KR20140046684A - Microtiter plate and detection method of antigen-antibody reaction using thereof - Google Patents

Abstract

The present invention provides a microtiter plate comprising metal nano point arrays indicating a local surface plasmon resonance phenomenon on a chamber of the microtiter plate for testing antigen-antibody reaction, combines with an antigen after immobilizing a capture antibody on the metal nano point arrays, and compares absorbance properties of before and after the antigen-antibody coupling, thereby quantifying antigen-antibody reaction; and a testing method of antigen-antibody reaction using thereof. According to the present invention, there is an effect of quantifying an antigen-antibody reaction by a simple method which does not perform marking steps such as a step of binding a detection antibody and applying substrates to an enzyme, etc.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microtiter substrate and an antigen-antibody reaction assay method using the microtiter substrate.

The present invention relates to a microtiter substrate and a method of assaying an antigen-antibody reaction using the microtiter substrate, and more particularly, to a method for assaying an antigen-antibody reaction without introducing a separate labeling step by introducing a metal nano- And a method for testing an antigen-antibody reaction using the microtiter substrate.

Microtiter plates are widely used in hospital diagnostic laboratories and life sciences laboratories. They are mainly used for enzyme-linked immunosorbent assay (ELISA).

Referring to FIG. 1, a microtiter substrate 100 includes a main body 110 and a plurality of chambers 120 arranged on the main body 110. The chamber 120 is a space having a microliter capacity formed downward from the upper surface of the main body 110, and serves as a separate reactor for various reactions. Since a plurality of chambers 120 are formed on the microtiter substrate 100, a plurality of antigen-antibody reaction tests can be simultaneously performed.

FIG. 2 is a flow chart of an antigen-antibody reaction test using a conventional microtiter substrate 100, and FIG. 3 is a diagram conceptually showing a reaction proceeding in this order.

 2 and 3, a procedure for inspecting the antigen-antibody reaction using the conventional microtiter substrate 100 will be described. First, a capture antibody 310 is attached to the chamber surface 300 of the microtiter substrate. (S210), and binds the antigen 320 to be detected to the immobilized capture antibody 310 (S220). In the next step, the detection antibody 330 for quantifying the antigen 320-antibody 310 reaction is bound to the antigen 320 (S230), and the enzyme 331 attached to the detection antibody 330 is immobilized on the substrate The byproducts 341 are formed by applying the catalysts 340 in step S240 and then the produced byproducts 341 are measured using the detector 350 in step S250.

An antigen-antibody reaction assay using the conventional microtiter substrate 100 is performed by labeling an antigen 320 bound to a capture antibody 310 using a detection antibody 330 and an enzyme reaction, In step S240, a substrate 340 is provided to the enzyme 331 to give a detectable property such as color or fluorescence property to the enzyme 331 instead of forming a by-product 341 A method may be used in which the substrate 340 capable of detecting characteristics such as hue or fluorescence is used.

However, in the conventional method of assaying an antigen-antibody reaction using a microtiter, it is necessary to continuously perform complicated multi-step reactions such as binding of a detection antibody and providing a substrate to an enzyme after the antigen-antibody reaction step (S220) Which requires a large amount of analysis time and cost, and requires a skilled workforce.

Accordingly, there is a need for a microtiter substrate and an antigen-antibody reaction assay method using the microtiter substrate, which can reduce the time and cost required for the antigen-antibody reaction test while maintaining the advantages of the microtiter-based assay method.

In addition, there is a need for a microtiter substrate and an antigen-antibody reaction assay method using the microtiter substrate to simplify the antigen-antibody reaction test by omitting a separate labeling step so that the antigen-antibody reaction test can be easily performed without skilled personnel .

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and it is an object of the present invention to provide a microtiter substrate capable of reducing the time and cost required for antigen- And to provide a method for testing an antigen-antibody reaction.

The present invention also provides a microtiter substrate capable of easily performing an antigen-antibody reaction test without skilled manpower by allowing the antigen-antibody reaction to be quantified by a simple method even if a separate labeling step is omitted, and an antigen- And a method for inspecting an antibody reaction.

According to an aspect of the present invention, there is provided a microtitre substrate including a main body, a plurality of chambers arranged on an upper surface of the main body, and a chamber including a metal nano dot array exhibiting local surface plasmon resonance . At this time, the metal nano-dot array may be directly attached to the chamber surface, but it may be formed on a separate substrate and the substrate may be attached to the chamber surface.

The metal nano-dot array may be formed of gold or silver nanoparticles, and may be formed by a patterning method or by directly attaching metal nanoparticles.

The method for testing an antigen-antibody reaction using a microtiter substrate according to the present invention includes a body and a plurality of chambers arranged on an upper surface of the body, wherein the chamber includes a metal nano-dot array exhibiting local surface plasmon resonance The method comprising: providing a microtiter substrate; immobilizing the capture antibody to the metal nano-dot array; binding the antigen to the capture antibody; and measuring the absorbance of each chamber using a detector . At this time, the metal nano-dot array may be directly attached to the chamber surface, but it may be formed on a separate substrate and the substrate may be attached to the chamber surface.

In addition, the method for testing an antigen-antibody reaction using a microtiter substrate according to the present invention can detect a wavelength at which the maximum absorbance is maximized by measuring the absorbance, By comparing with the wavelength at which the absorbance is maximized in the state where the capture antibody is immobilized in the nano-dot array, the change of the absorption characteristic by the antigen-antibody reaction can be detected, and the amount of the antigen bound to the capture antibody can be quantified.

Here, the change in the light-absorbing property is determined by the amount of the antigen bound to the capture antibody, and the change in the light-absorbing property according to the amount of the antigen bound to the capture antibody is preferably database .

According to the microtiter substrate and the antigen-antibody reaction assay method using the microtiter substrate according to the present invention, it is possible to reduce the time and cost required for the antigen-antibody reaction test while maintaining the advantages of the microtiter assay method .

In addition, according to the microtiter substrate and the antigen-antibody reaction assay method using the same according to the present invention, it is possible to quantify the antigen-antibody reaction by a simple method without performing a separate labeling step, - There is an effect that the antibody reaction test can be performed easily.

1 is a perspective view of a conventional microtiter substrate.
2 is a flow chart of an antigen-antibody reaction test using a conventional microtiter substrate
3 is a conceptual view of an antigen-antibody reaction test using a conventional microtiter substrate
FIG. 4 is a perspective view of a microtiter substrate according to the present invention and an electron micrograph of a metal nano-dot array formed on the surface of an individual chamber.
5 is a view for explaining the principle of examining an antigen-antibody reaction by a microtiter substrate according to the present invention;
6 is a flow chart of an antigen-antibody reaction assay method using a microtiter substrate according to the present invention
7 is a conceptual illustration of a method for testing an antigen-antibody reaction using a microtiter substrate according to the present invention
8 is a view illustrating a method of manufacturing a microtiter substrate according to an embodiment of the present invention
9 illustrates a method of manufacturing a microtiter substrate according to another embodiment of the present invention
10 is a flowchart of a method of forming a metal nano-dot array by the lift-off method according to the present invention
11 is a schematic view of a process of forming a metal nano-dot array according to the flow of FIG.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings, but the present invention is not limited to or limited by the embodiments.

FIG. 4 is a perspective view (FIG. 4A) of a microtiter substrate according to the present invention and an electron micrograph (FIG. 4B) of a metal nano dot array formed on the surface of an individual chamber. Referring to FIG. 4A, the microtiter substrate 400 according to the present invention includes a body 410 and a plurality of chambers 420 arranged on an upper surface of the body 410. The chamber 420 is a space having a microliter capacity formed downward from the upper surface of the main body 410, and serves as a separate reactor for various reactions. Since a plurality of chambers 420 are formed on the microtiter substrate 400, a plurality of antigen-antibody reaction tests can be performed simultaneously with the conventional microtiter substrate.

In the microtiter substrate 400 according to the present invention, a metal nano dot array 430 is formed on each chamber surface 421. The metal nano-dot array 430 means that nano-sized metal particles 431 are arranged as shown in the electron micrograph of FIG. 4B. The size of the nano-sized metal particles 431 is about 10 to 200 nm, And the kind of the metal is preferably noble metal such as gold or silver, but is not limited thereto.

When a metal becomes nano-sized, the surface plasmon resonance phenomenon appears by the behavior of free electrons in the metal. Plasmon, which is a similar particle in which free electrons in a metal oscillate collectively, is called a surface plasmon resonance phenomenon. Refers to a phenomenon in which light is absorbed by a specific wavelength band to become an excited state. In particular, unlike nano-sized metal thin films, in the case of nano-sized metal particles, the excited plasmons do not propagate but are attributed to the localized surface plasmon resonance phenomenon.

FIG. 5 is a view for explaining the principle of checking an antigen-antibody reaction without a separate labeling step by a microtiter substrate according to the present invention. 5, when the capture antibody 520 is immobilized on the metal nanoparticles 510 as shown in FIG. 5 (a) and the light absorption characteristics of the metal nano dot array are examined, local surface plasmon resonance And the light absorbing characteristic at which the absorbance becomes maximum at the wavelength lambda 1 (graph 540a in Fig. 5 (c)).

On the other hand, when the antigen 530 is bound to the capture antibody 520 as shown in FIG. 5 (b), the dielectric constant of the surface of the metal nanoparticles 510 changes and the wavelength band at which the maximum absorbance changes also changes. And the light absorbance characteristic at which the absorbance becomes maximum at the other wavelength? 2 (graph 540b in Fig. 5 (c)).

Here, since? 2 is determined by the amount of the antigen 530 bound to the capture antibody 520, using the microtiter substrate according to the present invention, the change of the absorption characteristics of the metal nanoparticles before and after the antigen- The antigen-antibody reaction can be quantified without the labeling step of < RTI ID = 0.0 >

Hereinafter, an antigen-antibody reaction assay procedure using the microtiter substrate according to the present invention will be described with reference to FIGS. 6 and 7. FIG. FIG. 6 is a flow chart of an antigen-antibody reaction test using a microtiter substrate according to the present invention, and FIG. 7 is a diagram conceptually showing a reaction proceeding in this order.

The antigen-antibody reaction assay using the microtiter substrate according to the present invention is performed by first immobilizing the capture antibody 720 on the metal nano-dot array formed on the chamber surface 700 of the microtiter substrate (S610). By this step, the capture antibody 720 is immobilized on the surface of the metal nanoparticles 710 as shown in FIG. 7 (a). As a method of immobilizing the capture antibody on the metal nano-dot array, a method of inducing thiol (-SH) to the antibody or forming a monolayer by self-assembly between metal and thiol can be used. It goes without saying that any physical or chemical immobilization method can be used without limitation.

The next step is to bind the antigen 730 to be immobilized to the immobilized capture antibody 720 at step S620 so that the antigen 730 is bound to the capture antibody 720 as shown in FIG. And the absorption characteristics of the metal nanoparticles 710 are changed by the local surface plasmon resonance phenomenon according to the combined amount.

In the next step, the changed absorption characteristic is detected by the detector 740 (S630). Here, the detector 740 may use a spectrometer used in spectroscopic analysis.

As described above, the use of the microtiter substrate according to the present invention can eliminate complicated labeling steps such as binding the detection antibody separately after antigen-antibody binding and providing a substrate to the enzyme, There is an advantage of being simple.

On the other hand, in order to quantify the antigen-antibody reaction by the step S630, since there is a change in the light-absorbing property before and after the antigen-antibody binding, in particular, wavelength change data that maximizes the absorbance, the capture antibody 720 is immobilized on the metal nano- And then detecting the light absorbing characteristic using the detector 740 even before the antigen 730 is bound. Preferably, the absorption characteristic data in the state where the capture antibody 720 is immobilized on the metal nano-dot array is constructed and provided in advance in a database, thereby detecting the absorption characteristic data after binding of the antigen and comparing this with the data provided in the database , The detection step before the antigen binding can be omitted.

In addition, it is preferable that data on the change of the light absorbance depending on the binding amount of the antigen, particularly the wavelength change data that maximizes the absorbance, is also built in advance in a database and is provided together. 6, the antigen-antibody reaction can be quantified by sequentially performing the steps S610 to S630 and comparing the detected absorption characteristics, in particular, the wavelength at which the maximum absorbance is maximum, with the data stored in the above database. Preferably, the control circuit is configured within the detector so that the comparison step of the detection data and the database is automatically performed, and the antigen-antibody reaction value extracted by the comparison step may be displayed through a predetermined display unit .

A method of manufacturing the microtiter substrate according to the present invention will be described with reference to FIGS. 8 and 9. FIG. 8 shows a method of manufacturing a metal nano-dot array substrate 830 in which a metal nano dot array is formed on a separate substrate 831, and then attaching the metal nano dot array substrate 830 to the chamber surface of a conventional microtiter substrate. The metal nano dot array substrate 830 is preferably formed by forming a metal nano dot array on a substrate having a size larger than the chamber, and then cutting the nano dot array according to the size of each individual chamber.

Here, the method of forming the metal nano dot array on the substrate 831 is not particularly limited, but a conventional patterning method, for example, a method in which a metal thin film is deposited on the substrate 831, and then a photoresist and a pattern mask A method of patterning a metal nano-dot array using a lithography and etching process using a metal nano-dot array or a lift-off method to pattern a metal nano-dot array, A metal nano dot array may be formed without a separate patterning process by directly attaching the metal nano dot array to the substrate 831 so that physical or chemical bonding is performed.

9 is a method of forming a metal nano-dot array directly on the microtiter chamber surface 920 without forming a separate metal nano array array substrate 830. [ As a method of forming the metal nano-dot array on the chamber surface 920, a conventional patterning method similar to that described with reference to Fig. 8, or a method of directly attaching the metal nanoparticles to the chamber surface 920, And the like may be used. However, the present invention is not limited thereto.

On the other hand, among the conventional patterning methods for forming the metal nano-dot array, it is preferable to use a lift-off method which is simple in process and can proceed at a low cost. Hereinafter, referring to FIGS. 10 and 11, A method of forming a metal nano dot array will be described.

FIG. 10 is a flow chart of a method of forming a metal nano-dot array by the lift-off method according to the present invention, and FIG. 11 is a schematic view of a process of forming a metal nano-dot array according to the flow of FIG.

First, as shown in FIG. 11A, a mask metal layer 1120 is deposited on the base substrate 1110 with a metal material (step S1010). In this embodiment, the base substrate may be a separate substrate 831 attached to the surface of the chamber thereafter, and may be the surface of the microtiter chamber in the embodiment of FIG. In addition, the mask metal layer should be a metal that can easily be selectively removed with a metal material forming a metal nano-dot array, and may be aluminum, for example.

Next, a plurality of pores 1122 are formed in the mask metal layer 1120 deposited on the base substrate 1110 to form the porous thin film 1121 as shown in FIG. 11 (b) As an example of the aluminum mask metal layer, a plurality of pores 1122 can be formed in the aluminum thin film through an anodizing process. Here, the anodizing process is a process of immersing the aluminum thin film in an acidic solution and then applying direct current. The size of pores formed at this time can be easily controlled by controlling the anodizing process conditions. The pore size can be controlled by adjusting the nanoscale size of the final metal nano- .

When the porous thin film 1121 is formed, a metal material 1130 for forming a metal nano-dot array is deposited on the porous thin film 1121 as shown in FIG. 11C (step S1030). Although not particularly limited, the metal material 1130 may be deposited by using E-beam evaporation, and the metal material 1130 may be deposited on the pores 1122 of the porous thin film 1121 and on the porous thin film 1121 Lt; / RTI >

Finally, as the step of removing the porous thin film 1121, that is, the mask metal layer (step S1040), the metal material 1130 deposited on the upper part of the mask metal layer is removed together with the removal of the mask metal layer, Only the metal material 1130 deposited on the portion remains on the base substrate, resulting in the formation of a nano-dot array. At this time, when the mask metal layer is made of aluminum metal, it is possible to remove only the mask metal layer through a wet etching process using a basic solution.

Although described above with reference to the limited embodiments and drawings, it will be apparent to those skilled in the art that various modifications are possible within the scope of the technical idea of the present invention. Therefore, the protection scope of the present invention should be defined by the description of the claims and their equivalents.

100, 400, 800, 900: Microtiter substrate
110, 410, 810, 910: body, 120, 420, 820, 920: chamber
310, 520, 720: capture antibody, 320, 530, 730: antigen
330: detection antibody, 331: enzyme, 340: substrate, 341: by-product
350, 740: detector
430: metal nano dot array
510, 710, 832, 930: metal nanoparticles
830: metal nano dot array substrate, 831: substrate

Claims (10)

A microtiter substrate for an antigen-antibody reaction test,
main body;
It includes a plurality of chambers arranged on the upper surface of the body,
And the chamber comprises a metal nanodot array exhibiting local surface plasmon resonance. The method of claim 1,
And a substrate on which the metal nano dot array is formed is attached to the chamber surface. 3. The method according to claim 1 or 2,
Wherein the metal nano dot array is made of gold or silver nanoparticles. 3. The method according to claim 1 or 2,
The metal nano-
Or may be formed by a patterning method,
or,
Wherein the metal nanoparticles are formed by directly attaching the metal nanoparticles. A method for testing an antigen-antibody reaction using a microtiter substrate,
Providing a microtiter substrate including a body and a plurality of chambers arranged on an upper surface of the body, wherein the chamber includes a metal nanopoint array exhibiting a local surface plasmon resonance phenomenon;
Immobilizing the capture antibody on the metal nano-dot array;
Binding an antigen to the capture antibody;
Measuring the absorbance of each of the chambers using a detector;
Antigen-antibody reaction test method using a microtiter substrate comprising a. 6. The method of claim 5,
The microtiter substrate,
The method of claim 1, wherein the substrate on which the metal nano dot array is formed is a microtiter substrate attached to the surface of the chamber. The method according to claim 5 or 6,
And measuring a wavelength at which the maximum absorbance is detected through the step of measuring the absorbance. 8. The method of claim 7,
By comparing the wavelength at which the absorbance becomes maximum with the wavelength at which the absorbance becomes maximum in the state that the capturing antibody is immobilized in the metal nano dot array, the change in the absorbance characteristic due to the antigen-antibody reaction is detected, Wherein the antigen-antibody reaction is quantified by quantifying the amount of the antigen. 9. The method of claim 8,
Wherein the change in the light absorption characteristic is determined by the amount of the antigen bound to the capture antibody. 10. The method of claim 9,
Wherein the change of the light absorbance characteristic according to the amount of the antigen bound to the capture antibody is databaseed before the test.

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