KR100496938B1 - Method for manufacturing nano bio-array - Google Patents

Abstract

The present invention comprises the steps of taking a sample containing the target material on the tip while applying a tip bias voltage of the opposite polarity to the target material to the STM tip; Moving the tip onto the substrate to be sampled, and then applying a sample bias of opposite polarity to the target material to generate a tunneling current to approach the tip to the substrate; And removing the tip bias voltage to drop a sample attached to the tip onto a substrate.

Description

Method for manufacturing nano bioarrays {Method for manufacturing nano bio-array}

The present invention relates to a method for manufacturing a bioarray, and more particularly, to a method for manufacturing a Baoarray with a high density of nanoscale.

Biochips, commonly referred to as biochips, are biological microchips that combine biological molecules such as DNA and proteins on small substrates to analyze gene binding, protein distribution, and reaction patterns. Biochips can be used in the fields of proteomics and genomics, especially in the diagnosis of genetic or pathogenic diseases, the study of protein expression and function, the study of protein interactions, and the development of new drugs. It is widely used in medicine, pharmacy, and life sciences.

Protein chips have a microarray of proteins that react with a substance to be detected (target protein), so that the presence or absence of a target protein in a sample can be determined using the protein chip. DNA chips can detect the presence of genetic diseases by identifying the DNA sequences involved in the expression of specific proteins. To this end, the DNA chip uses a polynucleotide complementary to the target DNA as a probe, and confirms the presence or absence of the target DNA in the sample by detecting the presence or absence of a hybrid bond between the target DNA and the probe in the sample. However, the hybrid binding of DNA probes to target DNAs and the reactivity of antibodies and antigens are significantly lower in vitro than when in vivo. Therefore, in order to improve detection sensitivity of biochips and further develop high-level technologies for manufacturing multi-detection biochips such as lab on a chip, there are methods of increasing the concentration of a target substance in a sample and highly integrated micro array. . For example, in the case of a DNA chip, the target DNA in the sample is amplified and used by PCR. Protein chips can be amplified using a rolling-circle-amplification technique.

On the other hand, one of the high integration method of the micro array is a method of minimizing the size of the spot. Spot forming methods of the micro array include a spray method and a tip method. However, this conventional method has a limit in reducing the spot size. For example, for protein chips, the spot size is usually hundreds of micros, and DNA chips have a spot size of tens of macros. Recently, methods for producing nanoscale probes using atomic force microscopy (AFM) techniques have been known. Specifically, the gold thin film and the organic monolayer are formed on the substrate, and the AFM is used to etch the organic monolayer in a predetermined pattern to expose the gold thin film, and then the DNA probe is attached to the exposed gold thin film region. The selected regions on the substrate are cut along the scanning track of the AFM tip, which removes the organic monolayer and immediately adsorbs DNA molecules into these regions (Production of Nano Structures of DNA on Surfaces, "Nano Letters, August 14, 2002). This method also did not reach the degree of high concentration of the sample.

Accordingly, the main object of the present invention is to provide a method for producing a nano bioarray that enables high concentration of a sample and high integration of a micro array.

In order to achieve the object of the present invention, the present invention comprises the steps of collecting a sample containing the target material on the tip, while applying a tip bias voltage of the opposite polarity to the target material to the STM tip; Moving the tip onto the substrate to be sampled, and then applying a sample bias of opposite polarity to the target material to generate a tunneling current to approach the tip to the substrate; And removing the tip bias voltage to drop a sample attached to the tip onto a substrate.

In the method for producing the array of the present invention, the target material is preferably a biological molecule, more preferably a protein, nucleic acid, or lipid.

In the method of manufacturing an array of the present invention, the method may further include coupling a thiol group to a target material before the sampling step.

In the method of manufacturing an array of the present invention, the method may further include pretreating the target material to exhibit a positive charge or a negative charge before the sampling step.

In the method of manufacturing the array of the present invention, the method may further include forming a metal thin film on the substrate before the sampling step, wherein the metal thin film is preferably formed of gold, silver, copper or platinum. The method may further include pretreating the substrate to form a positive or negative charge on the substrate after the metal thin film forming step.

Hereinafter, a method of fabricating an array on a substrate using the STM of the present invention will be described in more detail.

Most proteins have optimal pI values. By adjusting this value with a buffer solution, the net charge of a protein can be changed to either positive or negative charge. If the target material to be arrayed is DNA, it has a negative charge on its own. And since the STM tip itself acts as an electrode, most proteins and DNA can be concentrated and harvested. Therefore, the sample is collected at a high concentration on the STM tip by the electrical attraction force when applied to the tip bias of the charge opposite to the target material. By adjusting the sample bias, tip bias, and tunneling current, the size of the arrayed points can also be adjusted. The size of the arrayed spot is usually about 500-800nm.

First, STM (Scanning Tunneling Microscopy) used in the present invention is a surface analyzer. Unlike conventional microscopes, STM uses quantum mechanical electron tunneling to measure the surface geometry. Approaching the probe to the surface of the sample, which is a conductor, at an interval of about one-half size (about 0.5 nm), and applying a voltage between the two conductors, electrons penetrate through the energy wall and current flows through the quantum mechanical tunneling phenomenon. Happens. STM is a device that obtains a photograph showing the topography of a sample by placing a sample on a scanner and constantly measuring the tunneling between two electrodes to quantify the degree of change and display the brightness on a computer screen. Not only the shape of the surface structure of the sample, but also the degree of curvature of each part, as well as the cross-sectional view, three-dimensional view and various statistical data can be obtained.

1 is a view schematically showing a manufacturing principle of the nano bioarray using the STM tip. In FIG. 1B, reference numeral 1 is an STM tip, 2 is a target material, 3 is STM, 4 is a metal film, and 5 is a substrate.

First, the tungsten wire is etched in an oxidizing solution and then washed with distilled water to remove residual oxide to complete the STM tip 1 used as an array.

The thiol group can be bound to the target material before sampling. In the case where the substrate has the metal thin film 4 on its surface, particularly when using a substrate on which a gold film is formed, it is preferable to bond the thiol group to the target material. As a method of bonding a thiol group, any method conventionally used in the art by a thiol group attachment method can be used.

If the target material has a neutral charge, it is preferable to make the total charge positive or negative with a buffer.

The substrate 5 used in the present invention can be any one conventionally used in the art such as glass, silicon, and polymer, and a silicon or glass substrate having a gold film formed thereon is preferable.

Observe the STM tip (1) under a microscope and dip it so that the tip of the tip enters the sample slightly. Apply a tip bias voltage with a polarity opposite to the target material to allow the target material, eg negatively charged DNA (2), to adhere to the sample at the tip of the STM tip for 3 to 5 seconds. Soak.

After moving the STM to the spot to be spotted, the tip 1 is brought close to the substrate 5. In this case, the tip bias is applied as a charge of the same polarity as the target material, and the sample bias is applied as a charge of the opposite polarity to the target material.

In the present invention, "applying a sample bias" means applying a bias to a substrate.

Provide a tunneling current between 0.2 and 0.3 nA between the tip and the substrate. Tunneling current is used in the instrument's clearance control to bring the tip close to the sample. That is, if there is a voltage between the tip and the sample (i.e. the substrate) and the distance between the tip and the sample is very close (several to several nm), even if the tip and the sample are not connected in one circuit, the current between the tip and the sample Flows, which is the tunneling current. Therefore, if the same bias is applied, the smaller the input, the farther the distance between the tip and the sample. The gap control of this unit is controlled by a controller and a computer connected to the unit. The maximum bias of this instrument is ± 7V. Therefore, the voltage between tip and sample can be extended up to 14V, and automatic clearance control is only between 0.2 ~ 5nA. In order to reduce the size of the spot, the distance between the tip and the sample must be as wide as possible, so the voltage is ± 7 V, which is the limit of the instrument (actual voltage is 14 V), and the set point of the tunneling current is the smallest value (0.2 to 0.3 nA). The distance between the tip and the sample will then be the furthest from the instrument's acceptable range.

In the case of the automatic approach, when the tip stops, the tip moves with a slight vibration. Therefore, it is desirable to check the tunneling current to force or manually approach the tip.

The tip stops when the tunneling current reaches a constant value. At this time, if the tip bias is set to zero and the sample bias is applied at the opposite charge to the target material, the target material in the sample is dropped onto the substrate by the electrical attraction between the substrate and the sample material.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

1. Making the tip

After tungsten wire was etched into the oxidizing solution and washed with distilled water to remove the remaining oxide to produce an STM tip used as an array.

2. Preparation of Sample

A single-stranded DNA (20 μM) (genotech Co., Ltd.) labeled with a thiol group (-SH) was used.

3. Sample Collection Step

The STM used in this example was from PSI (Park Scientific Instruments). A sample was taken and placed on the substrate using an STM tip before loading onto the substrate. This is a preprocessing process to easily find the array points with AFM. The outer strand DNA to be used as a probe was dissolved in a 3% glycerol solution for moisturizing effect, and the sample was taken by dipping the tip in 5 ml of the solution. At this time, a tip bias of about + 5V was applied with a charge opposite to the target material of the STM, and the tip was immersed in the depressed sample solution for about 5 seconds using a microscope to sufficiently adhere the sample (external strand DNA) to the tip.

4. Tip approach steps

After DNA was attached to the tip, the tip was moved to a predetermined position on the substrate. At the end of the shift a tip bias was applied at + 7V and a sample bias at about -7V. The tunneling current gave 0.3 nA, so that the proximity between the tip and the substrate was as far as possible.

5. Unloading Step

After the proximity was complete, the tip bias was removed and the sample bias was + 7V. This control allowed a large amount of negatively charged DNA to be loaded onto the substrate. The array was completed by repeating the above process.

6. Identification of Nano Arrays

An image of the nanoarray was obtained by scanning using AFM (PSI, Model Name: CP) (using a 100 μm scanner) (see FIG. 3). The shape of the points arrayed using STM is well characterized when loaded. In other words, the part where the tip touches the middle is deep, and the sample is pushed to both sides. This is because the sample bias of the tip is strongly sucked by the electrical force into the substrate by applying a sample bias when loaded.

In situ experiments were performed using a surface plasmon resonance device (Optrel, model name: multiscope) to confirm that the strand DNA was fixed to the surface (see FIG. 2).

In FIG. 2, water was flowed initially (0 to about 900 seconds), and the reflectance intensity at this time was 0.32. After about 900 seconds, the stranded DNA was added, and the size of the reflectance changed to 0.45. However, after about 6000 seconds, the water was washed again, but the reflectivity did not change significantly from 0.45. As a result, it can be seen that the single stranded DNA is bound on the substrate.

As described above, according to the present invention, the high concentration of the target material in the sample and the high integration of the bioarray can be achieved by using the STM, thereby enabling the manufacture and miniaturization of the high sensitivity sensor and the multi-detection biochip in a simple method and at a low cost.

1 is a view schematically showing a manufacturing principle of the nano array using the STM tip.

2 is a graph confirming that the outer strand DNA, which is an immobilized sample material, is immobilized on the surface of the substrate on which the gold film is formed using the surface plasmon resonance apparatus.

3 is a diagram obtained by using an AFM after fabricating a nano-sized array using the STM tip.

<Description of the symbols for the main parts of the drawings>

1. Tips

2. Target substance

3. STM

4. Metal film

5. Substrate

Claims (8)

Collecting a sample containing a target material at the tip while applying a tip bias voltage of a polarity opposite to that of the target nucleic acid or protein to the scanning tunneling microscopy (STM) tip; Moving the tip onto the substrate to be sampled, and then applying a sample bias of opposite polarity to the target material to generate a tunneling current to approach the tip to the substrate: and Removing the tip bias voltage to drop a sample attached to the tip onto a substrate. delete delete The method of claim 1, further comprising binding a thiol group to a target material before the sampling step. The method of claim 1, further comprising pretreating the target material to exhibit a positive charge or a negative charge before the sampling step. The method of claim 1, further comprising forming a metal thin film on the substrate before the sampling step. The method of claim 6, wherein the metal thin film is formed of gold, silver, copper or platinum. The method of claim 6, further comprising pretreating the substrate to form a positive or negative charge on the substrate after the metal thin film forming step.