibited a stable Raman signal, but the sensitivity was low. In the case of silver, there was a problem that the sensitivity was high but the signal characteristics were unstable.

Therefore, there is an urgent need to study a biosensor using bimetallic nanodendrite capable of simultaneously increasing the sensitivity and stability of a Raman signal for high sensitivity detection of a specific DNA sequence.

KR 10-2007-0065063

The inventors of the present invention have found that when using gold and silver coated bimetallic nanodendrite having surface enhanced Raman scattering activity, the sensitivity and stability of the Raman signal are excellent and the biosensor And the target DNA is detected by a simple and economical method. Thus, the present invention has been completed.

Accordingly, the present invention provides a biosensor for detecting target DNA using gold and silver coated bimetallic nanodendrite having surface enhanced Raman scattering activity, and a method for producing the biosensor.

In order to achieve the above object,

The present invention

Hybridization of labeled DNA with probe DNA and Raman dye; And a bimetallic nanodendrite of gold and silver on which the probe DNA of the hybrid product is immobilized.

In addition,

(1) hybridizing a labeled DNA with a probe DNA and a Raman dye;

(2) immobilizing the hybridization probe DNA to gold and silver bimetallic nanodendrite;

(3) adding a target DNA to the bimetallic nanodendrite-immobilized hybrid, and replacing the labeled DNA with the target DNA; And

(4) measuring a surface enhanced Raman scattering (SERS) signal before and after replacing the labeled DNA of step (3) with the target DNA.

Hereinafter, the present invention will be described in detail.

The present invention relates to a hybrid of labeled DNA and probe DNA and Raman dye-bound labeled DNA; And a bimetallic nanodendrite of gold and silver on which the probe DNA of the hybrid product is immobilized.

The probe DNA of the present invention means a DNA hybridized by complementary binding of a base sequence with a labeled DNA to which a Raman dye is bound. By replacing the hybridized labeled DNA with the target DNA without Raman dye, the target DNA can be detected by changing the Raman signal from the Raman dye.

The hybridized probe DNA may be activated to fix the bimetallic nanodendrite surface. For example, the probe DNA can be immobilized on the surface of the nanodendrite by linking -SH, -CHO, -COOH, or -NH ₂ groups.

The labeled DNA is preferably hybridized from the 1 st to 10 th bases of the probe DNA from the nanodendrite, and more preferably hybridized from the 2 nd base.

According to one embodiment of the present invention, when the labeled DNA was hybridized from the nanodendrite to the second base of the probe DNA, it exhibited the largest Raman signal intensity.

The Raman dye means a Raman active organic compound, and any of them can be used without limitation as long as it is widely used in the technical field. Specific examples thereof include cyanine (Cy3, Cy3.5 or Cy5), xanthines, rhodamine 6G, rhodamine B isothiocyanate (RBITC), adenine, 4-amino-pyrazole azine-adenine, 8-aza-guanine, 6-benzoyladenine, 6-fluoroadenine, 6-benzoyladenine, kinetin, dimethyl-allyl-amino-adenine, zeatin, But are not necessarily limited to, mercaptopurine, 4-amino-6-mercaptopyrrazolo (3,4-d) pyrimidine, 8-mercaptoadenine, and 9-amino-acridine.

The gold and silver bimetallic nano-dendrites are preferably produced by an electrochemical deposition method using a galvanic substitution reaction of gold and silver, but are not limited thereto.

It is preferred that the gold and silver bimetallic nanodendrites comprise from 1 to 10 atomic percent gold and from 90 to 99 atomic percent silver and in accordance with one embodiment of the present invention 3.17 atomic percent, ) Of silver and 96.83 atomic percent (%) of silver. The silver standard intensity (RSV) of the silver nanodendrite (0 atomic percent gold) is about 17 times that of the gold nanodendrite, but the signal standard deviation (RSV) is 34.6%, compared to 9.2% of the gold nanodendrite It is very unstable. In addition, for bimetallic nanodendrite with 3.17 atomic percent gold and 96.83 atomic percent silver, the signal standard deviation (RSV) was 8.7%, which was four times more stable than silver nanodendrite.

The detection limit (LOD) of the target DNA of the biosensor is preferably 50 to 80 fM (femto-molar), and more preferably 62.5 fM according to one embodiment of the present invention.

Also, the present invention provides a method for producing a hybridoma cell, comprising the steps of: (1) hybridizing a labeled DNA with a probe DNA and a Raman dye; (2) immobilizing the hybridization probe DNA to gold and silver bimetallic nanodendrite; (3) adding a target DNA to the bimetallic nanodendrite-immobilized hybrid, and replacing the labeled DNA with the target DNA; And (4) measuring a surface enhanced Raman scattering (SERS) signal before and after replacing the labeled DNA of step (3) with the target DNA.

The surface enhanced Raman scattering (SERS) signal is preferably formed by laser light irradiation with a wavelength of 632 to 785 nm at an energy of 20 to 400 mW, but is not limited thereto.

In addition, the surface enhanced Raman scattering (SERS) signal preferably measures the vibration band of the Raman dye in the range of 530 to 1540 cm ^-1 , and the measurement time is 10 minutes to 120 minutes, but is not limited thereto.

The biosensor for detecting target DNA of the present invention has an excellent effect of detecting a target DNA at a femto-molar concentration level by using the SERS method.

The biosensor for detecting target DNA according to the present invention is manufactured using gold and silver coated bimetallic nanodendrite having surface enhanced Raman scattering activity, so that the sensitivity and stability of the Raman signal are excellent, When DNA is detected, it exhibits excellent target DNA detection effect in a simple and economical manner.

1 is a diagram showing a biosensor according to Example 1 and a detection method of a target DNA using the same.
2 is a scanning electron microscope (SEM) image of (a) gold nanodendrite and (b) gold (3.17 atomic%) and silver (96.83 atomic%) bimetallic nanodendrite.
3 shows a transmission electron microscope (TEM) image of a bimetallic nano-dendrite of gold (3.17 atomic%) and silver (96.83 atomic%) and a corresponding TEM image mapping image of gold (c) Fig.
FIG. 4 is a graph showing changes in the concentration of gold (blue circle, right y-axis) in gold and silver bimetallic nanodendrite according to the Galvanic reaction time and the Raman signal at 1378 cm ^-1 of the corresponding adsorbed 2-NpSH (Red circle with error bars, left y-axis). The illustration here shows the intensity of the Raman signal at 1378 cm ^-1 of adsorbed 2-NpSH using gold nanodendrite.
Figure 5 shows (a) three different cases (I, II, and III) of hybridization of probe DNA and labeled DNA, wherein the image in the circle represents the molecular structure of the 1-nucleic acid thiol and Raman dye Cy5, and (b ) Corresponding to the Raman spectrum I (red line), II (green line), and III (blue line) of Cy5. In the case of the above II, the illustration shows the Raman spectrum (black line) at 60 minutes after replacing the hybridized labeled DNA with 200 pM of the target DNA.
Figure 6 shows the Raman spectrum of the Raman dye Cy5 at 1468 cm < ^-1 > over time after replacing the hybridized labeled DNA with 200 pM of target DNA (tDNA). Here, the dashed line represents the band intensity before adding the target DNA.
7 is a graph showing the correlation of Raman signal intensity according to target DNA (tDNA) concentration.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example 1. Fabrication of biosensor using gold and silver coated nanodendrite

A bimetallic nanodendrite coated with gold and silver was prepared using an electrochemical deposition method using a galvanic substitution reaction, and a biosensor capable of effectively detecting a target DNA was prepared.

1-1. Preparation of hybridization of labeled DNA with probe DNA and Raman dye

Labeled DNA having 20 base sequences including a thiol functional group at the 5 'terminus and Cy5, a Raman dye, through a linking chain molecule at the 3' terminus, was stirred in a buffer solution To prepare complementary binding probe DNAs and labeled DNAs.

1-2. Preparation of gold and silver bimetallic nano-dendrite

First, a thin titanium foil was cut into 1 × 2 cm to deposit a silver nanodendrite structure, and then 10 mM nitric acid was immersed in 1 M citric acid solution to a depth of 1 cm. When a current of 450 uA is applied for 780 seconds while the titanium foil is placed at the working electrode, the platinum electrode is at the counter electrode, and the silver / silver chloride electrode is at the reference electrode position, silver ions are reduced to silver and a dendritic structure Respectively. When such a substrate is immersed in a 0.1 mM chloroauric acid solution being stirred, the silver on the surface of the structure is oxidized because gold is more reductive than silver, and gold is reduced to cause interstitial substitution reaction. This galvanic substitution reaction was maintained for 1 to 12 minutes to adjust the atomic ratio of gold in the structure.

1-3. Binding of DNA Hybrid to Bimetallic Nanodendrite

The gold and silver bimetallic nanodendrite prepared in Example 1-2 was immersed in the mixed solution of the probe DNA and the labeled DNA prepared in Example 1-1 so that the hybrid was spontaneously formed on the surface of the nanodendrite Lt; / RTI >

Example 2 Detection of Target DNA Using Biosensor

The target DNA was qualitatively and quantitatively detected using the biosensor and Raman signal prepared in Example 1 above.

2-1. Replace the labeled DNA in the biosensor with the target DNA

When the biosensor prepared in Example 1 is immersed in the target DNA solution, a substitution reaction occurs in the hybridization product of the probe DNA and the labeled DNA, and a target DNA is added to 6 to 7 single base chains at the 3 'end of the probe DNA Hybridization begins. Since the target DNA has a base sequence which is completely complementary to the probe DNA, the hybridization proceeds sequentially in the order of the bases, and the original labeled DNA is pushed out. This reaction is reversible, but when all the target DNA binds to the probe DNA, the substitution reaction becomes irreversible, so that the amount of the target DNA can be determined by checking the amount of the labeled DNA dropped.

2-2. Measurement of Raman signal

Raman measurements were made using an integrated Kaiser Optics Raman microscope system. As an excitation source having a laser output of 20 to 400 mW, a diode laser of? = 785 nm was used. A 10 × objective and a 10 × camera imaging system were used to focus the sample. After laser irradiation, scattered light scattered on the substrate was decomposed using a holographic diffraction grating and Raman spectra were collected using a thermoelectrically cooled solid state image sensor detector.

A method of detecting a target DNA using the biosensor is shown in FIG.

Experimental Example 1. Measurement of uniformity of each metal component of gold and silver bimetallic nano-dendrite

Scanning electron microscope (SEM) and transmission electron microscope (TEM) images were measured to confirm the uniformity of each metal component in gold and silver bimetallic nanodendrite in the biosensor prepared according to Example 1.

First, a scanning electron microscope (SEM) image of (a) gold nanodendrite and (b) gold (3.17 atomic%) and silver (96.83 atomic%) bimetallic nanodendrite was measured and shown in FIG.

(A) Transmission electron microscope (TEM) images of bimetallic nano-dendrite of gold (3.17 atomic%) and silver (96.83 atomic%) and corresponding TEM atomic mapping images of gold and (c) As shown in Fig.

As shown in FIG. 2 and FIG. 3, it can be confirmed that the gold and silver components are uniformly coated on the entire surface of the bimetallic nanodendrite of gold (3.17 atomic%) and silver (96.83 atomic%).

Experimental Example 2 Measurement of Stability of Raman Signal According to Gold Concentration of Gold and Silver Bimetallic Nano Dendrite

In order to determine the composition ratios of the respective metal components in the gold and silver bimetallic nanodendrite in the biosensor prepared according to Example 1, the degree of stabilization of the Raman signal according to the concentration of gold was measured. The intensity of the Raman signal at 1378 cm ^-1 of the corresponding 2-NpSH adsorbed (blue circle, right y-axis) and the change in the content of gold in the bimetallic nanodendrite of silver and silver according to the time of galvanic reaction A red circle having an error bar, and a left y-axis) are measured and shown in Fig. The illustration here shows the intensity of the Raman signal at 1378 cm ^-1 of adsorbed 2-NpSH using gold nanodendrite.

As shown in FIG. 4, the silver noddendrite (0 atomic% of gold) has a Raman signal intensity of about 17 times that of gold nanodendrite, but the standard deviation (RSV) of the signal is 34.6% Which is very unstable compared to 9.2% of dendrive. From the above measurement results, it can be seen that, in the case of the bimetallic nano-dendrite having 3.17 at.% Of gold and 96.83 at.% Of silver when the galvanic reaction time is 3 minutes, the RSV of the signal is 8.7% 4 times more stable.

Experimental Example 3. Determination of hybridization sites of probe DNA and labeled DNA

In order to determine the hybridization sites of the probe DNA and the labeled DNA hybridization in the biosensor prepared according to Example 1, (a) three different hybridization cases (I, II, and III) and (b) The Raman spectra of Cy5 (I (red line), II (green line), and III (blue line)) were measured and shown in FIG. Herein, in the case of the hybridization II, the illustration shows the Raman spectrum after elution of the hybridized labeled DNA with 200 pM of target DNA after 60 minutes (black line).

As shown in FIG. 5, in the case of hybridization II, that is, when the labeled DNA was hybridized from the nanodendrite to the second base of the probe DNA, the intensity of the largest Raman signal was exhibited.

Experimental example 4. Detection of target DNA of biosensor

In order to confirm the detection of the target DNA of the biosensor prepared according to Example 1, the change in the Raman signal after the hybridization of the labeled DNA with the target DNA (tDNA) was measured.

First, the hybridized labeled DNA in the biosensor was replaced with 200pM of target DNA, and the Raman spectrum of the Raman dye Cy5 at 1468 cm ^-1 with time was measured and shown in FIG. Here, the dashed line represents the band intensity before adding the target DNA.

In addition, the correlation of the Raman signal intensity according to the target DNA (tDNA) concentration was measured and shown in FIG.

As shown in FIG. 6, it can be seen that the Raman signal is gradually decreased as the hybridized labeled DNA in the biosensor is replaced with the target DNA.

In addition, as shown in FIG. 7, when the concentration of the target DNA was increased, the decrease rate (%) of the Raman signal was gradually increased, and the detection limit (LOD) of the target DNA was 62.5fM.

Claims (11)

Hybridization of labeled DNA with probe DNA and Raman dye; And a bimetallic nanodendrite of gold and silver on which the probe DNA of the hybrid product is immobilized. The method according to claim 1,
Wherein the probe DNA and the labeled DNA are hybridized by complementary binding of the base sequence. The method according to claim 1,
The probe DNA, target DNA biosensor for detection, characterized in that fixed to the surface by a nano dendrite -SH, -CHO, -COOH or -NH _2. The method according to claim 1,
Wherein the labeled DNA is hybridized from the 1 st to 10 th bases of the probe DNA from the nanodendrite. The method according to claim 1,
The Raman dye may be selected from the group consisting of cyanine (Cy3, Cy3.5 or Cy5), xanthines, rhodamine 6G, rhodamine B isothiocyanate (RBITC), adenine, d) pyrimidine, 2-fluoroadenine, N6-benzoyladenine, kinetin, dimethyl-allyl-amino-adenine, zeatin, bromo-adenine, 8- Amide-acridine, and a combination thereof, characterized in that it is any one selected from the group consisting of mercaptopurine, mercaptopurine, 4-amino-6-mercaptopyrrazolo (3,4-d) pyrimidine, 8-mercaptoadenine, Wherein the biosensor is for detecting a target DNA. The method according to claim 1,
The biosensor for detecting target DNA, wherein the gold and silver bimetallic nanodendrite are produced by an electrochemical deposition method using a galvanic substitution reaction of gold and silver. The method according to claim 1,
Wherein the gold and silver bimetallic nanodendrite comprises 1 to 10 atomic percent of gold and 90 to 99 atomic percent of silver. The method according to claim 1,
Wherein the detection limit (LOD) of the target DNA of the biosensor is 50 to 80 fM (femto-molar). (1) hybridizing a labeled DNA with a probe DNA and a Raman dye;
(2) immobilizing the hybridization probe DNA to gold and silver bimetallic nanodendrite;
(3) adding a target DNA to the bimetallic nanodendrite-immobilized hybrid, and replacing the labeled DNA with the target DNA; And
(4) measuring a surface enhanced Raman scattering (SERS) signal before and after replacing the labeled DNA of step (3) with the target DNA. The method of claim 9, wherein
Wherein the surface enhanced Raman scattering (SERS) signal is generated by laser light irradiation with a wavelength of 632 to 785 nm at an energy of 20 to 400 mW. The method of claim 9, wherein
Wherein the surface enhanced Raman scattering (SERS) signal measures the vibration band of the Raman dye in the range of 530 to 1540 cm ^-1 , and the measurement time is 10 minutes to 120 minutes.

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