KR20140127463A - Nanopatterned Biochip Kit based on Wavelength-Dependent Plasmon Resonance Scattering and Biomolecule Detection Method Using the Same - Google Patents

Abstract

The present invention relates to a nano-biochip kit, which detects the existence or content of target biomolecules included in an assay sample, using plasmon resonance scattering. Also, the present invention relates to a detecting device equipped with the nano-biochip kit and a target biomolecule detection method using the nano-biochip kit. The nano-biochip kit including metal nano-particles and metal nano-patterned biochip of the present invention minimizes a matrix effect through plasmon resonance scattering which is different among metal crystals because metals forming metal nano-patterns and metal nano-particles are different from each other, and increases detection sensitivity, thereby selectively detecting metal crystals. In other words, the metal nano-particles and the metal nano-pattern display unique wavelength-dependent selective plasmon resonance scattering, so biomolecules can be selectively detected without a matrix effect by a dark-field microscope. Therefore, accuracy and reliability of an analysis result can be improved, and the detection device can be widely used for gene, protein, and cell detection whose accuracy is considered important.

Description

[0001] The present invention relates to a nano-biochip kit using wavelength-dependent plasmon resonance scattering and a method for detecting biomolecules using the same,

The present invention relates to a nano biochip kit for detecting the presence or content of a target biomolecule contained in an assay sample using plasmon resonance scattering. The present invention also relates to a detection device equipped with the nano-biochip kit and a method for detecting a target biomolecule using the nano-biochip kit.

Nanobiochip is a nanotechnology integrated with biochip. Nano-biochip technology combines nanotechnology (NT) and biotechnology (BT) to produce a variety of genes (DNA, RNA), proteins, cells and various metabolites on a chip By sensing directly with various sensors and various preprocessing functions, it is possible to diagnose diseases faster and more accurately, thereby contributing to the eradication of human diseases and quality improvement of healthy life. This nano-biochip has advantages such as small size, low price, high performance, high integration, low power, relatively small amount of sample, fast reaction time and analysis time compared to micro-biochip. And is rapidly growing, expecting to make innovative changes in various fields such as life sciences, medicine, pharmacy, and clinical.

Nano-array biochips are used for various nanolithography technologies such as ink-jet nano-printing technology, e-beam lithography and atomic force microscope-dip pen nanolithography (AFM-DPN) Can be fabricated by arranging / arranging various materials in a size of several hundreds of nanometers on one chip substrate.

However, it is required to develop new detection methods and detectors that can detect analytes, target or marker molecules easily and quickly with high sensitivity because of using ultra-small chips and trace amounts of analytes .

On the other hand, the interaction between metal nanocrystals and organic adsorption is attracting interest as a phenomenon of very interest (eg, plasmon-enhanced fluorescence, fluorescence quenching, plasmon energy conversion, and plasmon resonance of molecules). In particular, metal nanoparticles have been attracting much attention over the past decade because of the unique size and shape of the nanoparticles and their plasmon resonance. Moreover, their nanoscale sizes have been able to induce high diffusivity and nanoparticles have been used as probes because they can be used in the detection process by chemically / physically combining well with photophysical properties. However, application of nanoparticles for discrimination of high sensitivity of biological samples or disease markers is still required to develop new detection techniques or to improve detection sensitivity through signal amplification.

Immunoassay for the diagnosis of disease on a metal thin film formed on a transparent substrate using a biomolecule or a fluorescent nanoparticle (for example, a quantum particle) combined with a fluorescent dye is already widely used by a device such as a fluorescence microscope or a total reflection fluorescence microscope (Talanta, 2013, 104, 32-38). Recently, the necessity of non-fluorescence detection has been greatly increased due to limitations such as sensitivity, stability, In addition, the efficiency of scattered light according to the size and shape of gold nanoparticles (GNPs) and silver nanoparticles (SNPs) under a common light source is 10 ⁶ times higher than the emission from fluorescent dyes (For SNPs) (Anal. Biochem., 1998, 262, 157-176).

Various types of sensors based on plasmon resonance have been developed so far. Optical plasmon sensors can be divided into localized surface plasmon resonance shift (LSPR-shift) and local electric field enhancement (LEF) mechanisms. Chemical sensors based on the LSPR-shift include aggregation sensors and dark field microscopy techniques using extinction technology and UV / Vis spectroscopy techniques, elastic scattering ) Technology using a local refractive index sensor. Chemical sensors based on local electric field enhancement include surface enhanced raman spectroscopy and metal enhanced fluorescence (MEF) based on enormous electromagnetic field enhancement near metal particles.

The Raman scattering theory comes from the same principle as the quantized oscillation change associated with infrared absorption. Although surface enhanced infrared absorption spectroscopy (SEIRAS) has been developed to improve infrared (IR) signals in molecules near conventional metal nanoparticles, the chemical absorption Surface enhanced raman scattering (SERS) as a sensor has the advantage of being able to analyze samples in aqueous solution because Raman scattering of water is very weak.

In 2011, the Korea Advanced Institute of Science and Technology (KAIST) developed surface enhanced Raman scattering using optical signals of hot spots generated by local surface plasmon resonance (LSPR) between nano-particles and nanoparticles of the same metal. (Korean Patent No. 10-1059596), which is applied to a sandwich immunoassay system as an analytical tool for detection of biomolecules such as genes and proteins. However, this is a detection method that directly utilizes light scattering of two identical materials of metal crystal (Au), so that it can select different unique wavelengths generated from two different metal crystals and can use wavelength- Is not based on the detection method.

MALDI-TOF-MS (immuno-matrix-assisted laser desorption / mass spectrometry), which contributes to minimizing the risk of cross-reactivity and enhancing the specificity of target antibodies by enabling simultaneous analysis of mass analysis and immunoaffinity, ionization time-of-flight mass spectrometry has recently been used to combine surface plasmon resonance (SPR) in 2012 to analyze multiple biomarkers on a gold-patterned chip (Analyst, 2012, 137, 386- 392). However, this method has limitations in the detection of single molecules, and there are many difficulties in obtaining accurate correlations between the sensitivity of mass peaks and antigen concentration under current experimental conditions. In addition, the SPR method in this study is a method for confirming whether or not an analyte interacts with a change in the resonance angle of light reflected from a metal substrate. In this method, plasmon resonance scattering of two metal crystals having different specific plasmon resonance scattering Has a different meaning from that proposed in the present patent for performing high sensitivity selective detection by minimizing the disturbing effect due to the difference.

The technique of dark-field scattering based on plasmon resonance has been utilized by von Leeuwenhoek, Hooke and Huygens in the 17th century. In 1903, Siedentopf and Zsigmondy first demonstrated the possibility of observing single gold nanoparticles smaller than 10 nm in size. The group of Vainrub succeeded in having a resolution of 90 nm with a cardioid annular condenser mounted on a general microscope. In recent years, it has been used for the detection of single-molecule interactions, intracellular viral particles, and nanostructures containing metals. In 2005, the Alivisatos team used the concept of darkfield technology to introduce the concept of molecular plasmon rulers based on plasmon binding of single nanoparticles of the same material (gold-gold, silver-silver) (Nature Biotechnology, 2005, 23, 741-745). This "plasmon ruler" provides higher sensitivity and lower background noise than a ruler based on a common fluorescence resonance energy transfer (FRET), and has an intermolecular distance limit of 70 nm to 10 nm for FRET And suggested applicability to hybridization studies of DNA molecules. In 2009, Ling's group attempted a sandwich immunoassay using a dark-field microscope coupled with a general spectrophotometer to collect plasmon resonance scattering (PRS) of silver and gold nanoparticles (Anal. Chem. 2009, 81, 1707-1714). However, this demonstrates that silver nanoparticles show stronger scattering than gold nanoparticles and are more useful for establishing an immunoassay system on a glass substrate by applying the PRS properties of only two metal particles individually. The actual disease marker analysis And no wavelength-dependence studies have been performed simultaneously applying two metal particles of different materials with distinct PRS signals. In addition, the interaction between dispersed biomolecules on a glass substrate merely affects the reproducibility and accuracy of the PRS signal, depending on the degree of agglomeration of the nanoparticles relative to the interaction on the patterned substrate on the nanoarray biochip There is a problem that can occur. Thus, high sensitivity selectivity detection of single nanoparticles using an improved dark-field scattering technique based on wavelength-dependence on metal-nanopatterned chips has not yet been reported.

Under these circumstances, the inventors of the present invention have developed a nano-biochip kit capable of detecting target biomolecules using metal nano-biochips and metal nanoparticles having inherent plasmon resonance scattering properties, and a plasmon resonance characteristic specific to each nano-biochip and metal nanoparticles A detection method using a high-sensitivity dark-field microscope capable of detecting scattering in a wavelength-dependent manner has been established and the present invention has been completed.

It is an object of the present invention to provide a nano biochip kit for detecting the presence or content of a target biomolecule contained in an assay sample using plasmon resonance scattering.

Another object of the present invention is to provide a nano-biochip kit of the present invention; A dark field microscope equipped with a dark field condensing device capable of collecting plasmon resonance scattering light; And a detecting unit for detecting the presence or content of the target biomolecule contained in the analysis sample using the plasmon resonance scattering.

It is still another object of the present invention to provide a method for detecting a target biomolecule using the nano-biochip of the present invention.

In order to solve the above problems, the present invention provides a nano biochip kit for detecting the presence or content of a target biomolecule contained in an assay sample using plasmon resonance scattering, comprising: (a) a substrate; A nano pattern located on the substrate and comprising a first metal; And a capture molecule connected to the first metal of the nanopattern; And (b) a second metal that emits plasmon resonance scattering light upon illumination; And a detection molecule connected to the nanoparticle, wherein the capture molecule and the detection molecule are each capable of binding to a target biomolecule, and the nano-biochip kit is a target molecule In the biomolecule binding, the detection molecule is bound to the bound target biomolecule and the nanoparticle complex is connected to the nanobiochip. Light emitted from the first metal due to plasmon resonance scattering during the light irradiation is converted into plasmon resonance scattering Wherein a material of the first metal and a material of the second metal are different from each other so as not to interfere with the light signal emitted from the second metal.

The term "nano-biochip kit" in the present invention includes nano-biochips and nanoparticles, and may further include other reagents and tools necessary for the target biomolecule detection process.

In the present invention, the term "nano-biochip" refers to a biochip produced by combining nanotechnology. The nanotechnology is a technology in which a size or a component of a chip itself is miniaturized, sensitivity is improved, It is a chip that enables smart functions that were impossible with technology. In the present invention, the nano-biochip may preferably be a bio-chip nanopatterned with a metal. The nano-biochip of the present invention can be bound with a target molecule bound to a detection molecule to which metal nanoparticles having a property of absorbing wavelength-dependently selectively at other wavelengths are bound.

In the present invention, the term "nanopattern" means a repeated pattern of nanometer units. Specifically, in the present invention, the nanopattern may have a diameter of preferably from 0.1 to 1000 nm, more preferably from 1 to 500 nm. The pitch of the nanopattern may be between 1 nm and 100 um, preferably between 10 nm and 10 um. In addition, the nanopattern of the present invention may be arranged in a lateral direction. Preferably, the nanopattern is a nanolithography technique such as inkjet nano-printing, e-beam lithography, and atomic force microscope-dip pen nanolithography (AFM-DPN) And is manufactured by either one. Preferably, the nanopattern may be nanopatterned with a metal. In addition, the shape of the nanopattern is not limited. Because the plasmon resonance can be varied depending on the shape of the metal nanopattern, it is possible to use patterns according to various shapes. Preferably a spherical metal nano-pattern. In the case of nanopatterning of a biochip with metal, a specific chemical reaction or binding occurs well depending on the characteristics of the metal, and it is easy to connect capture molecules such as an antibody or an aptamer. Accordingly, the nanopattern of the present invention may more preferably be a gold nanopattern that is easily bonded to a compound or the like using a thiol group (-SH).

According to a specific embodiment of the present invention, the nanobiochip is nanopatterned with gold in the present invention. Specifically, the gold nanopattern is 10 mm ² And processed on a glass substrate by electron beam nanolithography at the National Nanotechnology Fab Center (Daejeon, South Korea). Nano-arrays were fabricated by electron beam deposition using gold spots with diameters of 100 nm and 500 nm with a 4 x 5 nano array at 10 μm intervals (Fig. 2).

In the present invention, the term "nanoparticle" refers to particles of a small size. The nanoparticles are not limited in size, but may include particles larger than the nanoparticles, Any size particle that can cause plasmon resonance scattering is acceptable. Preferably nanoparticles made of metal. In the nano region, the color changes depending on the particle size. Therefore, it is an important problem in the nanoparticle field to manufacture and commercialize nanoparticles having a uniform size. In the present invention, the nanoparticles may preferably have a size of 1 to 200 nm. In addition, in the nano region, the color changes depending on the particle shape. Therefore, any particle can be used as long as it can cause plasmon resonance scattering. Preferably, the nanoparticles of the present invention can bind to a detection molecule that specifically reacts with a target biomolecule. In the present invention, the term "nanoparticle complex" refers to a complex comprising the nanoparticle and the detection molecule linked to the nanoparticle.

In the present invention, the shape of the nanoparticle including the first metal or the nanoparticle including the second metal may be selected from the group consisting of spherical, star-shaped, quadrangular, and cubic, It can be spherical.

According to a specific embodiment of the present invention, an antibody is bound to silver nanoparticles in the present invention. Specifically, silver nanoparticles (SNPs) were dissolved in 996 mL of high-purity ethanol. The MUA and MCH solutions were mixed with SNPs solution and sonicated for 10 minutes. The MUA-MCH was self- assembled monolayer, SAM). To bind the SNPs to the antibody, EDC and Sulfo-NHS solutions were first added to activate them. Activated SNPs were added to detection antibody (cTnI clone 16A11) solution and reacted. Finally, unbound antibodies were removed to produce SNP-antibodies.

In the present invention, the term "plasmon resonance scattering" means that when a metal or the like is prepared in a nanoscale, numerous free electrons in the metal, which is a conductor, are easily sensitized to a specific external stimulus. When light enters the metal nanocrystal, The resonance with the energy electromagnetic field causes the free electrons of the metal to vibrate collectively and scatter in this process. In particular, metal nanocrystals such as gold and silver strongly resonate with light in the visible light region, resulting in very strong absorption and scattering.

On the other hand, the frequency of plasmon resonance varies depending on the size and shape of the metal nanocrystals and the solvent in which the metal nanocrystals are dispersed. Accordingly, techniques for constantizing the size and shape of the metal nanocrystals have been continuously developed. The term "wavelength-dependent plasmon resonance scattering" of the present invention means that the size and shape of the metal nanocrystals are extremely uniform, and as a result, the phenomenon of absorption and scattering occurs specifically (i.e., wavelength- It says.

In the present invention, the term "biomolecule" refers to any substance that can be found in and / or out of a living body, and refers to any substance that generates a specific reaction in a living body or is caused by a specific state or reaction. Preferably, the biomolecule of the present invention may be a compound, a protein, a DNA, an RNA, or a cell. In the present invention, the term "target biomolecule" refers to a substance to which the presence or content of the nano-biochip of the present invention is to be detected. Specifically, a detection molecule bound to capture molecules and metal particles constituting the nano- Means a substance that reacts to a mutual reaction.

In the present invention, the term "capture molecule " means a molecule immobilized on the nanopattern of the nanobiochip, and means any molecule having a characteristic of binding to the target biomolecule. In the present invention, the term "detecting molecule" which binds to the metal nanoparticles means all kinds of molecules having a characteristic of binding to the target biomolecule. Preferably, the capture molecule or the detection molecule in the present invention may be any one of a protein, a gene, a lipid, an enzyme, an aptamer and a ligand. More preferably, the capture molecule or detection molecule may be an antibody.

In the present invention, a nano-biochip capable of simultaneously detecting or quantifying various antigens can be constructed by connecting capture molecules binding to different antigens to each nanopattern on a nanopattern basis or on a segment basis. In this case, the trapping molecules can be connected to each of the nanopatterns or the compartments through masking in general. As the capture molecules are varied, corresponding detection molecules and also for various antigens can be used.

In the present invention, the substrate may be composed of any one of glass, fused-silica, quartz, polydimethylsiloxane (PDMS) and polymethylmethacrylate (PMMA) . Preferably, the substrate may be a glass or polymeric material capable of transmitting light having a thickness of 10 mm or less, most preferably 10 nm to 1 mm.

The components commonly applied to the nano-biochip kit 100 of the present invention include trapping molecules 10 such as compounds or biomolecules that specifically react with a target biomolecule 20, which is a gene, a protein, or a cell, A metal nanoparticle (nanoparticle complex (30)) bonded with a detection molecule such as a compound or a biomolecule that specifically interacts with the target biomolecule (20), and a substrate (40) where the capture molecule (10) . On this substrate 40 a nanopattern is formed and the capture molecules 10 are located above the substrate 40 (FIG. 4A).

Specifically, the mechanism of detecting a target biomolecule is as follows. 1) First, the capture molecule 10 or the detection molecule can be composed of any one of a protein, a gene, a lipid, an enzyme, an aptamer and a ligand. A sample for confirming the presence or content of the target biomolecule 20 is treated on the nano-biochip including the capture molecule 10 attached on the substrate 40. 2) Then, the sample-treated nano-biochip is allowed to interact with the detection molecule 30 that specifically binds to the target biomolecule bound to the metal nanoparticle. Finally, the presence of the target biomolecule is finally detected by condensing the metal nanoparticle-specific plasmon resonance scattering through a dark field microscope equipped with a dark field condensing device capable of collecting the plasmon resonance scattering light.

Preferably, the nano-biochip is different in a first metal patterned on a substrate and a second metal constituting metal nanoparticles, and the two metal crystals have different plasmon resonance scattering wavelengths. By making the constituent metals of the two metal crystals different from each other, it is possible to select different intrinsic wavelengths occurring in the two metal crystals having different materials and to detect them without interfering with each other. That is, in the nano-biochip of the present invention, since the first metal and the second metal constituting the metal nanoparticles are different from each other, the metal crystals have different plasmon resonance scattering wavelengths, It is possible to selectively detect each metal crystal by increasing the detection sensitivity by minimizing the interference effect by the nano pattern.

Preferably, the first metal or the second metal constituting the metal nanoparticles or the metal nanoparticles may be selected from the group consisting of gold (Au), silver (Ag), platinum (Pt), and palladium (Pd) More preferably, the first metal constituting the metal nanopattern may be gold, and the second metal constituting the metal nanoparticles may be silver.

In a specific embodiment of the present invention, the nano-biochip kit of the present invention was prepared with an antibody bound to gold nanoparticle chips and silver nanoparticles. The efficacy of the nano-biochip kit of the present invention was confirmed through sandwich immunoassay using the nano-biochip kit. Specifically, a capture antibody (anti-cTnI, clone 19C7) was attached to a sandwich reaction, and a sample diluted with target antigen (cTnI standard protein) at various concentrations was reacted for 1 hour. The silver nanoparticle-antibody (anti-cTnI, clone 16A11) was reacted for about 2 hours, and the dark-field plasmon resonance scattering imaging was measured (FIGS. 5 to 9).

(Light blue) due to plasmon resonance scattering at a specific absorption wavelength of silver nanoparticles and a false color image (dark blue) after combining the detection antibody with these (FIG. 5). In addition, it was proved that a transmission lattice mirror can be mounted between the image processing unit and the base microscope to detect images due to the diffraction of light of silver nanoparticles and silver nanoparticle-antibodies (FIG. 6). (Blue) detected by selective wavelength-dependent plasmon resonance scattering after a gold nanopattern chip (yellow) and a disease-related sample are subjected to a sandwich immune response on the gold nanopattern chip (yellow), as well as qualitative analysis (Fig. 7). In addition, high-sensitivity detection cameras were used to quantitatively analyze their antigen concentrations (Fig. 8).

Deposition of Gold Nanopattern Chips After Sandwich Immunization with Gold Nanopatterned Chip on Nanoparticle-Immunoassay Various Bandpass Filters Included in Dark-Field Microscope with Improved Concentrator for Converging Plasmon Resonance Scattering Light it is possible to selectively detect at a desired absorption wavelength using a bandpass filter (Fig. 9).

In the present invention, the "analytical sample" includes any sample that can be processed into the nanobiochip of the present invention, and may be a liquid sample that can be uniformly treated on the nanobiochip. In the present invention, the analytical sample may be any substance capable of containing a target biomolecule, and may be various substances exposed to or exposed to the living body and substances separated from the living body. The substances separated from the living body may specifically be blood, urine, runny nose, cells, extracted DNA, RNA, protein, and the like.

In another aspect, the present invention provides a nano biochip kit according to the present invention; A dark field microscope equipped with a dark field condensing device capable of collecting plasmon resonance scattering light; And a detection unit for detecting the presence or content of target biomolecules contained in the analysis sample using the plasmon resonance scattering, the image processing unit being connected to the darkfield microscope and processing an image provided from the microscope.

The detection device of the present invention preferably includes a transmission grating; Metal halide light source; And a selective spectral bandpass filter for obtaining a wavelength-dependent resonance scattering light image.

At this time, the microscope is not limited to a size-type microscope, but a microscope capable of mounting an improved-condensing device capable of condensing wavelength-dependent plasmon resonance scattered light on an inverted microscope is all possible.

Here, the image processing unit may preferably be a three-dimensional image of non-fluorescent metallic nanoparticles as well as a four-dimensional image including various spatial multi-images and time concepts by mounting a z-motor . At this time, the space may be an interval of 10 nm, but is not limited thereto.

Preferably, the detection device can perform real-time color-real-time detection according to the influence of the absorption wavelength peculiar to the nanoparticles even in a biomolecule sample combined with metallic nanoparticles according to various shapes, sizes, and materials. In particular, the detection apparatus according to the present invention can immunoassay a non-fluorescent sample on the basis of the principle of plasmon resonance scattering on a bio-chip.

According to a specific embodiment of the present invention, the detection device of the present invention was produced as described below. It should be understood, however, that the following description is only illustrative of the invention and is not intended to limit the scope of the invention.

In the present invention, an upright type microscope detection apparatus (FIG. 1A) equipped with an improved condensing apparatus capable of collecting wavelength-dependent plasmon resonance scattering light, a transmission grating (FIG. 1B) , And a detection device of the present invention was fabricated using two image portions (a camera capable of detecting a false color and a high-sensitivity detection camera) (Fig. 1C) mounted on a dual port of the microscope.

A dark field detection apparatus equipped with an improved-light condensing device capable of condensing wavelength-dependent plasmon resonance scattering light according to the present invention includes an image processing section for processing an image provided from a microscope, and a wavelength-dependent plasmon resonance scattering light A dark field detection device equipped with an improved concentrator capable of focusing can be used as an investment grating mirror which is disposed between the image processing section and the base microscope and acquires an image of the sample using a transmission grating technique or a spectral selectable band pass filter bandpass filter).

On the other hand, the dark field detection apparatus equipped with the enhanced-light condensing device capable of condensing the plasmon resonance scattering light according to the present invention can use an inverted microscope or a fixed-size microscope without limitation. Generally, an object type lens is classified into an up-type or a down-type according to a position of an object lens on the basis of a object stand. In the object type detection apparatus, an objective lens is positioned in a lower direction of the light- .

The image processing unit according to the present invention is connected to a dark-field microscope equipped with an enhanced-light condensing device capable of condensing the wavelength-dependent plasmon resonance scattering light, processes the image provided from the microscope, And is not particularly limited as long as it is a typical image processing unit in the art having such a purpose. In a specific embodiment of the present invention, a camera capable of detecting false colors and a high-sensitivity detection camera are provided in duplicate (FIG. 1C). At this time, the image processing unit is preferably connected to a computer or a microprocessor that is programmed to process the image.

In another aspect, the present invention provides a method for detecting a target biomolecule using the nano-biochip kit of the present invention.

Preferably, the method further comprises: reacting the nano-biochip with an analytical sample; Reacting the nanobiochip with a detection molecule bound to the metal nanoparticle; And detecting the metal nanoparticles and the metal nanoparticles in the nanobiochip. More preferably, the detecting step comprises exposing the metal nanoparticles to a photodiode array (PDA), a charge-injection device (CID), a charge-couple device (CCD) Or by a method of one or more of digital single lens reflex cameras.

In the present invention, the nano-biochip, the assay sample, the capture molecule, and the target biomolecule are as described above.

The target biomolecule detection method of the present invention detects a metal nanoparticle and a metal nanoparticle using the nanobiochip arranged with the metal nanopattern of the present invention and the nanobiochip kit including the detection molecule and the metal nanoparticle. In the nano-biochip kit of the present invention, since the metal nanopattern and the metal constituting the metal nanoparticles are different from each other, the two metal crystals cause different wavelength-dependent plasmon resonance scattering, thereby minimizing the interference effect between the metal crystals It is possible to selectively detect each metal crystal by increasing the detection sensitivity when detecting the target biomolecules according to the present invention.

In another aspect, the present invention provides a method for providing disease diagnosis information using the nano-biochip kit of the present invention.

Preferably, the present invention provides a method for analyzing a nano-biochip, comprising: contacting a capture molecule of the nano-biochip kit with an assay sample; Contacting the capture molecule in contact with the analytical sample with metal nanoparticles bound to the detection molecule; The metal nanoparticles can be used in various applications such as photodiode arrays (PDAs), charge-injection devices (CIDs), charge-couple devices (CCDs), digital single lens reflex cameras ); ≪ / RTI > And diagnosing disease according to whether or not the target biomolecule is detected. The present invention also provides a method for providing information on disease diagnosis using a nano-biochip.

In the present invention, the nano-biochip, the assay sample, the capture molecule, and the target biomolecule are as described above.

In the present invention, the term "disease" includes, without limitation, diseases susceptible to detection of a target biomolecule by the nano-biochip of the present invention. Soon, the disease to be diagnosed is determined by the type of the target biomolecule.

Such diseases include not only disease but also all of the state of the individual, genetic characteristics, biostability of the sample and activation of a specific metabolic process. For example, the disease may include various genetic features, genetic diseases, mutations, etc., if the target biomolecule is for a particular genetic component, such as DNA, RNA, protein, and the like. In addition, if the target biomolecule is a protein specifically expressed or controlled in a specific disease or condition, it may include the state of the specific disease or individual. In addition, when the target biomolecule is a compound that specifically occurs or affects a specific disease or individual, or occurs in a specific metabolic process, it includes the activation of the specific disease, the condition of the individual, and the specific metabolic process .

As described above, the nano-biochip kit including the metal nanoparticles and the metal nanopatterned nanobiochip of the present invention has a structure in which the metal constituting the metal nanoparticles and the metal nanoparticles are different from each other, The effect can be minimized and the detection sensitivity can be increased to selectively detect each metal crystal. That is, as the metal nanoparticles and the metal nanopatterns exhibit unique wavelength-dependent selective plasmon resonance scattering, biomolecules can be selectively detected by a dark-field microscope without a matrix effect. Therefore, the accuracy and reliability of the analysis result are improved, and it can be widely used in the detection field of genes, proteins, cells, etc. in which the accuracy of detection is important.

1A through 1C illustrate an enhanced-light concentrator capable of collecting wavelength-dependent plasmon resonance scattering light according to the present invention, wherein FIG. 1A illustrates a non- FIG. 1B is a microscope showing an improved upright type microscope using plasmon resonance scattering for fluorescence detection. FIG. 1B is a schematic diagram of an upright type microscope employing an enhanced condensing device capable of condensing the wavelength-dependent plasmon resonance scattering light according to the present invention. FIG. 1C is a schematic view showing a transmission grating included in an upright type microscope detection apparatus. FIG. 1C is an image processing unit according to an embodiment of the present invention. (A camera capable of detecting a false color and a high-sensitivity detection camera) are mounted at the same time.
FIG. 2 is a schematic view (A), a DIC image (B), and an SEM image (C) of a gold nanopatterned biochip of the present invention.
FIG. 3 is a schematic diagram illustrating a process for producing a nanoparticle composite (silver nanoparticle-antibody) according to the present invention.
FIG. 4 is a schematic diagram showing elements constituting (A) a nano-biochip kit according to the present invention and (B) a sandwich ELISA method using a gold nanopattern chip and a silver nanoparticle-antibody.
Fig. 5 is a photograph showing the silver nanoparticles (A) and the silver nanoparticle-antibody conjugate (B) according to the present invention imaged.
6 is an image of a silver nanoparticle (A) and a silver nanoparticle-antibody conjugate (B) obtained through an apparatus including a transmission grating mirror according to the present invention.
FIG. 7 is a graph showing the wavelength dependence of the wavelength-dependent plasmon resonance scattering light of a gold nanopattern chip (B) after 100 nm and 500 nm gold nanopatterned chips (A) and a sandwich immunoassay using the silver nanoparticle- It is a false color image detected using a dark field microscope equipped with an improved condensing device.
FIG. 8 is a graph showing the results of the detection of a captured antibody, a target antigen, and a detection antibody (silver nanoparticle-antibody) on a gold nanopattern chip having diameters of 100 nm and 500 nm by a selective wavelength-dependent plasmon resonance .
FIG. 9 is a graph showing the results of an enhanced focusing to collect the wavelength-dependent plasmon resonance scattering light of a gold nanopattern chip including various bandpass filters after sandwiched immunity with a silver nanoparticle-antibody on 100 nm and 500 nm gold nanopatterned chips This is a false color image detected using a dark field microscope equipped with a device.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

Example  One : Nano biochip Of kit  making

1-1. Sample Preparation

1-ethyl-3- (3-dimethylamino-propyl) carbodiimide hydrochloride (EDC), dimethyl sulfoxide (DMSO, 99.5%), 11-mercaptoundecanoic acid (MUA, 95%), 6-mercapto- %), 2-mercaptoethanol, 2- (morphozlino) ethanesulfonic acid (MES), glycine, O - [2- (3-mercaptopropionylamino) ethyl] - O -methylpolyethylene glycol (HS-PEG, 5kDa), phosphate buffered saline (PBS ) Were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sulfo-NHS ( N- hydroxysulfosuccinimide, NHSS), dithiobis (succinimidyl propionate) (DSP) and Protein A / G were purchased from Pierce (Rockford, IL, USA). Tris (base) was purchased from Mallinckrodt Baker, Inc. (Phillipsburg, NJ, USA). StabilGuard was purchased from SurModics (Eden Prairie, MN, USA). 80 nm silver nanoparticles (SNPs, 1.83 pM, 1.10 x 10 ⁹ particles / mL) were purchased from BBI Life Sciences (Cardiff, UK). Monoclonal mouse anti-cardiac troponin I antibody (clone 19C7 and 16A11) and standard human cardiac troponin I (cTnI, clone 8T53) were purchased from HyTest (Turku, Finland). 1 mM sodium phosphate buffer (pH 7.4; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na ₂ HPO ₄ , 1.4 mM KH ₂ PO ₄ ) and 50 mM MES buffer were filtered using a 0.2-μm membrane filter, B (G15TE, 280-315 nm, Philips, Netherlands) lamp.

1-2. Fabrication of gold nanopattern chip

Gold nanopatterns were processed on a 10 mm ² glass substrate by electron beam nanolithography at the National Nanotechnology Fab Center (Daejeon, South Korea). Nano-arrays were fabricated by electron beam evaporation, and gold spots with diameters of 100 nm and 500 nm were arrayed in 4 x 5 nanometers at 10 μm intervals (Fig. 2). All chips 30 seconds deposited (deposition) of acetone (99.5% purity) before and 30 seconds with isopropyl alcohol (99.9% purity), and blood Rana solution (piranha solution, 1: 1 = H 2 SO 4: 30% H ₂ O ₂ ). Between each step was washed with high purity water, and the washed chips were dried with nitrogen gas and stored in a dryer.

1-3. Antibody to surface Combined silver nanoparticles  Produce

The binding process of the antibody and the silver nanoparticles (SNPs) is as shown in FIG. Specifically, 10 mM MUA (~ 2.2 mg) and 30 mM MCH (3.982 mL) dissolved in 996 mL of high purity ethanol were mixed with 3 mL (final 1.35 pM) of 80 nm SNPs and sonicated for 10 minutes And reacted for about 2 hours to form a self-assembled monolayer (SAM) of MUA-MCH on the SNP surface. And washed twice with high purity distilled water through a centrifuge (12 000 rpm, 90 min, 4 ^o C). The SNPs-MUA-MCH precipitate was re-dissolved in 50 mM MES and 0.1 M NaCl (pH 6.0).

EDC and Sulfo-NHS were used to bind the antibody and SNPs. To the 2 mL SNPs-MUA-MCH solution, 40 mg EDC (2 mg / mL in 50 mM MES pH 6.0, 20 mL) and 196 mg Sulfo-NHS (2 mg / mL 1xPBS pH 7.4, 98 mL) were added. After shaking at room temperature for 30-40 minutes, the SNPs-MUA-MCH-NHSS solution was washed by centrifuge (17,000 rpm, 30 min, 4 ^° C) and dissolved again in PBS buffer. Activated SNPs were added to 1 mL of 10 mg / mL detection antibody (cTnI clone 16A11), shaken at room temperature for 4 hours, ^o C stored. Add 10 mM Tris (pH 7.5) and 200 mL of 1 M glycine solution to the solution for 15 minutes to quench the excess unreacted hydroxylamine. Finally, the SNP-antibody suspension was centrifuged (17,000 rpm, ~ 90 min, 4 ^o C) was used to remove unbound antibody. The SNP-detection antibody (final concentration ~ 5.48 pM = 3.30 x 10 ⁹ particles / mL) was dissolved in PBS buffer ^o .

Example  2: Selective wavelength-dependent Plasmon  Dark-field microscope system for resonance scattering detection

The physical arrangement and schematic diagram of the wavelength dependent-enhanced dark-field microscopy (WD-EDFM) is shown in FIG. On the gold nanopattern chip, SNPs-cTnI antibodies were detected by using an improved dark field illumination system (CytoViva, Auburn, AL, USA) attached to a specularized Olympus BX51 microscope (Olympus Optical Co., Ltd., Tokyo, Japan) It was imaged by light scattering. The system, equipped with a CytoViva 1250 dark-light concentrator instead of a conventional microscope concentrator, was equipped with a Solarc 24 W metal halide light source (Welch Allyn, Skaneateles Falls, NY, USA). A 100 x oil type objective lens whose numerical aperture (NA) is adjustable from 0.6 to 1.3 was used. The image of the true resonance light scattering of the SNPs-cTnI antibody on the gold nanopattern chip was collected by a Nikon D3S digital camera (Tokyo, Japan). Data were collected using MetaMorph 7.0 (Universal Imaging Co., Downingtown) software.

An improved upright type microscope system using plasmon resonance scattering for non-fluorescence detection of clinical samples responsive to gold nanopatterned chips according to the present invention was used (Fig. 1a) (FIG. 1A) and a transmission grating (FIG. 1B) included therein, which are equipped with an improved condensing device capable of collecting the light, An improved image processing system capable of collecting wavelength-dependent plasmon resonance scattering light. An image processing unit for processing an image provided from a microscope by connecting to a dark field microscope equipped with a condensing device and outputting a desired image to a user. Two image processing units (a camera for detecting various colors and a high-sensitivity camera) were simultaneously mounted on a dual port 1c).

Example  3: Sandwich immunoassay using gold nanopattern chip

The basic procedure of sandwich immunoassay is as follows (Fig. 4B). The gold nanopattern chip is immersed in a 4 mg / mL DSP dissolved in DMSO for about 30 minutes. After washing with DMSO solution, it was washed again with distilled water. 0.1 mg / mL Protein A / G was reacted for about 1 hour to activate the gold surface. Unreacted succinimide groups were blocked with 10 mM Tris (pH 7.5) and 1 M glycine for approximately 30 minutes. The chip was stabilized by reacting with StabilGuard for about 30 minutes and was simply rinsed with distilled water. For the sandwich reaction, 20 mL of capture antibody (anti-cTnI, clone 19C7) was reacted for 1 hour and washed with 1 × PBS (pH 7.4). The target antigen (cTnI standard protein) diluted to various concentrations was reacted for 1 hour. After washing with 1xPBS (pH 7.4), 20 mL of SNPs-antibody (anti-cTnI, clone 16A11) was reacted for about 2 hours and the dark-field plasmon resonance scattering imaging was measured (FIG. 7). Antibodies associated with SNPs increased the reaction time than antibody molecules in consideration of molecular size. Between all reaction steps was washed with 1x PBS buffer and stirred at room temperature.

FIGS. 5 to 9 show the result of the above-mentioned method. As a result of the above-mentioned results, it was found that a false-color image (bright blue) due to plasmon resonance scattering at a specific absorption wavelength of 80 nm nano- ) Is shown in Fig. In addition, FIG. 6 demonstrates that a transmissive grating mirror can be mounted between an image processing unit and a base microscope to detect images of light diffraction of silver nanoparticles (A) and silver nanoparticle-antibody (B). FIG. 7 shows a false color image collected by an improved dark field detection apparatus by selective wavelength-dependent plasmon resonance scattering after sandwich immunity reaction of a gold nanopattern chip and a disease-related sample thereon. (A) is an image (gold spot, yellow) detected by selective wavelength-dependent plasmon resonance scattering on a gold nanopattern chip having diameters of 100 nm and 500 nm, B) was characterized by selective wavelength-dependent plasmon resonance scattering after sandwich immunization with the capture antibody, target antigen (cTnI), and detection antibody (silver nanoparticle-antibody) on gold nanopattern chips having diameters of 100 nm and 500 nm In the detected image (blue), the image processing unit proved that not only the qualitative analysis but also the mutual molecules were coupled due to the detection of the false color image. In addition, the possibility of quantitative analysis according to the antigen concentration thereof was confirmed using a high-sensitivity detection camera (FIG. 8). In addition, a dark-field microscope equipped with an improved condensing device capable of collecting wavelength-dependent plasmon resonance scattering light of a gold nanopattern chip after sandwich immune reaction using a silver nanoparticle-antibody on 100 nm and 500 nm gold nanopatterned chips It is possible to selectively detect at a desired absorption wavelength by using various bandpass filters included in the optical filter (FIG. 9).

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

10: capture molecule
20: Target biomolecule
30: Metal nanoparticles bound to the detection molecule
40: substrate
100: Nano Biochip Kit

Claims (17)

A nano biochip kit for detecting the presence or content of a target biomolecule contained in an assay sample using plasmon resonance scattering,
(a) a substrate; A nano pattern located on the substrate and comprising a first metal; And a capture molecule connected to the first metal of the nanopattern; And
(b) nanoparticles comprising a second metal that emits plasmon resonance scattering light upon irradiation; And a detection molecule coupled to the nanoparticle, wherein the nanoparticle complex comprises:
The capture molecule and the detection molecule are each capable of binding to the target biomolecule,
In the nano-biochip kit, when a target biomolecule is bound to a capturing molecule of a nano-biochip, a detection molecule is bound to the bound target biomolecule, and the nanoparticle complex is connected to the nano-
The material of the first metal and the material of the second metal are different so that light that can be emitted from the first metal by the plasmon resonance scattering at the time of light irradiation does not interfere with the light signal emitted from the second metal by plasmon resonance scattering In nano biochip kit.
The nano biochip kit according to claim 1, wherein the target biomolecule is a compound, a protein, a DNA, an RNA, or a cell.
The nano-biochip kit according to claim 1, wherein the capture molecule or the detection molecule is any one or more of a protein, a gene, a lipid, an enzyme, an aptamer, and a ligand.
The nano-biochip kit according to claim 3, wherein the capture molecule or the detection molecule is an antibody.
The method according to claim 1, wherein the substrate is any one of glass, fused-silica, quartz, polydimethylsiloxane (PDMS), and polymethylmethacrylate (PMMA) As a nano biochip kit.
The nano-biochip kit according to claim 1, wherein the substrate is a glass or polymer material capable of transmitting light having a thickness of 10 nm to 1 mm.
2. The method of claim 1, wherein the nanopattern is selected from the group consisting of nanolithography (AFM-DPN) including ink-jet nano-printing, e-beam lithography and atomic force microscope-dip pen nanolithography ) ≪ / RTI > technology. ≪ RTI ID = 0.0 > 8. < / RTI >
The nano-biochip kit according to claim 7, wherein the nanopattern has a diameter ranging from 1 to 500 nm, and the pitch of the nanopattern ranges from 10 nm to 10 μm.
The nano-biochip kit according to claim 1, wherein the first metal or the second metal is selected from the group consisting of gold (Au), silver (Ag), platinum (Pt), and palladium (Pd).
The nano biochip kit according to claim 9, wherein the first metal is gold and the second metal is silver.
2. The method of claim 1, wherein the nanoparticles comprising the nanopattern or the second metal comprising the first metal are selected from the group consisting of spherical, star-shaped, Nano Biochip Kit.
12. The nano-biochip kit according to claim 11, wherein the nanopatterns or nanoparticles have a spherical shape.
A nano biochip kit according to any one of claims 1 to 12;
A dark field microscope equipped with a dark field condensing device capable of collecting plasmon resonance scattering light; And
And an image processing unit connected to the dark field microscope and processing an image provided from the microscope, wherein the detection unit detects the presence or content of the target biomolecule contained in the analysis sample using plasmon resonance scattering.
14. The apparatus of claim 13, further comprising: a transmission grating; Metal halide light source; And a selective spectral bandpass filter for obtaining a wavelength-dependent resonance scattered light image.
A method for detecting a target biomolecule using the nano-biochip kit according to any one of claims 1 to 12.
16. The method of claim 15,
(a) reacting the nano-biochip with an analytical sample;
(b) reacting the nanobiochip according to step (a) with a detection molecule bound to the metal nanoparticle; And
(c) detecting the metal nanoparticles bound to the nanobiochip according to step (b).
17. The method of claim 16, wherein the detecting of the step (c) comprises the steps of: (a) forming a photodiode array (PDA), a charge-injection device (CID), a charge- , And a digital single lens reflex camera (digital single lens reflex camera).

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