WO2011149249A2 - Electric biosensor for detecting an infinitesimal sample - Google Patents

Abstract

The present invention relates to an electric biosensor for detecting an infinitesimal sample. More specifically, the present invention relates to a method for electrical detection of an infinitesimal sample, comprising the steps of: (a) preparing a biosensor by fixing, to an electrically insulated nano-electrode chip, a receptor molecule that is selectively coupled to a target substance to be subjected to an assay; (b) adding a sample that contains the target substance selectively coupled with the receptor molecule to the biosensor for reaction; (c) subjecting the nano-electrode chip that has been reacted with the target substance-containing sample to treatment and reaction with gold nanoparticles; (d) treating peripheral regions of the reacted gold nanoparticles with gold ions to induce reduction of the gold ions; and (e) measuring the electric conductivity or impedance of the nano-electrode chip where the gold ions have been reduced, thereby detecting a target substance that is specifically bonded to the receptor molecule. The method for electrical detection of an infinitesimal sample using the biosensor according to the present invention not only interconnects nano-electrodes through a single reduction reaction between gold nanoparticles and gold ions, but it also electrically interconnects the electrically insulated nano-electrodes such that the electrical conductivity is increased. As a result, the measurement sensitivity is high, the quantitative assay of an infinitesimal sample becomes possible, and a selective antigen-antibody bond can be measured with a very simple, electrical method.

Description

Electric Biosensor for Ultra Trace Sample Detection

The present invention relates to an electric biosensor for detecting a trace amount of a sample, and more specifically, (a) to fabricate a biosensor by immobilizing a receptor molecule that selectively binds to a target material of analysis on an electrically insulated nano-electrode chip. step; (b) adding and reacting a sample containing a target substance selectively binding to the receptor molecule to the biosensor; (c) treating and reacting the gold nanoparticles to the nano-electrode chip reacted with the sample containing the target material; (d) treating and reducing gold ions around the reacted gold nanoparticles; And (e) measuring an electrical conductivity or impedance of the nano-electrode chip of which the gold ions are reduced to detect a target substance that specifically binds to the receptor molecule. It is about.

Quantitative measurement of biomolecules such as trace proteins and DNA is important for clinical diagnosis and proteomics research.

In order to measure proteins, conventionally, an enzyme-linked immunosorbent assay (ELISA) method is mainly used, which first immobilizes an antibody on a matrix, and prepares a sample solution in which an antigenic protein is mixed on the surface where the antibody is immobilized. After reacting for a certain time, a detection antibody that selectively binds to the antigenic protein on the surface to which the antigenic protein is bound is bound, and then a reaction substance capable of causing the enzyme to develop color or fluorescence by binding an enzyme to the detection antibody. It is a method of quantitating a small amount of antigenic protein by adding a color or fluorescence reaction to occur. Although the ELISA method has been used as an effective method for measuring trace antigen proteins, the analysis range of antigen proteins is about 10 pg / ml ~ 1 ng / ml, and only one antigen protein can be analyzed at a time. The disadvantage is that a large amount of sample is required to analyze various antigenic proteins.

Recently, various methods for simply measuring antigen concentration by applying the principle of ELISA to biosensors and biochips have been developed. After the ELISA process, the enzyme attached to the surface in proportion to the antigen concentration induces a precipitation reaction to allow the enzyme reaction product to adhere to the surface, followed by surface plasmon resonance (SPR), quartz crystal microbalance, QCM), electrochemical sensors and other research results have been published. This method can not only measure trace amounts of antigens easily, but can also measure various kinds of antigens simultaneously (Kim et al., J. Immunol. Meth., 297: 125, 2005; Abad et al., Anal Chim.Acta, 368: 183, 1998). However, the method does not significantly improve the measurement sensitivity compared to the conventional ELISA.

Accordingly, the present inventors have diligently tried to develop a method for the electrical detection of a trace amount sample using a biosensor. As a result, the biosensor was manufactured by immobilizing a receptor molecule that selectively binds to a target material on an electrically insulated nano-electrode chip. Next, after processing the target material in the biosensor, by connecting the nano-electrodes through a gold nanoparticle-gold ion reduction reaction, by measuring the electrical conductivity between the nano-electrode, the trace amount of the target material is easily measured It was confirmed that the present invention can be completed.

Summary of the Invention

An object of the present invention is to provide a method for the electrical detection of the trace amount sample using a biosensor.

In order to achieve the above object, the present invention comprises the steps of: (a) manufacturing a biosensor by immobilizing a receptor molecule that selectively binds to the target material of analysis on the electrically insulated nano-electrode chip; (b) adding and reacting a sample containing a target substance selectively binding to the receptor molecule to the biosensor; (c) treating and reacting the gold nanoparticles to the nano-electrode chip reacted with the sample containing the target material; (d) treating and reducing gold ions around the reacted gold nanoparticles; And (e) measuring an electrical conductivity or impedance of the nano-electrode chip of which the gold ions are reduced to detect a target substance that specifically binds to the receptor molecule. To provide.

FIG. 1 is a schematic diagram illustrating a measurement method of a target material using a nano-electrode biosensor coupled by a gold nanoparticle-gold ion reduction reaction and a schematic diagram of a chromium electrode biosensor having an electrode interval of 400 nm.

2 is a process of forming a precipitate by gold ion reduction of gold nanoparticles in a liquid phase according to the present invention. (A: gold ion reduction solution without gold nanoparticles; B: gold ion reduction solution with gold nanoparticles bound to antibodies; C: comparison of absorbance over time at 970 nm)

3 is a scanning electron microscope (SEM) image of a silicon oxide substrate prepared by the method of Example 1 according to the reduction time of gold ions. (a: after 1 minute of reduction time; b: after 2 minutes of reduction time; c: after 5 minutes of reduction time; d: after 7 minutes of reduction time; e: after 10 minutes of reduction time; f: fixation of gold nanoparticles as a control After 10 minutes of reduction time on the silicon oxide substrate)

4 is an image (A), and SEM images (B and C) of a 400 nm chromium electrode made by the method of Example 2. FIG.

FIG. 5 shows the current of the electrode before the reduction of the gold ions to the electrode to which the gold nanoparticles were fixed for 0 pg / ml, 100 pg / ml, and 100 ng / ml of the IL5 antigen, by using a DC current-voltage method. Results (A: 0 pg / ml (…); 100 pg / ml (-); 100 ng / ml (−)) and current after gold ions were reduced (B: 0 pg / ml (…); 100 pg / ml (-); 100 ng / ml (-)).

6 is a result of measuring the current under 1 V voltage with respect to the concentration of 0 pg / ml ~ 100 ng / ml IL5 antigen.

7 is a Scanning Electron Microscope (SEM) image of the surface of silicon oxide between chromium electrodes prepared by the method of Example 2 for concentrations of 0 pg / ml, 100 pg / ml, 100 ng / ml IL5 antigen to be.

FIG. 8 shows the results of measuring the current of the H1N1 virus 0 PFU and 10 ³ PFU by using DC current-voltage method on the electrode before the reduction of gold ions to the electrode to which the gold nanoparticles were fixed (A: 0 PFU (…). 10 ³ PFU (-)) and the current after reduction of gold ions (B: 0 PFU (…); 10 ³ PFU (-)).

9 is a result of measuring the current at 1 V voltage after gold ion reduction with respect to H1N1 virus 0 PFU-10 ³ PFU concentration (0, 10 ¹ , 10 ² , 10 ³ PFU).

Detailed Description of the Invention and Specific Embodiments

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, (a) manufacturing a biosensor by immobilizing a receptor molecule that selectively binds to the target material to be analyzed on an electrically insulated nano-electrode chip; (b) adding and reacting a sample containing a target substance selectively binding to the receptor molecule to the biosensor; (c) treating and reacting the gold nanoparticles to the nano-electrode chip reacted with the sample containing the target material; (d) treating and reducing gold ions around the reacted gold nanoparticles; And (e) measuring an electrical conductivity or impedance of the nano-electrode chip of which the gold ions are reduced to detect a target substance specifically binding to the receptor molecule. It is about.

In the present invention, the nano-electrode chip can be produced by a process selected from the group consisting of photolithography, electron beam lithography, ion-focused lithography and nanoimprinting, the interval between the electrodes in the nano-electrode chip is As demonstrated in the experiment, any nano-electrode chip having a spacing of 1 μm or less may be applicable, for example, 10 nm to 1 μm, but is not limited thereto.

In the present invention, the term "nano-electrode chip" means that a gap between electrodes constituting a generally known biochip is 1 μm or less or 100 nm or less.

The method for measuring electrical conductivity or impedance in the present invention is not particularly limited, and any conventional electrical detection method may be applied to the present invention without limitation.

Studies using gold nanoparticle-silver reduction reactions on microelectrodes have already been published (Park et al., Science, 2002; Marcon et al., Biosensors and Bioelectronics, 2007). It requires a long time, and has a problem that if the chlorine ions such as sodium chloride in the previous solution caused by the chlorine ions and precipitates to be removed, the present invention is possible within 10 minutes using an electrode of less than a micron By using a gold reduction reaction, the above problems can be solved.

In the present invention, the substrate may be a silicon wafer, glass, or the like, and the electrode may be chromium, titanium, gold, or the like.

In addition, one embodiment of the present invention exemplifies IL5 and H1N1 viruses as target substances, but in addition to this, various cytokine, various enzymes, proteins (eg, interleukin), DNA, RNA, microorganisms, viruses, animal cells, plant cells , Organ cells and neurons may be applicable, and the receptor molecule may be selected from the group consisting of antibodies, DNA, aptamers, peptide nucleic acids (PNAs), and ligands.

In the present invention, the gold nanoparticles in the step (c) may be characterized in that the binding to the target material using the additional sensing receptor molecules capable of binding to the target material. For example, the receptor molecule may be an antibody and an antigen-binding agent, with the target substance as an antigen, and thus, detection using the sandwich method as exemplified in the examples may be performed, and at this time, an additional agent for detecting a target substance may be used. Sensing receptor molecules are needed. Such further sensing receptor molecules may also be selected from the group consisting of antibodies, DNA, aptamers, peptide nucleic acids (PNAs) and ligands.

In the present invention, the gold nanoparticles may be characterized in that the size of 2nm ~ 100nm. The gold nanoparticles can be prepared by mixing gold ions with a reducing agent, and are readily available commercially from reagent companies such as Sigma.

Much research is underway on the method of electrically connecting micro electrodes by deposit formation by enzymatic reactions (Mo1ller et al., Nano Lett. , 5: 1475, 2005; WO 02/103037). However, since the study uses a micro electrode, the amount of enzyme binding to the surface should be large, or an additional precipitation reaction must be induced after the enzyme precipitation reaction to connect the electrodes so that current flows.

On the other hand, the method of measuring the target material by the method of bonding the gold nanoparticles to the nano-electrode (Malaquin et al., Microelectron. Eng ., 73 ~ 74: 887, 2004), but the gold nanoparticles There is a disadvantage that the current increase is not large because it is not completely connected, and a method of measuring a target material using a nano-sized electrode has been reported (Kim et al., Nanotechnology , art.No. 455502: 20, 2009), which also has the disadvantage that the change of the signal does not change significantly.

Accordingly, the present invention provides the advantage that by using a gold nanoparticle-nano electrode, by reducing the gold ions, one gold nanoparticle can be connected to the electrode during the reduction reaction to increase the current intensity to increase the analysis sensitivity do. In addition, due to the increased sensitivity of the assay, according to the measurement method of the present invention, as shown in the following examples, it is possible to detect the target substance at a low concentration of about 1 pg / ml, or even in the case of viruses, 10 PFU. By confirming that the electrical detection is possible, it was confirmed that the detection of a very small amount of sample is possible.

In one embodiment of the invention, the surface of the silicon oxide substrate is modified with an amine group, and then the surface of the substrate modified with the amine group is modified with a biotin group. After streptavidin is immobilized on the surface of the substrate modified with the biotin group, the biotin-coupled antibody is immobilized. Then, the antigen is reacted, and the gold nanoparticle-bound antibody is reacted to measure sandwich immunity. Then, a gold reduction solution is induced by using a gold chloride solution and a hydroxylamine solution to generate a metal reduction product on the substrate surface. do. After washing the substrate, it can be observed by scanning electron microscopy (SEM) that the precipitate is formed by drying with nitrogen gas.

In one embodiment of the present invention, after fixing the gold nanoparticles, the gold ions are reduced to the gold ions and the reduced electrode respectively, the current flows after the current, after measuring the current, the gold ions are reduced It can be seen that the conductivity of the electrode is increased compared to the electrode that is not reduced, and the current value increases as the concentration of the antigen increases.

Therefore, in the present invention, after binding the gold nanoparticles to various binding reactions such as antigen-antibody, DNA-DNA, DNA-RNA, PNA-DNA, induce gold nanoparticle-gold ion reduction reaction, and then apply current or resistance. By the measuring method, the concentration of the target substance to be analyzed can be measured.

Through this measuring method, finally, a reducing material obtained through a reduction reaction of gold nanoparticles-gold ions to an electrically insulated nano-electrode chip fills the nano-gap to flow an electric current. There is provided a biosensor for detection (FIG. 1).

EXAMPLE

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1 Gold Ion Reduction over Time of Gold Nanoparticles Immobilized on Silicon Oxide Substrate

The silicon oxide substrate (16 × 16 mm) was immersed in a solution of 95% sulfuric acid and 30% hydrogen peroxide solution at a volume ratio of 3: 1 at 60 to 65 ° C. for 20 minutes, followed by distilled water and ethanol. The washed silicon oxide substrate was immersed in an ethanol solution dissolved in 1% 3-aminopropyltrimethoxysilane (3-aminopropyltrimethoxysilane, Aldrich, St. Louis, MO, USA) for 1 hour, then washed with ethanol, 100 Treatment at 1 ° C. for 1 hour allowed the substrate surface to be modified with an amine group.

A 10 nm gold nanoparticle (British Biocell International, Cardiff, UK) solution diluted 10,000-fold with distilled water was reacted on the surface modified with the amine group for 10 minutes. Next, a 10 mM site containing 25 mM gold tetrachloride (HAuCl ₄ , hydrogen tetrachlorideaurate (III), Aldrich, Millwauke, WI, USA) and 5 mM hydroxylamine (Sigma, St. Louis, MO, USA) The rate buffer solution (pH 4.0) was reacted with time.

FIG. 2 illustrates a process of forming a reduced product reacted with gold nanoparticles in a liquid phase by using the reducing solution prepared above. When 1 μl of a gold nanoparticle solution is added, gold ions are formed after 5 minutes and 10 minutes. It was confirmed that the maximum afterwards.

Figure 3 shows a scanning electron microscope (Scanning Electron Microscope, SEM) image of the silicon oxide substrate prepared above, as a result of confirming the size of the gold nanoparticles according to the reduction time (reduction time) of the gold ions, the reduction time will be longer As the size of the gold nanoparticles gradually increased, it was confirmed that the reduction reaction was about 1 μm after 7 minutes. This result means that nano-electrodes having a gap of 1 μm or less can be electrically connected by gold ion reduction.

Example 2: IL5 Detection Using Gold Nanoparticle-Gold Ion Reduction at 400 nm Chromium Electrode

A 400 nm chromium electrode was prepared by a stepper photolithography process and an electron beam deposition process (FIG. 4). After the silicon oxide film was formed on the silicon wafer by low pressure chemical vapor deposition (LPCVD), the pattern was first patterned by stepper photolithography to form a line having a 400 nm line width on the surface. Cr 100 nm was deposited by a thermal evaporator. Figure 4 shows an SEM image of the electrode (that is, interdigitated electrode) manufactured by the above process, it can be seen that the electrode insulated at intervals of 400 nm.

The prepared electrode was immersed in a solution of 95% sulfuric acid and 30% hydrogen peroxide solution at a volume ratio of 3: 1 at 60 to 65 ° C. for 20 minutes, and then washed with distilled water and ethanol. The washed electrode was immersed in an ethanol solution in which 1% 3-aminopropyltrimethoxysilane (Aldrich, St. Louis, MO, USA) was dissolved for 1 hour, washed with ethanol, and then washed at 100 ° C. After processing for 1 hour, the silicon oxide surface between the chromium electrodes was modified with an amine group.

In order to modify the surface of the amine group modified with a biotin group, NHS-LC-biotin (10 mg / ml) dissolved in dimethyl sulfooxide (DMSO, dimethy sulfoxide, SImga-Aldrich, St. Louis, MO, USA) at a concentration of 10 mg / ml ( NHS-LC-biotin, Thermo sciences, Rockford, IL, USA) was treated with a solution adjusted to a concentration of 1 mg / ml with PBS buffer, and reacted at room temperature for 1 hour to replace an amine group with a biotin group. After treating 0.1 mg / ml of streptavidin (Streptavidin, SImga-Aldrich, St. Louis, MO, USA) in PBS buffer (pH 7.4) on the surface of the biotin-modified substrate, By reacting for a time, the silicon oxide surface between the chromium electrodes was made into the streptavidin surface.

Thereafter, the biotin-bound IL5 antibody was immobilized on the surface of streptavidin, and the antigen-antibody reaction was performed at an concentration of 0 pg / ml-100 ng / ml. Next, the IL5 antibody with 10 nm gold nanoparticles was reacted with the antigen immobilized on the surface, followed by 25 mM gold tetrachloride (HAuCl ₄ , hydrogen tetrachlorideaurate (III), Aldrich, Millwauke, WI, USA) and 5 mM hydroxylamine. 10 mM citrate buffer (pH 4.0) dissolved in (hydroxylamine hydrochloride, Sigma, St. Louis, MO, USA) was reacted for 10 minutes to reduce gold ions to gold nanoparticles.

The currents of the electrodes in which the gold ions were not reduced and the electrodes in which the gold ions were reduced were measured.

As a result, in the case of the electrode without the reduction of gold ions, when the voltage of 1 V was applied, the current difference between the current at 0 pg / ml IL5 antigen concentration and the current at 100 ng / ml IL5 antigen concentration was 1 nA. For the reduced electrode it can be seen that the current difference of 300 μA (Fig. 5). This means that the electrode with reduced gold ions induces an increase in conductivity compared to the electrode which is not reduced.

6 is a graph showing the current value for 1 V after the gold ion reduction according to the concentration of IL5 antigen, it was confirmed that the current value increases as the concentration of the antigen increases.

In addition, a scanning electron microscope (Scanning Electron Microscope, SEM) was used to confirm the surface of the silicon oxide between the chromium electrode prepared above.

As a result, as shown in Figure 7, it was confirmed that the surface of the silicon oxide between the chromium electrode prepared above, the formation of a gold ion reducing material of several micrometers or more as the concentration of the antigen increases.

Example 3: H1N1 Virus Detection Using Gold Nanoparticle-Gold Ion Reduction at 400 nm Titanium Electrodes

A 400 nm titanium electrode was fabricated by a stepper photolithography process and an electron beam deposition process. A silicon oxide film was formed on the silicon wafer by low pressure chemical vapor deposition (LPCVD), and then patterned by stepper photolithography to form a line having a 400 nm line width on the surface. Titanium 30 nm was deposited by thermal evaporator.

In order to immobilize the antibody on the electrode, the silicon oxide surface between the titanium electrodes was made into the streptavidin surface in the same manner as in Example 2. Then, biotin-bound H1N1 virus antibody (rabbit polyclonal) was immobilized on the surface of streptavidin, 17 μl of H1N1 virus solution (suspended in pH 7.4 PBS buffer) was reacted on the sensor surface, and the amount of virus reacted was 0. , 10 ¹ , 10 ² , and 10 ³ PFU (plaque forming units). Subsequently, each electrode was washed with PBS and reacted with a H1N1 antibody (mouse monoclonal) with 10 nm gold nanoparticles followed by 25 mM gold tetrachloride (HAuCl ₄ , hydrogen tetrachlorideaurate (III), Aldrich, Millwauke, WI, USA). And 10 mM citrate buffer (pH 4.0) dissolved in 5 mM hydroxylamine (Sigma, St. Louis, MO, USA) were reacted for 10 minutes to reduce gold ions to gold nanoparticles.

As a result of measuring the currents of the electrode with no reduced gold ions and the electrode with reduced gold ions, the current at 0 PFU H1N1 virus concentration when a voltage of 1 V was applied in the case of the electrode with no reduced gold ions. The current difference at and 10 ³ PFU H1N1 virus concentration was 0.8 nA, whereas the gold ion-reduced electrode showed a difference of 96 μA (FIG. 8). This means that the electrode with reduced gold ions induces an increase in conductivity compared to the electrode which is not reduced.

9 is a graph showing the current value for 1 V after the gold ion reduction according to the concentration of the virus, it was confirmed that the current value increases as the concentration of the virus increases.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Explanation of the sign

11: electrode; 21: insulator surface; 31: antibody; 41: antigen; 51: detection antibody; 61: gold nanoparticles; 71: precipitate by reduction reaction

As described above, the electrical detection method of the trace amount sample using the biosensor according to the present invention can not only connect between the nano-electrodes by one gold nanoparticle-gold ion reduction reaction, but also between the electrically insulated nano-electrodes The electrical conductivity is increased by increasing the electrical conductivity, so that the measurement sensitivity is high, the trace sample can be quantitatively analyzed, and the selectively bound antigen-antibody binding can be measured very simply by an electrical method.

Claims (8)

1. Electrical detection method of trace amount sample using a biosensor comprising the following steps:

   (a) fabricating a biosensor by immobilizing a receptor molecule selectively binding to an analyte target material on an electrically insulated nano-electrode chip;

   (b) adding and reacting a sample containing a target substance selectively binding to the receptor molecule to the biosensor;

   (c) treating and reacting the gold nanoparticles to the nano-electrode chip reacted with the sample containing the target material;

   (d) treating and reducing gold ions around the reacted gold nanoparticles; And

   (e) detecting a target substance specifically binding to the receptor molecule by measuring the electrical conductivity or impedance of the reduced nano-electrode chip.
2. The method of claim 1, wherein the nano-electrode chip is manufactured by a process selected from the group consisting of photolithography, electron beam lithography, ion-focused lithography, and nanoimprinting.
3. The method of claim 1, wherein the spacing between electrodes in the nano-electrode chip is less than 1 μm.
4. The method of claim 1, wherein the target material is selected from the group consisting of enzymes, proteins, DNA, RNA, microorganisms, viruses, animal cells, plant cells, organ cells and neurons.
5. The method of claim 1, wherein the receptor molecule is selected from the group consisting of antibody, DNA, aptamer, PNA and ligand.
6. The method of claim 1, wherein the gold nanoparticles are 2nm ~ 100nm in size.
7. The method of claim 1, wherein in step (c), the gold nanoparticles are bound to the target substance using an additional sensing receptor molecule capable of binding to the target substance.
8. The method of claim 1, wherein said additional sensing receptor molecule is selected from the group consisting of antibody, DNA, aptamer, PNA and ligand.

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