Method for detecting unstable cell-free DNA and device using the same

Abstract

In one embodiment, the present invention relates to a technology in which small-sized cfDNA is detected from liquid samples such as urine, cerebrospinal fluid, plasma, blood, pleural fluid or body fluid with ultra-high sensitivity, concentrated and separated, and then analyzed for gene mutations without PCR. In particular, in the case of using positively charged nanostructures, the capture and detection rate of cfDNA can be improved. The detection method according to one embodiment of the present invention does not require PCR amplification reactions, which greatly shortens the time required to obtain results. In addition, since direct on-site analysis can be performed without the need for specific equipment, it is expected that the present invention can be used as a point-of-care test (POCT) that can simultaneously search for multiple genes in a short time.

Description

Technical Field

[0001] The present invention relates to a method and a device for detecting cell-free DNA with an unstable double helix structure without a gene amplification process. Background Art

[0002] Recently, the importance of early diagnosis of cancer diseases has become increasingly prominent worldwide. Therefore, there are more and more studies on early diagnosis methods for cancer. However, up to now, the diagnosis method of cancer is carried out by invasive methods such as collecting tissue samples and endoscopy. In particular, a histological examination is performed to extract a part of the suspected disease site and observe it under a microscope. Therefore, since the human body needs to be cut open in order to collect tissue samples using a needle, a punch, an endoscope or a laparoscope, not only does it make the patient feel quite uncomfortable, but it also leaves scars and a long recovery time.

[0003] As an alternative to invasive diagnostic and examination methods, molecular diagnostic methods using liquid biopsies have attracted attention. Since liquid biopsies use non-invasive methods, their results can be quickly identified. In addition, unlike tissue samples that can only analyze a part of the disease, liquid biopsies can perform multilateral analysis of the disease. In particular, liquid biopsies are expected to play an excellent role in cancer diagnosis. In particular, it is foreseeable that by simply examining body fluids such as blood and urine, DNA derived from cancer cells in various parts of the body present in the blood can be analyzed, making it possible to observe the occurrence and metastasis of cancer in detail.

[0004] Molecular diagnostic methods are representative technologies for in vitro diagnosis, which detect and diagnose changes in DNA or RNA from samples containing genetic information (such as blood and urine) through numerical or image detection. Due to its advantages of high accuracy and no need for histological examination, with the rapid development of genome analysis technology, attempts have been made to apply molecular diagnostic methods to cancer diagnostic technology based on its cost-saving advantages.

[0005] On the other hand, cell-free DNA (hereinafter referred to as cfDNA) refers to DNA present in plasma and derived from cells. cfDNA generally has a double helix structure. In addition, in many cases, cfDNA has a coiled coil structure. cfDNA can be derived from tumor cells. In addition, cfDNA derived from tumor cells can be found in biological samples such as blood, plasma or urine obtained from cancer patients.

[0006] The cfDNA found in cancer patients may be derived from necrotic cells, normal cells and / or cancer cells. Such cfDNA is released into urine, blood, etc. through various processes. Therefore, the development of technologies for separating and detecting cfDNA in biological samples such as blood, plasma or urine makes liquid biopsy a more effective and reliable tool for monitoring patients at risk of cancer. In particular, since urine, plasma, blood or body fluids are easily accessible samples, a large number of samples can be collected in a non-invasive manner.

[0007] However, given the current state of the art, there are many difficulties in early diagnosis of cancer, including analyzing cfDNA in liquid samples such as blood and urine and discovering mutations in genes. Therefore, it is necessary to develop a method for easily detecting cfDNA and a technology for improving detection sensitivity and accurately diagnosing cancer at an early stage.

[0008] Meanwhile, Korean Patent No. 10-1751962 discloses a technology capable of quantifying cfDNA by performing polymerase chain reaction (PCR) using primers to detect cfDNA. However, there are still problems that separate polymerase and experimental equipment are required for polymerase chain reaction, and there is a problem that accurate primers need to be generated for amplification and it is not easy to perform on-site diagnosis.

[0009] In addition, Korean Patent No. 10-1701618 discloses a nanostructure whose surface properties can be changed by changes in an electric field in order to effectively separate cfDNA. The nanostructure can bind or dissociate cfDNA by changes in the electric field, so that cfDNA can be easily separated from a sample. However, there is still a limitation, that is, polymerase chain reaction should be used to identify which cfDNA is present.

[0010] In order to amplify cfDNA by polymerase chain reaction, various types of primer pairs are required, and complicated steps are performed, which take a lot of time. Therefore, research has been conducted to overcome the limitation of having to perform PCR and to develop methods for analyzing cfDNA with high accuracy. Summary of the invention

[0011] Technical issues

[0012] Conventionally, in order to detect cfDNA, cfDNA must be denatured and amplified using primers complementary to cfDNA. However, the present inventors have identified the presence of unstable cfDNA in the blood. In addition, the present inventors have found that, unlike stable cfDNA, unstable cfDNA shows abnormal reactions with single-stranded probes. In particular, the present inventors have identified that this unstable cfDNA is derived from cells of individuals with cancer or infectious diseases. Based on these findings, the present inventors have completed the present invention.

[0013] Therefore, an object of the present invention is to provide a method for identifying the presence or absence of unstable cfDNA, and to provide a method for providing information for diagnosing cancer or various diseases by identifying such unstable cfDNA.

[0014] Solutions to the problem

[0015] In order to achieve the above-mentioned object, the present invention provides a method for detecting unstable cfDNA from a sample that has not been amplified. In addition, the present invention provides a method for providing information for diagnosing or predicting cancer and infectious diseases by detecting unstable cfDNA from a sample that has not been amplified. In addition, the present invention provides a device for detecting unstable cfDNA from a sample that has not been amplified.

[0016] Advantageous Effects of the Invention

[0017] When using the method for detecting unstable cfDNA of an embodiment, not only is there no need to amplify the unstable cfDNA process, but also the time for analyzing cfDNA can be shortened. In particular, unstable double-stranded cfDNA containing gene mutation parts can be effectively detected, thereby effectively diagnosing or predicting cancers or diseases related to gene mutations. In addition, when using the method of the embodiment of the present invention, the presence or absence of cfDNA with an unstable double helix structure can be quickly and accurately identified from a small amount of biological samples such as urine, cerebrospinal fluid, pleural fluid, ascites, plasma, blood or body fluids. In addition, since it has been confirmed that the unstable cfDNA thus detected is related to various cancers or diseases, the method according to the embodiment of the present invention can be effectively used for the diagnosis of cancer or the identification of prognosis after treatment. SEQUENCE LISTING <110> GENNO PSYCHOLOGY CO., LTD. <120> Method for detecting unstable cell-free DNA and device using the same <130> P20116737WP <150> KR 10-2018-0063258 <151> 2018-06-01 <160> 48 <170> PatentIn version 3.5 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> HPV 16-CP <400> 1 gaggaggagg atgaaataga tggt 24 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HPV 16-DP <400> 2 ttggaagacc tgttaatggg c 21 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HPV 18-CP <400> 3 cacattgtgg cacaatcttt ta 22 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HPV 18-DP <400> 4 gccatatcgc tttcatctgt 20 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EGFR 19-CP <400> 5 gaggle gaggle ctcc <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> EGFR 19-DP <400> 6 aacctcaggc ccacctttt <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> EGFR 21-CP <400> 7 ccaggaacgt actggtgaaa a <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> EGFR 21-DP <400> 8 ggaagagaa gaataccatg ca <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EGFR receptor19-link1 CP1 <400> 9 gaggle gaggle ctcc <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> EGFR Exon 19-Probe 1 DP <400> 10 aacctcaggc ccaccttt 18 <210> 11 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> EGFR exon 19-probe 2 CP2 <400> 11 aaaattcccg tcgctatcaa g 21 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> EGFR Exon 19-Probe 2 DP <400> 12 aacctcaggc ccacctttt 19 <210> 13 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> EGFR exon 19-probe 3 CP3 <400> 13 ggactctgga tcccagaagg tgag 24 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> EGFR exon 19-probe 3 DP <400> 14 aacctcaggc ccacctttt 19 <210> 15 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> EGFR exon 20-probe 1 CP1 <400> 15 ccatgagtac gtattttgaa actc 24 <210> 16 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> EGFR Exon 20-Probe 1 DP <400> 16 gcaagagttt gccatgggga tatg 24 <210> 17 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> EGFR exon 20-probe 2 CP2 <400> 17 ccaccgtgca gctcatcacg cagctca 27 <210> 18 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> EGFR Exon 20-Probe 2 DP <400> 18 gcaagagttt gccatgggga tatg 24 <210> 19 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> EGFR exon 20-probe 3 CP3 <400> 19 gaagcctacg tgatggccag cgt 23 <210> 20 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> EGFR exon 20-probe 3 DP <400> 20 gcaagagttt gccatgggga tatg 24 <210> twenty one <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> EGFR exon 21-probe 1 CP1 <400> twenty one ccaggaacgt actggtgaaa a 21 <210> twenty two <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> EGFR Exon 21-Probe 1 DP <400> twenty two ggaagagaaa gaataccatg ca 22 <210> twenty three <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> EGFR exon 21-probe 2 CP2 <400> twenty three aagatcacag attttgggcg gg 22 <210> twenty four <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> EGFR exon 21-probe 2 DP <400> twenty four ggaagagaaa gaataccatg ca 22 <210> 25 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> EGFR exon 21-probe 3 CP3 <400> 25 ggcatgaact acttggagga ccgt 24 <210> 26 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> EGFR exon 21-probe 3 DP <400> 26 ggaagagaaa gaataccatg ca 22 <210> 27 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> KRAS exon 2-probe CP1 <400> 27 aaatgactga atataaactt g 21 <210> 28 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> KRAS Exon 2-Probe DP <400> 28 gagtgccttg acgatacagc t 21 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALK-EML4 variant 1 - probe CP2 <400> 29 tagagcccac acctgggaaa 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALK-EML4 variant 1 - Probe DP <400> 30 cggagcttgc tcagcttgta 20 <210> 31 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> ALK-EML4 variant 3 - probe CP3 <400> 31 gcataaagat gtcatcatca accaag 26 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALK-EML4 variant 3 - Probe DP <400> 32 cggagcttgc tcagcttgta 20 <210> 33 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> BRCA1 exon 7 in normal cells <400> 33 caaagtatgg gctacagaaa ccgtgccaaa ag 32 <210> 34 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> BRCA1 exon 7 in cancer cells <400> 34 caaagtatgg gcttcagaaa ccgtgccaaa ag 32 <210> 35 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> BRCA1 exon 10 in normal cells <400> 35 tgggaaaacc tatcggaaga aggcaagcct cc 32 <210> 36 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> BRCA1 exon 10 in cancer cells <400> 36 tgggaaaacc tatcggtaga aggcaagcct cc 32 <210> 37 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> BRCA1 exon 11 in normal cells <400> 37 ggggccaaga aattagagtc ctcagaagag 30 <210> 38 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> BRCA1 exon 11 in cancer cells <400> 38 gggccaaga aaattagagt cctcagaaga g 31 <210> 39 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> BRCA1 exon 15 in normal cells <400> 39 atatacagga tatgcgaatt aagaagaaac aaa 33 <210> 40 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> BRCA1 exon 15 in cancer cells <400> 40 atatacagga tatgtgaatt aagaagaaac aaa 33 <210> 41 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> TP53 in normal cells <400> 41 taggaggccg agctctgttg cttcgaactc ca 32 <210> 42 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> TP53 in cancer cells <400> 42 taggaggccg agctctttgc ttcgaactcc a 31 <210> 43 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> MSH2 in normal cells <400> 43 tgaggaggtt tcgacatggc ggtgcagccg a 31 <210> 44 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> MSH2 in cancer cells <400> 44 tgaggaggtt tcgacctggc ggtgcagccg a 31 <210> 45 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> EGFR in normal cells <400> 45 aaaaagatca aagtgctggg ctccggtgcg tt 32 <210> 46 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> EGFR in cancer cells <400> 46 aaaaagatca aagtgctgag ctccggtgcg tt 32 <210> 47 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> FGFR3 in normal cells <400> 47 atcctctctc tgaaatcact gagcaggaga aag 33 <210> 48 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> FGFR3 in cancer cells <400> 48 atcctctctc tgaaatcact gcgcaggaga aag 33 BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Scanning electron microscopy (SEM) images of positively charged nanowires (PEI / Ppy NWs) are shown.

[0019] Figure 2 Transmission electron microscopy (TEM) images of positively charged nanowires (PEI / Ppy NWs) are shown.

[0020] Figure 3 Shown is a surface of a nanostructure (PLL / Ppy NW) represented by a scanning electron microscope image, to which polylysine (PLL) as a cationic polymer is bound.

[0021] Figure 4 Scanning electron microscopy images of HRP / streptavidin-conjugated nanoparticles are shown.

[0022] Figure 5 Scanning electron microscopy (SEM) images of PEI-bound nanoparticles (PEI / Ppy NPs) with a diameter of 50 nm are shown.

[0023] Figure 6 A schematic diagram of a method is shown, in which cfDNA is obtained from the patient's body fluid using nanowires with polyethyleneimine (PEI) bound to the surface (PEI / Ppy NW), and then analyzed for gene mutations within 60 minutes by reacting with probes and HRP / streptavidin-labeled nanoparticles (HRP / ST-labeled NPs).

[0024] Figure 7 A method for detecting labile cfDNA using nanowires, probes, and HRP / streptavidin-labeled nanoparticles is schematically shown.

[0025] Figure 8 A chronological flow chart illustrates a method for detecting labile cfDNA in a sample such as blood, cerebrospinal fluid, or pleural fluid.

[0026] Fig. 9 A method for detecting labile cfDNA in a sample such as urine is shown in a chronological flow chart.

[0027] Fig.10 Schematically shown are the differences in denaturation conditions depending on the state of cfDNA obtained from blood.

[0028] Fig.11 Schematically shown are the differences in denaturation conditions depending on the state of cfDNA obtained from urine, saliva, and sputum.

[0029] Fig.12 Shown are the results obtained by obtaining cfDNA from urine samples of cervical cancer patients and urine samples of normal subjects, and then detecting the presence or absence of gene mutations in the cfDNA using an HPV 18 or HPV 16 probe.

[0030] Fig.13 Shown are the results obtained by identifying by absorbance the binding between cfDNA present in the urine of HPV-positive cervical cancer patients (HPV16(+) and HPV18(+)) and HPV-negative healthy control patients (HPV-) and probes specific for HPV 18 or HPV 16.

[0031] Fig.14 Shown are the results obtained by sequentially reacting cfDNA isolated from the urine of cervical cancer patients with probes specific for HPV 16, EGFR19 deletion, HPV 18, and EGFR 21L858R, and then identifying the binding between cfDNA and each probe.

[0032] Fig.15Shown are the results obtained by adding low-range (10 bp-100 bp), mid-range (100 bp-2 kb), and high-range (3.5 kb-21 kb) DNA ladders to the plasma of normal subjects, and then evaluating the capture efficiency when the DNA ladders were captured using PEI / Ppy NW.

[0033] Fig.16 Shown are the results obtained by identifying the size of cell line genomic DNA (hereinafter referred to as gDNA) obtained by degrading A549 cell line (lanes 1 and 2), HCC2279 cell line (lanes 3 and 4), and H1975 cell line (lanes 5 and 6) without ultrasonic treatment, and the results obtained by identifying the size of fragmented DNA (hereinafter referred to as fDNA) obtained by ultrasonicating the gDNA of each cell line.

[0034] Fig.17 Shown are the results obtained by capturing gDNA obtained from A549 cell line, HCC2279 cell line, and H1975 cell line without sonication using PEI / Ppy NW, and fDNA obtained by sonicating the respective cell lines, and then evaluating the capture efficiency.

[0035] Fig.18 and Fig.19 The results obtained by capturing gDNA obtained from A549 cell lines, HCC2279 cell lines and H1975 cell lines without ultrasonic treatment and fDNA obtained by ultrasonic treatment of the respective cell lines, and then adding probes specific for EGFR 19 (19Del), EGFR 20 (T790M) and EGFR 21 (L858R) to identify color changes and visible spectrum changes of UV-Vis are shown. Only fDNA has been identified to selectively bind to the target probe.

[0036] Fig. 20 Shown are the results of obtaining fDNA of H1975 cell line, HCC2279 cell line, and A549 cell line by using PEI / Ppy NW and then analyzing gene mutations using DNA probes conjugated with fluorescent dyes.

[0037] Fig.21 The results obtained by using fDNA from HCC2279 (EGFR exon 19 deletion gene mutation) cells and a probe specific for EGFR exon 19 deletion and analyzing the binding of fDNA to the probe using ultraviolet absorbance are shown. By this method, detectable concentrations of cfDNA have been determined.

[0038] Fig. 22Results obtained by using fDNA of H1975 (EGFR exon 20 T790M and exon 21 L858R gene mutations) and a probe specific for EGFR exon 20 T790M and analyzing fDNA binding to the probe using UV absorbance (depending on the concentration of fDNA) are shown. By this method, detectable concentrations of cfDNA have been identified.

[0039] Fig.23 Results obtained by analyzing cfDNA obtained from 200 μL of plasma from lung cancer patients using PEIA / Ppy NW by Bioanalyzer are shown. According to the literature, cfDNA associated with cancer is known to have an average size of 166 bp. As shown in the Bioanalyzer data, it was determined that cfDNA obtained from lung cancer patient plasma using PEI / Ppy NW had a peak at 169 bp.

[0040] Fig.24 Shown are the results obtained by identifying cfDNA isolated from the plasma of lung cancer patients with EGFR gene mutations reacted with a probe that specifically binds to EGFR exon 19 deletion (Del) or EGFR exon 21 L858R, and the figure shows the same color change and UV absorbance of the tissue genotype.

[0041] Fig.25 Shown are the results obtained by identifying cfDNA isolated from the plasma of lung cancer patients with EGFR exon 19 deletion (Del), EGFR exon 20 T790M, and EGFR exon 21 L858R gene mutations reacting with probes that specifically bind to EGFR exon 19 Del, EGFR exon 20 T790M, or EGFR exon 21 L858R, and the figure shows the color change and UV absorbance of the same tissue genotype.

[0042] Fig.26 and 27 Shown are the results obtained by identifying the reaction of cfDNA isolated from the plasma of lung cancer patients with KRAS exon 2 gene mutation using PEI / Ppy NW with a probe that specifically binds to KRAS exon 2, and the figure shows the same color change and UV absorbance of the tissue genotype.

[0043] Fig.28 The results obtained by identifying cfDNA isolated from the plasma of lung cancer patients with ALK-EML4 fusion gene mutations reacted with a probe that specifically binds to ALK-EML4 are shown. The same color change and UV absorbance of the tissue genotype are shown.

[0044] Fig.29 It was shown that in the case where cfDNA isolated from the plasma of lung cancer patients with EGFR exon 19 deletion or EGFR exon 20 T790M gene mutation was reacted with various probes that specifically bind to EGFR exon 19 deletion or EGFR exon 20 T790M, both color change and ultraviolet absorbance were shown to match the tissue. However, it can be confirmed from the results that regardless of the three types of probes used, no color change was observed for the EGFR 21L858R mutation. As can be seen from the results, the reaction between the gene and the probe is not limited to a specific probe, and any probe can bind to a gene with a mutation as long as the probe specifically binds to the gene.

[0045] Fig.30 Results obtained by denaturing cfDNA isolated from the plasma of lung cancer patients without EGFR gene mutation (wild type, WT) at 95°C for 1 minute and 10 minutes, respectively, and then reacting with probes that specifically bind to EGFR exon 19 deletion, EGFR exon 20 T790M, or EGFR exon 21 L858R are shown. It was found that cfDNA extracted from the plasma of EGFR WT patients did not react with any probes when denatured at 95°C for 1 minute; on the other hand, cfDNA reacted nonspecifically with all probes after denaturation at 95°C for 10 minutes.

[0046] Fig.31 , Fig.32 and Fig.33 The nanowires were used to capture cfDNA from the plasma of lung cancer patients with EGFR exon 19 deletion and EGFR exon 20 T790M gene mutation and normal subjects, and the cfDNA was denatured at 95°C for 0 min ( Fig.31 )、1 minute( Fig.32 ) and 10 minutes ( Fig.33 ), and then identify the results after reacting it with a probe that specifically binds to EGFR 19 deletion, 20T790M or 21L858R.

[0047] Fig.34 Shown is a table obtained by using cfDNA obtained from the plasma of 151 lung cancer patients and analyzing gene mutations in lung cancer patients.

[0048] Fig.35The results are shown by obtaining cfDNA from the plasma of lung cancer patients without EGFR mutation (wild type), lung cancer patients with EGFR exon 19 deletion, and lung cancer patients with EGFR exon 21 L858R, mixing these cfDNAs with probes specific to EGFR exon 19Del, and then determining the gene mutations of lung cancer patients by analyzing UV spectral absorbance (ΔOD, 500nm-650nm) values.

[0049] Fig.36 shows the results obtained by obtaining cfDNA from the plasma of lung cancer patients with EGFR exon 19 deletion, mixing the cfDNA with a probe specific for EGFR exon 19Del, and then analyzing the specificity and sensitivity of the gene mutation.

[0050] Fig.37 The results are shown by obtaining cfDNA from the plasma of lung cancer patients without EGFR mutation (wild type), lung cancer patients with EGFR exon 19 deletion, and lung cancer patients with EGFR exon 21L858R, adding a probe specific to EGFR exon 21L858R, and then determining the gene mutations of the patients by analyzing the UV spectral absorbance (ΔOD, 500nm-650nm) values.

[0051] Fig.38 The results obtained by obtaining cfDNA from the plasma of lung cancer patients with EGFR exon 21L858R, adding a probe specific for EGFR exon 21L858R thereto, and then analyzing the specificity and sensitivity of the gene mutation in the patients are shown.

[0052] Fig.39 The results obtained by reacting cfDNA isolated from the plasma of lung cancer patients with EGFR exon 19 deletion gene mutation using PEI / Ppy NP with probes specific for EGFR exon 19Del (E19), EGFR exon 20T790M (E20), or EGFR exon 21L858R (E21) were shown, and it was found that UV absorbance matching that of tissue was observed.

[0053] Fig.40 The results are shown by obtaining fDNA of H1975 cell lines having EGFR exon 20T790M and EGFR exon 21L858R gene mutations using poly-lysine-modified PLL / Ppy NW, reacting the fDNA with probes specific to EGFR exon 19Del, EGFR exon 20T790M, or EGFR exon 21L858R, and then analyzing the gene mutations.

[0054] Fig.41 The sequences of CP and DP for EGFR exon 19 deletion are shown. In this study, cfDNA gene mutations of lung cancer patients were analyzed using CP_1 and DP. Here, CP refers to a probe that specifically binds to a sequence containing a mutation portion or adjacent to it, and DP is a probe that specifically binds to a portion spaced from the mutation sequence.

[0055] Fig.42 The sequences of CP and DP of EGFR exon 20T790M are shown. In this study, cfDNA gene mutations in lung cancer patients were analyzed using CP2 and DP.

[0056] Fig.43 The sequences of CP and DP of EGFR exon 21L858R are shown. In this study, cfDNA gene mutations in lung cancer patients were analyzed using CP2 and DP.

[0057] Fig.44 showed that cfDNA was obtained from the plasma of lung cancer patients with EGFR exon 19 deletion mutations and then used Figure 41-42 The results obtained by analyzing gene mutations in EGFR exon 19CP_1, exon 20CP2, and exon 21CP2 were obtained without using DP.

[0058] Fig.45 The results obtained by treating the plasma of lung cancer patients with EGFR exon 19 deletion with RNase and DNase and obtaining cfDNA by PEI / Ppy nanowires, and then detecting unstable cfDNA using a probe specific for EGFR exon 19 deletion are shown.

[0059] Fig.46 The results obtained by treating the plasma of lung cancer patients with EGFR exon 20T790M with RNase and DNase and obtaining cfDNA by PEI / Ppy nanowires, and then detecting unstable cfDNA using a probe specific for EGFR exon 20T790M are shown.

[0060] Fig.47The results are shown by reacting cfDNA obtained from the plasma of lung cancer patients with EGFR exon 19 deletion and EGFR exon 20 T790M gene mutation with probes specific to EGFR exon 19 deletion (Del19), EGFR exon 20 T790M, and EGFR exon 21 L858R, and then adding HRP / streptavidin nanoparticles (containing a large amount of HRP) thereto to detect cfDNA by color change and UV absorption.

[0061] Fig.48 Shows that by making Fig.47 cfDNA obtained from the plasma of the same lung cancer patients with EGFR exon 19 deletion and EGFR exon 20 T790M gene mutation was reacted with probes specific for EGFR exon 19 deletion (Del19), EGFR exon 20 T790M, and EGFR exon 21 L858R, and then HRP / streptavidin complex (complex obtained by 1:1 binding between HRP and streptavidin) was added thereto to detect cfDNA by color change and UV absorption. According to the results, noise was generated in the HRP / streptavidin complex compared with the HRP / streptavidin nanoparticles.

[0062] Fig.49 A graph shows two results obtained by extracting cfDNA from the plasma of five lung cancer patients with EGFR exon 19 deletion and exon 20 T790M gene mutation, and then reacting the cfDNA with probes specific to EGFR exon 19Del, EGFR exon 20T790M, and EGFR exon 21L858R, and HRP / streptavidin-labeled nanoparticles (HRP / st-labeled NPs), and reacting with probes specific to EGFR exon 19Del, EGFR exon 20T790M, and EGFR exon 21L858R, and HRP / streptavidin complex (in which HRP and streptavidin are bound to each other in a 1:1 ratio), thereby identifying and comparing the matches with the cancer tissue genotype.

[0063] Fig.50 Shown are the results obtained by reacting cfDNA obtained from the pleural fluid of lung cancer patients with EGFR exon 20T790M and 21L858R gene mutations with probes for EGFR exon 19 deletion (19Del), EGFR exon 20T790M, and EGFR exon 21L858R (to which HRP / st-labeled NPs had been conjugated), and then detecting gene mutations by UV absorption.

[0064] Fig.51 The results obtained by mixing cfDNA obtained from the plasma of lung cancer patients with EGFR exon 20T790M and EGFR exon 21L861Q gene mutations with probes specific to EGFR exon 19 deletion (19Del), EGFR exon 20T790M, EGFR exon 21L858R, and EGFR exon L861Q, and HRP / st-labeled NPs all at once for detecting gene mutations in cfDNA were shown, and the results identified by UV absorption that gene mutations were observed only in EGFR exon 20T790M and EGFR exon 21L861Q, as in cancer tissues.

[0065] Fig.52 The results obtained by detecting gene mutations in cfDNA by mixing cfDNA obtained from the plasma of lung cancer patients with gene mutations of ALK-EML4 fusion and ALK point mutation (I1171N / T) all simultaneously with probes specific to ALK-EML4 fusion and ALK point mutation (T1151, L1152P, L1152R, C1156Y, I1171N / T) and HRP / st-labeled NPs were shown, and the results showed that ALK-EML4 fusion and ALK point mutation (I1171N / T) genotypes were determined to be detected in cancer tissues.

[0066] Fig.53 The results obtained by detecting gene mutations in cfDNA obtained from the plasma of thyroid cancer patients with BRAF V600E gene mutations by mixing them once with a probe specific to BRAF V600E and HRP / ST-labeled NPs were shown, and the results identified that the BRAF V600E gene mutation identical to the patient's genotype was detected.

[0067] Fig.54 Shown are the results obtained by denaturing samples collected from blood of normal subjects under various temperature conditions and then detecting unstable cfDNA for each treatment condition.

[0068] Fig.55 Results obtained by denaturing samples collected from patient blood under various temperature conditions and then detecting labile cfDNA for each treatment condition are shown.

[0069] Fig.56 Shown are the results obtained by denaturing fDNA obtained from mutant cell lines under various temperature conditions and then detecting unstable cfDNA for each treatment condition.

[0070] Fig.57Shown are the results obtained by treating fDNA obtained from mutant cell lines with DNase for 30 minutes at 37°C and then detecting unstable cfDNA for treatment conditions.

[0071] Fig.58 Shown are the results obtained by treating fDNA obtained from mutant cell lines with DNase for 60 min at 37°C and then detecting unstable cfDNA for treatment conditions.

[0072] Fig.59 Shown are the results obtained by treating fDNA obtained from mutant cell lines with DNase for 120 min at 37°C and then detecting unstable cfDNA for treatment conditions.

[0073] Fig.60 Shown are the results obtained by treating unstable cfDNA and stable cfDNA with 1 μl or 2 μl of DNase at 24°C for 120 minutes in order to identify the difference between unstable cfDNA and stable cfDNA based on the activity of DNase.

[0074] Fig.61 Shown are the results obtained by treating unstable cfDNA and stable cfDNA with 1 μl or 2 μl of DNase at 3°C ​​for 120 minutes in order to identify the difference between unstable cfDNA and stable cfDNA based on the activity of DNase.

[0075] Best Mode for Carrying Out the Invention

[0076] <Definition of terms>

[0077] As used in the present invention, the term "cell-free DNA" is also referred to as cfDNA. In addition, cfDNA may also be circulating tumor DNA (ctDNA), which is cancer cell-derived DNA that can be found in biological samples such as urine, cerebrospinal fluid, plasma, blood or body fluids of cancer patients. In addition, cfDNA may also be present in biological samples such as urine, cerebrospinal fluid, pleural fluid, ascites, plasma, blood, saliva, sputum or body fluids. Here, the size of cfDNA can be 80bp-10kbp, 100bp-1kbp or 120bp-500bp. In addition, the size of cfDNA can be 150bp-200bp, and can typically have a size of 165bp-170bp.

[0078] As used herein, the term "unstable cfDNA" refers to cfDNA that is thermodynamically unstable compared to "stable cfDNA". In other words, unstable cfDNA may denature under less severe conditions than the conditions under which stable cfDNA denatures. Unstable cfDNA is generated because unstable cfDNA has an unstable double helix structure.

[0079] As used in the present invention, the term "cfDNA with an unstable double helix structure" is characterized in that it has a lower Tm value relative to cfDNA with a stable double helix structure, or it denatures under conditions where the cfDNA with a stable double helix structure does not denature. Tm refers to the melting temperature at which 50% of double-stranded DNA is converted into single-stranded DNA. The Tm value is proportional to the length of the DNA and may vary with changes in the nucleotide sequence. Since a large number of nucleotides in genomic DNA are bound to each other through hydrogen bonds, the genomic DNA must be heated at 92°C-95°C for 5 minutes or more, or at 98°C for 2 minutes or more. In addition, genomic DNA is not easily denatured at temperatures below 90°C. Since cfDNA with a stable double helix structure has an average of 170bp of nucleotides, it can have a Tm value similar to that of genomic DNA.

[0080] However, since "cfDNA with unstable double helix structure" has a lower Tm value than cfDNA with stable double helix structure, when cfDNA with stable double helix structure is placed under any one of the following conditions: i) at room temperature for 1-120 minutes; ii) at 90℃-95℃ for 1 second to 3 minutes; iii) at 75℃-90℃ for 1 second to 5 minutes; iv) at 60℃-75℃ for 30 seconds to 60 minutes; v) at 25℃-40℃ for 10-120 minutes; vi) at protease for 10 seconds to 30 minutes; and vii) at DNase for 10 seconds to 30 minutes, it will denature and then react with a probe of 15-mer to 30-mer, and cfDNA with stable double helix structure will not bind to the probe. Here, "room temperature" refers to the ambient temperature, which can be 18℃-25℃. Furthermore, in addition to the above conditions, a condition of heating at 40°C to 65°C for 5 to 80 minutes may be included.

[0081] However, it has been confirmed that when cfDNA with an unstable double helix structure is treated under any of the conditions i)-vii) above and then subjected to a binding reaction with a 15-mer to 30-mer probe, the cfDNA with an unstable double helix structure binds to the probe. Here, the probe can be 15-mer to 30-mer, or 20-mer to 25-mer, and can be a 21-mer, 22-mer, 23-mer or 24-mer probe.

[0082] Here, the cfDNA with an unstable double helix structure can be a circulating tumor DNA (hereinafter referred to as ctDNA). In addition, the cfDNA with an unstable double helix structure can be cfDNA derived from a damaged nucleic acid sequence that does not exist in a normal cell. Here, the damaged nucleic acid sequence that does not exist in a normal cell may include a structural abnormality caused by the deletion, duplication, inversion or translocation of a portion of the gene. In addition, the damaged nucleic acid sequence may include a structural abnormality caused by the mismatch of a portion of the nucleic acid, and may be a single nucleotide site variation (SNV) caused by a mutation of a partial sequence of the nucleic acid. The damaged nucleic acid sequence may have a mutant sequence of at least one gene, and the gene is selected from the group consisting of EGFR, KRAS, BRAF, TP53, PIK3CA, ROS1, RET, c-Met, PTEN, RB1, AR, BRCA, KIT, FGFR, IDH, ESR1, HER2, ALK-EML4 and TMPRSS2-ERG.

[0083] Specifically, the damaged nucleic acid sequence that does not exist in normal cells may be caused by one of the following reasons: 1) single-strand cleavage, 2) double-strand cleavage, 3) replication fork stalling, 4) nucleic acid mismatch, 5) chromosome aberration, 6) intrachain crosslinking, 7) interchain crosslinking, 8) foreign gene insertion, 9) partial gene deletion, 10) partial nucleic acid substitution, 11) nucleic acid inversion, 12) formation of thymidine dimers, 13) deamination, 14) gene duplication, 15) chromosome translocation, or 16) base deficiency (AP site). In particular, in tumor cells, double-stranded DNA (dsDNA) is often damaged, and the damaged dsDNA may be contained in a specific structure found in tumor cells. In particular, due to the mismatch of nucleic acids, the damaged nucleic acid sequence may have a wobble base pair.

[0084] As used in the present invention, the term "probe" refers to a DNA or RNA used to detect target cfDNA. The probe can be designed to have a sequence that can complementarily bind to unstable cfDNA. As used in the present invention, the term "probe having a sequence complementary to cfDNA" refers to a probe having a nucleic acid sequence that can specifically bind to cfDNA to be detected, which has a target double helix structure and is present in plasma.

[0085] Here, probes can be produced in two ways. One is a first probe designed to bind to a part of a gene that has been damaged (hereinafter referred to as CP), and the other is a second probe designed to bind to the periphery of a damaged part of a gene (hereinafter referred to as DP). DP can be designed to specifically bind to a sequence that is 10bp-100bp, or 20bp-50bp away from the target DNA sequence or the damaged area.

[0086] In this specification, it has been confirmed that damaged cfDNA can be effectively detected not only when the first probe and the second probe are used simultaneously, but also when the first probe or the second probe is used alone. In addition, the probe can be in a form that is combined with a substance such as biotin so as to bind to a marker. Alternatively, the probe can be directly combined with the marker or combined with the marker through a linker. Here, the marker can be a nanoparticle, a fluorescent dye, a fluorescent protein or an enzyme. In addition, the probe and the marker can be added simultaneously or sequentially.

[0087] In one embodiment of the present invention, the probe capable of complementary binding to the target cfDNA can specifically bind to a region containing sequences specific to the following various cancer cell types. For example, a sequence specific to ovarian cancer or breast cancer can be a SNP present in BRCA1 exon 7, BRCA1 exon 10, BRCA1 exon 11, or BRCA1 exon 15. In addition, a sequence specific to gastric cancer can be a SNP present in TP53, and a sequence specific to colorectal cancer can be a SNP present in MSH2. A sequence specific to lung cancer can be a SNP present in EGFR. In addition, a sequence specific to liver cancer can be selected from a SNP present in FGFR3.

[0088] [Table 1]

[0089]

[0090] As used in the present invention, the term "isolated biological sample" refers to a sample of urine, saliva, cerebrospinal fluid, pleural fluid, ascites, plasma, blood, sputum or body fluid that has been separated from the human body. The isolated biological sample can be a liquid sample separated from the human body. Here, plasma can be obtained from blood.

[0091] As used in the present invention, the term "positively charged substance" refers to a substance that can be used in the form of nanoparticles, nanowires, mesh structures or filters. However, the shape of the positively charged substance is not limited thereto. One embodiment of the "positively charged substance" may be a positively charged nanowire or a positively charged film. Conductive polymers may be used to produce nanowires. Conductive polymers may be any one selected from the group consisting of poly(acetylene), poly(pyrrole), poly(thiophene), poly(p-phenylene), poly(3,4-ethylenedioxythiophene), poly(phenylene sulfide), poly(p-phenylene vinylene) and polyaniline. Depending on different production methods, the length and diameter of the nanowires may be appropriately adjusted. In one embodiment, the nanowire may have a diameter of 200 nm and a length of 18 μm. In addition, the nanowires may contain biotin during the production process.

[0092] The surface of the nanowire can be modified with a cationic polymer. The type of cationic polymer is not limited. An embodiment of the cationic polymer can be polyethyleneimine (PEI) or polylysine (PLL). In addition, the cationic polymer can be a cationic branched polymer polyethyleneimine. The nanowire modified with this cationic polymer can have a positively charged surface.

[0093] In one embodiment, positively charged nanowires can successfully and effectively capture cfDNA even at low concentrations. In particular, positively charged nanowires can effectively capture cfDNA due to their features such as large surface area for binding to target molecules including DNA and increased mobility for facilitating interaction with DNA.

[0094] As used in the present invention, the term "marker" refers to a substance for effectively detecting cfDNA with an unstable double helix structure, and may specifically include quantum dots, substances that degrade specific substrates and cause color development reactions, and substances that cause luminescence when irradiated with light of a specific wavelength. Specifically, the marker is a fluorescent protein, and may be green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP) or cyan fluorescent protein (CFP). Alternatively, the marker may be a substance that can convert any substrate selected from the group consisting of ABTS, OPD, AmplexRed, DAB, AEC, TMB, homovanillic acid and luminol into a colored substance, such as horseradish peroxidase (HRP).

[0095] The marker may further include a substance that can bind to the probe. Specifically, when biotin binds to the probe, the marker may further include any one selected from the group consisting of avidin, streptavidin or a combination thereof. In one embodiment, this marker may be used in the form of nanoparticles combined with streptavidin and HRP, and the nanoparticles are composed of a conductive polymer and hyaluronic acid. Here, the conductive polymer is as described above, and may preferably be polypyrrole. In another embodiment, the marker may be used in the form of nanoparticles combined with streptavidin and fluorescent protein, and the nanoparticles are composed of a conductive polymer and hyaluronic acid.

[0096] <Methods for detecting unstable cfDNA>

[0097] In one aspect of the present invention, a method for detecting unstable cell-free DNA from a non-amplified sample is provided, the method comprising the step of mixing unstable cfDNA with a probe bound to a marker.

[0098] This unstable cfDNA can be circulating tumor DNA (ctDNA) derived from a tumor. Here, ctDNA may have a damaged gene sequence as described above. In addition, ctDNA can be isolated from highly active genes. Here, the sample can be a biological sample and can be a sample isolated from the human body. Specifically, the sample can be urine, cerebrospinal fluid, plasma, blood, pleural fluid, ascites, saliva, sputum or body fluid.

[0099] Therefore, unstable cfDNA can be distinguished from stable cfDNA in terms of reactivity with the probe by treating the sample under any of the following conditions. Specifically, the treatment can be performed under any one of the following conditions: i) a condition of standing at room temperature for 1-120 minutes; ii) a condition of heating at 90°C-95°C for 1 second to 3 minutes; iii) a condition of heating at 75°C-90°C for 1 second to 5 minutes; iv) a condition of heating at 60°C-75°C for 30 seconds to 60 minutes; v) a condition of heating at 25°C-40°C for 10-120 minutes; vi) a condition of treating with protease for 10 seconds to 30 minutes; and vii) a condition of treating with DNase for 10 seconds to 30 minutes, followed by a binding reaction with a probe of 15-mer to 30-mer, and cfDNA with a stable double helix structure does not bind to the probe. Here, "room temperature" refers to ambient temperature, which can be 18°C-25°C. The probe may contain 15-mer to 30-mer nucleotides, or 20-mer to 25-mer nucleotides. Here, the probe may be designed to be able to complementarily bind to the gene sequence of unstable cfDNA.

[0100] When stable cfDNA is treated under any of the above conditions, it does not denature and does not bind complementary to the probe because the stable cfDNA forms a strong double strand in it. However, in the case of treatment under any of the above conditions, unstable cfDNA may bind to the probe. This instability is due to the fact that some nucleotides in cfDNA cannot form complementary binding, so cfDNA has an altered double helix structure.

[0101] In another aspect of the present invention, a method for detecting cfDNA having an unstable double helix structure from a sample that has not been amplified is provided. Here, the method comprises: a) a step of mixing a sample containing cfDNA with a positively charged substance; b) a step of separating the positively charged substance bound to the cfDNA; c) a step of mixing the mixture with a probe and a marker; d) removing the probe and marker that do not bind to the cfDNA; and e) detecting the marker.

[0102] Specifically, the method may include the step of mixing a sample containing cfDNA with a positively charged substance.

[0103] The sample may be a biological sample as described above. One embodiment thereof may be plasma or urine. In plasma or urine, cfDNA having a normal double helix structure and cfDNA having an unstable double helix structure may coexist. In addition, the positively charged substance may specifically be a positively charged nanowire. The positively charged nanowire is as described above.

[0104] Specifically, the above steps can be performed in the following order and under the following conditions. First, upon receiving the patient's plasma, urine, saliva, sputum, etc., centrifuge at 3,000 × g for 10 minutes at 4 ° C. Afterwards, the patient's plasma, urine, saliva, sputum, etc. are diluted in DPBS in a certain proportion. Then, in the case where the sample is plasma, 30 μl of plasma is mixed with 120 μl of distilled water (DW) and placed in a spin column (G type or Q type). Polypyrrole (PEI / Ppy) nanowires (20 μl) whose surfaces are modified with polyethyleneimine are added thereto and mixed at a speed of 1200 rpm for 20 minutes using a thermal mixer at room temperature.

[0105] Next, the method may include the step of isolating the positively charged species bound to the cfDNA.

[0106] The method of separating positively charged substances can be performed by centrifugation or applying negative pressure such as vacuum. cfDNA binds to positively charged substances. Therefore, when a sample mixed with a positively charged substance is placed in a spin column or a vacuum column and centrifuged or negative pressure is applied, positively charged substances such as nanowires will not pass through the filter in the column, while other components in the biological sample will pass through the filter. Therefore, positively charged substances bound to cfDNA can be separated by methods such as centrifugation or vacuum. In addition, in order to remove impurities from such separated nanowires, a washing step can be performed additionally one to three times. Conventional methods can be used appropriately for washing.

[0107] Specifically, the above steps can be performed in the following order and under the following conditions. The spin column is mounted on a device for applying vacuum, and then suction is performed at a pressure of 550 mBar. When washing, 400 μl of 1l0 is added thereto, and suction is performed again. The same process can be repeated once more.

[0108] In one embodiment, when temperature treatment is performed, the spin column that has completed aspiration can be placed in a heat block preheated to 95°C, incubated at 95°C for 1 minute, and then immediately removed from it. If a temperature denaturation step is not required, the sample can be skipped.

[0109] Next, the method may include the step of mixing the mixture with probes and markers.

[0110] As described above, the probe can be composed of 15-mer to 30-mer nucleotides, or 20-mer to 25-mer nucleotides. The sequence of the probe can be designed to be able to complementarily bind to the cfDNA with an unstable double helix structure to be detected. In particular, the cfDNA with an unstable double helix structure can be ctDNA derived from a tumor. In addition, the cfDNA with an unstable double helix structure can be designed to complementarily bind to a gene called a cancer biomarker.

[0111] In addition, the marker can be a substance detected under specific conditions, such as fluorescent protein or HRP. Here, the marker can include a substance that can bind to the probe. Specifically, when biotin binds to the marker, avidin or streptavidin can bind to the marker. In one embodiment, in order to increase the sensitivity of detection, the marker can be in the form of nanoparticles, in which several HRP molecules and several streptavidin molecules are aggregated.

[0112] Specifically, the above steps can be performed in the following order and under the following conditions: The probe (200 μl) and HRP / STR nanoparticle solution (200 μl) suitable for each experiment are placed in a spin column, respectively, and mixed at a speed of 850 rpm to 1,000 rpm for 30 minutes at room temperature.

[0113] Next, the method may include the step of removing probes and markers that are not bound to the cfDNA.

[0114] This is a step to remove probes that are not bound to cfDNA. 15-mer to 30-mer probes are also negatively charged and can bind to positively charged nanowires. Therefore, after the mixing reaction is completed, the residual probes and markers should be removed from the reaction solution. Here, the probes and markers can be removed by centrifugation or negative pressure. Here, cfDNA can strongly bind to positively charged substances (especially positively charged nanowires) compared to short-chain probes. In the step of removing probes and markers, cfDNA binds to positively charged substances and therefore will not be removed.

[0115] The above steps can be specifically performed in the following order. After performing the above steps, negative pressure is applied to the spin column and suction is performed. Then, 400 μl of 1×DPBS is added thereto and suction is performed again. The same process can be repeated once more.

[0116] Finally, the method may include the step of detecting the marker.

[0117] Depending on the marker used, the method of detecting the marker can be performed in different ways. The detection of the marker can be measured by a change in color, a change in UV absorbance, a change in fluorescence response, or an electrochemical change. For example, when HRP is used as a marker, the marker can be detected by observing a color development reaction. In addition, when the marker is a fluorescent protein such as GFP, the presence or absence of the marker can be detected by observing the detected light after irradiating the marker with light of a specific wavelength.

[0118] The implementation scheme of the above steps can be specifically carried out in the following order. 200 μl sodium acetate buffer (0.2M, pH 7.0), 50 μl TMB (10mM) and 50 μl H2O2 (0.1M) are added to the centrifugal column in sequence. Then, incubate for 3 minutes. Afterwards, centrifuge at a speed of 3,500 rpm to 5,000 rpm for 30 seconds. The solution collected in the collection tube is transferred to a 96-well with 200 μl per well, and then the absorbance in the wavelength range of 500 nm to 850 nm is measured using a UV / VIS spectrophotometer.

[0119] In addition, the method can further include an additional processing process to increase the difference in reactivity between cfDNA having a normal double helix structure and cfDNA having an unstable double helix structure and the probe. The additional processing process can be performed after obtaining the sample or after separating the cfDNA.

[0120] In addition, before step c), the method may further include the following steps: a step of denaturing the sample or cfDNA bound to the positively charged substance under any condition selected from the following: i) a condition of standing at room temperature for 1-10 minutes; ii) a condition of heating at 90°C-95°C for 1 second to 1 minute; iii) a condition of heating at 75°C-90°C for 10 seconds to 3 minutes; iv) a condition of heating at 60°C-75°C for 1-30 minutes; v) a condition of heating at 25°C-40°C for 5-60 minutes; vi) a condition of treating with protease for 1-10 minutes; vii) a condition of treating with DNase for 1-10 minutes. Such a denaturation process will not denature stable cfDNA, and will denature unstable cfDNA more unstably, so that unstable cfDNA can be more easily bound to the probe. After obtaining the sample, denaturation can be performed under the above conditions. In addition, denaturation can be performed after obtaining cfDNA bound to a positively charged substance. In addition, the temperature, protease, and time of treatment with DNase can be appropriately adjusted as long as the stable cfDNA is not denatured.

[0121] In one embodiment of the present invention, it has been determined that the cfDNA to be detected has an unstable double helix structure and can be detected using a target probe without a separate denaturation step ( Fig.31 ). In addition, it has been determined that even if cfDNA is subjected to a temperature treatment process, cfDNA with a stable double helix structure and cfDNA with an unstable double helix structure still show different binding reactions with the same probe ( Fig.32 and 33 ).

[0122] <Method for providing diagnostic information by detecting unstable cfDNA>

[0123] In another aspect of the present invention, a method is provided, which provides information for diagnosing or predicting cancer and infectious diseases by detecting cfDNA with an unstable double helix structure from an unamplified sample. Here, the method includes: a) mixing a sample containing cfDNA with a positively charged substance; b) separating the positively charged substance bound to cfDNA; c) mixing the mixture with probes and markers; d) removing probes and markers that do not bind to cfDNA; e) detecting markers; f) when the markers are detected, determining the presence of a cancer or infectious disease associated with a gene corresponding to cfDNA with an unstable double helix structure. Specifically, the method for detecting unstable cfDNA is as described above.

[0124] One embodiment of cancer used in the present specification may be any one selected from the group consisting of bladder cancer, bone cancer, blood cancer, breast cancer, melanoma, thyroid cancer, parathyroid cancer, bone marrow cancer, rectal cancer, laryngeal cancer, laryngeal tumor, lung cancer, esophageal cancer, pancreatic cancer, colorectal cancer, stomach cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, head and neck cancer, gallbladder cancer, oral cancer, colon cancer, perianal cancer, central nervous system tumor, liver cancer and colorectal cancer. In particular, the cancer may be stomach cancer, colorectal cancer, liver cancer, lung cancer or breast cancer.

[0125] In one embodiment, the sequence specifically present in the above-mentioned cancer cells may be a SNP present in the cancer cells. For gastric cancer, one embodiment of the sequence specifically present in cancer may be a mutation in the tumor suppressor genes p53 and PTEN. In addition, for colorectal cancer, one embodiment thereof may be a mutation in the APC and MSH2 genes. In addition, for liver cancer, since its main cause is infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), HBV or HCV nucleic acid may become a target. In addition, for lung cancer, mutations in the epidermal growth factor receptor (EGFR) gene may become a target; for breast cancer, mutations in the BRCA1 / 2 gene may be the main target. In addition, for cervical cancer, cfDNA derived from human papillomavirus DNA (HPV DNA) may become a target.

[0126] Another embodiment of unstable dsDNA can be at least one gene mutation selected from the group consisting of EGFR, KRAS, BRAF, TP53, PIK3CA, ROS1, RET, c-Met, PTEN, RB1, AR, BRCA, KIT, FGFR, IDH, ESR1, HER2, ALK-EML4 and TMPRSS2-ERG.

[0127] <Device for detecting unstable cfDNA>

[0128] In another aspect of the present invention, a device for detecting cfDNA having an unstable double helix structure at room temperature is provided, which comprises: a) a mixing part for mixing a sample containing cfDNA with positively charged nanowires; b) an acquisition part for removing samples other than the nanowires bound to the cfDNA; c) a reaction part for adding a biotin-bound probe capable of complementary binding to the cfDNA and nanoparticles comprising streptavidin and a marker to the nanowires bound to the cfDNA; d) a detection part for detecting the marker; e) an information processing part for determining that the sample contains cfDNA, wherein the cfDNA has a sequence complementary to the detection probe and has a double helix structure that is unstable at room temperature according to the detection of the marker.

[0129] <Diagnostic device using unstable cfDNA detection>

[0130] In another aspect of the present invention, a device is provided, which provides information for diagnosing or predicting cancer or infectious diseases by detecting cell-free DNA with an unstable double helix structure from a sample that has not been amplified. Here, the device includes: a) a mixing part for mixing a sample containing cfDNA with a positively charged nanowire; b) an acquisition part for removing samples other than the nanowires bound to the cfDNA; c) a reaction part for adding a probe bound to biotin that can complementarily bind to the cfDNA and nanoparticles containing streptavidin and a marker to the nanowire bound to the cfDNA; d) a detection part for detecting the marker; e) an information processing part for determining that the sample contains cfDNA, the cfDNA having a sequence complementary to the detection probe and having a double helix structure that is unstable at room temperature according to the detection of the marker.

[0131] The present invention is based on the difference in reactivity of stable cfDNA and unstable cfDNA with the probe due to the difference in their thermodynamic stability. Here, positively charged nanowires can bind to genomic DNA and cfDNA. However, due to the difference in their binding force and size, genomic DNA will be separated from the positively charged nanowires during washing. In addition, in one embodiment, when the nanowires are modified, they can be made to contain biotin. However, in the process of modifying the surface of the nanowires with cationic polymer polyethyleneimine, the biotin contained in the nanowires and exposed on the surface of the nanowires is bound to the cationic polymer. In addition, the nanowires are coated with cationic polymers, so the marker containing streptavidin does not bind to the nanowires. In addition, since the probe is also negatively charged, it can bind to the positively charged nanowires. However, it has been found that since the binding force of the probe is weaker than that of cfDNA, it will be removed during the washing process.

[0132] In addition, stable cfDNA and unstable cfDNA were treated with a probe (CP) that can specifically bind to a region containing a damaged DNA sequence and a probe (DP) that can specifically bind to a peripheral region that does not contain a damaged DNA sequence. As a result, it is possible to identify different binding reactions with the probes between cfDNA with an unstable double helix structure and cfDNA with a stable double helix structure. From these results, it was found that cfDNA with an unstable double helix structure can bind not only to a probe specific for a damaged DNA sequence, but also to a probe that can specifically bind to a peripheral DNA sequence. In addition, it has been confirmed that unstable cfDNA can be detected using only one probe.

[0133] Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

[0134] I. Experimental Methods and Production of Nanowires, Markers, and Probes

[0135] Experimental Methods 1. Methods for Detecting Unstable cfDNA

[0136] Step 1: Sample preparation and addition of nanowires

[0137] After receiving the patient's plasma, urine, saliva, sputum, etc., centrifuge at 3,000×g for 10 minutes at 4°C. Dilute the patient's plasma, urine, saliva, sputum, etc. in DPBS at a certain ratio. When it is plasma, 30μl of plasma is mixed with 120μl of DW and placed in a centrifugal column (G type or Q type). PEI / Ppy nanowires (20 microliters) are added thereto and mixed at 1200rpm for 20 minutes at room temperature using a thermal mixer.

[0138] Step 2: Vacuum / cleaning / temperature denaturation

[0139] The spin column is mounted on a vacuum suction device and then suction is performed at 550 mBar. 400 μl of 1× DPBS is added thereto and suction is performed again. The same process is repeated once more. The nanowire-DNA complex obtained only by the two-step process is captured in the spin column. If temperature denaturation is required, the spin column that has completed suction is placed in a heating block preheated to 95°C, incubated at 95°C for 1 minute, and then immediately removed from it. Samples that do not require a temperature denaturation step do not undergo this process.

[0140] Step 3: Add probe and HRP / STR NP

[0141] Place the probe (200 μl) and solution (200 μl) of HRP / STR NP suitable for each experiment in a spin column. Use a thermomixer to mix at 850 rpm-1,000 rpm for 30 minutes at room temperature. Install the spin column on a vacuum device and then aspirate. Add 400 μl of 1× DPBS and aspirate again. Repeat the same process once more.

[0142] Step 4: TMB response to detect gene mutations

[0143] After replacing the old collection tube with a new one, 200 μl of sodium acetate buffer (0.2M, pH 7.0) and 50 μl of H2O2 (0.1M) were added to the centrifugal column in sequence using a syringe pump. Then, incubate for 3 minutes. At the end of the incubation, the centrifugal column was centrifuged at a speed of 3500-5,000 rpm for 30 seconds. The solution collected in the collection tube was transferred to 96 wells at 200 μl per well, and then the absorbance in the wavelength range of 500-850 nm was measured using a UV / VIS spectrophotometer.

[0144] Preparation Example 1. Preparation of positively charged nanowires

[0145] like Figure 1 As shown in , nanowires with polyethyleneimine (PEI) conjugated to the surface as a cationic polymer were produced. The Q150T modular coating system (Quorum Technologies, UK) was used at 5 × 10 -3 One side of the anodic aluminum oxide (AAO) was coated with a layer of gold (Au) (thickness of about 150 nm) at 100 mbar and 50 mA for 600 seconds. In all electrochemical experiments, a potentiostat / galvanostat (BioLogic SP-150) equipped with a platinum wire counter electrode and a Ag / AgCl (3.0 M NaCl type) reference electrode was used to measure the AAO template coated with a gold (Au) layer.

[0146] To prepare nanowires with cationic polymer surface treated (PEI / Ppy NWs), electrochemical deposition was performed by applying chronoamperometry at 1.0 V (vs. Ag / AgCl) for 7 min with a 0.01 M pyrrole solution containing 0.01 M poly(4-styrenesulfonic acid) and 1 mg / ml biotin to the pores of the AAO template.

[0147] The resulting AAO template was washed several times with distilled water, immersed in a 2M sodium hydroxide (NaOH) solution for 3 hours, and then placed in a Bioruptor UCD-200 (Diagenode) for ultrasonic treatment to obtain a self-standing Ppy NW doped with biotin molecules. Then, 30mM N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and 6mM N-hydroxysuccinimide (NHS) were added to the resulting nanowires to activate the carboxylic acid (-COOH) group. Subsequently, after adding the PEI solution, the reaction was allowed to proceed at room temperature for 1 hour and washed with water. The surface-bound nanostructures (PEI / Ppy NW) with polyethyleneimine were dispersed in deionized water and kept at room temperature until use.

[0148] Using this preparation method, each polypyrrole (Ppy) nanowire was released from the AAO template after selective dissolution of the AAO template, and cationic branched polyethyleneimine (PEI) (25 kDa) was coupled to the nanowire via biotin-streptavidin interaction.

[0149] Preparation Example 2. Preparation of Nanoparticles Surface Treated with Cationic Polymers (PEI / Ppy NPs)

[0150] To prepare surface-conjugated polyethyleneimine nanoparticles (PEI / Ppy NPs), 0.2 g of polyvinylpyrrolidone (PVP) was added to 12.5 ml of triple distilled water and stirred for 30 minutes. Then, 65 μl of pyrrole was added thereto, and the resultant was further stirred for 10 minutes. Then, 0.5 ml of FeCl with a concentration of 0.75 g / ml was added thereto. 3 The solution was added and the reaction was allowed to proceed for 10 minutes. Thereafter, 20 ml of an aqueous hyaluronic acid solution (400 mg / 20 ml) was added thereto, and the resultant was stirred for 3 hours.

[0151] Triple distilled water was dialyzed for 2 days using a membrane with a 50,000 MWCO pore size. Centrifuged at 1200 rpm for 3 minutes to remove large-sized particle aggregates, and the resulting product was lyophilized. 200 μg of Ppy-HA-NP was added to 1 ml of triple distilled water. Then, 100 mM EDC / 50 mM NHS solution was added thereto and reacted for 45 minutes to activate the carboxyl group of hyaluronic acid. Centrifuged at 15,000 rpm for 10 minutes to remove the supernatant, during which time two washes were performed.

[0152] Subsequently, 100 mg / ml of PEI solution (solvent: 0.2 M sodium bicarbonate) was added thereto and reacted overnight at 4°C. Then, the supernatant was removed by centrifugation at 15,000 rpm for 10 minutes, and the resultant was then kept in triple distilled water. The shape of the nanoparticles (PEI / Ppy NPs) conjugated with polyethyleneimine on their surfaces was observed using a scanning electron microscope. Figure 5 As shown, the shapes of nanoparticles (NPs) with an average size of 50 nm bound to PEI were examined using scanning electron microscopy images (scale bar, 200 μm).

[0153] Preparation Example 3. Preparation of polypyrrole nanoparticles labeled with HRP and streptavidin

[0154] To prepare nanoparticles containing HRP and streptavidin, 0.5 g of polyvinylpyrrolidone (PVP) was added to 12.5 ml of triple distilled water and stirred for 30 minutes. Then, 65 μl of pyrrole was added thereto and stirred for further 10 minutes. Thereafter, 0.5 ml of FeCl at a concentration of 0.75 g / ml was added thereto. 3 Then, 20 ml of a hyaluronic acid aqueous solution (400 mg / 20 ml) was added thereto, and the resultant was stirred for 3 hours.

[0155] Triple distilled water was dialyzed for 2 days using a membrane with a 50,000 MWCO pore size. Centrifuge at 1200 rpm for 3 minutes to remove large-sized particle aggregates, and the resultant was then freeze-dried. 200 μg of Ppy-HA-NPs containing polypyrrole and hyaluronic acid were added to 1 ml of triple distilled water. Then, 100 mM EDC / 50 mM NHS solution was added thereto and reacted for 45 minutes to activate the carboxyl groups of the hyaluronic acid. Centrifuge at 15,000 rpm for 10 minutes to remove the supernatant, during which two washes were performed. 1 mg HRP and 1 mg streptavidin were added to the Ppy-HA-NPs and mixed at a temperature of 4 ° C. Subsequently, centrifuge at 15,000 rpm for 10 minutes to remove the supernatant, and the resultant was then kept in triple distilled water. The shape of the nanoparticles (HRP / st-labeled NPs) with HRP and streptavidin on their surfaces was observed using a scanning electron microscope ( Figure 4 ).

[0156] Preparation Example 4. Preparation of probe

[0157] A probe is prepared to detect cfDNA with an unstable double helix structure. The preparation of the probe varies depending on the cfDNA to be detected. Here, the probe is prepared in a form in which biotin is bound thereto. The probe is divided into two types, namely, a first probe (hereinafter referred to as CP) that complementarily binds to a region containing damaged DNA that causes an unstable double helix structure, and a second probe (hereinafter referred to as DP) that complementarily binds to a peripheral region of the damaged DNA.

[0158] II. Detection of cfDNA using two probes

[0159] Example 1. Analysis of HPV-derived cfDNA in urine

[0160] In order to isolate cell-free HPV DNA from urine samples, the surface of the nanowire (PEI / Ppy NW) was treated with the cationic polymer produced in Example 1 at 10 μg / ml, and then added to 200 μl of urine obtained from an HPV-positive patient and mixed at room temperature for 30 minutes. The isolated cfDNA was denatured at 95°C for 1 minute. Then, 1 pM of the first probe (CP) and the second probe (DP) with biotin bound to their ends were added and reacted at 37°C for 1 hour. Then, polypyrrole nanoparticles labeled with horseradish peroxidase (HRP) and streptavidin (hereinafter referred to as HRP / st-labeled NPs) were added to the sample and incubated at 37°C for 30 minutes.

[0161] Subsequently, 25 μl of 10 mM 3,3',5,5'-tetramethylbenzidine (TMB), 25 μl of 0.1 M H2O2, and 200 μl of 0.2 M sodium acetate trihydrate buffer (pH 5.0) were added to the HPV-derived cfDNA. The reaction was carried out with the DNA sample in the dark at room temperature for 3 minutes. In order to identify the correlation between the concentration and absorbance of HPV DNA, UV-Vis (ultraviolet visible) detection was performed using a DU 730 UV-Vis spectrophotometer (Beckman Coulter, USA) at a wavelength of 652 nm.

[0162] The results showed that it was possible to identify the results obtained by amplifying the colorimetric signal to such an extent that the colorimetric signal could be visually observed. Table 2 below shows the probe sequences used for detecting HPV.

[0163] Table 2

[0164] Probe Label sequence SEQ ID NO HPV 16CP 5'-biotin-GAG GAG GAG GAT GAA ATA GAT GGT-3' SEQ ID NO:1 HPV 16DP 5'-TTG GAA GAC CTG TTA ATG GGC-Biotin-3' SEQ ID NO:2 HPV 18CP 5'-Biotin-CAC ATT GTG GCA CAA TCT TTT A-3' SEQ ID NO:3 HPV 18DP 5'-GCC ATA TCG CTT TCA TCT GT-Biotin-3' SEQ ID NO:4 EGFR 19 deletion CP 5'-GGAATTAAGAGAAGCAACATCTCC-3' SEQ ID NO:5 EGFR 19-deletion DP 5'-AACCTCAGGCCCACCTTTT-3' SEQ ID NO:6 EGFR 21L858R CP 5'-CCAGGAACGTACTGGTGAAAA-3' SEQ ID NO:7 EGFR 21L858R DP 5'-GGAAGAGAAAGAATACCATGCA-3' SEQ ID NO:8

[0165] In addition, a PCR-free colorimetric assay was performed to evaluate urine samples from HPV-positive cervical cancer patients (HPV16(+) and HPV18(+)) and HPV-negative healthy controls (HPV-). PBS was used as a negative control. Fig.12 As shown, the target HPV DNA separated using nanowires was mixed with CP and DP, and HRP / st-labeled NPs were added thereto. Then a color change was observed. The separated cfDNA was analyzed by denaturation at 95°C for 1 minute.

[0166] A total of 24 HPV-positive and HPV-negative urine samples were collected and tested. Results showed that cfDNA isolated from urine of HPV-positive cervical cancer patients (HPV16(+) and HPV18(+)) and HPV-negative healthy controls (HPV-) had different absorbance values ​​( Fig.13 Here, probes that specifically bind to HPV16 or HPV18 were used and denatured at 95°C for 1 min to analyze the isolated cfDNA.

[0167] In addition, no reaction was observed when non-HPV probes such as EGFR 19 and EGFR 21 were used. And it was found that the color change and UV absorbance change were caused by type-specific binding between the target HPV and its complementary probe ( Fig.14 ). Here, denaturation at 95°C for 1 min was performed to analyze the isolated cfDNA.

[0168] Example 2. Identification of EGFR gene mutations by DNA analysis of lung cancer cell lines

[0169] Example 2.1 Identification of cfDNA size by nanowires

[0170] DNA ladders of low range (10bp-100bp), mid range (100bp-2kb), and high range (3.5kb-21kb) were added to the plasma of normal subjects, and then the capture efficiency was examined using nanowires (PEI / Ppy NW). It was determined that the nanostructures can effectively capture small size DNA ( Fig.15 ).

[0171] Using nanowires, genes were obtained from H1975 (a cell line with EGFR exon 20 T790M, 21 L858R gene mutations), HCC2279 (a cell line with EGFR exon 19 deletion gene mutations), and A549 cell lines (a cell line without EGFR exon gene mutations). Then, genomic DNA (gDNA) that was not sonicated and fragmented DNA (fDNA) that was sonicated were used to compare the efficiency of nanostructure capture. It was found that when using nanostructures, the capture efficiency of fDNA was observed to be higher than that of gDNA ( Fig.16 and 17 ).

[0172] Example 2.2 Detection of EGFR mutations by analyzing unstable cfDNA

[0173] A PCR-free colorimetric assay was performed using the nanostructures to separate gDNA and fDNA from the EGFR exon 20, 21-positive H1975 cell line, the EGFR exon 19-positive HCC2279 cell line, and the A549 cell line without EGFR gene mutation. Afterwards, probes specific to EGFR exons 19, 20, and 21 were added thereto and mixed. HRP / st-labeled NPs were then added thereto, and a color change was observed. The results confirmed that the nanostructures were more effective in capturing small-sized DNA (i.e., fDNA) and had significant color changes and UV-Vis spectral changes compared to gDNA ( Fig.18 and Fig.19 ).

[0174] Example 2.3 Detection of cfFNA using fluorescent dyes

[0175] In order to identify whether gene mutations can be detected from plasma samples of lung cancer patients using target probes, this possibility was examined by using fluorescent dyes. H1975 (a cell line with EGFR exon 20T790M, 21L858R gene mutations), HCC2279 (a cell line with EGFR exon 19 deletion gene mutations), and A549 cell lines (a cell line without EGFR exon gene mutations) were sonicated, and the obtained DNA (fDNA) was captured by nanowires. Afterwards, the fDNA was denatured at 95°C for 1 minute and then mixed with probes specific for EGFR19, 20, and 21. Gene mutations in cell lines were examined using fluorescent dyes (Alexa488) bound to the probes.

[0176] The results showed that in the A549 cell line, no fluorescence was detected when reacting with probes specific for EGFR 19 and 21. However, in H1975 and HCC2279, fluorescence was detected with probes specific for EGFR 19 and 20 ( Fig. 20 ).

[0177] Example 2.4 Determination of the limit of detection (LOD) of EGFR gene mutations by in vitro cfDNA analysis

[0178] H1975 (a cell line with EGFR exon 20 T790M, 21L858R gene mutations) and HCC2279 (a cell line with EGFR exon 19 deletion gene mutations) were sonicated, and the obtained fragmented DNA was then added to healthy human plasma (200 μl) at different concentrations (fDNA; 1ag ml-1 to 100ng ml-1). Then, the fDNA was captured by the nanowire. Thereafter, it was denatured at 95°C for 1 min, and the limit of detection (LOD) was checked using probes specific for EGFR exon 19Del and EGFR exon 20T790M and HRP / st-labeled NPs. The results identified that when a 3-fold signal-to-noise ratio was applied, fDNA of the HCC2279 cell line could be detected up to 10ag ml-1, while fDNA of the H1975 cell line could be detected up to 100ag ml-1 ( Fig.21 and 22 ).

[0179] Example 3. Identification of mutations in EGFR, KRAS and ALK-EML4 genes by analyzing cfDNA present in plasma or cerebrospinal fluid samples (lung cancer patients)

[0180] Example 3.1 Identification of EGFR gene mutations by analyzing unstable cfDNA

[0181] In one embodiment, cfDNA is isolated from a body fluid sample of a cancer patient using the nanostructures and genetic mutations are detected by hybridization with probes and subsequent binding to HRP / ST-labeled nanoparticles, which takes a total of 60 minutes ( Figure 6 ).

[0182] To isolate cfDNA from plasma or cerebrospinal fluid samples, 200 μl of EGFR-positive patient plasma and 5 μg / ml PEI / Ppy NW were mixed at room temperature for 30 minutes to extract cfDNA from patient plasma. The size of cfDNA extracted from 200 μl of lung cancer patient plasma using PEI / Ppy NW was analyzed by Bioanalyzer ( Fig.23 ). In general, similar to the fact that the average size of cfDNA associated with cancer is known to be 166 bp, in this bioanalyzer experiment, it was determined that the results showed that a peak was observed at 169 bp for cfDNA extracted from the plasma of lung cancer patients using PEI / Ppy NW.

[0183] Subsequently, the DNA captured in the nanostructure was denatured at 95°C for 1 min, 1 pM of biotin-conjugated CP and biotin-conjugated DP were added thereto, and reacted for 1 h at 37°C. Then, polypyrrole nanoparticles labeled with HRP and streptavidin (HRP / st-labeled NPs) were added to the sample and reacted at 37°C for 30 min.

[0184] Thereafter, 25 μl of 10 mM TMB, 25 μl of 0.1 M H2O2 and 200 l of 0.2 M sodium acetate trihydrate buffer (pH 5.0) were added to the reaction solution. Then, the reaction was allowed to take place at room temperature for 3 minutes under light shielding. Afterwards, UV-Vis detection was performed using a DU 730 UV-Vis spectrophotometer (Beckman Coulter, USA) at a wavelength of 652 nm.

[0185] The color difference caused by the oxidation reaction of TMB was observed by naked eyes. The probe sequences used to capture and detect mutant cfDNA with EGFR exon 19 deletion, 20T790M and 21L858R are shown in Table 3 below.

[0186] Table 3

[0187]

[0188] In this experiment, EGFR exon 19 deletion-probe 1 (target specific), EGFR exon 20 T790M-probe 2 (target specific), and EGFR exon 21 L858R-probe 2 (target specific) were used as CP and DP unless otherwise specified. The results determined that the isolated cfDNA with EGFR mutations was detected according to the detection probe specificity ( Fig.24 and 25 It was determined that when cfDNA captured from the plasma of lung cancer patients with EGFR exon 19 deletion, 20T790M, or 21L858R gene mutations was reacted with probes targeting EGFR exon 19 deletion, 20T790M, or 21L858R and HRP / streptavidin-conjugated nanoparticles (NPs), color changes and UV-Vis spectral changes occurred when probes targeting the same gene mutations as those in the patient tissue were used ( Fig.24 ).

[0189] Additionally, cfDNA captured from a lung cancer patient with another EGFR mutation was reacted with the same probes targeting EGFR 19, 20, and 21. The results confirmed that when probes targeting the same gene mutation as in the patient tissue were used, color changes and UV-Vis spectral changes occurred ( Fig.25 ).

[0190] Example 3.2 Identification of KRAS and ALK-EML4 gene mutations by cfDNA analysis

[0191] In addition, similar to EGFR, to analyze mutations in KRAS and ALK-EML4 genes, cfDNA was isolated from plasma or cerebrospinal fluid samples, and the captured DNA was then denatured at 95°C for 1 minute. 1 pM of biotin-conjugated CP and biotin-conjugated DP were added, and the reaction was allowed to proceed at 37°C for 1 hour. Then, polypyrrole nanoparticles labeled with HRP and streptavidin were added to the sample and reacted at 37°C for 30 minutes.

[0192] The results showed that cfDNA isolated from the plasma of lung cancer patients with KRAS exon 2 gene mutation and ALK-EML4 fusion gene mutation could be identified as reactive to the probe for KRAS exon 2 and the probe for ALK-EML4, and thus showed color changes and UV absorption matching those of the patient's tissue. Figure 26-28 Table 4 below shows the methods for detecting KRAS exon 2 mutations ( Fig.26 and 27 ) and ALK-EML4 variants 1 and 3 ( Fig.28 )’s cfDNA probes (CP and DP).

[0193] Table 4

[0194]

[0195] Example 3.3 Identification of non-specific reactions by temperature denaturation

[0196] Each of the three probes in Table 3 (including target-specific or target-nonspecific probes) was added to the plasma of lung cancer patients with EGFR exon 19 deletion and 20T790M gene mutation and mixed. The results showed that after DNA denaturation at 95°C for 1 minute, all probes used (i.e., whether the probes were target-specific or target-nonspecific) showed changes in color and UV absorbance only for EGFR exon 19 deletion and 20T790M, similar to tissues, making it possible to identify specific gene mutations ( Fig.29 ). However, for EGFR exon 21, no changes in color and UV absorption were observed regardless of the probe type. This suggests that unstable cfDNA responds to specific probes for EGFR exon 19 and EGFR exon 20 mutations, allowing analysis of gene mutations.

[0197] In addition, cfDNA was obtained from the plasma of lung cancer patients without EGFR gene mutations. The binding between probes specific for EGFR 19, 20, and 21 mutations and unstable cfDNA was examined after denaturation at 95°C for 1 minute and 10 minutes, respectively. The results showed that in the case of denaturation at 95°C for 1 minute, no color change and UV absorbance change were observed in lung cancer patients without EGFR gene mutations. However, in the case of denaturation at 95°C for 10 minutes, color change and UV absorbance change caused by nonspecific hybridization were observed in all probes specific for EGFR exons 19, 20, and 21 ( Fig.30 ).

[0198] In addition, cfDNA was captured by nanowires from the plasma of other lung cancer patients with EGFR 19 deletion and 20T790M gene mutation and normal subjects. Then, its hybridization reactivity with probes specific for EGFR19, 20, and 21 was examined by denaturation at 95°C for 0, 1, and 10 minutes, respectively. The results are shown in Figure 2. Figure 31-33 As shown, in normal subjects without EGFR gene mutation, denaturation at 95°C for 0 min ( Fig.31 ) and 1 minute ( Fig.32 ), no color change and UV absorbance change were observed. However, by denaturing at 95°C for 10 min ( Fig.33 ), color changes and changes in UV absorbance due to nonspecific hybridization were observed in all probes specific for EGFR exons 19, 20, and 21. Therefore, it was possible to determine that gene mutations in cfDNA could be analyzed without denaturation.

[0199] Example 4. Identification of the accuracy of a method for detecting unstable cfDNA by analyzing samples from lung cancer patients

[0200] In one embodiment of the present invention, it has been determined that the results obtained by analyzing the specificity and sensitivity of gene mutations in cfDNA obtained from the plasma of 151 lung cancer patients match the results of gene mutations in the patient's cancer tissue ( Fig.34 ). A probe specific for EGFR exon 19Del was added to cfDNA of lung cancer patients without EGFR mutation (EGFR wild type), lung cancer patients with EGFR exon 19 deletion (Del), and lung cancer patients with EGFR exon 21L858R, and the resulting gene mutations were detected by analyzing the absorbance value of UV spectrum (ΔOD, 500nm-650nm). The results determined that the obtained results showed 98.4% consistency with the results of gene mutations in the patient's cancer tissue ( Fig.35 and 36 ).

[0201] In addition, a probe specific for EGFR exon 21L858R was added to cfDNA of lung cancer patients without EGFR mutation, lung cancer patients with EGFR exon 19 deletion (Del), and lung cancer patients with EGFR exon 21L858R, and the resulting gene mutations were detected by analyzing the absorbance value of UV spectrum (ΔOD, 500nm to 650nm). The results determined that the obtained results showed 98.0% consistency with the results of gene mutations in the patient's cancer tissue ( Fig.37 and 38 ).

[0202] Example 5. Detection of cfDNA using positively charged nanoparticles

[0203] Similar to the above EGFR experiment, in order to isolate cfDNA from plasma samples of lung cancer patients with EGFR 19 deletion (Del) gene mutation, the PEI-conjugated nanoparticles (PEI-Ppy NP, 5 μg / ml) prepared in the preparation example were added to 200 μl plasma of EGFR-positive patients and mixed at room temperature for 30 minutes. Thereafter, the captured DNA was either undenatured or denatured at 95°C for 1 minute. Then, biotin-conjugated CP and biotin-conjugated DP were added in an amount of 1 pM and incubated at 37°C for 30 minutes. Thereafter, HRP / st-labeled NPs were added to the sample and reacted at 37°C for 15 minutes. The absorbance changes in the UV-Vis spectrum were observed only in probes specific for EGFR 19, as identified by the oxidation reaction of TMB ( Fig.39 ).

[0204] Example 6. Identification of EGFR gene mutations by analyzing cfDNA present in plasma

[0205] Using H1975 cell line (having EGFR exon 20T790M and 21L858R gene mutations), fDNA was prepared by ultrasonic treatment. Thereafter, the surface-bound polylysine nanostructure (PLL / Ppy NW) prepared by the method in the preparation example was added thereto, and then a PCR-free colorimetric assay ( Fig.40 ). The fDNA of the EGFR exon 20, 21-positive H1975 cell line was separated using nanowires. Then, probes specific to EGFR exons 19, 20, and 21 were added thereto and allowed to react. Thereafter, HRP / st-labeled NPs were added thereto, and a color change was observed. Fig.40 As shown, clear color changes and UV-Vis spectral changes can be identified only in EGFR 20 and EGFR 21.

[0206] III. Detection of unstable cfDNA using a single probe

[0207] Example 7. Detection of unstable cfDNA using only probes that bind only to damaged parts

[0208] cfDNA was isolated from plasma samples of lung cancer patients. Then, two types of probes, CP and DP, were mixed and used to detect gene mutations. Figures 41-43 As shown, in order to detect EGFR exon 19 deletion, exon 20T790M and exon 21L858R gene mutations, probes CP_1 (EGFR exon 19 deletion), CP2 (EGFR exon 20T790M) and CP2 (EGFR exon 21L858R) specific to cfDNA containing the above regions were mixed with DP.

[0209] However, if Fig.44 As shown, even in the case of adding CP alone without adding DP, the same EGFR exon 19 deletion gene mutation result (EGFR exon 19 deletion) can be identified in the patient's cancer tissue.

[0210] IV. Detectability of unstable cfDNA based on sample processing methods

[0211] Example 8. Identification of the detectability of unstable cfDNA based on DNase or RNase treatment

[0212] Plasma samples from lung cancer patients were first treated with DNase or RNase, and then cfDNA was isolated from them using nanostructures to identify gene mutations. Fig.45 As shown, after the plasma of a lung cancer patient with EGFR exon 19 deletion gene mutation was pretreated with RNase A, cfDNA was obtained from it using nanowires and then reacted with a probe specific for EGFR exon 19del. The results identified the same UV-vis peak of the EGFR exon 19 deletion gene mutation as the patient's cancer tissue, similar to the control (control cfDNA). However, it was determined that after pretreatment with DNase I, no UV absorption was observed due to possible degradation of cfDNA ( Fig.45 ).

[0213] Similarly, if Fig.46 As shown, after pre-treating the plasma of a lung cancer patient with EGFR exon 20T790M gene mutation with RNase A, cfDNA was obtained from it using the nanowire and then reacted with a probe specific for EGFR exon 20T790M. The results identified the UV absorption of the same EGFR exon 20T790M gene mutation as that of the patient's cancer tissue. However, it was confirmed that no UV absorption was observed after treatment with DNase I ( Fig.46 ). This is because cfDNA may be degraded by the addition of DNase I to the patient's plasma.

[0214] V. Detectability of cfDNA by Markers

[0215] Example 9. Identification of detectability of cfDNA using HRP / streptavidin complex

[0216] like Fig.47 As shown, cfDNA was extracted from the plasma of lung cancer patients with EGFR exon 19 deletion and exon 20T790M gene mutation and reacted with probes specific to EGFR exons 19Del, 20T790M and 21L858R and HRP / st-labeled NPs, and then the results of EGFR exon 19 deletion and exon 20T790M gene mutation, which were the same as those in the patient's cancer tissue, were identified by UV absorption and color change.

[0217] However, if Fig.48 As shown, when cfDNA was extracted from the plasma of the same patient and then reacted with probes specific for EGFR exons 19Del, 20T790M, 21L858R and HRP-streptavidin complex (where HRP and streptavidin bind to each other at a ratio of 1:1) instead of HRP / st-labeled NPs, the results of gene mutations were completely different from those of the patient's cancer tissues. It was found that when HRP / streptavidin complex was used instead of HRP / streptavidin-labeled nanoparticles (NPs), inaccurate gene mutation results were observed due to increased nonspecific binding.

[0218] like Fig.49As shown, cfDNA was extracted from the plasma of 5 lung cancer patients with EGFR exon 19 deletion and exon 20T790M gene mutation, and then the cfDNA was reacted with probes specific to EGFR exons 19Del, 20T790M and 21L858R, and NPs labeled with HRP / st; and the results obtained by reacting with probes specific to EGFR exons 19Del, 20T790M and 21L858R, and HRP / streptavidin complex (where HRP and streptavidin are bound to each other in a ratio of 1:1) instead of HRP / st-labeled NPs were analyzed. The results showed that due to the improvement of reaction specificity caused by the combination of probes and HRP / st-labeled NPs, the cfDNAs obtained by the nanowires showed UV absorption matching that of cancer tissues. Furthermore, it was determined that HRP / st-labeled NPs played an important role in determining gene mutations even in the plasma of five lung cancer patients with EGFR exon 20T790M and 21L858R gene mutations.

[0219] VI. Detection of cfDNA using a complex of probe and marker

[0220] Example 10. Detection of gene mutation using probes conjugated to HRP / st-labeled NPs

[0221] like Fig.50 As shown, instead of reacting cfDNA with probes and HRP / st-labeled NPs sequentially, HRP / st-labeled NPs were first combined with probes specific to EGFR exons 19Del, 20T790M, and 21L858R to produce a binding product in the form of a probe-HRP marker, and cfDNA was reacted with this binding product; the results showed that the same genotype as that of the cancer tissue was detected even in the pleural fluid of lung cancer patients with EGFR exon 20T790M and 21L858R gene mutations.

[0222] VII. Hybrid Sequence-Based cfDNA Detection

[0223] Example 11. Detection of unstable cfDNA by simultaneous mixing of probes and markers

[0224] like Fig.51 As shown in the figure, instead of reacting cfDNA with CP and DP probes and HRP / st-labeled NPs sequentially, cfDNA was mixed with probes and HRP / st-labeled NPs at once and reacted; the results showed that the same genotype as that of cancer tissue was detected even in the plasma of lung cancer patients with EGFR exon 20T790M and 21L858R gene mutations. In addition, as Fig.52 As shown, cfDNA was mixed with CP and DP probes and HRP / st-labeled NPs at once and the reaction was allowed to occur. The results showed that even in the plasma of lung cancer patients with ALK-EML4 fusion and ALK point mutation (I1171N / T) gene mutations, the same genotype as that of cancer tissue was detected.

[0225] VIII. Detection of unstable cfDNA based on sample denaturation conditions

[0226] Example 12. Detection of cfDNA according to temperature conditions after sample denaturation

[0227] Experiments were performed to determine whether unstable cfDNA and stable cfDNA can be distinguished based on sample denaturation conditions. Specifically, plasma collected from normal subjects and lung cancer patients (0208-343, 20190311_LC#1, from tissue: E19del) was subjected to various denaturation conditions using a probe capable of detecting EGFR 19 deletion, and then examined whether unstable cfDNA and stable cfDNA could be distinguished from each other.

[0228] As the probe, the probe ggaattaaga gaagcaacat ctcc (SEQ ID NO: 9) capable of detecting EGFR exon 19 deletion was used. Here, a biotin-bound probe was used. PEI / Ppy nanowires were used, and nanoparticles aggregated with HRP / streptavidin were used as markers.

[0229] Specifically, before separating cfDNA with nanowires, the samples were treated under different conditions as follows. The samples were heated at 30°C for 15 minutes and 0 minutes, respectively. In addition, the samples were heated at 60°C for 5 minutes and 0 minutes, respectively. In addition, the samples were heated at 95°C for 1 minute and 0 minutes, respectively. The other steps were as follows: Figure 8 The method shown is carried out.

[0230] Results showed that in normal subjects without EGFR 19 deletion mutation, no unstable cfDNA was detected under any denaturing conditions ( Fig.54 However, in patients with E19del, it was determined that unstable cfDNA was detected under all different denaturation conditions ( Fig.55 ). Based on these results, the presence or absence of unstable cfDNA may provide information about the occurrence of E19del mutation in lung cancer patients.

[0231] Example 13. Detection of unstable cfDNA from cell lines according to temperature conditions

[0232] As with samples obtained from humans, experiments were performed to identify mutation locations present in cell lines. Specifically, fDNA having a size similar to cfDNA was obtained from each of HCC2279 (exon 19Del), HCC827 (exon 19Del), H1975 (T790M, L858R), and A549 (EGFR wild type). Specifically, fDNA was obtained by the method of Example 2.

[0233] The results showed that under denaturing conditions of heating at 95°C for 1 minute and 0 minutes, only unstable cfDNA specifically bound to the probe and was detected in a state bound to the marker ( Fig.56 ). From these results, it was confirmed that the presence or absence of unstable cfDNA can also be identified in samples obtained from cell lines.

[0234] Example 14. Detection of unstable cfDNA relying on DNase treatment

[0235] To identify whether the difference between unstable cfDNA and stable cfDNA depends not only on temperature conditions but also on DNA degrading enzymes, unstable cfDNA and stable cfDNA were treated with DNase and then examined for reactivity with the probe. Here, the samples were not denatured using high temperatures.

[0236] Specifically, fDNA obtained from HCC2279 (exon 19Del), HCC827 (exon 19Del), H1975 (T790M, L858R), and A549 (EGFR wild-type) using PEI / Ppy nanowires was suspended in PBS and then treated with 1 μl of DNase. The treatment was performed at 37°C for 30 minutes, and it was found that unstable cfDNA and stable cfDNA had different reactivity with the probe ( Fig.57 ). In addition, it has been confirmed that the same effect is shown even when DNase is treated at 37°C for 60 minutes ( Fig.58 ). Based on these results, it can be determined that stable cfDNA is not easily degraded by DNase enzymes.

[0237] However, when treated with 1 μl or 2 μl DNase at 37°C for 120 min, it was found that when the DNase treatment time was prolonged or the amount of DNase was increased, the stable cfDNA also responded to the probe ( Fig.59 ). From these results, the stability difference between unstable cfDNA and stable cfDNA can be determined.

[0238] In addition, to determine whether the difference between unstable cfDNA and stable cfDNA depends on DNase activity, Fig.60 Results obtained with 1 μl or 2 μl of DNase treatment for 120 min at 24°C are shown. The results show that although the activity of the enzyme is reduced at 24°C, it has been determined that unstable cfDNA and stable cfDNA differ in their reactivity with the probe ( Fig.60 ).

[0239] Furthermore, to determine whether the difference between unstable cfDNA and stable cfDNA depends on DNase activity, Fig.58 Results obtained with 1 μl or 2 μl DNase treatment for 120 min at 3°C ​​are shown. The results show that although the activity of the enzyme was reduced at 3°C, it was determined that unstable cfDNA and stable cfDNA differed in their reactivity with the probe ( Fig.61 ).

Claims

1. A use of a positively charged substance, a probe and a marker for in vitro detection of cell-free DNA (cfDNA) with an unstable double helix structure from a subject sample without amplification in the preparation of a preparation for diagnosing or predicting cancer, wherein: The detection is carried out according to the following method, including: a) mixing a sample containing cfDNA with a positively charged substance; b) separating positively charged substances bound to the cfDNA; c) mixing the resulting mixture with probes and markers; d) removing probes and markers that do not bind to the cfDNA; and e) detection markers; Among them, cfDNA with an unstable double helix structure is thermodynamically unstable compared to cfDNA with a stable double helix structure.

2. The use according to claim 1, wherein the cfDNA having an unstable double helix structure is characterized by: i) has a lower Tm value compared to cfDNA with a stable double helical structure; or ii) Denaturation occurs under conditions where cfDNA with a stable double helix structure does not denature.

3. The use according to claim 1, characterized in that Under any of the following conditions, cfDNA with an unstable duplex structure can bind to a 15-mer to 30-mer probe that can bind complementary to cfDNA: i) leaving at room temperature for 1 to 120 minutes; ii) heating at 90°C-95°C for 1 second to 3 minutes; iii) heating at 75°C to 90°C for 1 second to 5 minutes; iv) heating at 60° C. to 75° C. for 30 seconds to 30 minutes; v) heating at 25°C to 40°C for 10 to 120 minutes; vi) treatment with a protease for 1-30 minutes; and vii) Conditions of treating with DNase for 1-30 minutes.

4. The use according to claim 1, characterized in that The cfDNA with an unstable double helix structure is circulating tumor DNA.

5. The use according to claim 1, characterized in that The cfDNA with an unstable double helix structure has damaged nucleic acid sequences that are not present in normal cells.

6. The use according to claim 5, characterized in that The damaged nucleic acid sequence not present in normal cells comprises any one structural abnormality selected from the group consisting of deletion, duplication, inversion, translocation, mismatch and single nucleotide variation (SNV).

7. The use according to claim 1, characterized in that Prior to step c), the method further comprises binding the sample or cfDNA to a positively charged substance for denaturation under any one of the following conditions: i) leaving at room temperature for 1-10 minutes; ii) heating at 90°C-95°C for 1 second to 1 minute; iii) heating at 75°C to 90°C for 10 seconds to 3 minutes; iv) heating at 60°C to 75°C for 1 to 30 minutes; v) heating at 25°C to 40°C for 5 to 60 minutes; vi) treatment with a protease for 1-10 minutes; and vii) Conditions of treating with DNase for 1-10 minutes.

8. The use according to claim 1, characterized in that The probe consists of 15-mer to 30-mer nucleotides.

9. The use according to claim 8, characterized in that The probe is in a form bound to biotin.

10. The use according to claim 1, characterized in that The markers include horseradish peroxidase (HRP) or fluorescent protein.

11. The use according to claim 10, characterized in that The marker also includes any one selected from the group consisting of avidin, streptavidin and a combination thereof.

12. The use according to claim 1, characterized in that The marker is a nanoparticle comprising a conductive polymer; hyaluronic acid; avidin or streptavidin; and horseradish peroxidase (HRP) or a fluorescent protein.

13. The use according to claim 1, characterized in that The positively charged substance is a positively charged nanowire.

14. The use according to claim 13, characterized in that The nanowires include a conductive polymer.

15. The use according to claim 14, characterized in that The nanowires also contain biotin.

16. The use according to claim 14, characterized in that The conductive polymer is any one selected from the group consisting of poly(acetylene), poly(pyrrole), poly(thiophene), poly(p-phenylene), poly(3,4-ethylenedioxythiophene), poly(phenylene sulfide), poly(p-phenylenevinylene) and polyaniline.

17. The use according to claim 1, characterized in that The sample is selected from any one of the group consisting of urine, cerebrospinal fluid, plasma, blood, pleural fluid, ascites, saliva and sputum.

18. The use according to claim 5, characterized in that The damaged nucleic acid sequence is a sequence in which at least one gene selected from the group consisting of EGFR, KRAS, BRAF, TP53, PIK3CA, ROS1, RET, c-Met, PTEN, RB1, AR, BRCA, KIT, FGFR, IDH, ESR1, HER2, ALK-EML4 and TMPRSS2-ERG is mutated.

19. The use according to claim 1, characterized in that In step e), the marker is detected by a change in color, a change in ultraviolet absorbance, a change in fluorescence response or a change in electrochemistry.

20. The use according to claim 5, characterized in that The probe has a sequence that binds complementary to a damaged nucleic acid sequence in cfDNA.

21. The use according to claim 1, characterized in that Prior to step c), the method also includes denaturing the sample or the cfDNA bound to the positively charged substance, wherein the cfDNA with an unstable double helix structure is denatured, while the cfDNA with a stable double helix structure is not denatured.

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