KR101015434B1 - Dna-based biosensor for the detection of mercury ions using surface plasmon resonance measurements - Google Patents

Abstract

The present invention provides a transparent support layer, a thin layer of gold (Au) coated on the transparent support layer, the self-assembled monolayer and the self-assembled monolayer bonded to the gold thin film layer through the thiol group (-SH) and the self-assembled monolayer By including a gold nanoparticle layer bonded to single-stranded DNA (ssDNA) that can selectively bind to mercury ions, it has high selectivity for mercury ions, which is a target detection material, and enables high sensitivity detection in real time even at very low concentrations. It is an object of the present invention to provide a biosensor for detecting molecular levels of mercury ions. The biosensor for detecting mercury ions of the present invention has high selectivity with respect to mercury ions, which are target detection substances, and enables high sensitivity detection in real time even at very low concentrations, and thus can be used in surface plasmon resonance (SPR) devices. It is effective to provide a biometric sensor of molecular recognition.

Gold nanoparticles, gold thin film layer, biosensor, mercury ion, self-assembled monolayer, surface plasmon resonance

Description

DNA-based biosensor for mercury ion detection using surface plasmon resonance device {DNA-BASED BIOSENSOR FOR THE DETECTION OF MERCURY IONS USING SURFACE PLASMON RESONANCE MEASUREMENTS}

The present invention relates to a biosensor for detecting mercury ions in an aqueous solution using surface plasmon resonance (SPR) measurements, and in particular, a sensing layer using gold nanoparticles in which synthetic DNA is bonded to a film coated with a gold thin film. By forming a sensing layer, the present invention relates to a biosensor for detecting mercury ions having an excellent detection degree of mercury ions.

The surface plasmon resonance (SPR) phenomenon is a collective vibration of electrons occurring on the surface of the metal thin film, and the surface plasmon wave generated by this is a surface electromagnetic wave propagating along the interface between the metal and the adjacent dielectric material. To detect specific contaminants in surface plasmon resonance (SPR) devices, one of the signal conversion techniques to analyze the level of interfacial phenomena.

Recently, cases of mercury damage have been reported as environmental pollutants. Mercury can exist in various forms, especially inorganic compounds, ions in solution, and solids. Among these, mercury is known to have the most stable structure and cause fatal environmental pollution and human damage. Therefore, there is a need for the development of a highly sensitive detection sensor with high selectivity under various conditions.

As an example of a high-sensitivity sensor for mercury detection, research on the development of optical sensors, especially fluorescent sensors ( Anal. Chem. 2008, 80, 3716-3721) has recently been reported. In particular, as disclosed in Korean Patent No. 10-0862606, a fluorescent sensitive portion is introduced to a compound capable of selectively binding with mercury ions to disclose a selective and highly sensitive fluorescent sensitive chemical sensor. However, there is a limit in lowering the detection limit, and there is a problem in that it is not possible to overcome the direct detection of label-free and photo-bleaching due to the introduction of a fluorescent substance that is a labeling in the signal conversion step.

Development of biosensor using local surface plasmon phenomenon using gold nanoparticles ( Angew. Chem. Int. Ed . 2007, 46, 4093-4096; Gold Blutein 2008, 41/1, 37-41) It is reported by the academic community. These studies succeeded in developing a selective sensor through color change using agglomeration of biomaterials and gold nanoparticles that can selectively bind with mercury ions, but it is less sensitive because it depends on the color change of gold nanoparticle solution. There was a problem that the stability of the sensor in terms of use in the fall.

 Therefore, it is required to develop a sensor that can selectively detect mercury ions in an aqueous solution state, and research on sensor chips with high stability and small scale that can be utilized in various industries in accordance with the development trend of nanotechnology It is required.

Therefore, the present invention for solving this problem, a transparent support layer, a gold (Au) thin film layer coated on the transparent support layer, the self-assembled monomolecular layer is bonded through the gold thin film layer and the thiol group (-SH) and the Located on the self-assembled monolayer, it comprises a gold nanoparticle layer bonded to single-stranded DNA (ssDNA) that can selectively bind to mercury ions, providing high selectivity to target mercury ions and real-time at very low concentrations. The present invention aims to provide a biosensor for detecting mercury ions at a nano level by detecting high sensitivity.

The mercury ion detection biosensor according to the present invention for achieving the above object is a transparent support layer, a gold (Au) thin film layer coated on the transparent support layer, the gold thin film layer is bonded through a thiol group (-SH) Located on the self-assembled monolayer and the self-assembled monolayer, characterized in that it comprises a gold nanoparticle layer bonded to a single strand DNA (ssDNA) capable of selectively binding to mercury ions.

The biosensor for detecting mercury ions according to the present invention can be analyzed using surface plasmon resonance, and the first attempt to change the resonance phenomenon of the sensor surface using single-stranded DNA that selectively interacts with mercury ions. It was measured by, to provide a molecular recognition biosensor.

Here, the surface plasmon resonance (SPR) phenomenon is a collective vibration of electrons occurring on the surface of the metal thin film as described above, and the surface plasmon wave generated by the surface plasmon resonance is a surface electromagnetic wave propagating along the interface between the metal and the adjacent dielectric material. The present invention aims to detect specific contaminants in a surface plasmon resonance device, which is one of signal conversion techniques for analyzing nanoscale interface phenomena.

Specifically, a single-stranded DNA containing a large number of thymine groups, which is a type of amino acid capable of molecularly recognizing mercury ions, is formed on a thin film of gold known as a metal that generates surface plasmon waves. By using a surface plasmon resonance (SPR) device to detect trace levels of mercury ions dissolved in aqueous solution using surface plasmon resonance (SPR) devices, It is.

More specifically, the single-stranded DNA that can selectively bind to the mercury ions preferably includes a plurality of thymine groups (hereinafter referred to as T) symmetrically. The reason for this is that the thymine group induces T-Hg (II) -T structure through interaction with mercury ions (hereinafter referred to as Hg (II)). This is to induce the change in loop form. Plasmon resonance phenomenon on the surface of the sensor is changed due to the change of permittivity due to the structural change of DNA and the binding of mercuric ions, and the mercury ion is detected through the signal change. You can do it.

Here, single-stranded DNA containing a large number of thymine groups is preferably bonded to gold nanoparticles and fixed to the surface of the sensor. The reason for this is to dramatically increase the sensitivity of the sensor. If gold nanoparticles are present in the range where the surface plasmon waves are excited on the gold thin film, the plasmon couple is caused by the dipole effect between the gold thin film and the gold nanoparticles. This is because ringing occurs and amplification of the SPR signal is expected.

Such gold nanoparticles are preferably evenly dispersed in a solution for use as the biosensor of the present invention. Specifically, the shape may be in the form of a rod of a spherical shape, a dumbbell shape, a triangle shape, a rhombus shape, a rod shape, and the state of the solution may be an aqueous solution or an organic solvent.

However, in order to self-assemble the nanoparticles into biomolecules and monomolecular layers, the activity and stability of the biomolecules must be maintained, so that a solution in which the nanoparticles are dispersed is preferably an aqueous solution. Do.

Methods of preparing gold nanoparticles in a liquid phase include a method of reducing metal salts at high temperature without using a template, and a method of synthesizing at room temperature using a template. In the method without using a template, metal salts are obtained by dissolving a metal salt in a solvent and adding a strong reducing agent such as citrate under a strong stirring condition at a boiling point or higher.

Alternatively, surface stabilizers or templates may be used to overcome the thermodynamic instability of gold nanoparticle formation. In some cases, hard templates such as medium pore alumina, silica, and carbon nanotubes (CNT) and soft templates such as surfactants are used. Methods for preparing nanoparticles using a template include seed-mediated methods using a seed and growth solution, and polyol processes.

It is preferable to use a citrate reduction method that does not use a template in order to use the biosensor of the present invention among these gold nanoparticle manufacturing method. The reason is that the manufacturing method is relatively simple, and the size of the spherical nanoparticles utilized in the present invention can be easily controlled. In addition, the manufacturing method and the environment is not harshed (harsh) is easy to conjugation (conjugation) with biomolecules sensitive to denaturation.

Hereinafter, the configuration of the present invention in more detail with reference to the drawings as follows.

The transparent support layer included in the present invention is preferably a silica material, in particular, when used in the surface plasmon resonance (SPR) device is preferably a glass of SF10 or BK7 material. The reason is that the material is the same as the prism of the surface plasmon device to be used in the present invention. When the material is used, the surface plasmon (SPR) resonator is used in various media (air, aqueous solution, organic solvent, etc.). This is because the surface plasmon resonance (SPR) angle can be measured.

Specifically, in the present invention, it is preferable to use gold (Au, purity of 99.9% or more) as a metal capable of generating surface plasmon waves and measuring surface plasmon resonance angles. This is because gold thin films are easy to form self-assembled monolayers for organic functional groups that are selective for the detection of contaminants and are advantageous metals for generating surface plasmon waves.

Herein, the gold thin film layer may be directly deposited on the transparent support or, if desired, on the chromium layer, and the thickness thereof is preferably 10 to 100 nanometers. If the thickness is smaller than 10 nanometers, the thickness of the thin film is difficult to form, and if larger than 100 nanometers, there is a problem that is not economical in the absence of an improved effect. For this reason, the thickness of the gold thin film layer is preferably 40 to 60 nanometers.

The gold thin film layer is stacked on one side of the transparent support layer, and thermal evaporation is preferable. This is a suitable method for coating a metal such as gold at the nano level, because it is a suitable method for measuring the surface plasmon resonance angle when using a surface plasmon resonance (SPR) device.

Meanwhile, the self-assembled monomolecular layer introduced on the thus formed gold thin film layer includes a self-assembled monomolecular layer formed of an alkane compound including a thiol group (SH) at one end and an amine group (NH ₂ ) at the other end, as shown in FIG. 1. It is desirable to. The self assembled monolayer (SAM) refers to a monomolecular layer in which the organic molecules constituting the alkane compound are regularly well aligned by spontaneous chemical bonding on the surface of the substrate.

It is preferable to use an alkane compound containing a thiol group (SH) at one end, because one of the thiol groups (SH) has a magnetic structure on the gold thin film layer because it shows a well-ordered surface structure with a crystalline metal such as gold (Au). It is because it is suitable to form an assembled monolayer. In addition, the other amine group (NH ₂ ) is to induce electrical bonding of gold nanoparticles introduced for high sensitivity signal amplification in the present invention. Here, when the alkaline compound including the amine group (NH ₂ ) is self-assembled on the gold thin film, it has a (+) property in its natural state, which is the surface of the gold nanoparticle having the (-) property prepared by the citrate reduction method. It can be combined with strong electrical attraction.

Specifically, an alkane compound containing a thiol group (SH) at one end and an amine group (NH ₂ ) at the other end is selected from the group consisting of 2-aminoethane thiolhydrochloride, 6-amino-1-hexanethiolhydrochloride and 4-aminothiolphenol. It is preferable to use at least one material as the self-assembled monolayer (SAM) of the present invention. This is because the alkane compound is an appropriate carbon chain length in which self-assembly is stable when the self-assembled monomolecular layer is formed on the gold thin film layer.

This self-assembled monolayer is formed by preparing the appropriate alkane compound in an aqueous solution or an ethanol solution and soaking the glass coated with the gold thin film layer for several hours. Specifically, the concentration of the aqueous solution and the ethanol solution is preferably 1 to 10 mM, and the production time is preferably 5 hours to 20 hours.

This is because the stability of the self-assembled monolayer in the above time range is the best. This manufacturing method is a chemical bond is formed by the self-assembly of the alkane compound, and does not require an additional chemical process, and is formed at room temperature and normal pressure, and thus undergoes a very simple process compared to the conventional technology.

A biosensor for detecting mercury ions is produced by introducing and fixing an aqueous gold nanoparticle solution in which single-stranded DNA is bonded onto a sensor surface on which a self-assembled layer of the alkane compound is stacked. In order to bond single-stranded DNA to the surface of well-dispersed gold nanoparticles, it is also preferable that one end (5 'or 3') of the DNA contains a thiol group (SH) because it induces a chemical bond on the gold surface. For the same reason as above.

The mercury ion detection biosensor of the present invention manufactured as described above has a random coil-like single-stranded DNA bonded to gold nanoparticles when exposed to mercury ions, as shown in FIG. As mercury ions are bonded between the symmetrically present thymine groups (T), they change to a loop shape. In order to further stabilize the loop shape, it is preferable to design the remaining bases of the single-stranded DNA in addition to the thymine group symmetrically to induce a duplex.

When the biosensor for detecting mercury ion prepared as described above is analyzed using the surface plasmon resonance device, the interaction between the molecules of the nanoscale level occurring on the surface is very sensitive and selectively detected through the signal change of the surface plasmon resonance. In particular, the introduction of gold nanoparticles has significantly improved the sensitivity (sensitivity) compared to the existing SPR sensor.

As described above, the biosensor for detecting mercury ion of the present invention is a transparent support layer, a gold (Au) thin film layer coated on the transparent support layer, and the gold thin film layer and the thiol group (-SH) are bonded through The self-assembled monolayer and the gold nanoparticle layer on the self-assembled monolayer and bonded to the single-stranded DNA (ssDNA) that can selectively bind to the mercury ions, including a high selectivity for the target detection material mercury ions, It is possible to provide a biosensor for detecting nanoscale molecular recognition mercury ions, which can be used for surface plasmon resonance (SPR) devices at high sensitivity even in very low concentrations in real time.

Hereinafter, the present invention will be described in more detail with reference to the accompanying preferred examples and examples. The following examples are intended to illustrate the invention, but are not limited to these.

[Production Example 1]

To prepare 150 ml of 20 nm spherical gold nanoparticle solution, precursor hydrogen tetrachloroaurate (III) trihydrate (HAuCl ₄ , 3H ₂ O, Aldrich) and reducing agent sodium citrate (Na ₃ C ₆ H ₅ O ₇ , 2H ₂ O , Mallinckrodt) stock solutions were prepared using ultrapure water as a solvent at concentrations of 0.05 g / ml and 0.01 g / ml, respectively.

0.2 ml of the prepared stock solution of HAuCl ₄ was diluted in 200 ml of ultrapure water to prepare an aqueous solution of HAuCl _{4 having} a mass ratio of 0.005%, followed by vigorous stirring while heating to 150 ° C. in a flask connected to a reflux condenser. When the aqueous solution starts to boil, add 1.53 ml of stock solution of sodium citrate to continue heating and stirring.When the color of the solution begins to change from pale yellow to gray after a few minutes, the heating is stopped and cooled at room temperature for one day. .

The TEM image and UV-visible spectrum of the gold nanoparticles thus prepared are shown in FIG. 2. As shown here, it was confirmed that the spherical nanoparticles having a size of 20 nm were well formed, and the absorption pick of the UV-visible spectrum also appeared in the wavelength band near 533 nm.

[Production Example 2]

Single-stranded DNA consisting of symmetrical plural thymine groups capable of selective interaction with mercury ions

After preparing and synthesizing with 5'-GGCTTCAACTGTGAGGCTTCGAGCTTTTGCTCGTTGCCTCTCTGTTGTTGCC-SH-3 'and preparing an aqueous solution of 0.1 mg / ml concentration, 300 μl of single stranded DNA solution was added to 3 ml of the gold nanoparticle solution prepared in Preparation Example 1 (Several solution samples were prepared for Preparation Example 3 below). The before and after comparison of the gold nanoparticle solution thus prepared was confirmed through a UV-visible spectrum, the results were proved through the change in absorption pick as shown in FIG.

[Manufacture example 3]

A 50 nm thick gold was deposited by thermal evaporation (using AUTO306 Edwards) on a SF10 (Fisher Scientific) high clear glass support measuring 1.5 cm wide by 1.5 cm long. The self-assembled monolayer was formed on the gold thin film thus prepared using 18 ml of 1 mM 2-aminoethane thiolhydrochloride ethanol solution for 18 hours at room temperature. Thereafter, the resultant was washed with ethanol and dried with a nitrogen gun, and soaked for 10 hours in 10 ml of the single-stranded DNA conjugated gold nanoparticle solution prepared in Preparation Example 2, and taken out to form a gold nanoparticle layer. After washing with ultrapure water and dried with a nitrogen gun to prepare a biosensor for detecting mercury ions.

Example 1

After the chip was fixed to the surface plasmon resonance apparatus using index matching fluid, 100 μM of aqueous solution of mercuric sulphate was injected into the sample cell portion, and then the plasmon resonance angle was fixed and the surface of the chip was fixed in real time. The change of the reflected light amount (Delta Reflectance) according to the interaction with mercury ions was measured.

This result is shown in FIG. 3. As shown in FIG. 3, it can be seen that the signal change reaches equilibrium at about 10 minutes, which means an equilibrium time according to the interaction between the surface of the manufactured biosensor and mercury ions.

[Example 2]

Except that 10 μM aqueous solution of mercury ion was used as the target detection solution, the biosensor of the present invention was prepared in the same manner as in Example 1, and then the same process was performed, and the results are shown in FIG. 3.

Example 3

A biosensor of the present invention was manufactured in the same manner as in Example 1, except that 5 μM of aqueous mercury ion solution was used as a target detection solution, and the same process was performed as follows.

Example 4

A biosensor of the present invention was manufactured in the same manner as in Example 1, except that 1 μM of aqueous mercury ion solution was used as a target detection solution, and the same process was performed as follows.

Example 5

A biosensor of the present invention was manufactured in the same manner as in Example 1, except that 0.1 μM of aqueous mercury ion solution was used as a target detection solution, and the same process was performed as follows.

As shown in FIG. 3, it can be seen that the biosensor of the present invention can be detected in real time within a short time up to a concentration of mercury ion of 0.1 μM. Based on this result, the SPR signal change according to mercury ion concentration is shown in FIG. 4. As shown here, it was confirmed that a form well suited to the Langmuir adsorption isotherm in the concentration range of the selected mercury ion. This makes it possible to track the concentration of mercury ions of the target substance whose concentration is unknown by using the biosensor of the present invention.

As described above, if the biosensor for detecting mercury ions is used, it is possible to detect in real time for a short time with high sensitivity to very low concentration. In addition, since the structure of T-Hg (II) -T induced by selective binding with mercury ions was used, it was confirmed that the selectivity was very high for other environmental pollutants. Therefore, it is expected to be very effective as a small industrial sensor based on nanotechnology.

The present invention described above is capable of various substitutions, modifications, and changes without departing from the technical spirit of the present invention for those skilled in the art to which the present invention pertains. Of course, it is not limited to, but should be determined to include the claims and equivalents as well as the claims to be described later.

1 is a schematic diagram showing the shape of the biosensor according to the present invention and the principle of mercury ion detection.

Figure 2 is a graph showing the UV-visible spectral change of the gold nanoparticles prepared according to the present invention and the gold nanoparticles aqueous solution conjugated with a single DNA.

3 is a graph showing the results of detecting mercury ions in different concentrations in real time using the biosensor according to the present invention.

Figure 4 is a graph showing the results of the surface plasmon resonance (SPR) signal obtained in Figure 3 according to the mercury ion concentration using the biosensor according to the present invention.

Claims (4)

Transparent support layer; A gold (Au) thin film layer coated on the transparent support layer; A self-assembled monolayer bonded to the gold thin film layer through a thiol group (-SH); And A gold nanoparticle layer positioned on the self-assembled monolayer and having a single stranded DNA (ssDNA) bonded thereto to selectively bind with mercury ions; Mercury ion detection biosensor comprising a. The method of claim 1, The self-assembled monolayer consists of an alkane compound containing a thiol group at one end and an amine group (-NH ₂ ) at the other end, and the alkane compound is 2-aminoethane thiolhydrochloride, 6-amino-1-hexanethiolhydrochloride and 4- A biosensor for detecting mercury ions, characterized by being formed from one or more substances selected from the group consisting of aminothiolphenol. The method of claim 1, Single-stranded DNA (ssDNA) capable of selectively binding with the mercury ion, mercury ion detection biosensor comprising a plurality of thymine (represented by T.) groups symmetrically. The method of claim 1, The gold nanoparticles are biosensors for mercury ion detection, characterized in that the spherical particles having an average particle diameter of 3 to 100 nanometers (nm).

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