KR20160009357A - Method for simultaneously detecting tumor-specific mutation and epigenetic changes of circulating tumor DNA(ctDNA) using Rayleigh light scattering - Google Patents

Abstract

Provided is a method for simultaneously detecting tumor-specific mutation and epigenetic changes of a circulating tumor DNA (ctDNA) using localized surface plasmon resonance (LSPR) and plasmon coupling of metal nanoparticles. According to the present invention, the method not only simultaneously detects tumor-specific mutation and epigenetic changes of a circulating tumor DNA existing by low concentration in the blood, but also is applied to simultaneously detection of various biomarkers existing by low concentration in the body.

Description

A method for simultaneous detection of tumor-specific mutations and epigenetic mutations of circulating tumor DNA (ctDNA) using Rayleigh scattering using Rayleigh light scattering (ctDNA) using simultaneous detection of tumor-specific mutations and epigenetic changes (ctDNA)

The present invention relates to a method for simultaneous detection of tumor-specific mutations and epigenetic mutations of circulating tumor DNA (ctDNA) using localized surface plasmon resonance (LSPR) and plasmon coupling phenomena of gold nanoparticles .

Circulating tumor DNA (ctDNA) is a double-stranded DNA that is isolated from cancer cells and is present in the blood. It has both tumor-specific mutations and epigenetic mutations, and is a very small amount of whole circulating DNA in the blood (Sunami et al., Methods in Molecular Biology, 507: 349-356, 2009; Chimonidou et al., Clinical Chemistry, 57 (8): 1169-1177, 2011). Two hotspot mutations in the ctDNA of the phosphatidylinositol 3-kinase catalytic subunit (PIK3CA) gene, phosphatidylinositol 3-kinase catalase subunit, at position 70271 of the exon 9 coding for mutation of the -542th glutamic acid to lysine (E542K) (A) of guanine (G) at position 70282 of exon 9 encoding a mutation of adenine (A) to adenine (A) of 545 th glutamic acid and lysine (Banas et al., 2002), and is a well-known mutation in several types of cancer, including cancer, brain cancer, liver cancer, stomach cancer and lung cancer (Andreas et al., PNAS, -409, 2012; Dawson et al., New England Journal of Medicine, 368: 1199-1129, 2013).

The development of a platform that can simultaneously detect genetic and epigenetic mutations can increase the sensitivity and specificity of diagnostic methods based on CTC DNA. The size of the circulating tumor DNA varies from 50 bp to 250 bp and since the circulating tumor DNA is floating in the blood, the ability to inhibit the binding to nonspecific circulating DNA, and to capture and concentrate only specific circulating tumor DNA, It is essential.

Neutral peptide nucleotide (PNA) is characterized by specific fusion with double-stranded DNA. Due to this characteristic, PNA has excellent performance as a probe which can be used in biosensor detecting strand-specific double-stranded DNA (Bhaskar et al., Journal of the Amercian Chemica Society, 123: 9612-9619, 2001; Zohar et al., Nanoletter, 10: 4697-4701, 2010). PNA has a strong affinity for DNA and RNA by minimizing electrical repulsion since the basic backbone is electrically neutral in binding to complementary nucleic acids (Zohar et al., Nanoletter, 10: 4697-4701, 2010; Bentin et al. , Biochemistry, 42: 13987-13995, 2003). In particular, PNA / DNA duplexes have poor thermal stability compared to DNA / DNA mismatches in mismatch. In the single base mismatch, the Tm values of PNA / DNA and DNA / DNA double complexes decrease by 15 ° C and 4 ° C respectively (Igloi et al., PNAS, 95: 8562-8567, 1998; Ray et al., The Journal Park et al., Bio-interphases, 1 (4): 113-122, 2006). Thus, PNA molecules are ideal probes for eliminating false positives in temperature-controlled environments.

Nanoplasmonic detection platforms based on gold nanoparticles have excellent sensitivity, accuracy and specificity. Many research groups are studying biosensing platforms that detect DNA targets using localized surface plasmon resonance (LSPR) phenomena of various nanoparticles ranging from silver to gold (Truong et al., Lab on a chip, 11: 2591-2597, 2011; Willets et al., The Annual Review of Physical Chemistry, 58: 267-297, 2007). These biosensors vary in intensity and frequency of surface plasmon absorption bands of specific metal nanoparticles depending on the type of material, and depending on the substance in which the nanoparticles are placed and the size, shape and size distribution of the nanoparticles, the surface plasmon frequency This characteristic changes the binding of the biomaterial into a measurable optical signal. Therefore, it is important to maintain a constant local refractive index around the nanoparticles in fabricating such an LSPR biosensor.

For LSPR biosensors, the degree to which the maximum wavelength changes is proportional to the sensitivity of the sensor. Recently, a method of amplifying the maximum wavelength change of LSPR using a plasmon coupling mode of gold nanoparticles and immunogold colloids has been known (Truong et al., Small, 9 (20): 3485-3492 , 2013; Hall et al., The Journal of Physical Chemistry C, 115 (5): 1410-1414, 2011). It is also known that the maximum wavelength of the LSPR due to the plasmon-binding mode changes to an observable red line (Lange et al., Langmuir, 28: 8862-8868, 2012).

Currently, there are only two methods to quantitatively analyze tumor-specific mutations: digital PCR and targeted deep seqeuncing (Banerji et al., Natere, 486: 405-409, 2012; Board et al., Clinical Chemistry, : 754-760, 2008). The disadvantage of this method is the need for labeling, the complexity of the detection method, and the inability to simultaneously detect genetic variation and subsequent genetic variation. Therefore, there is a need for a detection method capable of simultaneously detecting tumor-specific mutations and epigenetic mutations of the label-free circulating tumor DNA molecules that require minimal sample processing.

The present inventors have made intensive efforts to develop a nanoplasmonic biosensor capable of simultaneously detecting tumor-specific mutations and epigenetic mutations of circulating tumor DNA with high sensitivity and accuracy without labeling. As a result, And a plasmon coupling mode, and the sensor can simultaneously detect tumor-specific mutations and epigenetic mutations of circulating tumor DNA in blood with very high sensitivity and accuracy. And completed the present invention.

It is an object of the present invention to provide a method and apparatus for detecting tumor-specific genetic variation of circulating tumor DNA (ctDNA) in blood using a localized surface plasmon resonance phenomenon and a plasmon coupling mode, A method for simultaneously detecting genetic mutations is provided.

Another object of the present invention is to provide a PNA capable of detecting tumor-specific mutations of the phosphatidylinositol 3-kinase catalytic subunit (PIK3CA), and a nanoparasite And to provide a nickel biosensor.

In order to achieve the above object, the present invention provides a method for detecting tumor-specific DNA mutation, comprising the steps of: (a) injecting a blood sample into a nanoflashmonic biosensor immobilized with gold nanoparticle immobilized thereon, Amplifying a detection signal by injecting immobilized gold colloid immobilized with an antibody for detecting a genetic mutation in the host; (b) measuring the light scattering spectrum using a dark-field microscope and a resonance Rayleigh scattering microspectroscopy system and analyzing the maximum wavelength mobility; ciruller tumor DNA, ctDNA) in tumor-specific mutations and epigenetic mutations.

The present invention also relates to a mutant (E542K) substituted with a lysine of a phosphatidylinositol 3-kinase catalytic subunit (PIK3CA) at position 542 of glutamic acid and / or a mutant substituted with lysine of a 545th glutamic acid (E545K). ≪ / RTI >

The present invention also relates to a mutant (E542K) substituted with a lysine of a phosphatidylinositol 3-kinase catalytic subunit (PIK3CA) at position 542 of glutamic acid and / or a mutant substituted with lysine of a 545th glutamic acid (E545K), and a nanoflashmonic biosensor including the gold nanoparticle.

The nanoplasmonic biosensor according to the present invention is capable of simultaneously detecting mutation and epigenetic mutation of circulating tumor DNA present in blood at a very low concentration at a high sensitivity in a short time, and is useful for diagnosis of disease.

FIG. 1 is a conceptual diagram of a tumor-specific mutation of a circulating tumor DNA (ctDNA) using Rayleigh scattering and a method for detecting a metastatic genetic mutation.
FIG. 2 (A) is a transmission electron microscope (TEM) image of gold nanoparticles formed to have a size of 50 nm, and FIG. 2 (B) is a dark background image of gold nanoparticles at a magnification of 1,500.
FIG. 3 (A) is a transmission electron microscope (TEM) image of gold nanoparticles of 20 nm in size produced to produce immunoglobulin colloids, and FIG. 3 Fig.
FIG. 4A is a graph showing the change in LSPR maximum wavelength due to fusion of the circulating tumor DNA molecule and the PNA probe according to the temperature, FIG. 4B is a graph showing the binding of ctDNA to gold nanoparticles and the methyl cytosine FIG. 5 is a graph showing the maximum wavelength change of the Rayleigh light scattering spectrum when immobilized immunoglobulin colloids bound with cytosine antibody are bound. FIG.
The black column in FIG. 5 (A) is the maximum wavelength change of the Rayleigh light scattering spectrum according to the ctDNA concentration, and the red column is the result of measuring the amplified LSPR change using the immunoglobulin colloid binding to the mCpG site of ctDNA, (B) is a graph showing the linear correlation of the change of LSPR according to the concentration and methylation state of ctDNA, (C) is a dark field microscope photograph of gold nanoparticles bonded with ctDNA before injecting immunoglobulin colloid, (E) is a graph showing changes in LSPR according to the concentration of normal circulating DNA (ncDNA). FIG.
6 is a graph of the maximum wavelength change of the Rayleigh light scattering spectrum showing the detection results of E542K and E545K 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.

The present invention aims to develop a nanoplasmonic biosensor capable of simultaneously detecting mutations and epigenetic mutations of circulating tumor DNA in a blood sample.

 In the present invention, mutation and epigenetic mutation of circulating tumor DNA in blood were detected using a nanoflashmonic biosensor utilizing local surface plasmon resonance and plasmon-binding mode (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, the mutation of the 542-th glutamic acid, which is a tumor-specific mutation in the blood, circulating tumor DNA of the phosphatidylinositol 3-kinase catalytic subunit (PIK3CA) (G) of 70282th guanine (G) of exon 9 encoding a mutation of 70271th guanine (G) to adenine (A) of exon 9 coding for mutation of lysine to lysine (E545K) (Fig. 2), and a methyl cytosine antibody capable of detecting CpG methylation, which is a genetic mutation, is prepared by using a nanoparticulate biosensor containing the PNA-immobilized gold nanoparticle A fixed immune gold colloid was prepared (FIG. 3), and the prepared PNA probe was immobilized on a nanoplasmonic biosensor containing immobilized gold nanoparticles and immobilized gold colloid immobilized with a methyl cytosine antibody Using grade was confirmed to be capable of detecting a tumor specific mutation and epigenetic mutations of the circulating tumor DNA at the same time with high sensitivity and accuracy.

Accordingly, in one aspect, the present invention provides a method for detecting a tumor-specific DNA mutation, comprising: (a) injecting a blood sample into a nanoplasmonic biosensor immobilized with gold nanoparticle immobilized with a nucleic acid probe for detecting tumor- Amplifying a detection signal by injecting immunoglobulin colloid immobilized with an antibody for detecting a mutation in a mutation; (b) measuring the light scattering spectrum using a dark-field microscope and a resonance Rayleigh scattering microspectroscopy system and analyzing the maximum wavelength mobility; cirulating tumor DNA, ctDNA), and a method for simultaneous detection of tumor-specific mutations and epigenetic mutations.

In the present invention, the nucleic acid probe may be selected from the group consisting of DNA, aptamer, RNA, PNA and LNA.

In the present invention, the tumor-specific DNA epigenetic mutation may be DNA methylation, and the antibody may be a methyl cytosine antibody.

In the present invention, the mobility of the maximum wavelength is represented by the density of circulating tumor DNA bound to the surface of the nanoflashmonic biosensor, and is amplified by the plasmon binding phenomenon of the immunoglobulin colloid .

In the present invention, the nucleic acid probe is substituted with lysine (E542K) substituted with lysine of the phosphatidylinositol 3-kinase catalytic subunit (PIK3CA) at position 542 and / or lysine at position 545 of glutamic acid Or a PNA capable of detecting the mutant (E545K), can be used without limitation, and can be preferably a PNA represented by SEQ ID NO: 1 or 2.

In the present invention, the reaction temperature may be kept constant in the step (a). In the step (b), a spectrograph and a CCD camera may be mounted on the resonance Rayleigh scattering micro spectroscopy system And the maximum wavelength is measured.

In the present invention, the average particle size of the gold nanoparticles is preferably 11 to 70 nm, more preferably about 50 nm, and the average particle size of the gold colloid is preferably 10 to 90 nm, more preferably 10 to 30 nm Is more preferable.

In another embodiment of the present invention, a mutant (E542K) substituted with lysine of 542th glutamic acid, which is a tumor-specific mutation in the blood of circulating tumor DNA in the blood of phosphatidylinositol 3-kinase catalytic subunit (PIK3CA) (G) of 70282th guanine (G) of exon 9 coding for a mutation of 70271th guanine (G) to adenine (A) of exon 9 coding for mutation of lysine (E545K) substituted with lysine of 545th glutamic acid ) Was prepared, and the PNA-immobilized gold nanoparticles were prepared (FIG. 2), and a nanoflashmonic biosensor using the same was prepared (FIG. 1). As a result of the detection of circulating tumor DNA using the nanoflashmonic biosensor, it was confirmed that ctDNA having a concentration of 200 fM (2.0 * 10 ^-13 M) could be detected (FIG. 4).

Accordingly, the present invention provides, in a different aspect, a mutant (E542K) substituted with a lysine of a phosphatidylinositol 3-kinase catalytic subunit (PIK3CA) at position 542 of glutamic acid and / or a lysine And a gold nanoparticle immobilized with the PNA and a nanoflashmonic biosensor comprising the same.

In the present invention, the PNA may be characterized by being represented by SEQ ID NO: 1 or 2.

According to the present invention, it is possible to detect circulating tumor DNA in the blood quickly and easily with a very high sensitivity, so that it can be applied not only to the detection of circulating tumor DNA, but also to the detection of other biomarkers existing at a low concentration 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: Design of PNA probes and capture of ctDNA using temperature control

Two hotspot mutations of the PIK3CA gene ctDNA are already well known (Andreas et al., PNAS, 103 (5): 1475-1479, 2006; Dawson et al., New England Journal of Medicine, 368: 1199-1209, 2013). The ctDNA containing the two mutants was synthesized to a length of 69 bp, and the PNA probes of SEQ ID NO: 1 and SEQ ID NO: 2 were prepared so as to match 100% of each ctDNA.

SEQ ID NO: 1: 5'-HS-AGTGATTTTAGAGAG-3 '(E542K probe)

SEQ ID NO: 2: 5'-HS-CCTGCTTAGTGATTT-3 '(E545K probe)

PNA / DNA duplexes have poor thermal stability compared to DNA / DNA mismatches in mismatch. In single base mismatch, the Tm values of PNA / DNA and DNA / DNA double complexes decrease by 15 ° C and 4 ° C respectively (Igloi et al., PNAS, 95: 8562-8567, 1998). The binding between the PNA probe and the ctDNA molecule in the thermostable reaction chamber was measured for LSPR changes. The LSPR changes were measured while changing the temperature from 40 ^° C to 76 ^° C.

As shown in FIG. 1, when 200 bM of 69 bp ctDNA was injected into an image chamber (RC-30HV, Warner instruments) immobilized with gold nanoparticles immobilized with a PNA probe, LSPR changes were observed by binding of PNA probe and ctDNA molecules do. This is because the LSPR-based platform is well suited to the light scattering of gold nanoparticles and is highly sensitive to changes in the reflection index of the surrounding environment. The large difference in melting temperature between PNA probes and normal circulating DNA or circulating tumor DNA complexes is an important factor in specifically fusing ctDNA only to the surface of gold nanoparticles. After normal circulating DNA is washed, the concentration of the fused ctDNA can be calculated by measuring the change in the LSPR wavelength of each nanoflashmonic biosensor.

Example 2: Fabrication and functionalization of gold nanoparticles

To a reaction solution containing 10 ml of 0.1 M cetyltrimethylammonium bromide (CTAB) and 0.5 ml of 0.01 M hydrogen chloride solution (HAuCl ₄ ), 1.0 ml of cold 0.01 M sodium borohydride sodium borohydride, NaBH4) was added thereto, followed by rapid stirring. Thereafter, the mixture was further stirred for about 10 minutes and then cooled at room temperature. Gold nanoparticles having an average size of about 50 nm of brown were prepared by the same method as described above, and confirmed by transmission electron microscopy and dark field microscopy (FIG. 2).

The prepared gold nanoparticles were modified with thiol-modified PNA probes using well-known gold-sulfur chemistry and UV / Vis spectroscopy (UV-3600, Shimadzu) and transmission electron microscope (JEM-2100F, 200 kV, JEOL) ) (Fig. 2). Gold nanoparticles immobilized on each PNA probe were immobilized on a cover glass of a closed reaction chamber.

Example 3: Fabrication of nanoplasmonic biosensor

It is well known that LSPR systems consist of darkfield microscopes, spectrographs and CCD cameras. In the present invention, Rayleigh light scattering generated from each gold nanoparticle was observed using a Nikon Eclipse TE2000-U under a dark-field microscope. The spectrophotometer was photographed with Rayleigh light scattering spectrum using Microspec 2300i manufactured by Roper Scientifics, and PIXIS: 400B manufactured by Princeton Instruments was used as a CCD camera (FIG. 1).

Gold nanoparticles functionalized with PNA probes were immobilized on thiol-modified cover glasses treated with mercaptopropyltriethoxysilane (MPTES). The cover glass was placed in an RC-30HV reaction chamber, and the reaction chamber was connected to a TC-324C temperature controller manufactured by Warner Instruments. The reaction chamber was fixed to a platform of a dark-field microscope, combined with a syringe pump (Havard Apparatus), and finally washed with deionized water to prepare a ctDNA target.

The LSPR shift is caused by a change in the refractive index (RI), and the unit is nm / RIU (RIU = refractive index unit) It happens by change. Thus, the LSPR changes reflect the sensitivity of the nanoflashmonic biosensor. According to the reaction step was calculated by measuring the Rayleigh scattering spectra of each of the gold nanoparticles, calculation is LSPR _shift ?? = LSPR _{afterbinding beforebinding} LSPR. We used Origin Pro 8.6 software with the Lorentz algorithm to remove noise and match the spectrum.

Example 4: Detection of circulating tumor DNA (ctDNA)

The overall step of detecting the circulating tumor DNA is shown in Fig. First, synthesized ctDNA from 50 fM to 3200 fM was prepared in a solution of 1 × hybridization buffer (PerfectHyb ^™ buffer, Sigma) and 1 × human plasma (Sigma-Aldrich). This was injected into a reaction chamber containing gold nanoparticles functionalized with PNA and the change in LSPR was measured while changing the temperature for 30 minutes. As a result, it was confirmed that the optimum reaction temperature was 62 ^° C (FIG. 4). In order to obtain the limit of detection (LOD), a control experiment was carried out using 200 ul of 69 bp PIK3CA gene ncDNA and human genome manipulation (G1521, Promega, USA), and LSPR changes (Fig. 4).

Example 5: Preparation of immunoglobulin colloids immobilized with methyl cytosine antibody

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 OCH 2 COOH (HS-OEG6-COOH) / HS (CH 2) 11 (OCH 2 CH 2) 3 OH (HS-OEG3- OH) solution (1: 9 molar ratio, 0.5 mM) was added thereto, followed by culturing for 10 hours. Thereafter, the unbound OEG was removed by centrifugation at 10,000 rpm for 20 minutes. Thereafter, a solution of 1M EDC / NHS (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride / Nhydroxylsuccinimide) was added to the gold colloid solution to fix the gold nanoparticles to the methyl cytosine antibody, The reaction was stopped by the addition of 1.4 ul of 20 mM 2-mercaptoethanol. Then, 500 μl of a methyl cytosine antibody (1 μg / ml) was mixed and cultured for 3 hours. The resulting immunoglobulin colloid obtained by centrifugation at 10,000 rpm for 10 minutes was suspended in 50 mM phosphate buffered saline (PBS, pH 7.4) , And confirmed by a transmission electron microscope (Fig. 3). As a result of measuring the LSPR changes at each reaction step, when the surface was modified by treatment with thiols-oligo ethylene glycol (OEGs) at a maximum wavelength of 521 nm, when methylcytosine antibody was fixed with EDC / NHS at 530 nm, 534 nm (Fig. 3).

Example 6: Simultaneous detection of mutation and methylation using nanoplasmonic biosensor and immunoglobulin colloid

There are two methylated mCpG sites in each strand of ctDNA. After the first detection of ctDNA, the methylation of ctDNA was detected and the immune gold colloid immobilized with methyl cytosine antibody was injected into the nanoplasmonic biosensor to amplify the detection signal. It has recently been known that immunoglobulin colloids amplify the resonance signals of very low concentrations of analytical samples (Truong et al., Small, 9 (20): 3485-3492, 2013; Hall et al., The Journal of Physical Chemistry C , ≪ / RTI > 115 (5): 1410-1414, 2011).

As shown in FIG. 1, an immune gold colloid solution by injection of the biosensor it was incubated for 1 hour, rinsed the surface by using the back of deionized water. The amplified signal was observed by measuring the change in LSPR. As a result, the immunoglobulin colloid solution immobilized with the methyl cytosine antibody binds to the mCpG site of the ctDNA and exhibits a plasmon binding mode to amplify the LSPR change signal (Fig. 4), and its width increases from 4.3 nm to 11.4 nm (Fig. 5A). After injection of the immunogold colloid, detection of ctDNA methylation on the surface of the nanoplasmonic biosensor was directly observable through a dark-field microscope (Fig. 5C, Fig. 5D). When normal circulating DNA was used, no amplification of the LSPR change signal with concentration was observed (Figure 5E)

In addition, since circulating tumor DNA in the blood is present in an amount of 5-702 copies per milliliter of plasma, that is, about 0.01% to 25% of normal circulating DNA (Dawson et al., New England Journal of Medicine, 368: 1199-1209 , 2013), very high sensitivity is required. A sample was prepared by mixing Nanoplasmonic biosensor containing PNA-immobilized gold nanoparticles capable of detecting E542K or E545K of PIK3CA with ctDNA with E542 and E545K mutations at 50 fM in human plasma (H4522, Sigma Aldrich) As a result of amplification of the LSPR changes using immune gold colloids immobilized with a methyl cytosine antibody at 62 ° C, LSPR changes of 11.5 nm for E542K ctDNA and 15.7 nm for E545K ctDNA were observed, and blood circulating tumor DNA (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.

<110> Korea University <120> Method for simultaneous detection of tumor-specific mutation and          epigenetic changes of circulating tumor DNA (ctDNA) using Rayleigh          light scattering <130> P14-B208 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> E542K probe_PNA <400> 1 hsagtgattt tagagag 17 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> E545K probe_PNA <400> 2 hscctgctta gtgattt 17

Claims (15)

Simultaneous detection of tumor-specific mutations and epigenetic mutations in circulating tumor DNA (ctDNA) using Rayleigh scattering including the following steps:
(a) A blood sample is injected into a nanoplasmonic biosensor immobilized with a gold nanoparticle immobilized with a nucleic acid probe for detecting tumor-specific DNA mutation, and then an antibody for detecting tumor-specific DNA mutation is immobilized Injecting an immunoglobulin colloid to amplify the detection signal;
(b) Dark-field microscopy and resonance Rayleigh scattering A step of measuring the light scattering spectrum and analyzing the maximum wavelength mobility using a micro-spectroscopy system.
2. The method of claim 1, wherein the nucleic acid probe is selected from the group consisting of DNA, aptamer, RNA, PNA, and LNA.
2. The method of claim 1, wherein the tumor-specific DNA epigenetic variation is DNA methylation.
2. The method of claim 1, wherein the antibody is a methyl cytosine antibody.
The method according to claim 1, wherein the mobility of the maximum wavelength is represented by the density of circulating tumor DNA bound to the surface of the nanoflashmonic biosensor and is amplified by the plasmon binding phenomenon of the immunoglobulin colloid How to.
3. The nucleic acid probe according to claim 1 or 2, wherein the nucleic acid probe is a mutant (E542K) substituted with lysine at position 542 of glutamate of phosphatidylinositol 3-kinase catalytic subunit (PIK3CA) and / Wherein the mutant (E545K) substituted with lysine of glutamic acid can be detected.
3. The method according to claim 1 or 2, wherein the nucleic acid probe is a PNA represented by SEQ ID NO: 1 or 2.
The method according to claim 1, wherein the reaction temperature is kept constant in the step (a). The method according to claim 1, wherein the spectroscope and the CCD camera are mounted on the resonant Rayleigh scattering micro spectroscopy system in step (b), and the maximum wavelength is measured.
The method of claim 1, wherein the average particle size of the gold nanoparticles is from 11 to 70 nm, and the average particle size of the gold colloid is from 10 to 90 nm.
The method of claim 1, wherein the average particle size of the gold nanoparticles is about 50 nm and the average particle size of the gold nanoparticles is about 10 to 30 nm.
(E542K) substituted with lysine at position 542 of phosphatidylinositol 3-kinase catalytic subunit (PIK3CA) and / or mutant (E545K) substituted with lysine at position 545 of glutamic acid can be detected in the phosphatidylinositol 3-kinase catalytic subunit The PNA.
12. The PNA according to claim 11, which is represented by SEQ ID NO: 1 or 2.
A gold nanoparticle to which the PNA of claim 11 or 12 is immobilized.
The nanoflashmonic biosensor according to claim 13, wherein the gold nanoparticles are immobilized.

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