KR20170010616A - Method for Detecting Epigenetic Changes of DNA using Plasmon Coupling dependent Surface Enhanced Raman Spectroscopy - Google Patents
Method for Detecting Epigenetic Changes of DNA using Plasmon Coupling dependent Surface Enhanced Raman Spectroscopy Download PDFInfo
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Abstract
The present invention relates to a method and apparatus for detecting epigenetic mutations in DNA in a sample using surface-enhanced Raman spectroscopy (SERS) -based plasmon coupling phenomena of immunoglobulin colloids.
According to the present invention, not only high-sensitivity and accurate detection of epigenetic variation of DNA (for example, circulating tumor DNA, etc.) existing at a very low concentration in a sample, but also various biomarkers existing in a low concentration in the body .
Description
The present invention relates to a method and apparatus for detecting the epigenetic mutation of DNA in a sample using surface enhanced Raman spectroscopy (SERS) -based plasmon coupling of gold nanoparticles.
Surface-enhanced Raman spectroscopy (SERS) exhibits 104 to 109-fold greater sensitivity in a variety of fields (e.g., food safety and medical diagnostics, etc.) than conventional Raman spectroscopy (Craig AP et al. , 2013, Surface-enhanced Raman spectroscopy applied to food safety Annu. Rev. Food Sci. Technol. 4 369-80, Vo-Dinh T et al. Spectrosc. 36 640-64.). When the analyte is adsorbed on or near the plasmonic nanoparticles, the local electromagnetic field strength of the nanoparticle surface is increased and the surface plasmon of the plasmonic nanoparticles is excited to generate the SERS signal (Stiles PL et al. , 2008, Surface-enhanced Raman spectroscopy Annu. Rev. Anal. Chem. (Palo Alto Calif.) 1 601-26.). SERS is influenced by the type of substrates, the shape of the nanoparticles and the spacing between the gold nanoparticles in the bonding mode, and the excitation of the localized surface plasmon resonance (LSPR) (Sharma B et al., 2012, SERS: Materials, applications, and the future Mater. 15-16-25). The fabrication of SERS substrates is fabricated using a top-down self-assembly or top-down approach based on hot-spot formation, and substrates produced in this manner are known to have optical properties and excellent sensitivity that are adjustable by the nature of the plasmonic material (Sharma B et al., 2013, High-performance SERS substrates: Advances and challenges MRS BULLETIN 38 615-24.). Plasmonic nanoparticles on different substrates can be used to create a large number of hot spots for SERS scattering. Gold nanoparticles (AuNPs) and silver nanowires (AgNWs) as SERS substrates are attractive as a diagnostic platform due to their high roughness and excellent optical properties (polarization dependence) in a simple manufacturing process (Wei H et al , 2008, Polarization dependence of surface-enhanced Raman scattering in gold nanoparticle-nanowire systems Nano Lett. 8 2498-502.). It is known that silver nanowires operate as a plasmonic nano-antenna in their electromagnetic coupling mode, and the development of plasmons of each gold nanoparticle and silver nanowire is caused by an electric field enhancement mechanism by nanoparticle-wire bonding (Hao F et al., 2006, Plasmonic coupling between a metallic nanosphere and a thin metallic wire, Appl. Phys. Lett. 89 103101-03.). If a chemical reaction occurs in a narrow space between two plasmonic nanostructures, Raman probes or targets can dramatically amplify the SERS signal through field enhancement.
Local surface plasmonic resonance plays an important role in enhancing the electromagnetic field around each gold nanoparticle (Hutter E and Fendler J H 2003 Exploitation of localized surface plasmon resonance, Adv. Mater. 16 1685-1706). Conduction electrons created between two or more gold nanoparticles with electromagnetic field enhancement effects of different lengths are subjected to strong signals observed in SERS. This concept is a recognized concept explaining the electromagnetic enhancement of the SERS phenomenon (Sharma B et al., 2012, SERS: Materials, applications, and the future Mater.
DNA methylation can be induced by DNA methyltransferase and S-adenosyl methionine (SAM) on the cytosine of C-phosphate-G dinucleotides (CpG) (Keith D. Robertson, 2005, DNA Methylation and Human Disease, Nat. Rev. Genet. 6, 597-610). DNA methylation is a genetic variation that is not caused by a mutation that occurs within the gene base sequence. In cancer cells, DNA methylation causes abnormal gene suppression of tumor suppressor genes, such as methylation of the promoter region of a particular gene, which prevents the gene from being transcribed, resulting in gene suppression (Keith D et al., 2005, DNA Methylation and Human Disease, Nat. Rev. Genet., 6, 597-610.). In fact, DNA methylation can occur locally (eg, CpG island) or broadly (whole genome). Extensive DNA methylation is a very important epigenetic change in genomes in cancer development, X chromosome and Down syndrome, embryogenesis, transcription, X chromosome inactivation, genetic imprinting, and chromosome stability (Suzuki MM and Bird A 2008, DNA methylation landscapes: provocative insights from epigenomics Nat. Rev. Genet. 9 465-76.). In addition, it is known that many types of human diseases are associated with false DNA methylation. Several methods for measuring a wide range of DNA methylation have been reported, including high performance liquid chromatography (HPLC), HPLC-tandem mass spectrometry (LC-MS / MS) KC et al., 1980, Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA Nucleic Acids Res. 8 4763 76), two dimensional thin layer chromatography (Wilson VL et al , 1986, Genomic 5-methylcytosine determination by 32P-postlabeling analysis, Analytical Biochem. 152 275. 84.) and high performance capillary electrophoresis (Berdasco M et al., 2009, Quantification of global DNA methylation by capillary electrophoresis and mass spectrometry methods Mol. Biol. 507 23-34.) have been used as standard. However, these methods are disadvantageous in that they require a large amount of DNA samples and numerous steps that need to be optimized. In contrast, the PCR-based method or the luminometric methylation assay (LUMA) method after pyrosequencing can be carried out with only a very small amount of DNA. However, the PCR-based method described above is limited to specific genes such as Alu or LINE1, and it is known that extensive methylation can not be measured (Yang AS et al., 2004, A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements Nucleic Acids Res. 32 e38.). Therefore, there is a need for a method capable of rapidly detecting a wide range of DNA methylation with only a small amount of DNA sample.
Accordingly, the present inventors have made intensive efforts to develop a method capable of rapidly detecting the epigenetic mutation even at a very low concentration of DNA with high sensitivity and accuracy. As a result, plasmon coupling phenomena and surface enhanced Raman spectroscopy SERS), and confirmed that the DNA of the present invention can detect the epigenetic mutation of DNA only by the DNA of the unit of femtogram (10 -15 g). Thus, the present invention has been completed.
It is an object of the present invention to provide a method and apparatus for detecting the epigenetic variation of DNA in a sample using a plasmon coupling mode and Surface Enhanced Raman Spectroscopy (SERS) phenomenon .
It is another object of the present invention to provide a method for aligning immune gold nanocolloids bound to methylated portions on DNA to measure surface enhanced Raman spectroscopy based plasmonic binding phenomena.
In order to accomplish the above object, the present invention provides a method for detecting a biological sample, comprising the steps of: (a) injecting a sample containing DNA into a nanoplatemonic biosensor and then injecting immune gold colloid immobilized with an antibody for detecting a genetic mutation, step; And (b) measuring and analyzing a light scattering spectrum using a dark-field microscopy or Raman-Rayleigh scattering microscopic spectroscopy system. The surface enhanced Raman spectroscopy-based plasmonic binding The present invention provides a method for detecting a mutation in a gene.
(A) a multi-channel chamber in which a nanoplasmonic biosensor is immobilized; (b) a light source for irradiating the chamber with light; And (c) a Raman-Rayleigh spectroscope that measures Rayleigh-Raman spectroscopy of the light reflected from the chamber. The present invention provides an apparatus for detecting epigenetic variation in DNA using a surface enhanced Raman spectroscopy-based plasmonic binding phenomenon.
(A) injecting a sample containing DNA into a nanoplatemonic biosensor, and then performing an injection of immune gold colloid immobilized with an antibody for detecting a genetic mutation; And (b) measuring a plasmonic binding phenomenon with a dark-field microscopy or Raman-Rayleigh scattering microscope spectroscopy system. In the measurement of the surface-enhanced Raman spectroscopy-based plasmonic binding phenomenon, Provides an alignment method for immunoglobulin colloids.
The method using the surface enhanced Raman spectroscopy based plasmonic binding according to the present invention is useful for diagnosing diseases because it can detect the epigenetic variation of DNA present in a very low concentration in a sample with high sensitivity in a short time.
FIG. 1 is a conceptual diagram of a method for detecting the epigenetic mutation of DNA using plasmonic binding and surface enhanced Raman spectroscopy, wherein (a) is a conceptual diagram of optical equipment for measuring Raman spectroscopy and Rayleigh scattering, (b) (c) is a general model for measuring extensive methylation on the basis of SERS, and (d) is a model for binding to 5 mCpG sites (E) is a dark field micrograph of immune gold colloid bound to the DNA of HeLa cells.
(A) is a transmission electron microscope (TEM) photograph of gold nanoparticles prepared in a size of 50 nm, (b) is a photograph of a gold nanoparticle (C) is a transmission electron microscope (TEM) photograph of the prepared silver nanowire, and (d) shows the immunogold nanoparticles (1), Nanowire 2 and CTAB functionalization are the results of measuring the Zeta potential of the
FIG. 3 is a graph showing the results of measurement of the SERS signal using plasmonic binding according to the intervals of nanoparticles. FIG. 3 (a) is a schematic diagram showing a change in plasmonic binding according to the intervals of nanoparticles, (C) is the exponential decay curve of the SERS signal using plasmonic binding, and (d) the relationship between the interval between mCpG sites and the SERS signal is measured Graph.
FIG. 4 shows a result of extensive DNA methylation measurement using a SERS system using a glass substrate. FIG. 4 (a) is a schematic diagram showing the interaction between immunoglobulin colloid and DNA on the SERS system, (C) shows the result of measuring the concentration of DNA extracted from the glass substrate S1; Unmethylated DNA (S2); A genomic DNA (S3); And (d) is a linear regression curve between the DNA concentration and the SERS signal. FIG. 4 (b) is a graph showing the SERS signal in the case of the DNA + immune gold colloid (S4).
FIG. 5 is a schematic diagram showing a result of extensive DNA methylation measurement using a SERS system including a glass substrate and a silver nanowire, wherein (a) shows the interaction between the immunoglobulin colloid and DNA on the SERS system, (b) (C) is a microscopic photograph of the immune gold colloid bound to the methylated DNA absorbed in the silver nanowire, and (D) is a micrograph of the immunogold colloid absorbed in the nanowire. Unmethylated DNA (S1); And methylated DNA (S2) to measure the SERS signal. (E) is a graph showing the SERS signal change according to the concentration of the methylated DNA.
FIG. 6 is a photograph showing a device of the present invention (a) showing a result of extensive DNA methylation measurement in an actual sample using the apparatus of the present invention, wherein (b) shows a SERS signal (C) is a graph showing the relationship between the SERS method of the present invention and the 5mCpG ELISA assay, and (d) is a graph of SERS signal measurement showing the detection result of ctDNA mixed with human plasma.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.
In the present invention, a nanoplasmonic biosensor capable of simultaneously detecting the epigenetic variation of DNA in a sample was developed.
In the present invention, the detection of epigenetic mutation of DNA in a sample was carried out using a nanoplasmonic biosensor utilizing surface-enhanced Raman spectroscopy (SERS) plasmonic binding phenomenon (FIG. 1). As a result, it was confirmed that the nanoplasmonic biosensor has very high sensitivity and accuracy.
That is, in one embodiment of the present invention, a silver nanowire capable of binding to DNA in a sample and a nanoparticular biosensor containing the same and an immune immobilized with a methyl cytosine antibody capable of detecting a CpG methylation, Gold colloid was prepared (Fig. 2). Using the nanoplasmonic biosensor containing the silver nanowire and the immune gold colloid immobilized with the methyl cytosine antibody, the epigenetic variation of the DNA in the sample was detected with high sensitivity and accuracy (Fig. 3, Fig. 4 and Fig. 5).
Accordingly, in one aspect, the present invention provides a method of detecting a biological sample, comprising the steps of: (a) injecting a sample containing DNA into a nanoplatemonic biosensor, then injecting immune gold colloid immobilized with an antibody for detecting a genetic mutation, ; (b) a step of measuring and analyzing the light scattering spectrum using a dark-field microscopy or Raman-Rayleigh scattering microscopic spectroscopy system, wherein the surface enhanced Raman spectroscopy- And a method for detecting a genetic mutation.
In the present invention, the nanoplasmonic biosensor may be composed of a substrate and a metal nanowire modified with a cationic surfactant.
In the present invention, the substrate may be selected from the group consisting of glass, Si / SiO2, PET, PDMS, polyimide (PI) and Ecoflex, but the present invention is not limited thereto.
In the present invention, the metal of the metal nanowire may be selected from the group consisting of gold, silver and copper.
In the present invention, the cationic surfactant is selected from the group consisting of Cetryltrimethylammonium Bromide (CTAB), Benzalkonium chloride, Benzethonium chloride and Cerimonium chloride. .
In the present invention, the DNA epigenetic mutation may be DNA methylation, and the antibody may be a methyl cytosine antibody.
In the present invention, the light scattering spectrum is proportional to the methylation density of the DNA bound to the surface of the nanoplasmonic biosensor, and is amplified by plasmonic binding of the immunoglobulin colloid.
In the present invention, the immunoglobulin colloid may be prepared by treating a surface of gold nanoparticles with a thiol-oligoethylene glycol and functionalizing the gold nanoparticle, followed by fixing a methyl cytosine antibody and a Raman dye And the like.
In the present invention, the Raman dye may be any fluorescent material exhibiting a change in the SERS signal, but is preferably an anthraquinone, flavone, arylmethane, protoberberine, protoberberine) and
In the present invention, the shape of the gold nanoparticles of the immunoglobulin colloid may be selected from the group consisting of nanospheres, nanorods, and nanostars.
In the present invention, a spectrograph and a CCD camera may be mounted on the Raman scattering spectroscopy system in the step (b) to measure a light scattering spectrum.
In the present invention, the gold nanoparticles may have an average particle size of 11 to 70 nm, preferably about 50 nm, and the silver nanowire may have an average particle size of 1 to 5 μm, preferably about 1.5 μm And the average particle size of the immunoglobulin colloid is 80 nm.
In another embodiment of the present invention, a DNA methylation detection apparatus including a multi-channel chamber in which a nanoplasmonic biosensor is immobilized was manufactured, and it was confirmed that DNA methylation can be detected with very high sensitivity when the apparatus is used .
Accordingly, in another aspect, the present invention provides a multi-channel apparatus comprising: (a) a multi-channel chamber to which a nanoplasmonic biosensor is immobilized; (b) a light source for irradiating the chamber with light; And (c) a Raman-Rayleigh spectrometer for measuring Rayleigh-Raman spectroscopy of the light reflected from the chamber. BACKGROUND OF THE INVENTION
In the present invention, the epigenetic variation of the DNA may be DNA methylation.
In the present invention, it was confirmed that the surface enhanced Raman spectroscopy-based plasmonic coupling phenomenon signal increased in inverse proportion to the distance of nanoparticles, and it was confirmed that the nanoparticle spacing can be controlled by DNA methylation (FIG. 3).
Accordingly, in another aspect, the present invention provides a method for detecting an immunoglobulin, comprising the steps of: (a) injecting a sample containing DNA into a nanoplatemonic biosensor, and then performing immunization with an immunoglobulin colloid immobilized with an antibody for detecting a genetic mutation; And (b) measuring a plasmonic binding phenomenon with a dark-field microscopy or Raman-Rayleigh scattering microscope spectroscopy system. In the measurement of the surface-enhanced Raman spectroscopy-based plasmonic binding phenomenon, Lt; RTI ID = 0.0 > colloid. ≪ / RTI >
In the present invention, the substrate may be selected from the group consisting of glass, Si / SiO2, PET, PDMS, polyimide (PI) and Ecoflex, but the present invention is not limited thereto.
In the present invention, it may be characterized in that all or a portion of the CpG site of the DNA is methylated.
In the present invention, the immunoglobulin colloid may include a methyl cytosine antibody.
In the present invention, the immunoglobulin colloid binds to the methylated (mCpG) portion of the DNA and is aligned with the DNA.
The method according to the present invention can detect the epigenetic variation of DNA in the sample quickly and easily with a very high sensitivity and can be applied not only to detection of DNA mutation but also to detection of other biomarkers existing at low concentrations in vivo .
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.
Example 1: Fabrication and functionalization of gold nanoparticles, immunoglobulin colloids and silver nanowires
Example 1-1: Fabrication of gold nanoparticles
The gold nanoparticles were prepared by a known method (Kimling J et al., 2006, Turkevich Method for Gold Nanoparticle Synthesis Revisited J. Phys. Specifically, 10 ml of 1 M hydrogen chloride solution (HAuCl 4 ) was boiled, and 1 ml of 0.4% sodium citrate was added and the mixture was further boiled for 5 minutes. The mixture was cooled at room temperature for 15 minutes. After removing the aggregates using a 0.2-μm filter, the size, shape and surface functionalization were measured using a scanning transcription microscope (HRTEM, JEOL JEM-3011, 300 kV) and a UV-vis spectrometer (Shimadzu UV3600 UV-VIS-NIR) (Fig. 2).
Example 1-2: Preparation and functionalization of immunoglobulin colloid
Thiol-oligo ethyleneglycol (OEGs) was treated in the following manner to form a self-assembled monolayer (SAM) of gold nanoparticles. First, HS (CH 2) in the gold colloid solution of 8ml 11 (OCH 2 CH 2) 6
Example 1-3: Fabrication and functionalization of silver nanowires
Silver nanoparticles were prepared by a known method (Coskun S et al., 2011, Polyol synthesis of silver nanowires: an extensive parametric study Cryst. Growth Des., 11 4963-69). Specifically, a solution of 3.5 mg of sodium chloride and 0.45 M of poly (vinylpyrrolidone), Sigma, USA) -ethylene glycol (Sigma, USA) was heated to 170 ° C. Using a pump, 5 ml of 0.6 M silver nitrate (AgNO 3 ) -EG solution was added dropwise to the heated solution at a rate of 3 ml / h. The remaining PVP was rinsed with acetone at a ratio of 1:10 and centrifuged at 6000 rpm for 20 minutes to prepare silver nanowires (AgNWs). The silver nanowires were mixed with ethanol and mixed at 4500 rpm For 3 minutes for 30 minutes to remove silver nanoparticles, and the shape was confirmed by a scanning electron microscope (FIG. 2).
Silver nanowires were added to a 10 mM solution of Cetryltrimethylammonium Bromide (CTAB) to modify the surface of the silver nanoparticles into cations. The silver nanowires were mixed for 12 hours and then centrifuged at 3000 rpm for 20 minutes to remove the CTAB. And washed three times to complete functionalized silver nanowires.
Example 2 Measurement of SERS Signal Using Plasmonic Binding of Immunogold Colloid Bonded to Methylated DNA
Gold nanoparticle pairs have recently been known as "plasmonic rulers" in electromagnetic fields generated by the spacing between internal particles (Jain PK et al., 2005, On the universal scaling behavior of the distance decay of plasmonic coupling in metal nanoparticle pairs: a plasmonic ruler equation Nano Lett. 5 2246- 52.). Based on this fact, we tried to confirm whether the SERS signal using the plasmonic binding phenomenon can be measured using the methylated DNA having the mCpG site correctly defined. LSPR and SERS spectra were measured 30 minutes after injecting the immunoglobulin colloid synthesized in Example 1-2 into DNA (bioneer) having mCpG sites at intervals of 68 nm, 34 nm, 17 n, and 3.4 nm (FIG. 3) .
As shown in FIG. 3, it can be seen that the LSPR and SERS signals increase in inverse proportion to the distance between the mCpG sites.
Example 3: Construction of SERS system
Example 3-1: Production of DNA sample
In HeLa cells (ATCC CCL-2 TM) and HEK-293 cells (ATCC ® RL-1573) in order to prepare a wide variety of DNA sample Blood & Cell Culture DNA Mini Ki Cat . Genomic DNA was extracted using No13323 (Qiagen, USA). The concentration of the extracted DNA was determined using the Quant-iT TM PicoGreen ® dsDNA assay kit (Invitrogen, USA), genomic DNA is extracted and stored is the division in TE buffer with 100ul (100ng).
Example 3-2: Preparation of DNA bound to a glass substrate
DNA was bound to the glass substrate using a known method (Nanashi O Z et al., 2007, Capture of genomic DNA on glass microscope slides, Anal. Biochem., 365 2405.). Specifically, 100ul of DNA was mixed with binding buffer (5M guanidine thiocyanate, 20mM EDTA and 1% Triton X-100 in 0.1M Tris-chloride pH 6.4), and then 20ul of the solution was injected into a glass substrate and cultured at room temperature for 30 minutes After washing twice with 3 ml of wash buffer (10 mM Tris-chloride pH 7.4, 2.5 mM EDTA, 50 mM NaCl and 50% ethanol), it was used for further experiments.
Example 3-3: Production of Circulating Tumor DNA Mimetic Sample
The genomic DNA prepared in Example 3-1 was digested with restriction enzymes HinfI (5'-GANTC-3 '), AluI (5'-AGCT-3') and BssECI (5'-CCNNGG- Biolabs, USA) to prepare a ctDNA pool. The pools were then mixed with binding buffer and mixed with human plasma (Sigma Aldrich, H4522) at a ratio of 2: 1 for further experiments.
Example 3-4: Construction of Raman-Rayleigh scattering microscopic spectroscopy system for SERS measurement and optical measurement
The SERS signal measurement was measured using a Raman-Rayleigh scattering microscope spectroscopy system. Specifically, a laser (785 nm HeNE) was projected onto the sample through an X100 air objective (TU Plan ELWD 50x, Nikon, 0.6 NA, 0.56 mm WD) of a sieving microscope (Eclipse Ni-U, Nikon). The direction of polarization was controlled using a linear polarizer (PRM1 / M, Thorlabs). The intensity of the laser was set to 8.5 mW using a digital power meter (PM100, Thorlabs). Before measuring the Raman signal, the multi-chamber was washed with PBS containing 0.3 M NaCl. The data were analyzed with a spectrograph Monora 500i and a CCD camera, and Rayleigh scattering with a Monora 322i (Andor Technology) and a CCD camera.
Example 3-4: Derivation of the detection limit of the SERS system
The detection limit of methylation DNA in the SERS system was calculated by a known method (Ma W et al., 2013, Attomolar DNA detection with nanorod assemblies Nat. Commun 4: 2689). The relationship between the SERS signal intensity (y-axis) and the concentration of methylated DNA (x-axis) was calculated in the form of a linear graph y = ax + b where a and b are constants and y values are calculated as Y = y blank + 3SD Respectively. SD is the standard deviation calculated by the following equation, and y blank is the SERS signal value of the unmethylated DNA.
SD =
Where N is the sum of the samples, X i is the SERS value measured at the i-th order DNA concentration, and X average is the average value of the SERS values.
Finally, the limit of detection (LOD) of extensive methylation was calculated by the following equation.
LOD =
Example 4: Detection of DNA methylation using SERS system using only glass substrate
The SERS system using the plasmonic coupling phenomenon was constructed by using the multi-channel chamber including the glass substrate according to the method of Example 3.
After the sample DNA was bound to the glass substrate by the method of Example 3-2, non-specific binding of the antibody was prevented by using 0.5% BSA for 30 minutes. Then, the immune gold colloid (3.95 × 10 10 nanoparticles), incubated for 30 minutes, and washed with buffer containing 0.1
As a result of measuring the SERS signal according to the method of Example 3-4, it was confirmed that the SERS signal was amplified only when the methylated DNA and the immunoglobulin colloid were simultaneously present as shown in FIG. 4. As a result of the detection, the detection limit was 195 fg / ml.
Example 5 Detection of DNA methylation using the SERS system using a nanoplasmonic biosensor
The SERS system using the plasmonic binding phenomenon by the method of Example 3 was constructed using a multi-channel chamber including a nanoplasmonic biosensor (FIG. 6).
The nanoplasmonic biosensor was prepared by binding the silver nanowires functionalized with the CTAB to the glass substrate of Example 3 to DNA, and then the immunogold colloid was injected by the method of Example 4. Then, SERS Signal was measured.
As a result, as shown in Fig. 5, it was confirmed that the immunoglobulin colloid was bound to a nanosplasmaic biosensor in which the positive charge of the DNA and the positive charge of the silver nanowire were bonded to each other in a "beads on a string" structure, , Which was 100 times superior to the conventional method (Lisanti S et al., 2013, Comparison of methods for quantification of global DNA methylation in human cells and tissues, PloS one 8.).
Example 6: Detection of methylation of an actual DNA sample
Methylation of the DNA extracted from the actual cells and the circulating tumor DNA (ctDNA) was performed using the DNA methylation detection apparatus using the surface enhanced Raman spectroscopy (SERS) -based plasmonic binding phenomenon constructed in Example 5 (Fig. 6) .
As shown in FIG. 6, it was confirmed that the level of methylation of HeLa cells was 2.5 times higher than that of HEK293 cells. It was confirmed that the SERS signal was changed according to each case when treating methylase or dimethylase, (R 2 = 0.97, p <0.0001), indicating that the SERS signal measured using the 5-mc DNA ELISA kit (Zymo resarch, Ca, USA)
Also, it was confirmed that 10 pg of circulating tumor DNA methylation mixed with human plasma prepared in Example 3-3 can be detected with high accuracy (Fig. 6).
While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
Claims (21)
(a) injecting a sample containing DNA into a nanoplasmonic biosensor, and then injecting immune gold colloid immobilized with an antibody for detecting a genetic mutation to amplify a detection signal;
(b) measuring and analyzing the light scattering spectrum using a dark-field microscopy or a Raman-Rayleigh scattering microscope spectroscopy system.
(a) a multi-channel chamber to which a nanoplasmonic biosensor is immobilized;
(b) a light source for irradiating the chamber with light; And
(c) Raman-Rayleigh spectroscope measuring Rayleigh-Raman spectra of light reflected from the chamber.
(a) performing the step (a) of claim 1; And
(b) Determining the immunochromic colloids aligned on the DNA by measuring the plasmonic binding phenomenon with a dark-field microscopy or Raman-Rayleigh scattering microscopic spectroscopy system.
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CN108827935A (en) * | 2018-06-08 | 2018-11-16 | 南京师范大学 | It is a kind of based on the DNA methylation Surface Enhanced Raman Scattering Spectrum detection method of gold nano hole array and its application |
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