KR101514894B1 - A method and system for fluorecence-free detection of a target biomolecule using wavelength-dependent differential interference contrast microscopy - Google Patents

Abstract

The present invention relates to: a method for fluorescence-free detection of a target biomolecule; and a system for differential interference contrast signal detection. More specifically, according to the present invention, a differential interference contrast (DIC) signal of a nanoparticle including a second metal is detected by using a nanobiochip including first-nanopatterned substrates having absorption spectrums, which are not overlapped by each other, and a detection molecule, which is labeled with a fluorescence-free nanoparticle incluidng a second metal and is specifically bound to the target biomolecule. In the method for fluorescence-free detection of a target biomolecule, the metal nanopatterned nanobiochip bound with captured molecules, and a detection molecule including metal nanoparticles are used. According to the method, absorption spectrums of the metal-nanopattern and the metal nanoparticle are designed to be not overlapped by each other in order to minimize optical interference. A detection limit is lowered by increasing a signal-to-noise (S/N) ratio of the metal nanoparticle bound to the biomolecule; and thus a small amount of a sample can be efficiently detected on a pattern in the nanosize. Moreover, wavelength-dependent spectroscopy can be performed by combining an analysis system for the method with a band-pass filter. Therefore, the method can increase precision and reliability of the analysis. Accordingly, the method can be widely used for analyzing a certain type of a biosample such as genes, protein, and cells which require high sensitivity and precision.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a non-fluorescent detection method and system for target biomolecules using wavelength-dependent differential interference microscopy,

The present invention relates to a non-fluorescent detection method and system of target biomolecules.

Array technology, an assay on solid substrates for the detection of biomolecular interactions, is of great interest today for research aimed at disease or therapeutic applications. The use of micro-arrays for screening biomolecular profiles has become an industry standard, such as the identification of certain biomolecules or drug development. However, the micro-array not only requires a relatively large amount of sample and a long reaction time but also has a limitation in detection sensitivity. Nano-arrays have been developed for high-throughput screening (HTS) as well as for increasing the accuracy of the analysis results by overcoming these problems. In the 20th century, nano-array chips used nano-etching technology to reduce the spot size of conventional micro-arrays to nanometer size, and to fabricate carbon nanotube nanodevice arrays using carbon nanotubes. In recent years, plasmon nanoarray biochips have been developed to enable non-fluorescent detection without complicated procedures such as the use of fluorescent dyes or enzymes (IEEJ Transactions on Sensors and Micromachines 131, 425-426, 2011; Talanta 104, 32-38, 2013 ). However, a non-fluorescent detection method capable of selectively detecting a specific biomolecule with high sensitivity without spectroscopic interference on the nano-biochip has not yet been established.

As a result of efforts to find a method for quantitatively analyzing a trace amount of target biomolecules by a non-fluorescent detection method using a metal nano-biochip, the present inventors have found that metal nanoparticles formed on a bio-chip and metal nanoparticles A detection signal indicative of differential interference is detected using a microscope having two Ulster prisms and a selective band pass filter and the plasmon resonance scattering intensity of the metal nanoparticles is measured, The present inventors have confirmed that biomolecules can be quantitatively analyzed and completed the present invention.

The present invention relates to a non-fluorescent detection method for a target biomolecule, comprising the steps of: (a) reacting a nano-biochip comprising a first metal-nanopatterned substrate comprising a capture molecule specifically binding to the target biomolecule with an analytical sample ; (b) reacting the nano-biochip prepared in step (a) with a detection molecule labeled with a non-fluorescent nanoparticle containing a second metal and specifically binding to the target biomolecule; And (c) detecting a differential interference contrast (DIC) signal of the nanoparticles containing the second metal from the nanobiochip according to the step (b).

The present invention also provides a non-fluorescent detection method for a target biomolecule, comprising the steps of: (a) reacting an assay sample with a detection molecule labeled with a non-fluorescent nanoparticle containing a second metal and specifically binding to the target biomolecule step; (b) reacting a nano-biochip comprising a first metal-nanopatterned substrate comprising a capture molecule that specifically binds to the target biomolecule with the analytical sample prepared in step (a); And (c) detecting a differential interference interference signal of the nanoparticles containing the second metal from the nanobiochip according to the step (b).

Furthermore, the present invention provides a system for detecting differential interference due to detection of a target biomolecule using the nano-biochip, comprising: a light source, which is sequentially arranged at regular intervals; Polarizer; A first Wollaston prism; A nano-biochip comprising a first metal-nanopatterned substrate immobilized on a metal pattern and including capture molecules that specifically bind to a target biomolecule; An objective lens; A second Wollaston prism; And a detection unit.

Hereinafter, the present invention will be described in detail.

In the detection method according to the present invention, in detecting the non-fluorescent nanoparticles containing the second metal, in order to minimize the optical interference of the first metal-nanopattern, the non-fluorescent nanoparticles containing the second metal and the non- 1 metal-nanopatterns do not overlap with each other. Therefore, selective detection of the nanoparticles is possible by detecting differential interference contrast signals at different wavelengths.

Therefore, it is preferable that detection of the differential interference signal for non-fluorescence detection of the target biomolecule according to the present invention is performed through a selective band pass filter for wavelength-dependent signal detection.

The term "nano biochip" as used herein refers to a biochip produced by combining nanotechnology. The nanotechnology is a technology that reduces the size and components of a chip itself, increases sensitivity, 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.

The term "fluorescence-free detection" of the present invention collectively refers to detection methods using means other than fluorescence, and typically includes detection methods using absorption, scattering, and the like. Fluorescence is a widely used detection method, but it has a problem of low sensitivity and / or stability, and it is difficult to detect for a long time due to photobleaching or extinction phenomenon. Therefore, there is a great need for a detection method that can replace the fluorescence.

The term "differential interference contrast (DIC)" of the present invention is an optical microscopy illumination technique used to improve contrast in an unstained transparent sample. The DIC is based on the principle of the interference method to obtain information about the optical path length of the sample, thereby visualizing the invisible form by other methods. The resulting image will appear in black and white on a gray background. This image is similar to that obtained by phase contrast microscopy, except that there is no bright diffraction halo. The DIC works by separating the polarized light source into two orthogonally polarized, mutually regular portions that are spatially displaced (sheared) and sheared (spotted) at the sample plane and recombined before observation. The interference of the two parts upon recombination is sensitive to their optical path difference caused by the refractive index or the geometric path length.

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.

The term "biomolecule" of the present invention refers to a substance that can be found in and / or out of a living body, and refers to any substance that generates a specific reaction to a living body or is caused by a specific state or reaction. The biomolecule may be DNA, RNA, an antigen, an antibody, a ligand, a chelate, a receptor, a polymer, an organic compound, a metal ion and a polypeptide. The term "target biomolecule" of the present invention means a substance to be present or to be detected by the detection method of the present invention, specifically, a substance that specifically interacts with the capture molecule and the detection molecule . Conversely, molecules that specifically interact with the target biomolecule to be analyzed can be used as capture molecules and detection molecules.

The term "capture molecule" of the present invention means a molecule immobilized on a metal nano-pattern of a nano-biochip in the detection method of the present invention, and specifically interacts with a target biomolecule to be analyzed, The substance is immobilized in a fixed position where the capture molecules are detectable. Thus, the capture molecule refers to any kind of molecule capable of binding with the target biomolecule. Further, the "detecting molecule" labeled with metal nanoparticles in the present invention means any kind of molecule capable of binding with a target biomolecule in the same manner as the capturing molecule. 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.

Preferably, the capture molecule may be an antigen, an antibody, a ligand, a receptor, a chelate, an aptamer, a DNA and combinations thereof, and the detection molecule may also be an antigen, an antibody, a ligand, a receptor, a chelate, But is not limited thereto.

The substrate for a nano-biochip used in the detection method according to the present invention may be glass, fused silica, quartz, polydimethylsiloxane (PDMS), poly (methyl methacrylate ), And PMMA). Preferably, the substrate may be glass or a polymeric material capable of transmitting light having a thickness of 10 nm to 1 mm.

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 a pattern according to various shapes. Preferably a circular or rectangular metal nano pattern. In the case of nanopatterning a biochip with a metal, a specific chemical reaction or binding can be easily performed depending on the characteristics of the metal. In particular, it is easy to connect capturing molecules such as an antibody and an aptamer. Accordingly, the nanopattern of the present invention may more preferably be a gold nanopat pattern that facilitates covalent bonding 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, 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 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. At this time, the nanoparticles are not limited in size but may include particles larger than the nanoparticles, , Any particle that can cause specific 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 an average diameter of 1 to 200 nm. And more preferably an average diameter of 10 to 100 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.

Preferably, the first metal that is patterned on the nano-biochip or the second metal that constitutes nanoparticles labeled on the detection molecule is selected from gold (Au), silver (Ag), platinum (Pt), or palladium . More preferably, gold is used as the first metal, and silver is used as the second metal, but the present invention is not limited thereto.

In the detection method according to the present invention, the non-fluorescent nanoparticles including the first metal-nanopatterned substrate and the second metal are selected so that the absorption spectra do not overlap with each other. As described above, the absorption spectrum of the metal nanostructure generally varies depending on the size and / or shape of the structure as well as the type of the metal. Therefore, it is necessary to combine the absorption spectrum of the metal nanostructure with the kind and pattern of the first metal and the second metal, You can choose the type. Accordingly, the non-limiting forms of nanopatterns and / or nanoparticles according to the present invention may be circular (or spherical), star-shaped, rectangular, triangular, and the like.

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 prepared by mixing MUA and MCH solutions dissolved in ethanol with SNPs solution and dispersing them for 10 minutes through sonication. The MUA-MCH was self-assembled monolayer on SNP surface, SAM) for about 2 hours. 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.

Quantitative analysis of target biomolecules is possible using the detection method according to the present invention. Detecting a DIC signal with respect to a series of standard samples including a standard biomolecule at a known concentration using the detection method of the target biomolecules described above to prepare a calibration curve of the DIC signal according to the concentration; And comparing the DIC signal measured for the unknown sample with a calibration curve prepared from the standard sample to deduce the concentration, the target biomolecules in the unknown sample can be quantitatively analyzed.

At this time. It is preferable to use metal-nanopatterned biochips of the same size as the nano-biochips used for the DIC measurement of the series of standard and unknown samples.

Further, a step of detecting the plasmon resonance scattering signal of the nanoparticles can be further performed.

According to a specific embodiment of the present invention, a DIC signal is measured using a series of samples with different concentrations ranging from 85 aM to 10 fM on gold nanopatterned nano-biochips with a diameter of 500 nm and plotted against the concentration And showed linear proportional relationships on gold-nano patterns of the same size (FIG. 10).

The non-fluorescence detection of the target biomolecule according to the present invention can be accomplished by using a system for differential signal detection for detecting target biomolecules using the nano-biochip.

Preferably, the system for signal detection of differential interference for detecting a target biomolecule using the nano-biochip includes: a light source, which is vertically and periodically arranged at a predetermined interval; Polarizer; A first Wollaston prism; A nano-biochip comprising a first metal-nanopatterned substrate immobilized on a metal pattern and including capture molecules that specifically bind to a target biomolecule; An objective lens; A second Wollaston prism; And a detection unit.

The system for differential signal detection for detecting target biomolecules using the nano-biochip according to the present invention may further include a polarizer, a first and a second Ulster prism, and a detector based on a microscope commonly used in the art And the like. Or may be fabricated on its own by arranging each optical component in a suitable position. In the case of a microscope-based microscope, the microscope can be of any size or type, as long as the components are arranged in order along the path of light.

The term "light source" of the present invention refers to a light source that provides light for optical analysis As a source, it refers to a device capable of providing light of a specific wavelength range, such as X-rays, UV, and visible light, at a constant intensity, depending on the type of analysis desired. In the present invention, since absorption and scattering in the visible light region are utilized, a halogen lamp that provides white light can be used as a light source. Therefore, when a commercially available microscope is used, a light source of the microscope itself can be used, so that a separate light source is not required.

The term "polarizer" of the present invention is an optical filter that transmits light of a specific polarization and blocks the wavelength of another polarized light. The polarizer is capable of converting undefined or mixed polarized light into well-defined polarized light. Typical types of polarizers are linear polarizers and circular polarizers.

The term "wollaston prism" of the present invention is an optical device for manipulating polarized light, designed by William Hyde Wollaston. The Wollaston prism separates the randomly polarized unpolarized light into two orthogonal linearly polarized outgoing beams. The Wollaston prism is made up of two orthogonal calcite prisms and two right triangle prisms with perpendicular optic axes bonded together on the bottom. The emission beam diverges from the prism to form two polarized lights having an angle of divergence determined by the wedge angle and the wavelength of the prism. Commercially available prisms generally have a divergence angle of 15 to 45 degrees.

Wherein each component of the detection system is arranged in a row from the light source to the detection unit, wherein the spacing between each component is selected from the group consisting of the focal length of the lens and / or the concentrator, the divergence angle of the prism and the beam deviation angle and the like, it is desirable that the optical signal is fixed at a specific position that can provide the best signal considering the optical factor of each component. Therefore, spacing apart at the predetermined intervals does not mean that the components are arranged at the same interval.

Preferably, the system for detecting differential interference signals for detecting a target biomolecule using the nano-biochip may further include a condenser between the first Ulston prism and the nano-biochip.

It is also desirable to further include a selective bandpass filter for wavelength-dependent signal detection prior to said detector.

The term "bandpass filter" of the present invention refers to a device that passes only frequencies (or wavelengths) within a certain range and blocks or attenuates frequencies (or wavelengths) outside the range. The present invention specifically refers to an optical bandpass filter.

For sensitive detection, a band-pass filter capable of passing light in the range of ± 10 nm from the maximum plasmon resonance scattering wavelength of nanoparticles containing the second metal in the present invention, that is, a 20 nm band pass filter . More preferably, a 10 nm band-pass filter can be selected.

In a specific embodiment of the present invention, a gold-nanopatterned substrate of 100 nm or 500 nm diameter and silver nanoparticles of 80 nm diameter were used, respectively. In order to selectively detect the plasmon resonance scattering from silver nanoparticles, the gold nanoparticles have a maximum absorption wavelength of about 650 nm and the silver nanoparticles have a maximum absorption wavelength of about 456 nm, Alternatively, a 10 nm bandpass filter can be used.

The term "detection unit" of the present invention includes an apparatus capable of detecting and digitizing a signal provided from a microscope connected to the above-mentioned wavelength-dependent differential interference microscope. For example, the wavelength-dependent differential interference is connected to a microscope to process an image provided from a microscope to output a desired image to a user. The image processing unit is not particularly limited as long as it has such an object. As such, the detection unit may include a computer having a program for image processing or an image processing unit including a microprocessor. The present invention can be used in a system for signal detection of differential interference for detecting target biomolecules using a nano-biochip according to the present invention Non-limiting examples of the detection unit include a photodiode array (PDA), a charge-injection device (CID), a charge-couple device (CCD), and a digital single- lens reflex).

In a specific embodiment of the present invention, a system for signal detection versus differential interference is constructed by using a standard type microscope generally used in a laboratory. The configuration of the system according to one embodiment of the present invention is schematically shown in Fig.

The non-fluorescent detection method of the target biomolecule using the detection molecule including the metal-nanopatterned nanobiochip and the metal nanoparticle to which the capturing molecule of the present invention is bound is characterized in that in order to minimize optical interference, By reducing the detection limit by improving the signal-to-noise ratio (S / N) of the metal nanoparticles bound through the target biomolecule by designing the absorption spectra of the nanoparticles A small amount of sample can be efficiently detected. In addition, a wavelength-dependent spectroscopy method can be performed by combining a band-pass filter with an analytical system for this purpose, thereby improving the accuracy and reliability of the analysis. Therefore, it can be widely used in the analysis of specific biological samples such as genes, proteins, cells and the like where high sensitivity detection and accuracy are important.

FIG. 1 schematically shows a gold-nanopatterned biochip for detecting non-fluorescence of biological samples according to the present invention and a differential interference contrast (DIC) microscope upright type and a principle of analyzing a sample using the microscope. . The left side shows scattering and scattering wavelength shifts in the case of sample reaction and unreacted on the gold pattern of center type and right side, and the path difference of the light DIC microscope structure and light. HL, halogen lamp; P, polarizer; WP, Wollaston prism; C, condenser; GNC, gold-nano patterned chips; O, an objective lens; A, analyzer; F, filter cube; A CCD, a charge-coupled device; SNP, silver nanoparticles.
Figure 2 is a SEM image of a gold-nanopatterned chip with diameters of 100 nm and 500 nm according to the present invention.
FIG. 3 is a diagram illustrating differential interference contrast and SEM images of gold-nanopatterned chips with diameters of 100 nm and 500 nm according to the present invention.
FIG. 4 is a graph showing intensity of dynamic light scattering according to the SEM image (A) of the 80 nm diameter silver nanoparticles bound to the detection antibody and (B) the size of the nanoparticles.
FIG. 5 is an AFM image and cross-sectional profile of a silver nanoparticle-antibody molecule on gold-nanopatterned chips according to the present invention before and after a sandwich immuno-reaction. FIG. (A) and (B) illustrate images before and after the immune response on gold-nanopatterned chips, respectively, with diameters of 100 nm, (C) and to be.
Figure 6 shows AFM images and cross-sectional profiles after reacting silver nanoparticle bound antibodies on a gold-nanopatterned chip with (A) 100 nm and (B) 500 nm diameters according to the present invention.
FIG. 7 is a graph showing a wavelength-dependent differential interference contrast image at different wavelengths before and after a sandwich immuno-reaction of silver nanoparticle-antibody molecules on a gold-nano-patterned chip according to the present invention. (A) and (B) illustrate images before and after the immune response on gold-nanopatterned chips, respectively, with diameters of 100 nm, (C) and to be.
FIG. 8 is a view showing a resonance scattering spectrum of gold-nanopatterned chips according to the present invention and absorption spectra of 80 nm diameter silver nanoparticles labeled with a detection antibody. (A) and (B) are the resonance scattering spectra of gold-nanopatterned chips of diameters of 100 nm and 500 nm, respectively, (C) the diameter of 80 nm labeled on antibodies bound to the gold- The absorption spectrum of the particles.
FIG. 9 is a diagram illustrating differential interference versus sensitivity for a wavelength-dependent differential interference contrast image indicated by an arrow in FIG. (A) and (B) show the results before and after the immune response on gold-nanopatterned chips with diameters of 100 nm, (C) and (D) to be.
Figure 10 shows a calibration curve of sensitivity to concentration-dependent differential interference prepared using a standard antigen (cTnI) at 85 aM to 10 fM concentration immunoreacted on a gold-nanopatterned chip at 500 nm diameter according to the present invention .

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited by these examples.

gold- Nano-patterned  Manufacture of nanobiochip

Nanometer-scale gold-nanopatterns were formed on a glass substrate of 10 mm ² area at the National Nanotechnology Fab Center (Daejeon, Korea) using electron beam nanolithography. Gold spots having diameters of 100 nm and 500 nm were formed through electron beam deposition to form a 4x5 type nano array at intervals of 10 占 퐉 (FIG. 2). All chips are dipped 30 seconds in acetone (purity: 99.5%) prior to (deposition), isopropyl alcohol 30 seconds (purity 99.9%) and blood Rana solution _{(Piranha solution, H 2 SO 4} : 30% H 2 O 2 = 1: 1). Between each step, it was washed with high purity water, and the final washed chip was dried with nitrogen gas and stored in a dryer.

Silver nanoparticles Combined  Preparation of antibodies

Silver nanoparticles (SNPs, 1.10 x 10 ⁹ particles / ml) were purchased from BBI Life Sciences (Cardiff, UK). The silver nanoparticles were bound to a detection antibody (monoclonal mouse anti-cardiac troponin I antibody, clone 16A11) for use as a non-fluorescent probe for immunoassay. The binding process between the SNP and the antibody is as follows: 11-mercaptoundecanoic acid (MUA, 95%) and 6-mercapto-1-hexanol %) Was dissolved in ethanol to prepare 10 mM and 30 mM of each concentration, and then SNP was added. Dispersed through sonication for 10 minutes and allowed to react for about 2 hours and 30 minutes to form a MUA-MCH self-assembled monolayer (SAM) on the SNP surface. Thereafter, the cells were centrifuged at 12,000 rpm for 90 minutes at 4 DEG C and washed twice with high purity distilled water. The SNPs-MUA-MCH precipitate was re-dissolved in 50 mM MES (2- (N-morpholino) ethanesulfonic acid) and 0.1 M NaCl solution (pH 6.0).

(1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide) and sulfo-N-Hydroxysuccinimide (EDC) were used to bind the antibody and SNPs. 40 ug EDC (2 mg / ml in 50 mM MES, pH 6.0) and 196 술 sulfo-NHS (2 mg / ml in 1 x PBS, pH 7.4) were added to the SNPs-MUA-MCH solution. After stirring for 30 to 40 minutes at room temperature, the SNPs-MUA-MCH-NHS solution was washed by centrifugation at 17,000 rpm for 30 minutes at 4 DEG C and resuspended in PBS. Activated SNPs were added to 1 ml of 10 μg / ml detection antibody (cTnI clone 16A11), reacted by stirring at room temperature for 4 hours, and stored at 4 ° C for 12 hours. 10 mM Tris (pH 7.5) and 200 ml of 1 M glycine solution were added and reacted for 15 minutes to quench excess unreacted hydroxylamine. Finally, unbound antibody was removed by centrifugation at 4 ° C and 17,000 rpm for 90 minutes. The SNP-antibody was dissolved in PBS and stored at 4 ° C.

gold- Nano-patterned  Analysis of sandwich immune response on biochip

The gold-nanopatterned chip prepared as described above was dipped in 4 mg / ml DSP (dithiobis succinimidyl propionate) dissolved in DMSO (diethylthyl sulfoxide, 99.5%, Sigma-Aldrich) for about 30 minutes. Followed by washing with DMSO solution and washing again with distilled water. And reacted with 0.1 mg / ml protein A / G (Pierce) for about 1 hour to activate the patterned gold surface on the chip. Unreacted succinimide groups were blocked by treatment with 10 mM Tris (pH 7.5) and 1 M glycine for about 30 minutes. The chip was treated with StabilGard for 30 minutes to stabilize and briefly wash with distilled water. The cells were reacted with 20 ml of capture antibody (anti-cTnI, clone 19C7) for 1 hour and washed with 1 × PBS (pH 7.4). The target antigen (cTnI standard protein) was diluted to various concentrations and reacted with the standard sample for 1 hour. After washing with 1 x PBS, the cells were reacted with 20 ml of SNPs-antibody solution for about 2 hours and then observed. Considering the size of the molecule, the antibody bound to SNPs increased the reaction time compared to the case of reacting with the single antibody molecule. Between all the reaction steps, washing with 1 x PBS was further carried out with stirring at room temperature.

Configuration of wavelength-dependent differential interference microscope

A schematic diagram of a wavelength-dependent differential interference contrast microscopy is shown in Fig. 1 (left). On the gold-nanopatterned biochip, the SNPs-cTnI antibody is ligated to a light source (HL) and a gold-nano-particle (HL) to be observed on a standardized Olympus BX51 microscope (Olympus Optical Co., Ltd., Tokyo, Japan) The polarizer P and the Wollaston prism W1 are sequentially mounted between the patterned chips and another Wollaston prism W2 is mounted after the objective lens O to measure the wavelength-dependent differential interference contrast . A 100 W halogen lamp was used as the light source, and a 100 × oil type lens whose numerical aperture (NA) was adjustable from 0.6 to 1.3 was used as the objective lens. Differential interference signals of SNPs-cTnI antibodies on gold-nanopatterned chips were measured using an electron multiplying cooled charge-coupled device (EM-CCD) camera (512 x 512, QuantEM 512 SC, Photometrics, ). MetaMorph 7.0 (Universal Imaging Co., Downingtown) and METLAB software were used for data collection and analysis.

FIGS. 2 to 10 show results according to the above-described embodiment. FIG. 4 shows SEM images of 80 nm diameter silver nanoparticles bound to the detection antibody and results of measurement of dynamic light scattering. The data measured by the present inventors for the average size (ie, 80 ± 5 nm) of the silver nanoparticles provided by the supplier showed a size of 26.3 ± 1.6 nm and 85.0 ± 1.4 nm.

The gold-nanopatterned chips were arranged at 100 nm and 500 nm spot sizes and have gold / chrome thicknesses of 14.5 nm and 30.5 nm, respectively (FIGS. 5A and 5C). On the gold-nanopatterned chip, the step differences in the substrate after the reaction of the 85 nM cTnI protein antigen and 80 nM diameter silver nanoparticle-detecting antibody molecules were increased by 76.9 nm and 80.4 nm, respectively, which were measured using AFM Figures 5B and 5D). As described above, the silver nanoparticles bound to the detection antibody may not be uniform in size, or may be inferred as a result of forming two molecular sieves or multi-molecular sieves, as described above. (Fig. 6). However, even though they showed different step differences, we could confirm that the sandwich immune response occurred on the gold / nanopatterned chip due to the step difference before / after the reaction.

Differential interference contrast microscope can be explained as follows.

Where _b is the bias retardation (in degrees or radians), Δ _b is the length, and λ is the wavelength of light (Optics Express, 16, 19462-19479, 2008).

According to the above equation,? _B varies depending on the wavelength, so that a unique DIC image can be obtained at a specific wavelength. In the wavelength-dependent differential interference microscope according to the present invention, the polarizer, the analyzer, the first Ulster prism (WP1) is fixed to a specific position, and the second Ulster prism (WP2) is designed to be adjustable. However, at the time of image acquisition versus differential interference of gold-nanopatterned chips at various wavelengths, only the components of all DIC devices were fixed to observe the DIC for wavelength dependence.

FIG. 7 shows the DIC according to the wavelength change (406 nm to 670 nm) before and after the immune response to the 85 aM cTnI protein. Gold spots showed images with contrast in terms of lightness / darkness at a wavelength of 670 nm (Figs. 7A and 7C). As the wavelength of light decreased, this contrast was only detected on the dark side of the 500 nm gold spot, and on the 100 nm gold spot, the naked eye identification became impossible. In the 100 nm gold-nanopattern chip, the DIC was identifiable after the reaction that was not identifiable prior to the immune response at the 473 nm wavelength (FIGS. 7A and 7B). In case of the 500 nm gold-nanopattern chip, only a strong dark nodule was observed in the gold nano-spot prior to the immune reaction, but the light / dark contrast coexisted after the reaction (Figs. 7C and 7D). This is because as shown in the spectrum of FIG. 8, the noble metal (maximum absorption wavelength ~ 650 nm) and silver (maximum absorption wavelength ~ 456 nm) have inherent absorption spectra, (Gold bars) of two kinds of noble metal materials (gold bars) differing in the light path difference using the non-fluorescent sandwich immunoassay method. FIG. 9 is a graphical representation of the results of a sandwich immunoassay of silver nanoparticle-antibody molecules on 100 nm (A, B) and 500 nm (C, D) gold-nanopatterned chips for the wavelength- Left) / rear (right).

Quantitative analysis of cTnI protein using non-fluorescent detection device versus selective wavelength-dependent differential interference was possible. In particular, the sensitivity of the immune sensor was enhanced using protein A / G as a method for homogeneous arrangement of the capture antibody on the chip surface substrate to reduce nonspecific binding or steric hindrance in the reaction of the capture antibody with the antigen. The gold-nanopatterned chip was able to detect non-fluorescence with high sensitivity at low detection limits (85 aM cTnI) by detection antibodies coupled with nanoparticles using a wavelength-dependent differential interference contrast system. As shown in Fig. 10, a linear calibration curve with a high correlation coefficient was obtained on a 500 nm gold-nanopattern chip at a concentration range of 85 to 10 fM, which enables quantitative analysis, and furthermore a high sensitivity And is applicable to the clinical diagnosis required.

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.

Claims (21)

In a non-fluorescent detection method of a target biomolecule,
(a) reacting a nano-biochip comprising a first metal-nanopatterned substrate comprising a capture molecule that specifically binds to the target biomolecule with an assay sample;
(b) the nano-biochip prepared in the step (a) is labeled with a non-fluorescent nanoparticle including a second metal having an absorption spectrum that does not overlap with an absorption spectrum of the first metal-nanopattern in order to remove optical interference With a detection molecule that specifically binds to the target biomolecule; And
(c) detecting a differential interference contrast (DIC) signal of the nanoparticles containing the second metal from the nanobiochip according to the step (b) through a selective bandpass filter ≪ / RTI >
In a non-fluorescent detection method of a target biomolecule,
(a) detection that is labeled with a non-fluorescent nanoparticle comprising a second metal having an absorption spectrum that does not overlap an absorption spectrum of the first metal-nanopattern to remove optical interference and specifically binds to the target biomolecule Reacting the molecule with the analytical sample;
(b) reacting a nano-biochip comprising a first metal-nanopatterned substrate comprising a capture molecule that specifically binds to the target biomolecule with the analytical sample prepared in step (a); And
(c) detecting a differential interference interference signal of the nanoparticles containing the second metal from the nanobiochip according to the step (b) through a selective band pass filter.
delete 3. The method according to claim 1 or 2,
Wherein detection of the differential interference contrast signal is performed by wavelength-dependent signal detection.
3. The method according to claim 1 or 2,
Wherein the target biomolecule is selected from the group consisting of DNA, RNA, antigens, antibodies, ligands, chelates, receptors, polymers, organic compounds, metal ions and polypeptides.
3. The method according to claim 1 or 2,
Wherein the capture molecule is selected from the group consisting of an antigen, an antibody, a ligand, a receptor, a chelate, an aptamer, DNA, and combinations thereof.
3. The method according to claim 1 or 2,
Wherein the detection molecule is selected from the group consisting of an antigen, an antibody, a ligand, a receptor, a chelate, an aptamer, DNA, and combinations thereof.
3. The method according to claim 1 or 2,
Wherein the substrate is a transparent substrate selected from the group consisting of glass, fused silica, quartz, polydimethylsiloxane (PDMS), poly (methyl methacrylate) Lt; / RTI > is a solid.
3. The method according to claim 1 or 2,
Wherein the substrate is a glass or polymer material capable of transmitting light having a thickness of 10 nm to 1 mm.
3. The method according to claim 1 or 2,
The nanopattern may be formed by any one of nanolithography techniques including inkjet nano-printing, e-beam lithography, and atomic force microscope-dip pen nanolithography (AFM-DPN) ≪ / RTI >
3. The method according to claim 1 or 2,
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.
3. The method according to claim 1 or 2,
Wherein the first metal or the second metal is selected from the group consisting of gold (Au), silver (Ag), platinum (Pt), and palladium (Pd).
13. The method of claim 12,
Wherein the first metal is gold and the second metal is silver.
3. The method according to claim 1 or 2,
Wherein the nanoparticles have an average diameter of 10 nm or more and 100 nm or less.
3. The method according to claim 1 or 2,
For quantitative analysis of target biomolecules,
Detecting a DIC signal for a series of standard samples containing standard biomolecules at a known concentration to produce a calibration curve of the DIC signal according to the concentration; And
And comparing the DIC signal measured for an unknown sample with a calibration curve prepared from the standard sample to infer the concentration.
16. The method of claim 15,
Wherein the nano-biochip used for the DIC measurement of the series of standard and unknown samples uses metal-nanopatterned biochips of the same size.
16. The method of claim 15,
And detecting a plasmon resonance scattering signal of the nanoparticle.
A system for signal detection of differential interference for detecting target biomolecules using the nano-biochip according to claim 1 or 2,
Which are sequentially arranged at regular intervals,
Light source; Polarizer; A first Wollaston prism; Condenser; A nano-biochip comprising a first metal-nanopatterned substrate immobilized on a metal pattern and including capture molecules that specifically bind to a target biomolecule; An objective lens; A second Wollaston prism; A selective bandpass filter; And a detector.
delete delete 19. The method of claim 18,
The detector may be a photodiode array (PDA), a charge-injection device (CID), a charge-couple device (CCD) and a digital single lens reflex And a detector selected from the group consisting of.

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