KR101745897B1 - Method for Quantitative Screening of Viruses Using Total Internal Reflection Scattering - Google Patents

Abstract

The present disclosure describes a method for detecting viral genes on a detection platform with at least two metals containing probes specific for viral genes such as influenza virus and having an absorption spectrum that does not overlap with each other with plasmonic nano- There is provided a disease diagnosis screening method and detection system capable of directly detecting and quantitatively detecting a single molecule at a single molecule level.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a method of screening for viruses,

The present disclosure relates to a quantitative screening method of a virus (viral gene) using total-reflection scattering. More particularly, the present disclosure relates to a detection platform comprising at least two metals having an absorption spectrum that contains a probe specific for a viral gene such as an influenza virus and has plasmon nanometallic scattering properties and does not overlap with each other, And more particularly, to a disease diagnosis screening method capable of directly detecting and quantifying the gene at a single molecule level without amplification of the gene.

Recently, researches are being actively applied to diagnosis fields such as biochips using nanotechnology. As such, by integrating the nanotechnology, miniaturization of the biochip, enhancement of sensitivity, simple sample preprocessing, and smart functions that are difficult to implement in the prior art can be performed. Therefore, in the existing microbiological bio-diagnosis technology, It has become possible to overcome a variety of problems related to labor, field diagnosis, and detection limitations.

We are trying to apply these nano-based technologies to the field of diagnosis of influenza viruses, especially influenza viruses such as swine flu.

Influenza viruses are causing serious threats to public health worldwide, and the recent epidemic of the highly virulent algae H5N1 strain has raised concerns about the possibility of transmission to humans, and the emergence of the pandemic new A / H1N1 rapidly spreading worldwide This is an increasing concern. In particular, as a new type of influenza virus is found and the possibility of transmission among humans increases, a method for detecting and diagnosing influenza viruses is required more rapidly and precisely.

In addition, considering the high infectivity of influenza virus, a method of accurately diagnosing even a trace amount of sample is required.

The influenza virus rapid antigen method is known as a universal test method for diagnosing (or confirming) a suspected patient of influenza virus. Quadidel's Sofia POCT products, which are currently on the market, are expected to achieve high sensitivity as an automated inspection system. ClearView and Veritor products are known as other products, but there is a disadvantage that only a swab specimen is used. In particular, there is a problem that Clearview and Bioline products of SD do not have a high sensitivity and thus do not contribute to actual patients (Eurosurveillance, Volume 18, Issue 21, May 23, 2013). For this reason, real-time polymerase chain reaction (RT-PCR) technique is mainly used as a specific test for the diagnosis of influenza virus such as confirmation of a new influenza virus.

In this regard, clinicians generally diagnose disease using DNA amplifiers such as enzymatic polymerase chain reaction (PCR). This technology is not only widely used today, but also has a relatively high efficiency. However, it has disadvantages such as false positives, false negatives, and time and sample consumption. There are also technical limitations due to specificity, low clinical sensitivity, and low accuracy.

In addition, ligase chain reaction (LCR) and ligase detection reaction (LDR) are known, but all of them involve amplification reaction, so that the target nucleic acid is amplified in an exponential manner The above-described problem is implied.

 Therefore, there is a continuing need to develop a detection protocol for single-molecule spectroscopy that excludes the amplification process.

Amplification-free detection methods for gene detection include fluorescence, photonic colonic arrays, electrochemical methods, dark-field sensors using plasmon nanoparticles, and piezoelectric plate sensors. Recently, Mayr group has developed a microfluidic chip and succeeded in detecting non-amplified genes at the level of peptomol (fM = 10-15 M). However, there is still a need for improved single molecule level gene quantification techniques.

As described above, there is known a method of detecting fluorescence by fluorescence spectroscopy after directly binding the fluorescent label to detect the fluorescence signal at a single molecule level without amplification of the gene, but time-dependent fluctuations of fluorescence signal with time, there is a problem that it is difficult to detect for a long time due to a phenomenon such as photostability, photoblinking, and photobleaching, and an interest in a fluorescence-free detection method is increasing as an alternative thereto (V. Espina et al. , J. Immunol. Methods , 2004, 290: 121-133).

On the other hand, the inventors of the present invention have developed a non-fluorescent detection method of target biomolecules using wavelength-dependent differential interference versus microscopy (Japanese Patent No. 1514894), a nano-biochip kit using wavelength-dependent plasmon resonance scattering, Detection method (Korean Patent No. 1470730), and the like. Specifically, the wavelength-dependent differential interference effect detection method is a microscopic method which can observe the step of colorless transparent cells or metal surfaces using the polarization and coherence of light, by a polarizing element and a Wollaston prism The light is dispersed and transmitted to the sample surface to change the optical path difference value generated in the sample to the contrast on the sample surface to obtain the image of the sample.

Further, the present inventors have developed a nanotag / nanoprobe platform using at least two metals, and have developed an evanescent field formed on a biochip surface using total internal reflection (TIR) A scattering signal by a selective band-pass filter.

However, it is not an easy task to accurately distinguish a signal or an image derived from each of the nano probe (first metal) and the nano tag (second metal) from the signal or image collected by the total reflection scattering. The band-pass filter used in the above-described conventional techniques has a function of passing only frequencies (or wavelengths) within a specific range and blocking or attenuating frequencies (or wavelengths) outside the range, but the first and second metals It is difficult to collect a signal originating from a single shot on the screen. Especially, in order to accurately calculate the distance between pixels in the data processing process in order to quantify the virus, in addition to accurately detecting the virus from the scattering signals of the two metals described above, it is necessary to accurately check the center position information of the scattering signal, The above-mentioned prior arts are difficult to implement effectively.

Therefore, there is an increasing demand for a screening method for effectively detecting viruses and quantifying them through more precise spectroscopic images without amplification of viral genes.

Thus, in this disclosure, a platform comprising at least two metals having the property of plasmon nanometallic scattering containing an oligomer that specifically reacts with a viral gene can be used to amplify viral genes And a simultaneous quantification of the scattered signal generated from the virus-related virus gene, thereby providing a screening method capable of achieving high sensitivity detection and simultaneous quantification even for a trace amount of disease-associated virus gene.

According to one aspect of the present disclosure,

As a method for quantitative screening through non-amplification of a disease-associated target virus and non-fluorescence detection method,

a) a capture nano pad having a probe oligomer specifically binding to the target virus immobilized on the first metal-nanopatterned substrate, and a detection oligomer capable of specifically binding to the target virus, Providing a platform comprising a combined detection tag, wherein the first metal and the second metal are selected such that the absorption spectra do not overlap with each other;

b) hybridizing the target virus in the assay sample with the detection oligomer of the detection tag and the probe oligomer of the capture nano pad;

c) forming a desiccation field by totally reflecting the light emitted from the light source, and generating a total reflection scattering signal generated from the detection tag and the capturing nano pad by the desorption field; And

d) dividing the generated total reflection scattering signal into a scattering signal originating from the first metal and a scattering signal originating from the second metal by passing the signal through a transmission grating mirror (TR), whereby a non-dispersed or zero- Generating and collecting a spectral image and a dispersed or first order spectroscopic image;

/ RTI >

Here, a method of measuring the center position information or the interval between the orders of each of the divided scattered signals through data processing by Gaussian fitting is provided.

According to an exemplary embodiment, the step d) may comprise the step of firstly reacting the target virus in the assay sample with the detection oligomer of the detection tag, and reacting the probe oligomer of the capture nano-pad with the first reaction product .

According to an illustrative embodiment, the first metal-nanopattern of the platform has a non-dispersed or zero order spectroscopic image of the total reflection scattering signal generated from the first and second metals passing through the transmission grating mirror, So that the spectroscopic images of the difference are not overlapped with each other.

In an exemplary embodiment, the non-dispersed or zero order spectroscopic images and the dispersed or primary or more spectroscopic images may be collected into a single shot.

According to the exemplary embodiment, the distance between the pixels of the spectroscopic image can be calculated by measuring the center position information or the interval between the orders for each of the divided scattering signals during data processing.

According to an exemplary embodiment, the light source may be a single wavelength light source.

In an exemplary embodiment, the target virus may be an influenza virus, for example, a particular type of influenza virus.

According to another aspect of the present disclosure,

A detection system for quantitatively screening disease-associated target viruses in a single shot through non-amplification and non-

A single wavelength laser light source;

A Dove prism configured to totally reflect the incident light irradiated from the light source to form a desiccation field;

A capture nano pad onto which a probe oligomer that specifically binds to a target virus is immobilized on a first metal-nanopatterned substrate, and a detection oligonucleotide coupled with a detection oligomer to which the second metal-containing nanoparticle specifically binds to the target virus Wherein the first metal and the second metal are selected so that their absorption spectra do not overlap with each other, the trapping nano-pads are located in a collapsing region on the dope prism, and the probe oligomer of the trapping nano- Generating a total internal scattering signal derived from the first metal and the second metal when the detection oligomer of the detection tag hybridizes and hybridizes with the target virus;

An objective lens spaced above the trapped nano-pads and arranged to collect the total reflection scattering signal; And

A transmission grating mirror disposed above the objective lens to divide (disperse) the collected total reflection scattering signal into a scattering signal originating from a first metal and a scattering signal originating from a second metal; And

A detector for measuring the center position information or the interval between the orders of the divided scattered signals through data processing by gauss fitting and imaging the measured scattered signals;

Is provided.

According to the present disclosure, spectroscopic images (zero-order and first-order images) between at least two types of metals having different plasmon resonance scattering absorption spectra using a transmission grating mirror (TG) can be collected in a single shot on a screen have. As a result, it is possible to effectively distinguish the two scattering signals, which are difficult to distinguish from the nondispersive (0th order) image, without disturbing effect, and to obtain an optimum background line-to-signal ratio (S / N) Can be increased.

Further, more accurate quantitative screening can be achieved by measuring the center position information or the interval between the orders of the scattered signals divided by the data processing by Gaussian fitting of the scattered signal (or image) divided from the transmission grating mirror TG . The above detection or analysis characteristics can minimize single-molecule counting, rapid screening, and preprocessing of samples in a wide linear range of quantification curves (calibration curves), thereby enabling rapid quantification of disease-related viral genes requiring quantitative analysis. And can be diagnosed by a non-amplification method with high reproducibility.

FIG. 1A is a schematic diagram of an embodiment of the present invention, in accordance with one embodiment of the present disclosure, using a total reflection scattering detection system equipped with a transmissive grating mirror and a platform for the diagnosis of unamplified and non- FIG. 2 is a schematic diagram schematically illustrating a process of hybridization; FIG.
FIG. 1B and FIG. 1C are schematic diagrams of an upright type microscope detection apparatus equipped with a high-sensitivity CCD camera capable of detecting plasmon resonance scattered light, two light sources having different wavelengths, A prism-type full-scattering-scattering-type microscope photograph and a configuration diagram for a non-fluorescence detection equipped with a mirror (TG) and an objective lens;
1D is a differential interference contrast (DIC) image and a scanning electron microscopy (SEM) image of a gold (Au) nano pattern of 100 nm size arranged on a substrate (glass substrate);
FIGS. 2A and 2B are transmission electron microscope (TEM) images of silver (Ag) nanoparticles and silver (Ag) nanoparticles combined with nanoparticles for total reflection scattering according to Example 2, Image and its UV-VIS absorption spectrum;
Figure 3 is a graphical representation of the results obtained in Example 2 of Example 2, wherein (a) 20 nm silver (Ag) nanoparticles, (b) samples centrifuged in the final stage, (c) samples excluded from the centrifugation step, and ≪ RTI ID = 0.0 > zeta < / RTI > potential for each of the separated samples and the color of the sample;
FIG. 4 is a graph showing the results of measurement of the transmittance of gold (Au) -nano-pads, (b) probe-oligomer bonded gold (Au) (D) a gold (Au) -nano pad in which only MCH is present;
FIG. 5 is a graph showing the relationship between (a) 20 nm total scattering scattering of (Ag) nanotag and (b) single stranded DNA combined with double stranded DNA in Example 2, IR spectrum;
FIG. 6 is a schematic diagram of a scattering image obtained from a hybridization system of H7N9 viral genes on gold (Au) -nano-pads in Example 2, (a) a scattered image obtained from a transmission scattering detection system without a transmission grating mirror (TG) b) Scattering image from a full-acid scattering detection system equipped with a transmission grating mirror (TG). (c) graph showing the relative distance between the 0th and the 1st order of the nanotag that is bound to gold (Au) -nano-pad and double-stranded DNA through relative sensitivity, and (d) Gaussian alignment for accurate distance measurement ≪ / RTI >
FIG. 7 is a spectroscopic image of a target gene before hybridization of (a) a target gene and (b) after hybridization of a target gene on a gold (Au) -nano pad under irradiation of a 671 nm light source and a 405 nm light source in Example 2; And
8 is a graph showing a quantitative curve graph of a scattering signal according to various concentrations of a target gene upon irradiation with a 405 nm light source in Example 2, and (b) a blank control method in which a target sample is not present for investigation of specificity, (H5N1) having a non-complementary base sequence and a target sample (H7N9) having a complementary base sequence.

The present invention can be all accomplished by the following description. The following description should be understood to describe preferred embodiments of the present invention, but the present invention is not necessarily limited thereto.

The terms used in this specification can be defined as follows.

The term "bio-chip" refers to a nano-biochip produced by combining nanotechnology. At this time, "nanotechnology" It is possible to enable smart functions that are not possible. As a whole, the biochip of the present invention is a biochip in which a metal is nanopatterned, and at least two kinds of metal nanostructures having scattering characteristics at different wavelengths are bound (mediated) with a target virus or a genetic material It can be understood in the form of.

The term " fluorescence-free detection "may generally mean a detection method using means other than fluorescence, and typically includes a detection method using absorption, scattering, and the like.

The "total internal reflection scattering (TIRS)" is a phenomenon in which, when light enters a specific angle through a first medium in a system in which two media having a different refractive index (a first medium and a second medium) are in contact with each other, The incident light can not be transmitted to the second medium due to the refractive index difference between the mediums, and may be reflected back to the first medium. That is, when the refractive index of the other surface at the interface is lower and the incident angle is larger than the critical angle, the light can not pass through and the whole reflection is formed. At this time, since an evanescent field is formed at a predetermined width at the interface, it is possible to detect light scattered by an optically active substance in a corresponding range, that is, a substance causing scattering. Thus, when located near the small field layer (EFL), the scattering intensity depends on the distance from the interface to the particle. The scattering intensity can be expressed by the following equation (1).

[Equation 1]

I (h) = I ₀  exp (-βh)

Where h is the distance between particle and surface, I ₀ is the scattering intensity measured when h is 0, and β is the decay index.

As shown in Equation (1), the intensity of the dissipative field depends on the intensity of the incident light and is exponentially attenuated according to the distance.

The "analytical sample" is not particularly limited as long as it is applicable to the nano-biochip or detection platform set forth in the present disclosure, but may be a liquid sample that can be uniformly treated on the nano-biochip. In particular, it may be any substance or component capable of containing a target virus type gene, and further may be a substance separated from a living body and various substances exposed to or treated by the living body. Examples of substances or components separated from the living body can be blood, urine, runny nose, cells, extracted DNA, RNA, protein, and the like.

A "virus" may refer to a microinfectant that is less invasive than bacteria and is located at an intermediate stage between the organism and the inanimate object. Since the virus alone is not viable, it is replicable only in other living cells. Specifically, the term "virus" refers to adenovirus 7, PRRSV, influenza including salmonid, salmon anemia virus, classical swine fever, hepatitis C virus, , Hepatitis E virus, African swine fever virus, Puumala virus, infectious bronchitis virus, transmissible gastroenteritis, SARS CoV, Mraburg virus, Virus, ebola virus, West Nile virus, yellow fever virus, tick-borne encephalitis virus, bovine diarrhea virus, parvovirus, Coxsackie B3, Coxsackie B5, avian reovirus, , Rotavirus, Semliki forest virus, and swine type 2 circo type 2 virus.

"Influenza virus" may generally refer to a pathogenic agent of influenza which causes respiratory diseases by invading the mucous membrane. Influenza viruses are classified into three types of A-B-C according to the difference of complement-binding antigens. There are four species of NA-N2 in NA of A type and HAO-HA1-HA2-HA3 in HA. Type A infects horse - swine - birds. The new subtype is a recombinant form of the animal virus, and there are other subtypes within the same subtype. Types B and C are generally not infected except the human body and have the property of aggregating red blood cells of chicken. In addition, influenza viruses may include influenza A-B-C, as well as live virus and tgootovirus, and may be understood to include especially avian influenza and swine influenza.

"Nanopattern" can refer to a repeating pattern of nanometer units. According to one embodiment, the nanopattern may have a diameter of, for example, about 0.1 to 1000 nm, more specifically about 1 to 500 nm. In addition, the pitch of the nanopattern may be in the range of, for example, about 1 nm to 100 占 퐉, more specifically about 10 nm to about 10 占 퐉.

In an exemplary embodiment, the nanopattern is nanopatterned with a metal and may be formed, for example, in a laterally arranged form. For this purpose, one of nanolithography techniques such as inkjet nano-printing, e-beam lithography, atomic force microscope-dip pen nanolithography (AFM-DPN) can do.

By "nanoparticles" is meant, by way of example, particles having a size or average diameter in the range of about 1 to 200 nm, specifically about 10 to 100 nm, . ≪ / RTI > In particular, it can be understood as including the particles having any size that can cause plasmon resonance scattering under the purport of this disclosure. In an exemplary embodiment, the nanoparticles may be of a metallic material, and in particular in the nanoscale region, the color may vary depending on the particle size. In addition, the nanoparticles can bind detectable oligomers that specifically react with the target virus or genetic material.

"Oligomer" can refer to a multimer protein that contains a relatively small number of identical subunits (i.e., protomers).

A "target genetic material" can consist of several, hundreds, thousands or millions of nucleotides, and fragments of DNA, RNA, and the like. The base sequence of such a target genetic material (or a fragment thereof) has a complementary relationship with the base sequence of the detection oligomer and the probe oligomer and can be hybridized.

The term "complementary" means capable of hybridizing a nucleotide or base pair between nucleic acids, usually A and T (or U), or C and G.

 The term "probe" means that it contains a base sequence complementary to a specific base sequence contained in the target nucleic acid, and can be hybridized under appropriate conditions, in order to evaluate the presence, absence and / or amount of the target nucleic acid.

"Capture" can mean, for example, being immobilized on a metal nanopattern, specifically interacting with the target virus or viral genetic material to be analyzed specifically, The substance is immobilized on the metal nanopattern substrate (substrate) on which the probe oligomer is immobilized so that it can be detected.

"Probe oligomers" and "detecting oligomers" may refer to oligomers that are capable of specifically binding to a target virus or genetic material. At this time, the probe oligomer and the detection oligomer may be the same or different from each other.

"Hybridization" may mean that a single-stranded nucleic acid made from denaturation of a double-stranded nucleic acid produces a double-stranded nucleic acid by interaction with another type of nucleic acid having a complementary base sequence. General aspects of hybridization are known to those skilled in the art and are described, for example, in a number of documents, such as U.S. Patent No. 4,647,529, which is incorporated herein by reference.

A "light source" is a source for supplying light for optical analysis, and it can be used to provide light of a specific wavelength range, such as X-ray, ultraviolet (UV) And the like. A single wavelength or a multi-wavelength light source may be used as the light source. However, as a specific example of the light source, it may be advantageous to use a laser of a specific wavelength (single wavelength) as a light source in order to easily adjust the angle of light incident on the prism.

"Transmission grating mirror (TG)" means that light passing through a sample is decomposed by a grating into zero-order, first-order, and secondary- (Dispersed) and can be imaged.

The "detection unit" may mean a device capable of sensing and digitizing and / or imaging a total reflection scattering signal derived from a sample. And an image processing unit connected to the microscope for processing an image provided from a microscope and having a computer or a microprocessor having a program for image processing. For example, an image provided from a microscope can be connected to a total reflection scattering system and output to a desired image. However, the image processing unit is not limited to any particular image processing unit commonly used in the art.

As described above, in the present specification, based on spectral resolution, a method of quantitatively screening a trace amount of diseased virus or gene by a single shot method without amplification such as PCR, and a method of performing quantitative screening using a transmission grating mirror A non-fluorescent total reflection scattering detection system is provided to provide a method for non-amplified and non-fluorescent detection of a viral gene such as influenza virus. For this purpose, at least two kinds of metal nanostructures which do not overlap with each other in absorption spectrum and preferably have plasmon scattering properties are selected.

Specifically, a total reflection scattering signal of at least two kinds of metals which are difficult to distinguish in a nondispersive (0-th order) image is dispersed using a platform including at least two kinds of metal nanostructures (or particles) having different absorption wavelengths For example, the distinction effect of the scattering signal of the metal nanoparticles used as the base material (substrate) is removed by distinguishing the scattered signals of the metal nanoparticles used as the detection tag, and the detection sensitivity is increased Single molecule level screening is possible.

FIG. 1A is a schematic diagram of an embodiment of the present invention, in accordance with one embodiment of the present disclosure, using a total reflection scattering detection system equipped with a transmissive grating mirror and a platform for the diagnosis of unamplified and non- The process of hybridization is illustrated. In the following description, the first metal is specified as gold (Au) while the second metal is specified as silver (Ag), but this should be understood as an example for convenience of explanation.

In the above-described process, first, single-stranded DNA (ssDNA) capable of specifically binding to a target virus (for example, H7N9 virus) is used as a detection oligomer, and silver (Ag) Metal nanoparticles) to form a detection tag. The detection tag thus formed is brought into contact with the target virus (PC; for example, H5N1 virus) contained in the assay sample, and specifically bound through hybridization reaction with the double PC.

Separately, a capturing nanopad or a biochip on which a probe oligomer that specifically binds to the target virus is immobilized is provided on a substrate on which gold (Au) is nanopatterned as a first metal.

According to exemplary embodiments, in the case of a substrate usable in the capture nano-pads or bio-chips, a substrate, such as silicon, glass, fused silica, quartz, polydimethylsiloxane (PDMS) And may be a light transmitting solid structure material such as polymethyl methacrylate (PMMA). Here, the thickness of the substrate may be in the range of, for example, about 10 nm to 1 mm, specifically about 100 nm to 500 μm, more specifically about 1 to 100 μm, but this should be understood in an exemplary sense.

According to one embodiment of the present invention, at least two kinds of metals which do not overlap with each other as the first metal and the second metal and can exhibit the plasmon scattering property can be used in combination. .

In this regard, the first metal and the second metal may be specifically selected from gold (Au), silver (Ag), platinum (Pt), palladium (Pd), and combinations thereof. However, in the case of the illustrated embodiment, gold (Au) and silver (Ag) are selected as the first metal and the second metal, respectively. In addition, the nanoparticles containing the first metal and / or the nanoparticles containing the second metal may be spherical, star-shaped, quadrature, triple or the like, and more specifically, .

In this regard, the scattered light absorption spectrum of the metal nanostructure usually changes not only depending on the type of metal but also on the size or shape of the structure. Therefore, it is possible to combine all of them to form a substrate for nanoprobe formation, The size, shape, and / or material of each metal to be used can be appropriately selected by the tag. For example, the plasmon resonance may be varied depending on the shape of the metal nanopattern, so that a pattern according to various shapes can be used. Specifically, the metal nanopattern may be a circular or rectangular metal nanopattern.

According to the exemplary embodiment, when the captured nanopads are nanopatterned with metal, specific chemical reactions or bonds can be easily performed depending on the metal characteristics. In particular, it is easy to connect (fix) capturing molecules such as antibodies and aptamers Do. Illustratively, the nanopattern of the capture nanopads may be a gold (Au) -nano pattern treated to facilitate covalent bonding using a thiol group (-SH).

In an exemplary embodiment, to immobilize the probe oligomer on the first metal-nanopatterned substrate, the surface of the first metal nanopattern is immersed in a buffer solution (e. G., Potassium phosphate salt buffer) containing a probe oligomer Chemical adsorption reaction, and blocking may be carried out in order to suppress nonspecific binding prior to the hybridization reaction with the target virus described later. At this time, for blocking, for example, mercaptohexanol (specifically, 6-mercapto-1-hexanol) may be used.

Further, the surface of the second metal nanoparticles of the detection tag may be modified before the hybridization reaction. A representative example of such a surface treatment method is to form a single layer of alkanethiol (HS (CH ₂ ) _n X) (where n is 1 to 15), by a functional group X present at the terminal position of the alkanethiol, The interface properties can be controlled by changing the metal surface structure. More specifically, it can be modified in such a manner as to form a monolayer of carboxy group-terminal alkanethiol. For this purpose, mercapto carbonic acid represented by the formula [HS- (CH ₂ ) _n -COOH] (wherein n is 1 to 15) and the like can be used as the carboxyl group-containing compound (that is, Mercapto alcohol, specifically, mercaptohexanol (more specifically, 6-mercapto-1-hexanol) can be used as the alkane thiol-containing compound (that is, X is -OH ). The modified metal nanoparticles can be activated using, for example, EDC (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide) and NHSS (N-hydroxysulfosuccinimide) Tag can be formed.

As described above, the first hybridization reaction product between the detection tag and the target virus (PC) is brought into contact with the trapped nanopads to perform a second hybridization reaction. As a result, the target virus is reacted with the first metal (Au) And the nanoparticles of the second metal (Ag). On the other hand, in the case of NC, it is not combined with both the capture nano pad and the detection tag.

In an exemplary embodiment, the primary and secondary hybridization reactions are limited to specific conditions as long as they are carried out under conditions suitable for the hybridization reaction between the target virus and the detection oligomer in the assay sample, and the primary hybridization reaction product and the probe oligomer no. However, it is also possible to appropriately adjust the reaction temperature within a controlled temperature range of, for example, about 50 to 80 ° C (specifically about 60 to 70 ° C) and within a reaction time range of, for example, about 20 to 120 minutes Can be selected.

As such, when the target virus (or viral genetic material) is present in the assay sample, it specifically binds to the detection oligomer bound (labeled) with the second metal-containing nanoparticle (non-fluorescent nanoparticle) The metal-containing nanoparticles generate a total reflection scattering signal at a wavelength different from that of the first metal-containing nano-pattern of the trapped nanopads by total reflection scattering.

As described above, the target virus combined with the captured nano-pads and the detection tag is subjected to a non-amplified and non-fluorescent detection and quantification screening process (presence or absence of target virus, etc.) through a total internal reflection scattering detection system.

FIGS. 1B and 1C are diagrams for explaining an example of a method in which two light sources (lasers) having different wavelengths are used in an upright type microscope detection apparatus equipped with a high-sensitivity CCD camera capable of detecting plasmon resonance scattered light, , A transmission grating mirror, and a non-fluorescence detection prism type full-scattering type scattering type microscope having an objective lens.

In the case of the above-described detection system, an erecting type microscope in which components are sequentially arranged along an optical path is used as a basis, but an inverted microscope can also be used. In an exemplary embodiment, the detection system is based on a microscope commonly used in the art, and includes a light source, a prism that induces total reflection scattering, an optical member such as a numerical aperture controllable objective lens, a transmission grating mirror (TG) It is possible to arrange the detecting section provided with the fitting function at an appropriate position.

The members constituting the detection system (specifically, the members from the prism to the detection portion) may be arranged in a sequential contact or spaced apart arrangement, wherein the spacing between the respective members is such that the focal length of the lens and / The divergence angle of the prism, the beam deviation angle, and the like are taken into consideration in consideration of the optical characteristics of the prism. Therefore, when the respective members are spaced apart, it does not necessarily mean that they are arranged at the same interval.

According to the illustrated embodiment, the light emitted from each of the two laser light sources is incident on the prism at a predetermined angle. In this regard, a prism may be mounted on the bottom of a substrate that can be used in the trapped nanopads or biochips to cause total scattering and to form a desiccation field.

At this time, when the light source is irradiated with the metal nanoparticles, the plasmon resonance scattering is caused, and the light of a specific wavelength range can be provided with a constant intensity according to the characteristics of the metal nanoparticles. According to an exemplary embodiment, a laser for emitting light having a short wavelength can be used. Optionally, at least one mirror or mirror manipulator may be provided in front of the light source to adjust the angle of incidence of light.

At this time, the prism includes a first surface (in the illustrated example, two first surfaces are formed corresponding to two light sources) on which light is incident, and a second surface formed so that incident light is totally reflected to form a depressed surface. A typical example of this type of prism is a dove prism (DP). The Dove prism does not change the direction of parallel light but is known as a prism that forms an upside down image.

According to the alternative embodiment, a total reflection type objective lens can be used in place of the prism, and in this case, a separate objective lens described later may be omitted.

In one embodiment, the target virus or target gene may specifically be an influenza virus, and the capture nano-pads (biochip) sandwiched with the detection tag in the presence of the target virus may be generated in the cow- And generates a total reflection scattering signal.

Specifically, the second metal-containing nanoparticles (non-fluorescent nanoparticles) generate a total reflection scattering signal of the second metal at a wavelength different from the total reflection scattering signal of the first metal of the trapped nanopads. According to the illustrated embodiment, an oil layer having a refractive index similar to that of a glass slide is applied to the upper and lower surfaces of a capturing nano pad (e.g., a gold nano-biochip (GNC)) in order to selectively minimize light refraction phenomenon )can do.

The total reflection scattering signals generated from the two kinds of metals thus generated are collected through an objective lens (OL) spaced apart at a predetermined interval from the top of the captured nanopads (biochips). According to the exemplary embodiment, the objective lens can be configured to adjust the numerical aperture (NA) to adjust the amount of light irradiated on the sample. At this time, the numerical aperture of the objective lens may be in the range of, for example, 0.6 to 1.3.

According to an exemplary embodiment, the detection system may optionally include a rotating stage between the prism and the capture nano pad (or bio-chip). The rotating stage is a component that can rotate 360 revolutions of the platform where the analytical sample is located. It can rotate in the desired direction at intervals of 1 검출 and detects polarizations in different directions at various angles (0 to 360 ㅀ) , Which makes it possible to easily analyze the properties or properties of asymmetric metals.

According to this embodiment, the total reflection scattering detection system is configured so that the total reflection scattering (plasmon resonance scattering) signal that has passed through the objective lens passes through the transmission grating mirror (TG) as the optical adjustment device. The transmission grating is a beam of monochromatic light having a specific wavelength that is incident on the grating at an angle to the grating vertical plane and is diffracted along a plurality of mutually distinct paths corresponding to the diffraction order m. In this case, d in the figure means the spacing between the grooves of the lattice.

Thus, by combining the total reflection scattering and the transmission grating mirror in a manner of interposing a transmission grating mirror between the objective lens and the detection portion, it is possible not only to collect the total reflection scattering signals derived from the two kinds of metals simultaneously on one screen, It is possible to easily distinguish the total reflection scattering signal generated from the two kinds of metals through the image (primary or more). That is, the total reflection scattering signal transmitted through the transmission grating mirror can be divided into spectral imaging or dispersed image, primary image (n = +1), and original image or nondispersed image (transmission grating mirror (N = 0), which is the same image as the image in the absence of the image (n = 0).

According to one embodiment, the total reflection scattering signal divided by the transmission grating mirror is imaged by the detector. The detector may be a photodiode array (PDA), a charge-injection device (CID), a charge-couple device (CCD), or a digital single lens reflex < / RTI > In the figure, L ₁ is the distance between the transmission grating mirror and the detector (for example, CCD), and L ₂ is the displacement distance between the zero-order image and the primary image.

On the other hand, it is desirable to obtain the high-resolution image by accurately calculating the distance between the pixels in the data processing by collecting the center position information of the divided scattered signals described above. For this, Gaussian fitting is applied in this specific example. That is, for the graph showing the relative distances between the 0th-order and first-order images of the nano-tag coupled with the first metal-nanopad under the intervention of the double-stranded DNA (mediated), a Gaussian spelling method To be applied.

 When the Gaussian spelling method is applied to the divided total reflection scattering signal, the center position of the total reflection scattering signal derived from each of the two kinds of metals can be found, and the error of the interval calculation between the orders can be reduced to a meaningful level can do. As a result, it is possible to easily find the center position of the dispersed (primary or higher) spectroscopic image even when using various detection apparatuses using a multi-wavelength light source as well as a total reflection scattering measurement method using a single wavelength light source do.

As described above, the main features of the quantitative screening method of viruses in the sample according to this embodiment are as follows.

1) Total light scattering can be detected by the non-fluorescence detection system through the scattering detection system. Therefore, it is possible to overcome the technical limitations of the conventional fluorescence detection technique. In particular, by introducing the transmission grating mirror (TG) The two kinds of metal scattering signals generated from the substrate (substrate) and the detection tag can be screened by the single shot method, so that the overlapping or interference effect of the two different metal scattering signals can be eliminated.

2) Precise quantitative analysis can be performed through hybridization screening for disease genes, particularly viral genes, in a substrate on which first metal (trapped) nanopads are arranged.

3) Since only the scattering signal of the metal in the detection tag is collected without the influence of the metal scattering signal used as the substrate (substrate), and the detection sensitivity can be increased by using a single wavelength light source, the quantitative analysis efficiency can be increased.

4) Gaussian spelling is applied as a method of collecting the center position information of the scattering signal in order to accurately calculate the distance between pixels in data processing.

By combining the above-mentioned plurality of technical features, the viral genetic material (especially, influenza virus), which is increasing in demand through non-amplification and non-fluorescence methods, differs from the prior art which requires an amplification process in diagnosing disease using a trace amount of sample Can be efficiently performed at a single molecule level.

 The present invention can be more clearly understood by the following examples, and the following examples are merely illustrative of the present invention and are not intended to limit the scope of the invention.

Example

The materials used in this example are as follows.

Mercapto-decanoic acid (MUA), 95%), 6-mercapto-1-hexanol (MCH), 97% 3-dimethylamino-propyl carbodiimide hydrochloride (EDC), dimethyl sulfoxide (DMSO) (99.5%), 2 (3-dimethylamino-propyl) carbodiimide hydrochloride - (morphoslino) ethanesulfonic acid (MES), glycine, ethylene diamine tetra acetic acid (EDTA), sodium citrate tribasic dehydrate, , And 20-nm silver nanoparticles (SNPs) were purchased from Sigma-Aldrich (St. Louis, Mo., USA).

- N-hydroxysulfosuccinimide (NHSS), tris-HCl is known as J.T. Baker.

- Potassium phosphate monobasic (KH2PO4) and sodiumchloride (NaCl) were purchased from Junsei.

- Potassium phosphate dibasic (K2HPO4) was purchased from Samchun Pure Chemical.

- Dithiothreitol (DTT) solution was purchased from Biosolution.

The nucleotide sequence was designed by Bioneer as follows.

- Probe oligomer (H7N9): 5'-GCT ATT CCA ATG-T10-SH-3 '

Detecting oligomer (H7N9): 5'-NH2-T10-AAT CCT AGG TTT-3 '

- Target Oligomer (H7N9): 5'-CAT TGG AAT AGC AAA CCT AGG ATT-3 '

- Target oligomer (H5N1; non-complementary oligomer for H7N9): 5'-AGA CCC AAA GTA AAC GGG CAA-3 '.

Example 1

Fabrication of detection platform

end. Fabrication of gold (Au) nanopads

The substrate on which gold (Au) nanopads were arranged was processed on a 10 mm 2 glass substrate by electron beam nanolithography at the Korea Nano Technology Institute (Suwon, Korea). At this time, gold (Au) nano-pads having a diameter of 100 nm were arrayed in a 4 × 4 array at intervals of 5 μm through electron beam deposition (Analyst, 2013, 138, 3478-3482).

I. The combination of silver (Ag) nanoparticles and detection oligomers

The binding reaction of the detection oligomer of 20 nm silver (Ag) nanoparticles is as follows.

Ethanol in which 10 mM MUA and 30 mM MCH were dissolved was added to the 20 nm nanoparticle solution and sonicated for 10 minutes and reacted at room temperature for 2 hours and 30 minutes. MUA-MCH was bound to the surface of the nanoparticles, and then 0.31 mmol EDC was added thereto, followed by reaction for 15 minutes, followed by addition of 77.2 nmol NHSS for 30 minutes. A detection oligomer having 74 pM amine groups was added and allowed to react at room temperature for 15 hours. After centrifugation at 12000 rpm for 90 minutes, the supernatant was removed and diluted in 10 mM Tri-HCl and 0.1 mM EDTA (pH 8.0, TE).

All. Immobilization of probe oligomers on gold (Au) nanopads

A 1M potassium phosphate buffer (pH 7.0) solution containing a probe oligomer having a thiol group and MCH was immersed in a cleaned gold (Au) nano pad and subjected to chemical adsorption reaction at room temperature for 15 hours. The reacted gold (Au) nanopads were washed with 10 mM NaCl and 5 mM Tris (pH 7.4) for about 5 seconds. Prior to the hybridization reaction, 1 mM MCH solution was reacted for 1 hour to block the non-specific binding other than the target oligomer to be immobilized on the gold (Au) nano pad.

la. Sandwich hybridization reaction

- Primary hybridization reaction

The detection oligomer coupled with 20 nm silver (Ag) nanoparticles present in the buffer for hybridization (1 μ SSC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) and the target gene were reacted at 65 ° C for 30 minutes To carry out a primary hybridization reaction.

- Secondary hybridization reaction

The first hybridization reaction product was contacted with the gold (Au) nano pad on which the probe oligomer was immobilized, and the second hybridization reaction was performed under the reaction conditions of 65 ° C and 1 hour. After the reaction, the gold nanopads were washed with high purity water to remove unbound oligomers and then used in detection and quantitative screening experiments.

Example 2

Detection and quantitative screening using a detection system equipped with a transmission grating mirror (TG)

The detection and quantitative screening experiments of the sandwich hybridization reaction products obtained in Example 1 were performed using a total reflection scattering detection system equipped with a transmission grating mirror (TG) shown in FIG. 1B. In particular, the non-fluorescent total scattering method based on a standard type Olympus BX51 microscope (Olympus Optical Co., Ltd., Tokyo, Japan) was used for disease diagnosis screening of trace amounts of unamplified genes hybridized on gold (Au) Respectively.

As the light source, silver (Ag) nanoparticles were irradiated with 40-nm laser (SOL-405-LM-020T, Shanghai laser century Co., Ltd., China) Two solid-state 671-nm laser (SDL-671-040T, Shanghai laser century Co., Ltd.) with a power of 30 mW were used.

(Ag) nanotags and gold (Au) nanoparticles of the second hybridization reaction product were attached to the front of the EMCCD camera using a 70 g / mm product (Edmund Optics Inc., Barrington, NJ, USA) as a transmission grating mirror ) Scattered total reflection scattering signals derived from the nanopads were collected. At this time, a holder was manufactured to adjust the distance between the camera and the transmission grating mirror from 23 mm to 85 mm, and all the images were originally divided into a zero-order and a first-order A test was performed by fixing the transmission grating mirror at a position spaced 85 mm from the camera so as not to overlap.

The total reflection scattering signal was obtained using an objective lens (UPLANFLN, × 100) whose numerical aperture was adjustable from 0.6 to 1.3.

Total scattering images of the hybridization reaction products were collected through an electronic coupling device (512 ㅧ 512 pixel imaging array, QuantEM 512SC, Photometrics, AZ, USA). At this time, the exposure time was adjusted to 50 ms through the shutter control. Images were also collected and analyzed using MetaMorph 7.5 software.

The spectroscopic images (0th and 1st order) obtained through the transmission grating mirror (TG) were obtained by Gaussian fitting through the ThunderSTORM plug-in of Image J (National Institutes of Health, Bethesda, MD, USA) I got information about the location (center localization).

DNA hybridization was carried out by using 20 nm silver (Ag) nanoparticles (SNP, nanotag), detection oligomer (SS), target virus (PC, H7N9) and probe oligomer (PS) . ≪ / RTI > At this time, half of the base sequence of the target virus (H7N9) was hybridized with the detection oligomer, and the other half was designed to hybridize with the probe oligomer. For the control experiment, the non-complementary target sample (NC, H5N1) was designed not to hybridize. The sandwiched hybrid gold (Au) nanopads (nano-biochips) were screened using the transmission grid mirror-total reflection scattering (TG-TIRS) imaging system shown in FIGS. 1B and 1C. The screening results are shown in Figs. 2 to 8. Fig.

2A is a TEM image of 20 nm diameter silver (Ag) nanoparticles and pure silver (Ag) nanoparticles coupled with a detection oligomer. As shown in the figure, the surface of the silver (Ag) nanoparticles to which the detection oligomer is bonded shows a cloudy thin layer shell.

Figure 2b is the UV-Vis spectra of the detection oligomers and 20 nm nanoparticles present in the aqueous solution. According to the figure, the silver (Ag) nanoparticles bound to the detection oligomer are red-shifted by 10 nm, unlike the initial pure silver (Ag) nanoparticles having an absorption wavelength of 395 nm, Respectively. In addition, silver (Ag) nanoparticles can be confirmed to bind oligomers (detection tags) to the nanoparticles by confirming the spectrum of the detected oligomer at around 260 nm after hybridization with the detection oligomer.

Figure 3 is a graphical representation of the results of (a) 20 nm silver (Ag) nanoparticles, (b) samples centrifuged at the final stage, (c) samples excluded from the centrifugation step, and (d) And the zeta potential for each and the color of the sample.

Generally, the zeta potential of a nanoparticle and a detection oligomer has a negative charge. This is due to the presence of carboxy ions on the surface of the nanoparticles present in the citrate buffer.

In this connection, according to FIG. 3, it can be seen that the zeta potential value change after centrifugation and washing step has a strong negative charge of -50.6 ± 8.3 mV at -38.4 ± 1.7 mV. From this, it was confirmed that the silver (Ag) nanoparticles to which the detection oligomer was bonded were more stable.

On the other hand, after the probe oligomer having a thiol group was reacted with gold (Au) nano-pads, the contact angle was measured to confirm immobilization.

As shown in the figure, the contact angle (a) of gold (Au) nano-pads before the reaction with the probe oligomer was 57.8 °. The contact angles (b) of the gold (Au) nano-pads reacted with the probe oligomer are 42.6 °, which indicates that the measured angle is relatively hydrophilic. In addition, the contact angle c of the gold (Au) nano pad contacted with MCH as the blocking solution was 47.9 °. On the other hand, it can be seen that the contact angle (d) of gold (Au) -nano pad which is in contact with only the MCH without reacting with the probe oligomer is 68.6 °, which is different from the contact angle of the gold (Au) -nano pad. Thus, it can be confirmed that the probe oligomer was successfully immobilized on the gold (Au) -nano pad.

The hybridization reaction between the target oligomer and the detection oligomer was confirmed by FT-IR spectroscopy, and the results are shown in FIG.

According to the figure, in the case of the Ag nanotag (a) with a double-stranded DNA 20 nm total reflection scattering, a peak appeared at a wavelength of 1655 cm ^-1 by the hybridization reaction, whereas silver (Ag) Ag) nanoparticle (b) showed no characteristic peaks due to the hybridization reaction. As described above, it was confirmed that a hybridization reaction between the target oligomer and the detection oligomer occurred as a spectrum originating from the double-stranded DNA molecule.

FIG. 6 is a graph showing scattering images obtained from a first-order scattering system in which a transmission grating mirror (TG) is not mounted, (b) a scattering image obtained by performing a hybridization of H7N9 viral genes on a gold Scattering images from a full - scale scattering system with a transmission grating mirror (TG). (c) a graph showing relative distances between the zero-order and the first-order distances of the gold nanopads and the nanoparticles to which the double-stranded DNA is bound, and (d) graphs showing Gaussian fit for accurate distance measurement. .

6A, it is difficult to distinguish signals derived from gold (Au) -nano-pad and silver (Ag) -containing detection tags from images collected by total reflection scattering (TIRS) without a transmission grating mirror TG . On the other hand, as shown in FIG. 6B, scattering signals derived from the two metals can be simultaneously collected on a single screen through the combination of transmission grating mirror-total reflection scattering (TG-TIRS) The scattering signals of the two metals can be easily distinguished.

On the other hand, the scattered signal transmitted through the transmission grating mirror (TG) is a spectral imaging primary image (n = +1) and an original image (the same image as the image not passed through the transmission grating mirror) (Separated or dispersed) into the 0th order image (n = 0).

The transmission grating mirror TG is obliquely mounted in order to avoid superposition between the primary image of the yellow circle and the green circle shown in the region (ii) and the region (iii) in FIG. 3c. Then, a Gaussian fitting was applied to determine the center position of the scattering signal originating from each metal, and the distance between the 0th and first images was calculated without overlapping in the x-direction (Fig. 6d). The distances between the zero-order and primary images between the yellow circle and the detection tag were 248.4 ± 0.1 and 149.0 ± 0.4, respectively. Thus, the spectral resolution of the detection system was 2.7 nm / pixel.

FIG. 7 is a spectroscopic image of a target gene before hybridization with (a) a target gene and (b) after hybridization of a target gene on a gold (Au) -nano pad under irradiation of light sources of 671 nm and 405 nm in this embodiment.

In the case of FIG. 7 (a), a detection system equipped with a transmission grating mirror (TG) at a position spaced 85 mm from the camera was used to detect the gold (Au) -nano This is the image of the pad. When a 671 nm laser is irradiated, the zero-order and primary images of the gold (Au) -nano-pads appeared in regions (i) and (ii). However, when irradiating a 405 nm light source, zero-order and primary images of gold (Au) -nano-pads were not observed (region (iii) and region iv).

It should be noted in the above observation result that as shown in the region (iv) of FIG. 7 (b) as a gold (Au) -nano pad image after the hybridization reaction, the scattering signal generated from the gold (Au) It is possible to obtain the scattering signal of the detection tag-DNA without interference.

Furthermore, when 405 nm light is irradiated to the gold (Au) -nano pad after the hybridization reaction, the scattered signal obtained in the 0th order image (region (iii) in FIG. 7B) (iv)) was about 10 times higher than the scattered signal. This supports the ability to detect individual monomolecules or monolayers at trace levels without the interference of the substrate due to the use of a transmission grating mirror (TG).

A quantitative clinical screening test was carried out by hybridization to target genes (H7N9 viral gene) at various concentrations when irradiating a 405 nm light source using the detection system used in the present example. As a result, a target gene (DNA) A linear curve (74 zM to 7.4 fM, R = 0.9928) with various concentrations versus sensitivity is shown in Fig. 8a.

In the case of the H7N9 viral gene, it is common to detect or analyze after amplification. However, as in the present embodiment, a detection system using a combination of transmission grating mirror-total reflection scattering (TG-TIRS) is a system that is effective for detection and quantitative analysis of non-amplified hybridization as a result of screening test of hybridized gold (Au) .

In addition, gold (Au) -nano pad reacted in the absence of the target gene, and probe oligonucleotide in place of the H7N9 target gene and other influenza virus DNA (H5N1) as an unrecognized viral gene in the detection oligomer The same procedure was performed to confirm the specificity. In addition, a scattering signal of a target sample (H7N9) having a complementary base sequence was shown.

According to FIG. 8B, in the case of the control group, the scattering sensitivity was about 6 times lower than that in the case of applying the target sample H7N9 target gene. Thus, it can be seen that the detection platform provided in this disclosure is effectively applicable to viral genes.

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (19)

As a method for quantitative screening through non-amplification of a disease-associated target virus and non-fluorescence detection method,
a) a capture nano pad having a probe oligomer specifically binding to the target virus immobilized on the first metal-nanopatterned substrate, and a detection oligomer capable of specifically binding to the target virus, Providing a platform comprising a combined detection tag, wherein the first metal and the second metal are selected such that the absorption spectra do not overlap with each other;
b) hybridizing the target virus in the assay sample with the detection oligomer of the detection tag and the probe oligomer of the capture nano pad;
c) forming a desiccation field by totally reflecting the light emitted from the light source, and generating a total reflection scattering signal generated from the detection tag and the capturing nano pad by the desorption field; And
d) dividing the generated total reflection scattering signal into a scattering signal originating from the first metal and a scattering signal originating from the second metal by passing the signal through a transmission grating mirror (TG), whereby a non-dispersed or zero- Generating and collecting a spectral image and a dispersed or first order spectroscopic image;
/ RTI >
Here, the center position information or the interval between the orders of the divided scattered signals is measured through data processing by Gaussian fitting. The method according to claim 1, wherein the step (d) comprises: a first hybridization reaction of the target virus in the assay sample with the detection oligomer of the detection tag; and a second hybridization reaction of the probe oligomer of the captured nanopads with the first hybridization reaction product And a hybridization reaction step. 2. The method of claim 1, wherein the first metal-nanopattern of the platform comprises a non-dispersed or zero order spectroscopic image of the total reflection scattering signal generated from the first and second metals passing through the transmission grating mirror, Wherein the spectroscopic images are arranged so as not to overlap each other. The method according to claim 1, wherein the light source is irradiated through a prism mounted on a lower portion of the trapping nano pad in order to form a dead space for causing total reflection scattering. 2. The method of claim 1, wherein the non-dispersed or zero order spectroscopic images and the dispersed or primary or more spectroscopic images are collected into a single shot. The method of claim 1, wherein the light source is a single wavelength light source. The method according to claim 1, wherein the distance between the pixels of the spectroscopic image is calculated by measuring the center position information or the interval between the degrees of each of the divided scattered signals through data processing by Gaussian fitting. 2. The method of claim 1, wherein the target virus is adenovirus 7; PRRSV; Influenza virus; Salmon anemia virus; Classical swine fever; Hepatitis C virus; Hepatitis E virus; African swine fever virus; Puumala virus; Infectious bronchitis virus; Transmissible gastroenteritis; SARS CoV; Mraburg virus; Ebola virus; West Nile virus; Yellow fever virus; Tick-borne encephalitis virus; Bovine diarrhea virus; Parvovirus; Coxsackie B3; Coxsackie B5; Avian reovirus; Rotavirus; Semliki forest virus; Or a porcine type 2 circo type 2 virus. 9. The method of claim 8, wherein the target virus is an influenza virus. 3. The method of claim 2, wherein each of the first hybridization reaction and the second hybridization reaction is performed at a temperature selected from the range of 50 to 80 DEG C and a reaction time period selected from the range of 20 to 120 minutes. The method of claim 1, wherein the first metal is gold (Au) and the second metal is silver (Ag). 12. The method of claim 11, wherein the nanopattern of the trapped nanopads is treated to facilitate covalent bonding using a thiol group (-SH). 12. The method of claim 11, wherein, in order to immobilize the probe oligomer on the first metal-nanopatterned substrate, the surface of the first metal nano-pattern is chemically adsorbed and reacted with a buffer solution containing a probe oligomer, Further comprising the step of blocking to inhibit non-specific binding prior to the hybridization reaction. 14. The method of claim 13, wherein the blocking step is performed using mercaptohexanol. 12. The method of claim 11, further comprising modifying the surface of the second metal nanoparticles of the detection tag prior to the hybridization reaction between the detection oligomer of the detection tag and the target virus, CH ₂ ) _n X) single layer.
Wherein X is -COOH or -OH, and n is from 1 to 15. The method according to claim 15, wherein the alkanethiol monolayer is formed by using a mercaptocarboxylic acid represented by the formula [HS- (CH ₂ ) _n -COOH] (wherein n is 1 to 15), or by using a mercapto alcohol Lt; / RTI > 16. The method of claim 15, wherein the modified second metal-containing nanoparticles react with the detection oligomer after being activated using EDC and NHSS to form a detection tag. 10. The method of claim 9, wherein the influenza virus is the H7N9 virus. A detection system for quantitatively screening disease-associated target viruses in a single shot through non-amplification and non-
A single wavelength laser light source;
A Dove prism configured to totally reflect the incident light irradiated from the light source to form a desiccation field;
A capture nano pad onto which a probe oligomer that specifically binds to a target virus is immobilized on a first metal-nanopatterned substrate, and a detection oligonucleotide coupled with a detection oligomer to which the second metal-containing nanoparticle specifically binds to the target virus Wherein the first metal and the second metal are selected such that the absorption spectra do not overlap with each other, the trapping nano-pads are arranged to be located in a collapsing region on the dope prism, and the probe oligomer And generating a total internal scattering signal derived from the first metal and the second metal when the detection oligomer of the detection tag hybridizes and hybridizes with the target virus;
An objective lens spaced above the trapped nano-pads and arranged to collect the total reflection scattering signal; And
A transmission grating mirror disposed above the objective lens and dividing the collected total reflection scattering signal into a scattering signal derived from a first metal and a scattering signal derived from a second metal; And
A detector for measuring the center position information or the interval between the orders of the divided scattered signals through data processing by gauss fitting and imaging the measured scattered signals;
≪ / RTI >

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