Cancer diagnostic method using CFDNA

Abstract

Provided are methods for detecting biomarkers that are specifically expressed or overexpressed in cancer cells present in a liquid sample without a PCR or nucleic acid amplification step, or for detecting mutations (e.g., SNPs) in cancer cell biomarkers without a PCR or nucleic acid amplification step. [Solution] The diagnostic method of the present invention relates to a technology for concentrating and isolating small-sized cfDNA from a liquid sample such as urine, cerebrospinal fluid, plasma, blood, pleural effusion, or other body fluid, and then detecting biomarkers overexpressed in specific cancers with ultrahigh sensitivity without PCR. Because the detection method according to one embodiment of the present invention does not require PCR amplification, it significantly reduces the time it takes to diagnose cancer. Furthermore, it allows for immediate analysis on-site, making it suitable for point-of-care testing (POCT), which allows for simultaneous detection of multiple genes in a short period of time.

Description

[Technical Field]

[0001] The present invention relates to a method for diagnosing cancer using cell-free DNA with a double helix structure, and more particularly to a method for detecting biomarker genes that are specifically expressed or overexpressed in cancer without the need for amplification, and an apparatus for using the method. [Background technology]

[0002] Recently, the importance of early cancer diagnosis has increased worldwide. Accordingly, research into early cancer diagnosis methods has been intensifying. However, cancer diagnosis methods to date have been invasive, such as tissue sampling and endoscopy. In particular, tissue examination involves removing a portion of the suspected area and examining it under a microscope. Therefore, the use of needles, punches, endoscopes, or laparoscopes to collect tissue samples requires incisions, which not only cause considerable inconvenience to patients but also leave scars and long recovery times.

[0003] Molecular diagnostics using liquid biopsies are gaining attention as an alternative to invasive diagnostic and testing methods. Because liquid biopsies use non-invasive methods, test results can be confirmed quickly. Furthermore, unlike tissue samples, which can only analyze a portion of a disease, liquid biopsies can analyze a disease from multiple angles. Liquid biopsies are particularly expected to be extremely useful in cancer diagnosis. In particular, they are expected to enable detailed observation of cancer development and metastasis by analyzing DNA derived from cancer cells present in the blood of various body parts using only body fluid tests such as blood and urine.

[0004] Molecular diagnostics is a representative in vitro diagnostic technique that detects changes in DNA or RNA through numerical values ​​or images from samples containing genetic information such as blood or urine. This method has the advantage of being highly accurate and does not require tissue testing, and with the rapid development of genome analysis technology, attempts are being made to apply it to cancer diagnosis technology based on its cost-saving advantages.

[0005] Cell-free DNA (hereinafter referred to as cfDNA) refers to DNA derived from cells present in plasma. The cfDNA generally has a double helix structure, but often also a coiled-coil structure. The cfDNA may be derived from tumor cells. Furthermore, cfDNA derived from tumor cells can be found in body fluids such as blood, plasma, and urine collected from cancer patients.

[0006] cfDNA found in cancer patients often originates from cell necrosis, cell death, or normal and / or cancer cells in the urinary tract. Such cfDNA is released into urine, blood, and other fluids through a variety of processes. Therefore, as technologies for isolating and detecting cfDNA in biological samples such as blood, plasma, and urine develop, liquid biopsies are expected to become more effective and reliable tools for monitoring cancer-risk patients. In particular, urine, cerebrospinal fluid (CSF), plasma, pleural effusion, ascites, blood, and other body fluids are easily obtainable samples, allowing for the collection of large quantities of samples through repeated sampling in a simple and noninvasive manner.

[0007] However, with current technology, there are many difficulties in analyzing cfDNA in liquid samples such as blood and urine to detect genetic mutations and diagnose cancer early. Therefore, there is a need not only to develop a method for easily detecting cfDNA, but also to develop technology that improves detection sensitivity and enables accurate early cancer diagnosis.

[0008] In addition, Korean Patent No. 10-1751962 discloses that a chain polymerization reaction is performed using a primer to detect cfDNA, and that cfDNA can be quantified using a probe that can complementarily bind to cfDNA. However, there are still problems with the chain polymerization reaction, such as the need for a separate polymerase and laboratory equipment, and the difficulty of on-site diagnosis.

[0009] In addition, Korean Patent No. 10-1701618 discloses a nanostructure whose surface properties can be changed by changing the electric field to effectively separate cfDNA. The nanostructure can bind and dissociate cfDNA through an electric change, making it easy to separate cfDNA from a sample. However, there is a limitation in that a chain reaction must still be used to identify the type of cfDNA present.

[0010] Amplifying cfDNA through chain polymerization requires not only various primer sets but also complex steps, making it time-consuming. Therefore, ongoing research is being conducted to overcome the limitations of PCR and develop methods for analyzing cfDNA with high accuracy. Furthermore, because cfDNA is found at extremely low levels in liquid samples, ongoing research is being conducted to develop DNA extraction and analysis methods with high accuracy and small sample volumes, as well as efficient analysis methods. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Korean Patent No. 10-1751962 [Patent Document 2] Korean Patent No. 10-1701618 Summary of the Invention [Problem to be solved by the invention]

[0012] Conventionally, detecting cfDNA requires a denaturation process to convert double-stranded cfDNA into single-stranded DNA so that complementary primers can bind to it. Therefore, detecting cfDNA requires a process of applying heat and reacting with enzymes such as polymerase.

[0013] However, the inventors discovered that the DNA transcription process occurs actively in cancer cells, and that there are many DNA sections in which double-stranded DNA dissociates into single strands during the transcription process, and that probes can bind to cfDNA released from such cancer cells without the need for a denaturation process, which led to the present invention.

[0014] Thus, one aspect of the present invention provides a method for detecting biomarkers that are specifically expressed or overexpressed in cancer cells present in a liquid sample such as plasma or urine using probes having sequences complementary to cfDNA without a PCR or nucleic acid amplification step.

[0015] According to another embodiment, a method is provided for detecting mutations (eg, SNPs) in cancer cell biomarkers without a PCR or nucleic acid amplification step. [Means for solving the problem]

[0016] To achieve the above-mentioned object, one aspect of the present invention provides a method for diagnosing cancer by detecting genes derived from cancer cells from the sample without amplification, the method comprising the steps of: a) mixing a biological sample isolated from an individual containing cell-free DNA (hereinafter referred to as cfDNA) with a positively charged substance; b) separating the positively charged substance bound to the cfDNA; c) sequentially or simultaneously mixing a probe and a marker having a sequence complementary to the cfDNA with the mixture; d) removing the probe and marker that do not bind to the cfDNA; and e) detecting the marker, wherein the cfDNA is derived from cancer cells and the probe having a sequence complementary to the cfDNA complementarily binds to a gene known to be a cancer biomarker. [Effects of the Invention]

[0017] The cancer diagnostic method of the present invention relates to a technology for isolating small-sized cfDNA from a liquid sample such as urine, cerebrospinal fluid, plasma, blood, pleural effusion, or other body fluid, and then detecting biomarkers that are specifically or overexpressed in cancer with ultrahigh sensitivity without PCR. Because the detection method according to one embodiment of the present invention does not require PCR amplification, it significantly reduces the time required to diagnose cancer. Furthermore, it can be used for point-of-care testing (POCT), which allows for immediate analysis on-site and simultaneous detection of multiple genes in a short period of time. In particular, the method of the present invention can distinguish between cfDNA present in the blood of normal individuals and cfDNA present in the blood of cancer patients, thereby effectively detecting various cancers. [Brief explanation of the drawings]

[0018] [Figure 1a] FIG. 1 shows a scanning electron microscope (SEM) image of positively charged nanowires (PEI / Ppy NW). [Figure 1b] FIG. 1 shows a scanning electron microscope image of HRP / streptavidin-conjugated nanoparticles. [Figure 2a] Figure 1 shows a conceptual diagram of the fabrication of a nanostructure (PEI / mPpy NW) with the cationic polymer polyethyleneimine (PEI) attached to its surface, and the method of using it to detect and recover cfDNA. [Figure 2b] This figure shows photographs of the process of detecting and recovering cfDNA using a magnetic nanostructure (PEI / mPpy NW) with the cationic polymer polyethyleneimine (PEI) attached to its surface. [Figure 3] This is a diagram illustrating the process of collecting cfDNA using an Eppendorf tube. [Figure 4] This figure shows the level of PD-L1 DNA expression and PD-L1 mRNA expression measured from cfDNA of PD-L1-positive or PD-L1-negative cancer cell lines. [Figure 5] This figure shows the level of PD-L1 DNA expression and PD-L1 mRNA expression measured from cfDNA of PD-L1-positive or PD-L1-negative cancer cell lines. [Figure 6] This figure shows the level of EpCAM DNA expression and EpCAM mRNA expression measured from cfDNA of EpCAM-positive or EpCAM-negative cancer cell lines. [Figure 7] This figure shows the level of EpCAM DNA expression and EpCAM mRNA expression measured from cfDNA of EpCAM-positive or EpCAM-negative cancer cell lines. [Figure 8] This figure shows the level of FOLR1 DNA expression and FOLR1 mRNA expression measured from cfDNA of FOLR1-positive or FOLR1-negative cancer cell lines. [Figure 9]This figure shows the level of FOLR1 DNA expression and FOLR1 mRNA expression measured from cfDNA of FOLR1-positive or FOLR1-negative cancer cell lines. [Figure 10] FIG. 1 shows the levels of EGFR DNA expression and EGFR mRNA expression measured from cfDNA of EGFR-positive or EGFR-negative cancer cell lines. [Figure 11] FIG. 1 shows the levels of EGFR DNA expression and EGFR mRNA expression measured from cfDNA of EGFR-positive or EGFR-negative cancer cell lines. [Figure 12] FIG. 1 shows the levels of ERBB2 DNA expression and ERBB2 mRNA expression measured from cfDNA of ERBB2-positive or ERBB2-negative cancer cell lines. [Figure 13] FIG. 1 shows the levels of ERBB2 DNA expression and ERBB2 mRNA expression measured from cfDNA of ERBB2-positive or ERBB2-negative cancer cell lines. [Figure 14] FIG. 1 shows the level of OGT DNA expression measured from cfDNA of OGT-positive or OGT-negative cancer cell lines. [Figure 15] FIG. 1 shows the level of OGT DNA expression measured from cfDNA of OGT-positive or OGT-negative cancer cell lines. [Figure 16] FIG. 1 shows the level of CEA DNA expression measured from cfDNA of CEA-positive or CEA-negative cancer cell lines. [Figure 17] FIG. 1 shows the level of CEA DNA expression measured from cfDNA of CEA-positive or CEA-negative cancer cell lines. [Figure 18] FIG. 1 shows the level of CEA DNA expression measured from cfDNA of CEA-positive or CEA-negative cancer cell lines. [Figure 19] FIG. 1 shows the level of PSA DNA expression measured from cfDNA of PSA-positive or PSA-negative cancer cell lines. [Figure 20] FIG. 1 shows the level of PSA DNA expression measured from cfDNA of PSA-positive or PSA-negative cancer cell lines. [Figure 21] This is a graph showing the level of CA19-9 DNA expression measured from cfDNA of CA19-9-positive or CA19-9-negative cancer cell lines. [Figure 22] This is a graph showing the level of CA19-9 DNA expression measured from cfDNA of CA19-9-positive or CA19-9-negative cancer cell lines. [Figure 23] This is a graph showing the level of CA125 DNA expression measured from cfDNA of CA125-positive or CA125-negative cancer cell lines. [Figure 24] This is a graph showing the level of CA125 DNA expression measured from cfDNA of CA125-positive or CA125-negative cancer cell lines. [Figure 25] FIG. 1 shows the level of AFP DNA expression measured from cfDNA of AFP-positive or AFP-negative cancer cell lines. [Figure 26] FIG. 1 shows the level of AFP DNA expression measured from cfDNA of AFP-positive or AFP-negative cancer cell lines. [Figure 27]FIG. 1 shows the DNA expression levels of PSA, PSMA, PAP, and PAC3 measured using plasma obtained from prostate cancer patients. [Figure 28] FIG. 1 shows the DNA expression levels of PSA, PSMA, PAP, and PAC3 measured using plasma obtained from prostate cancer patients. [Figure 29] FIG. 1 shows the DNA expression levels of PSA, PSMA, PAP, and PAC3 measured using plasma obtained from prostate cancer patients. [Figure 30] FIG. 1 shows the DNA expression levels of PSA, PSMA, PAP, and PAC3 measured using plasma obtained from normal humans. [Figure 31] FIG. 1 shows the DNA expression levels of PSA, PSMA, PAP, and PAC3 measured using plasma obtained from normal humans. [Figure 32] FIG. 1 shows the DNA expression levels of PSA, PSMA, PAP, and PAC3 measured using plasma obtained from normal humans. [Figure 33] FIG. 1 shows the DNA expression levels of NSE, SCC, CEA, Cyfra21-1 and TPA measured using plasma obtained from lung cancer patients. [Figure 34] FIG. 1 shows the DNA expression levels of NSE, SCC, CEA, Cyfra21-1 and TPA measured using plasma obtained from lung cancer patients. [Figure 35] FIG. 1 shows the DNA expression levels of NSE, SCC, CEA, Cyfra21-1 and TPA measured using plasma obtained from normal humans. [Figure 36] FIG. 1 shows the DNA expression levels of CEA, NSE, TG, and CALCA measured using plasma obtained from thyroid cancer patients. [Figure 37] FIG. 1 shows the DNA expression levels of CEA, NSE, TG, and CALCA measured using plasma obtained from thyroid cancer patients. [Figure 38]FIG. 1 shows the DNA expression levels of CEA, NSE, TG, and CALCA measured using plasma obtained from thyroid cancer patients. [Figure 39] FIG. 1 shows the DNA expression levels of CEA, NSE, TG, and CALCA measured using plasma obtained from normal humans. [Figure 40] FIG. 1 shows the DNA expression levels of CEA, NSE, TG, and CALCA measured using plasma obtained from normal humans. [Figure 41] FIG. 1 shows the DNA expression levels of OGT, FGFR3, TP53, NMP22, and Cyfra21-1 measured using urine collected from bladder cancer patients. [Figure 42] FIG. 1 shows the DNA expression levels of OGT, FGFR3, TP53, NMP22, and Cyfra21-1 measured using urine collected from bladder cancer patients. [Figure 43] FIG. 1 shows the DNA expression levels of OGT, FGFR3, TP53, NMP22, and Cyfra21-1 measured using urine collected from patients with cystitis. [Figure 44] FIG. 1 shows the DNA expression levels of OGT, FGFR3, TP53, NMP22, and Cyfra21-1 measured using urine collected from patients with cystitis. [Figure 45] FIG. 1 shows the DNA expression levels of OGT, FGFR3, TP53, NMP22, and Cyfra21-1 measured using urine collected from normal humans. [Figure 46] FIG. 1 shows the DNA expression levels of OGT, FGFR3, TP53, NMP22, and Cyfra21-1 measured using urine collected from normal humans. [Figure 47] FIG. 1 shows the DNA expression levels of CA27-29, CA15-3 and CEA measured using plasma obtained from breast cancer patients. [Figure 48] FIG. 1 shows the DNA expression levels of CA27-29, CA15-3 and CEA measured using plasma obtained from breast cancer patients. [Figure 49]FIG. 1 shows the DNA expression levels of CA27-29, CA15-3 and CEA measured using plasma obtained from normal humans. [Figure 50] FIG. 1 shows the DNA expression levels of CA27-29, CA15-3 and CEA measured using plasma obtained from normal humans. [Figure 51] FIG. 1 shows the DNA expression levels of CEA and CA19-9 measured using plasma obtained from colon cancer patients. [Figure 52] FIG. 1 shows the DNA expression levels of CEA and CA19-9 measured using plasma obtained from colon cancer patients. [Figure 53] FIG. 1 shows the results of measuring the DNA expression levels of CEA and CA19-9 using plasma obtained from normal humans. [Figure 54] FIG. 1 shows the results of measuring the DNA expression levels of CEA and CA19-9 using plasma obtained from normal humans. [Figure 55] FIG. 1 shows the results of measuring the DNA expression levels of CEA and CA19-9 using plasma obtained from normal humans. [Figure 56] FIG. 1 shows the DNA expression levels of CA19-9, CA125, and CEA measured using plasma obtained from bile duct cancer patients. [Figure 57] FIG. 1 shows the DNA expression levels of CA19-9, CA125 and CEA measured using plasma obtained from normal humans. [Figure 58] FIG. 1 shows the DNA expression levels of CA19-9, CA125 and CEA measured using plasma obtained from normal humans. [Figure 59] FIG. 1 shows the DNA expression levels of CEA, CA19-9, CGB and Cyfra21-1 measured using plasma obtained from gastric cancer patients. [Figure 60] FIG. 1 shows the DNA expression levels of CEA, CA19-9, CGB and Cyfra21-1 measured using plasma obtained from normal humans. [Figure 61]FIG. 1 shows the DNA expression levels of CEA, CA19-9, CGB and Cyfra21-1 measured using plasma obtained from normal humans. [Figure 62] FIG. 1 shows the DNA expression levels of CA19-9, CA125 and CEA measured using plasma obtained from pancreatic cancer patients. [Figure 63] FIG. 1 shows the DNA expression levels of CA19-9, CA125 and CEA measured using plasma obtained from pancreatic cancer patients. [Figure 64] FIG. 1 shows the DNA expression levels of CA19-9, CA125 and CEA measured using plasma obtained from pancreatic cancer patients. [Figure 65] FIG. 1 shows the DNA expression levels of CA19-9, CA125 and CEA measured using plasma obtained from pancreatic cancer patients. [Figure 66] FIG. 1 shows the DNA expression levels of CA19-9, CA125 and CEA measured using plasma obtained from normal humans. [Figure 67] FIG. 1 shows the DNA expression levels of CA19-9, CA125 and CEA measured using plasma obtained from normal humans. [Figure 68] FIG. 1 shows the DNA expression levels of CA19-9, CA125 and CEA measured using plasma obtained from normal humans. [Figure 69] FIG. 1 shows the DNA expression levels of CA19-9, CA125 and CEA measured using plasma obtained from normal humans. [Figure 70] FIG. 1 is a graph showing the DNA expression level of CPT1A measured using plasma obtained from lung cancer patients. [Figure 71] FIG. 1 shows the results of measuring the level of CPT1A DNA expression using plasma obtained from normal humans. [Figure 72] FIG. 1 is a graph showing the DNA expression level of CPT1A measured using urine collected from bladder cancer patients. [Figure 73]FIG. 1 shows the results of measuring the DNA expression level of CPT1A using urine collected from normal humans. [Figure 74] FIG. 1 shows the level of PD-L1 DNA expression measured from cfDNA of PD-L1-positive cancer cell lines or PD-L1-negative cancer cell lines not treated with IFN-γ, and the presence or absence of PD-L1 detection measured using a method according to one embodiment of the present invention. [Figure 75] This figure shows the level of IFN-γ DNA expression measured from cfDNA of PD-L1-positive cancer cell lines or PD-L1-negative cancer cell lines that were not treated with IFN-γ. [Figure 76] This figure shows the level of DNA expression of IFNR1 (IFN-γ receptor) measured from cfDNA of PD-L1-positive cancer cell lines and PD-L1-negative cancer cell lines that were not treated with IFN-γ. [Figure 77] This figure shows the DNA expression levels of PD-L1, IFNG, and IFNR1 measured from cfDNA of PD-L1-positive or PD-L1-negative cancer cell lines treated with IFN-γ. [Figure 78] This figure shows the DNA expression levels of PD-L1, IFNG, and IFNR1 measured from cfDNA of PD-L1-positive or PD-L1-negative cancer cell lines treated with IFN-γ. [Figure 79] This figure shows the DNA expression levels of PD-L1, IFNG, and IFNR1 measured from cfDNA of PD-L1-positive or PD-L1-negative cancer cell lines treated with IFN-γ. [Figure 80] This figure shows the level of PD-L1 DNA expression measured from cfDNA of PD-L1-positive and PD-L1-negative cancer cell lines, with or without IFN-γ treatment. [Figure 81] This is a graph showing the level of IFN-γ DNA expression measured from cfDNA of PD-L1-positive cancer cell lines and PD-L1-negative cancer cell lines with or without IFN-γ treatment. [Figure 82]This figure shows the level of IFNR1 DNA expression measured from cfDNA of PD-L1-positive cancer cell lines and PD-L1-negative cancer cell lines, with or without IFN-γ treatment. [Figure 83] This figure shows the level of PD-L1 DNA expression measured from cfDNA of PD-L1-positive and PD-L1-negative cancer cell lines, with or without IFN-γ treatment. [Figure 84a] Figure 84a is a diagram illustrating the detection steps of the present invention. Figure 84a is a diagram illustrating a method for collecting cfDNA from a patient's body fluid using nanowires (PEI / Ppy NW) with polyethyleneimine (PEI) attached to their surface, and then analyzing gene mutations within about 60 minutes through a reaction with a probe and HRP / streptavidin-nanoparticles (HRP / st-tagged NP). [Figure 84b] Figure 84b is a schematic diagram of a method for detecting unstable cfDNA using nanowires, probes, and HRP / streptavidin nanoparticles. [Figure 84c] 84c shows a process for detecting gene mutations using a spin column with nanowires that do not contain magnetic nanoparticles. In one embodiment of the present invention, a step of treating the lysis buffer may be additionally included. [Figure 84d] Figure 84d shows a timeline of a method for detecting unstable cfDNA in samples such as blood, cerebrospinal fluid, or pleural effusion. [Figure 84e] Figure 84e is a timeline showing a method for detecting unstable cfDNA in a sample such as urine. [Fig. 84f] Figure 84f is a diagram illustrating the differences in denaturing conditions depending on the state of cfDNA obtained from blood. [Figure 84g] Figure 84g is a diagram illustrating the differences in denaturing conditions depending on the state of cfDNA obtained from urine, saliva, and sputum. [Figure 85]This figure shows the separation of cfDNA using a spin column with nanowires that do not contain magnetic nanoparticles. The top photo is an SEM image of the spin column before centrifugation, and the bottom photo is an SEM image of the spin column with separated cfDNA after centrifugation. [Figure 86] This is a graph showing the DNA expression levels of cancer-related biomarkers such as AKL Fusion and PIK3CA measured from blood collected from lung cancer patients using a syringe. [Figure 87] This is a diagram showing the DNA expression levels of cancer-related biomarkers such as AKL Fusion and PIK3CA measured from blood collected from lung cancer patients using a lancet. [Figure 88] This is a graph showing the DNA expression levels of cancer-related biomarkers such as AKL Fusion measured from blood collected from normal subjects using a syringe needle. [Figure 89] This is a diagram showing the DNA expression levels of cancer-related biomarkers such as AKL Fusion measured from blood collected from normal subjects using a lancet. [Figure 90] This figure shows the expression level of EML4-ALK confirmed by RT-PCR from cfDNA of EML4-ALK variant 3a / b positive cell (H2228) and EML4-ALK negative cell (A549, H1993, PC9, RT4) cancer cell lines. [Figure 91] This figure shows the level of EML4-ALK expression confirmed by Western blotting from cfDNA of EML4-ALK variant 3a / b positive cell (H2228) and EML4-ALK negative cell (A549, H1993, PC9, RT4) cancer cell lines. [Figure 92]This is a graph showing the results of confirming the level of EML4-ALK expression from cfDNA of EML4-ALK variant 3a / b positive cell (H2228) and EML4-ALK negative cell (A549, H1993, PC9, RT4) cancer cell lines using RT-PCR and Western blotting. [Figure 93] This figure shows the level of DNA expression of EML4-ALK fusion var.1 or EML4-ALK fusion var.3 measured from cfDNA of EML4-ALK variant 3a / b positive cell (H2228) and EML4-ALK negative cell (A549, H1993, PC9, RT4) cancer cell lines. [Figure 94] This is a graph showing the level of DNA expression of EML4-ALK fusion var.1 or EML4-ALK fusion var.3 measured from cfDNA of EML4-ALK variant 3a / b positive cell (H2228) and EML4-ALK negative cell (A549, H1993, PC9, RT4) cancer cell lines. [Figure 95] This is a graph showing the DNA expression levels of cancer-related biomarkers, such as EML4-ALK fusion var.3, KRAS, SYP, NCAM1, and NKX2-1, in blood collected from patients with small cell lung cancer. As a result, EML4-ALK fusion was found in both cancer tissue and blood ctDNA, and the ctDNA results showed that the EML4-ALK fusion was var.3, not var.1, indicating a poor response to the ALK TKI crizotinib. [Figure 96]This is a diagram showing the DNA expression levels of cancer-related biomarkers, such as EML4-ALK fusion var. 1, measured in blood collected from cancer patients. As a result, EML4-ALK fusion was found to be present in both cancer tissue and blood ctDNA, and the ctDNA results showed that the EML4-ALK fusion was var. 1, not var. 3, indicating a good response to the ALK TKI crizotinib, resulting in a partial response (PR) for the patient. [Figure 97] This is a diagram measuring the DNA expression level of cancer-related biomarkers such as EML4-ALK fusion var. 3 in blood collected from cancer patients. As a result, EML4-ALK fusion was found to be present in both cancer tissue and blood ctDNA. Since the ctDNA results showed that the EML4-ALK fusion was var. 3, not var. 1, and the patient did not respond well to the ALK TKI crizotinib, alectinib was prescribed from the start, and the patient's response was awaited. [Figure 98] This is a diagram showing the DNA expression levels of cancer-related biomarkers, such as EML4-ALK fusion var.3, BRAFV800E, and TP53, in blood collected from cancer patients. As a result, EML4-ALK fusion was found in both cancer tissue and blood ctDNA, and the ctDNA results showed that the EML4-ALK fusion was var.3, not var.1, indicating a poor response (PD) to the ALK TKI crizotinib. [Figure 99] This is a diagram showing the DNA expression levels of cancer-related biomarkers, such as EML4-ALK fusion var. 1, measured in blood collected from cancer patients. As a result, EML4-ALK fusion was found to be present in both cancer tissue and blood ctDNA, and the ctDNA results showed that the EML4-ALK fusion was var. 1, not var. 3, indicating a good response to the ALK TKI crizotinib, resulting in a partial response (PR) for the patient. [Figure 100]This is a graph showing the DNA expression levels of cancer-related biomarkers, such as EML4-ALK fusion var. 1, measured in blood collected from cancer patients. As a result, EML4-ALK fusion was found to be present in both cancer tissue and blood ctDNA, and the ctDNA results showed that the EML4-ALK fusion was var. 1, not var. 3, indicating a good response to ALK TKI and a partial response (PR) in the patient. [Figure 101] This figure shows the level of OGT protein expression confirmed in vitro using Western blotting from cfDNA of each cancer cell line. [Figure 102] This figure shows the level of OGT mRNA expression confirmed in vitro using RT-PCR from cfDNA of each cancer cell line. [Figure 103] This is a graph showing the results of confirming the level of OGT mRNA expression from cfDNA of each cancer cell line in vitro using RT-PCR. [Figure 104] This is a diagram showing the level of OGT DNA expression measured in vitro from cfDNA of each cell line. [Figure 105] This is a graph showing the results of measuring the level of OGT DNA expression from cfDNA of each cell line in vitro. [Figure 106] This figure shows the results of confirming the level of OGT expression in each cell line in vitro through Western blot, RT-PCR, and cfDNA detection. [Figure 107] This is a photograph of OGT cfDNA detected in vitro from each cell line on nanowires that do not contain magnetic nanoparticles. [Figure 108] This is a graph showing the quantification of cfDNA obtained from urine of normal subjects, cystitis patients, and bladder cancer patients. [Figure 109] This is a graph showing the analysis of the DNA expression level of OGT from cfDNA obtained from the urine of normal subjects, cystitis patients, and bladder cancer patients. [Figure 110]This is a graph showing the analysis of the DNA expression level of OGT from cfDNA obtained from the urine of normal subjects, cystitis patients, and bladder cancer patients. [Figure 111] This is a graph showing the analysis of OGT DNA expression levels in cfDNA obtained from urine samples of various cancer patients in a blind test. [Figure 112] This is a graph showing the analysis of OGT DNA expression levels in cfDNA obtained from urine samples of various cancer patients in a blind test. [Figure 113] This is a graph showing the analysis of OGT DNA expression levels in cfDNA obtained from urine samples of various cancer patients in a blind test. [Figure 114] This is a diagram showing the analysis of the DNA expression levels of BRAF V600E and TERT C250T from cfDNA obtained from the tissues of thyroid cancer patients. [Figure 115] This is a diagram showing the analysis of the DNA expression levels of BRAF V600E and TERT C250T from cfDNA obtained from the tissues of thyroid cancer patients. [Figure 116] This is a diagram showing the analysis of the DNA expression levels of BRAF V600E and TERT C250T from cfDNA obtained from the tissues of thyroid cancer patients. [Figure 117] This is a graph showing the DNA expression levels of cancer-related biomarkers such as SYP, CgA, NCAM1, and NKX2-1 in blood collected from patients with small cell lung cancer. In particular, the patient in Figure 120 was diagnosed with small cell lung cancer (SCLC) at Korea Atomic Energy Hospital and prescribed crizotinib, but it was found to be ineffective. [Figure 118] This is a graph showing the DNA expression levels of cancer-related biomarkers such as SYP, CgA, NCAM1, and NKX2-1 in blood collected from patients with small cell lung cancer. In particular, the patient in Figure 120 was diagnosed with small cell lung cancer (SCLC) at Korea Atomic Energy Hospital and prescribed crizotinib, but it was found to be ineffective. [Figure 119]This is a graph showing the DNA expression levels of cancer-related biomarkers such as SYP, CgA, NCAM1, and NKX2-1 in blood collected from patients with small cell lung cancer. In particular, the patient in Figure 120 was diagnosed with small cell lung cancer (SCLC) at Korea Atomic Energy Hospital and prescribed crizotinib, but it was found to be ineffective. [Figure 120] This is a graph showing the DNA expression levels of cancer-related biomarkers such as SYP, CgA, NCAM1, and NKX2-1 in blood collected from patients with small cell lung cancer. In particular, the patient in Figure 120 was diagnosed with small cell lung cancer (SCLC) at Korea Atomic Energy Hospital and prescribed crizotinib, but it was found to be ineffective. [Figure 121] This is a graph showing the DNA expression levels of cancer-related biomarkers such as SYP, CgA, NCAM1, and NKX2-1 in blood collected from patients with small cell lung cancer. In particular, the patient in Figure 120 was diagnosed with small cell lung cancer (SCLC) at Korea Atomic Energy Hospital and prescribed crizotinib, but it was found to be ineffective. [Figure 122] FIG. 1 shows the results of measuring the DNA expression levels of SYP, CgA, NCAM1, and NKX2-1 using blood obtained from non-small cell lung cancer patients. [Figure 123] FIG. 1 shows the level of CEA DNA expression in blood collected from lung cancer patients before and after anti-cancer treatment, and the prognosis of the patients. [Figure 124] This is a graph showing the DNA expression levels of cancer-related biomarkers such as NSE and CEA measured using blood collected from lung cancer patients. [Figure 125] This is a graph showing the DNA expression levels of cancer-related biomarkers such as NSE and CEA measured using blood collected from lung cancer patients. [Figure 126] This is a graph showing the DNA expression levels of cancer-related biomarkers such as NSE and CEA measured using blood collected from lung cancer patients. [Figure 127] This is a graph showing the DNA expression levels of cancer-related biomarkers such as NSE and CEA measured using blood collected from lung cancer patients. [Figure 128]This is a graph showing the DNA expression levels of cancer-related biomarkers such as NSE and CEA measured using blood collected from lung cancer patients. [Figure 129] This is a graph showing the DNA expression levels of cancer-related biomarkers such as NSE and CEA measured using blood collected from normal subjects. [Figure 130] This is a graph showing the DNA expression levels of cancer-related biomarkers such as NSE and CEA measured using blood collected from normal subjects. [Figure 131] This is a graph showing the DNA expression levels of cancer-related biomarkers such as NSE and CEA measured using blood collected from normal subjects. [Figure 132] This is a graph showing the DNA expression levels of cancer-related biomarkers such as NSE and CEA measured using blood collected from normal subjects. [Figure 133] This is a graph showing the DNA expression levels of cancer-related biomarkers such as NSE and CEA measured using blood collected from normal subjects. [Figure 134] This is a graph showing the DNA expression levels of cancer-related biomarkers such as PSA, PSMA, PAP, and PCA3 measured using blood collected from prostate cancer patients. [Figure 135] This is a graph showing the DNA expression levels of cancer-related biomarkers such as PSA, PSMA, PAP, and PCA3 measured using blood obtained from normal subjects. [Figure 136] FIG. 10 shows the results of measuring the DNA expression level of TMPRSS2-ERG fusion using blood collected from prostate cancer patients and normal subjects. [Figure 137] This is a graph showing the DNA expression levels of CEA, NSE, TG (Thyroglobulin), and CALCA measured using blood obtained from thyroid cancer patients. [Figure 138] This is a graph showing the DNA expression levels of CEA, NSE, TG (Thyroglobulin), and CALCA measured using blood obtained from normal humans. [Figure 139]This is a graph showing the DNA expression levels of BRAF mutation (V600E) and TERT promoter mutation (C228T, C250T) measured using blood collected from thyroid cancer patients and normal subjects. [Figure 140] This is a diagram showing the DNA expression levels of OGT, FGFR3, TP53, NMP22, and Cyfra21-1 measured using urine collected from bladder cancer patients, hematuria patients, and normal subjects. [Figure 141] FIG. 1 shows the DNA expression levels of CA27-29 and CEA measured using blood collected from breast cancer patients and normal subjects. [Figure 142] FIG. 1 shows the DNA expression levels of CEA and CA19-9 measured using blood collected from colon cancer patients and normal subjects. [Figure 143] FIG. 1 shows the results of measuring the DNA expression levels of CA 19-9, CEA, and CA123 using blood collected from bile duct cancer patients and normal subjects. [Figure 144] FIG. 1 shows the results of measuring the DNA expression levels of CA 19-9, CEA, and CA123 using blood collected from bile duct cancer patients and normal subjects. [Figure 145] This is a graph showing the DNA expression levels of CEA, CA19-9, CGB and Cyfra21-1 measured using blood collected from gastric cancer patients and normal subjects. [Figure 146] FIG. 1 shows the DNA expression levels of CA125 and CEA measured using blood collected from ovarian cancer patients and normal subjects. [Figure 147] FIG. 1 shows the results of measuring the DNA expression levels of CEA, CA19-9, and CA125 using blood obtained from pancreatic cancer patients. [Figure 148] FIG. 1 shows the DNA expression levels of CEA, CA19-9 and CA125 measured using blood obtained from normal subjects. [Figure 149]These are the results of early diagnosis conducted by measuring the DNA expression level of cancer-related biomarkers using blood collected from normal individuals (PC: Positive control, PC is a tool to confirm whether the early cancer diagnosis experiment was carried out correctly and has no relation to the early cancer diagnosis results). [Figure 150] These are the results of early diagnosis conducted by measuring the DNA expression level of cancer-related biomarkers using blood collected from normal individuals (PC: Positive control, PC is a tool to confirm whether the early cancer diagnosis experiment was carried out correctly and has no relation to the early cancer diagnosis results). [Figure 151] FIG. 1 is a diagram summarizing biomarkers by cancer type used in one example of the present invention. [Figure 152] FIG. 1 is a diagram summarizing biomarkers by cancer type used in one example of the present invention. [Figure 153] This figure shows the absorbance of cfDNA present in the urine of HPV-positive cervical cancer patients (HPV16(+) and HPV18(+)) and HPV-negative healthy controls (HPV-) to confirm the presence or absence of binding of probes specific to HPV18 or HPV16. [Fig. 154] This figure shows the results of sequentially reacting cfDNA isolated from the urine of cervical cancer patients with probes specific to HPV16, EGFR19 deletion, HPV18, and EGFR21 L858R, and then confirming whether or not each probe bound to the cfDNA. [Figure 155] This table shows the analysis of gene mutations in lung cancer patients using cfDNA obtained from the plasma of 151 lung cancer patients. [Figure 156] cfDNA was collected from the plasma of lung cancer patients without EGFR mutations (wild type), with EGFR exon 19 deletion, and with EGFR exon 21 L858R. A probe specific to EGFR exon 19 Del was then mixed with cfDNA, and the gene mutations in the lung cancer patients were confirmed through analysis of UV spectrum absorbance (ΔOD, 500nm~650nm). [Figure 157]This figure shows the specificity and sensitivity of gene mutations after cfDNA was collected from the plasma of a lung cancer patient with EGFR exon 19 deletion and mixed with a probe specific to EGFR exon 19 Del. [Figure 158] cfDNA was collected from the plasma of lung cancer patients without EGFR mutations (wild type), with EGFR exon 19 deletion, and with EGFR exon 21 L858R. A probe specific to EGFR exon 21 L858R was added, and the patient's gene mutations were confirmed through analysis of UV spectrum absorbance (ΔOD, 500nm~650nm). [Figure 159] This figure shows the specificity and sensitivity of the patient's gene mutation after collecting cfDNA from the plasma of a lung cancer patient with EGFR exon 21 L858R and adding a probe specific to EGFR exon 21 L858R. [Figure 160] This figure shows the CP and DP sequences of the EGFR exon 19 deletion. In this study, we analyzed cfDNA mutations in lung cancer patients using CP_1 and DP. CP is a probe designed to complement the sequence containing or adjacent to the mutation site, and DP is a probe designed to complement the sequence distant from the mutation site. [Figure 161] Figure 1 shows the CP and DP sequences of EGFR exon 20 T790M. In this study, we analyzed cfDNA gene mutations in lung cancer patients using CP2 and DP. [Figure 162] Figure 1 shows the CP and DP sequences of EGFR exon 21 L858R. In this study, CP2 and DP were used to analyze cfDNA mutations in lung cancer patients. [Figure 163]cfDNA obtained from the plasma of a lung cancer patient 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. HRP / streptavidin nanoparticles (containing a large amount of HRP) were then added, and the detection of cfDNA was confirmed by color change and UV absorbance. [Fig. 164] cfDNA collected from the plasma of a lung cancer patient with the same EGFR exon 19 deletion and EGFR exon 20 T790M genetic mutation as in Figure 163 was reacted with probes specific for EGFR exon 19 deletion (Del19), EGFR exon 20 T790M, and EGFR exon 21 L858R. Then, HRP / streptavidin complex (a 1:1 complex of HRP and streptavidin) was added, and the detection of cfDNA was confirmed by color change and UV absorbance. This figure confirms that the HRP / streptavidin complex generates more noise than HRP / streptavidin nanoparticles. [Figure 165] cfDNA was extracted from the plasma of five lung cancer patients with EGFR exon 19 deletion and exon 20 T790M gene mutations. The results were then reacted with probes specific for EGFR exon 19 Del, EGFR exon 20 T790M, and EGFR exon 21 L858R and HRP / streptavidin nanoparticles (HRP / st-tagged NPs), and with probes specific for EGFR exon 19 Del, EGFR exon 20 T790M, and EGFR exon 21 L858R and HRP / streptavidin complexes (HRP and streptavidin bound in a 1:1 ratio). The results were compared to confirm the concordance between the cancer tissue and genotype. [Figure 166]To detect gene mutations in cfDNA collected from the plasma of a lung cancer patient with EGFR exon 20 T790M and EGFR exon 21 L861Q gene mutations, probes specific for EGFR exon 19 deletion (Del19), EGFR exon 20 T790M, EGFR exon 21 L858R, and EGFR exon L861Q were mixed with HRP / st-tagged NP. As a result, gene mutations were observed only in EGFR exon 20 T790M and EGFR exon 21 L861Q, as confirmed by UV absorbance, just like in the cancer tissue. [Figure 167] To detect gene mutations in cfDNA obtained from the plasma of lung cancer patients with ALK-EML4 fusion and ALK point mutation (I1171N / T) gene mutations, probes specific for ALK-EML4 fusion and ALK point mutation (T1151, L1152P, L1152R, C1156Y, I1171N / T) and HRP / st-tagged NP were mixed together. As a result, it was confirmed that the ALK-EML4 fusion and ALK point mutation (I1171N / T) genotypes were detected in the same way as in the cancer tissue. [Figure 168] To detect gene mutations in cfDNA obtained from the plasma of thyroid cancer patients with the BRAF V600E gene mutation, a BRAF V600E-specific probe and HRP / st-tagged NP were mixed together. As a result, it was confirmed that the BRAF V600E gene mutation was detected in the same manner as the patient's genotype. [Figure 169] This figure shows the results of detecting unstable cfDNA depending on the treatment conditions after samples collected from normal human blood were denatured under various temperature conditions. [Figure 170] This figure shows the results of detecting unstable cfDNA depending on the processing conditions after samples collected from patient blood were denatured under various temperature conditions. [Figure 171]This figure shows the results of detecting unstable cfDNA depending on the treatment conditions after denaturing fDNA obtained from mutant cell lines under various temperature conditions. [Figure 172] This figure shows the results of detecting unstable cfDNA depending on the treatment conditions after treating fDNA obtained from mutant cell lines with DNase at 37°C for 30 minutes. [Fig. 173] This figure shows the results of detecting unstable cfDNA depending on the treatment conditions after treating fDNA obtained from mutant cell lines with DNase at 37°C for 60 minutes. [Fig. 174] This figure shows the results of detecting unstable cfDNA depending on the treatment conditions after treating fDNA obtained from mutant cell lines with DNase at 37°C for 120 minutes. [Figure 175] This figure shows the results of treating cfDNA with 1 μl or 2 μl of DNase at 24°C for 120 minutes to confirm the difference between unstable cfDNA and stable cfDNA due to DNase activity. [Figure 176] This figure shows the results of treating 1 μl or 2 μl of DNase at 3°C ​​for 120 minutes to confirm the difference between unstable cfDNA and stable cfDNA due to DNase activity. [Figure 177] FIG. 10 is a diagram showing a specific example of a cutoff value when detecting the EML4-ALK fusion gene using cfDNA from the plasma of a lung cancer patient in one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0019] <Terminology> As used herein, the term "cell-free DNA" is also referred to as cfDNA. cfDNA may refer to circulating tumor DNA (ctDNA), which is cancer cell-derived DNA found in biological samples such as urine, cerebrospinal fluid, plasma, blood, or body fluids derived from cancer patients due to tumor cells. cfDNA may also be present in biological samples such as urine, cerebrospinal fluid, pleural effusion, ascites, plasma, blood, saliva, sputum, or body fluids. In this case, cfDNA may have a size of about 80 bp to about 10 kbp, about 100 bp to about 1 kbp, or about 120 bp to about 500 bp. cfDNA may have a size of about 150 bp to about 200 bp, typically about 165 bp to about 170 bp. Furthermore, the cfDNA may include cfDNA of a size of about 80 bp or less.

[0020] As used herein, the term "unstable cfDNA" refers to cfDNA that is thermodynamically less stable than "stable cfDNA." That is, unstable cfDNA can be denatured under conditions that are slightly harsher than those under which stable cfDNA is denatured. The reason why unstable cfDNA is generated is that unstable cfDNA has an unstable double helix structure. Specifically, cfDNA derived from genes that are overexpressed in cancer cells can be a specific example of unstable cfDNA.

[0021] As used herein, the term "cfDNA with an unstable double helix structure" refers to DNA that has a lower Tm value than cfDNA with a stable double helix structure or that denatures under conditions that do not denaturate cfDNA with a stable double helix structure. The Tm refers to the melting temperature, or the temperature at which 50% of double-stranded DNA is converted to single-stranded DNA. The Tm value is proportional to the length of DNA and may vary depending on the nucleotide sequence. However, because genomic DNA contains many hydrogen-bonded nucleotides, it must be heated at approximately 92°C to approximately 95°C for 5 minutes or more, or at approximately 98°C for 2 minutes or more. Furthermore, genomic DNA does not readily denature at temperatures below approximately 90°C. In this regard, cfDNA with a stable double helix structure may have a Tm value similar to that of genomic DNA, assuming an average of approximately 170 bp of nucleotides.

[0022] However, the "cfDNA with an unstable double helix structure" has a lower Tm value than cfDNA with a stable double helix structure. Therefore, cfDNA with a stable double helix structure can be prepared under the following conditions: (i) leaving it at room temperature for about 1 minute to about 120 minutes; (ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes; (iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; (iv) heating at about 60°C to about 75°C for about 30 seconds to about 60 minutes; (v) heating at about 25°C to about 40°C for about 10 minutes to about 120 minutes; (vi) treating with protease for about 10 seconds to about 30 minutes. When cfDNA is denatured under any one of the following conditions: (vii) treatment with DNase for about 10 seconds to about 30 minutes; and (viii) treatment with a chemical (e.g., sodium hydroxide, DMSO, surfactant, etc.), and then subjected to a binding reaction with a probe of about 15 mer to about 30 mer having a sequence complementary to a partial sequence of cfDNA, cfDNA having a stable double helix structure does not bind to the probe. In this case, "room temperature" refers to room temperature, which may be about 18°C ​​to about 25°C. In addition to the above conditions, the method may further include heating at about 40°C to about 65°C for about 5 minutes to about 80 minutes.

[0023] However, when cfDNA with an unstable double helix structure was treated under any one of the conditions i) to viii) above and then subjected to a binding reaction with a probe of about 15mer to about 30mer, it was confirmed that the probe bound to the probe. In this case, the probe was about 15mer to about 30mer or about 20mer to about 25mer, and could be a probe of about 21mer, about 22mer, about 23mer, or about 24mer.

[0024] In this case, the cfDNA having the unstable double helix structure may be circulating tumor DNA (hereinafter referred to as ctDNA).

[0025] As used herein, the term "probe" refers to DNA or RNA for detecting target cfDNA. The probe can have a sequence designed to complementarily bind to unstable cfDNA. As used herein, the term "probe having a sequence complementary to cfDNA" refers to a probe having a nucleic acid sequence that can complementarily bind to the target double-stranded cfDNA that is present in a liquid sample such as plasma.

[0026] In this case, the probes can be prepared in two ways. One is a first probe (hereinafter referred to as CP) designed to bind to the damaged portion of the gene, and the other is a second probe (hereinafter referred to as DP) designed to bind to the area surrounding the damaged portion. The DP can be designed to complementarily bind to a target DNA sequence or a sequence located about 10 bp to about 100 bp, or about 20 bp to about 50 bp away from the damaged region.

[0027] Here, complementary binding means that the probe can bind to the target cfDNA under appropriate hybridization conditions to form a duplex, and has a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% complementary to the target sequence of cfDNA.

[0028] Hybridization conditions, including, for example, probe length, probe complementarity, and salt concentration (i.e., ionic strength) in the hybridization buffer, can be experimentally determined by those skilled in the art. Generally, stringent hybridization conditions are those under which a polynucleotide can bind preferentially to its complementary sequence and with higher affinity than any other region on the target. Exemplary stringent conditions for hybridization to the complement of a 20-base polynucleotide sequence can be approximately 50% G+C content, 50 mM salt (Na+), and an annealing temperature of 60°C. For longer sequences, hybridization can be performed at higher temperatures. Generally, stringent conditions are those under which annealing occurs at approximately 5°C below the melting temperature of the polynucleotide. The "melting temperature" is the temperature at which 50% of polynucleotides complementary to a target polynucleotide can bind complementarily at a given ionic strength, pH, and polynucleotide concentration.

[0029] In the present specification, it has been confirmed that damaged cfDNA can be effectively detected by using the first and second probes simultaneously, or by using the first or second probes separately. Furthermore, the probe may be bound to a substance such as biotin to bind to a marker. Alternatively, the probe may be bound to a marker directly or via a linker. In this case, the marker may be a nanoparticle, a fluorescent dye, a fluorescent protein, or an enzyme. Furthermore, the probe and the marker may be added simultaneously or sequentially.

[0030] In one embodiment of the present invention, the probe capable of complementary binding to the target cfDNA can complementarily bind to a region containing the following cancer cell-specific sequence. For example, in the case of a sequence specific to ovarian cancer or breast cancer, the SNPs may be SNPs present in BRCA1 exon 7, BRCA1 exon 10, BRCA1 exon 11, or BRCA1 exon 15. In the case of a sequence specific to gastric cancer, the SNPs may be SNPs present in TP53, and in the case of colon cancer, the SNPs may be SNPs present in MSH2. In the case of a sequence specific to lung cancer, the SNPs may be SNPs present in EGFR. In the case of a sequence specific to liver cancer, the SNPs may be selected from SNPs present in FGFR3.

[0031] Biomarker genes derived from and specific to cancer cells are known to those skilled in the art. For example, the following publications can be referenced: Circulating Cell-Free DNA in Plasma / Serum of Lung Cancer Patients as a Potential Screening and Prognostic Tool, Pathak et al., Clinical Chemistry, October 2006, vol. 52, no. 10, pp. 1833-1842; Cell-free Tumor DNA in Blood Plasma as a Marker for Circulating Tumor Cells in Prostate Cancer, Schwarzenbach et al., Clin Cancer Res, February 1, 2009, pp. 15-1032; Cell-free DNA: Measurement in Various Carcinomas and Establishment of Normal Reference Range, Wua et al., Clinica Chimica Acta, Volume 321, Issues 1-2, July 2002, Pages 77-87; Detection of Circulating Tumor DNA in the Blood (Plasma / Serum) of Cancer Patients, Anker et al., Cancer and Metastasis Reviews 1999,Volume 18,Issue 1,pp 65-73;Cell-free nucleic acids as biomarkers in cancer patients,Schwarzenbach et al,Nature Reviews Cancer 11,426-437(June 2011);Circulating Tumor-Specific DNA:A Marker for Monitoring Efficacy of Adjuvant Therapy in Cancer Patients,Fiegl et al,Cancer Res Feb.15,2005 65;1141.

[0032] In one embodiment of the present invention, a probe capable of complementary binding to a target cfDNA can complementarily bind to a region overexpressed in cancer cells, as described below. Such a region overexpressed in cancer cells can be a biomarker for cancer cells. Such cancer cell biomarkers can be, but are not limited to, the genes shown in Figures 151 and 152. Furthermore, throughout this specification, various biomarker genes for specific tumors / cancer cells and exemplary probes that complementarily bind to biomarker genes are described in the Examples. Furthermore, the probe can further comprise biotin or an avidin-based protein. Specifically, the marker can further comprise any one selected from the group consisting of avidin, streptavidin, or a combination thereof. Preferably, the probe can be in a form bound to biotin.

[0033] As used herein, the term "isolated biological sample" refers to a sample of urine, saliva, cerebrospinal fluid, pleural effusion, ascites, plasma, blood, sputum, or other body fluids isolated from a human body. The isolated biological sample may be a liquid sample isolated from a human body. In this case, plasma may be obtained from blood.

[0034] The term "positively charged substance" used in this specification refers to a material that is positively charged and can be used in the form of nanoparticles, nanowires, a network structure, or a filter, but is not limited to these shapes. A specific example of the "positively charged substance" may be a nanowire or membrane whose surface is positively charged. The nanowire or membrane can be manufactured using a conductive polymer. Examples of the conductive polymer include poly(acetylene), poly(pyrrole), poly(thiophene), poly(para-phenylene), poly(3,4-ethylenedioxythiophene), poly(phenylene sulfide), poly(paraphenylene vinylene), and poly(para-phenylene vinylene). The nanowire may be any one selected from the group consisting of vinylene and polyaniline. The length and diameter can be adjusted appropriately depending on the production method, but in the case of nanowires, the diameter can be selected from the range of about 50 nm to about 500 nm, about 100 nm to about 500 nm, about 100 nm to about 400 nm, about 150 nm to about 350 nm, about 200 nm to about 400 nm, or about 100 nm to about 300 nm, and the length can be selected from the range of several μm to about 100 μm, about 10 μm to about 100 μm, about 15 μm to about 50 μm, about 15 μm to about 40 μm, or about 15 μm to about 30 μm.

[0035] In one embodiment, the nanowire may have a diameter of about 200 nm and a length of about 18 μm. The nanowire may also be fabricated in a form in which biotin is bound.

[0036] The surface of the nanowire or membrane may be modified with a cationic polymer. The type of cationic polymer is not limited. Specific examples of cationic polymers include polyethyleneimine (PEI) and polylysine (PLL). Another example is the cationic branched polymer polyethyleneimine. The nanowire or membrane modified with such a cationic polymer may have a positively charged surface. To capture cfDNA, the surface charge of the nanowire or membrane may be about 20 mV to about 80 mV, about 30 mV to about 60 mV, or about 35 mV to about 50 mV. The surface charge may be about 36 mV, about 37 mV, about 38 mV, about 39 mV, about 40 mV, about 41 mV, about 42 mV, about 43 mV, or about 44 mV.

[0037] In one embodiment, positively charged nanowires can effectively capture cfDNA even at low concentrations, particularly due to nanowire characteristics such as a large surface area for binding to target molecules such as DNA and enhanced mobility for facilitating interaction with DNA.

[0038] As used herein, the term "marker" refers to a substance for effectively detecting and / or quantifying cfDNA having a double helix structure derived from cancer cells. Specifically, the marker may be a quantum dot, a substance that degrades a specific substrate to produce a color reaction, or a substance that emits light when irradiated with light of a specific wavelength. Specifically, the marker may be a fluorescent protein such as GFP (Green Fluorescent Protein), YFP (Yellow Fluorescent Protein), RFP (Red Fluorescent Protein), or CFP (Cyan Fluorescent Protein). Alternatively, the marker may be a chromogenic or bioluminescent enzyme such as alkaline phosphatase (AP), HRP (Horseradish peroxidase), or β-galactosidase (BGAL).

[0039] The chromogenic enzyme mediates a color-developing or luminescent reaction by reacting with a substrate. Examples of such substrates that can be used for HRP include ABTS, OPD, AmplexRed, DAB, AEC, TMB, homovanillic acid, and luminol. Examples of such substrates that can be used for AP include BCIP (5-Bromo-4-Chloro-3-Indolyl Phosphate) / NBT (nitriblue tetrazolium), pNPP (p-Nitrophenyl Phosphate), Fast Red TR / Naphthol AS-MX, and CDP-Star (Disodium 2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2'-(5-chlorotricyclo[3.3.1.1]). 3.7]decane])-4-yl]-1-phenyl phosphate) can be used, and in the case of BGAL, any one substrate selected from the group consisting of X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) or ONPG (ortho-nitrophenyl-β-galactoside) can be used together. The bioluminescent enzyme can be luciferase derived from firefly (Photinus pyralis), sea pansy (Renilla sp.), copepod Metridia longa, Vibrio bacteria, or dinoflagellate.

[0040] The marker may further include a substance capable of binding to the probe. Specifically, when biotin is bound to the probe, the marker may further include an avidin-based protein. Specifically, the marker may further include any one selected from the group consisting of avidin, streptavidin, or a combination thereof.

[0041] The marker may further include biotin. In this case, the probe may further include an avidin-based protein. Specifically, the marker may further include any one selected from the group consisting of avidin, streptavidin, or a combination thereof.

[0042] One specific example of such a marker is nanoparticles formed by conjugating streptavidin and HRP to nanoparticles composed of a conductive polymer and hyaluronic acid. In this case, the conductive polymer is as described above, preferably polypyrrole. Another specific example is nanoparticles formed by conjugating streptavidin and a fluorescent protein to nanoparticles composed of a conductive polymer and hyaluronic acid. The size of the HRP nanoparticles may be about 20 nm to about 150 nm, about 30 nm to about 120 nm, or about 40 nm to about 100 nm. The HRP nanoparticles may also be about 50 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, or about 80 nm.

[0043] A substrate compatible with the marker can be used to induce a color reaction. The substrate can be added simultaneously with the marker or before or after the marker. The marker and substrate can be used in a known manner. For example, when HRP is used as a marker, ABTS (2,2'-Azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt), OPD (o-Phenylenediamine dihydrochloride), Amplex Red, DAB (3,3'-diaminobenzidine tetrahydrochloride), AEC (3-Amino-9-ethylcarbazole), TMB (3,3',5,5'-Tetramethylbenzidine), homovanillic acid, or luminol can be used as a substrate. For example, when a fluorescent protein is used, the marker can be detected by detecting the presence or absence of light emitted after irradiation with light of a specific wavelength, rather than by a substrate.

[0044] According to one embodiment, the diagnostic method can detect target cfDNA with high precision and accuracy, even when the target cfDNA is present in extremely small amounts in a biological sample, and thus can be useful for detecting cancer cells at an early stage.

[0045] By detecting the presence or absence of specific abnormal cells / tissues, such as cfDNA encoding a biomarker for a specific cancer, in a biological sample, it is possible to understand the diagnosis, prognosis, or metastasis status of the cancer, and it is also possible to predict resistance / tolerance to conventional treatment methods.

[0046] <Prostate cancer> How prostate cancer is diagnosed One aspect of the present invention provides a method for diagnosing prostate cancer by detecting genes derived from prostate cancer cells from the sample without amplification, the method comprising the steps of: a) mixing a biological sample isolated from an individual containing cell-free DNA (hereinafter referred to as cfDNA) with a positively charged substance; b) separating the positively charged substance bound to the cfDNA; c) sequentially or simultaneously mixing a probe and a marker having a sequence complementary to the cfDNA with the mixture; d) removing the probe and marker that do not bind to the cfDNA; and e) detecting the marker, wherein the probe having a sequence complementary to the cfDNA complementarily binds to a gene known to be a prostate cancer biomarker. In this case, the gene known to be a prostate cancer biomarker may be a gene encoding a protein overexpressed in prostate cancer.

[0047] Specifically, the probe having a sequence complementary to the cfDNA may be one that binds complementarily to at least one gene selected from the group consisting of KLK3, FOLH1, PCA3, PDE4D7, SFMBT2, EFEMP1, RETN, ACADL, AGR2, COL1A1, FAM13C, GPX8, GRHL2, HNF1A, HOXB13, KLK2, MYBPC1, NR0B1, PITX2, SFRP4, SLCO1B3, TMEFF2, TMPRSS2-ERG, and combinations thereof.

[0048] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in the sample can be separated and / or concentrated using various methods. As a specific example, a nitrocellulose membrane, which has a strong affinity for nucleic acids, can be used. As another specific example, a positively charged substance can be used to capture negatively charged cfDNA. The positively charged substance can be, but is not limited to, nanoparticles, nanowires, a mesh structure, or a positively charged filter. A specific example of the "positively charged substance" can be a positively charged nanostructure or a positively charged membrane.

[0049] An example of the nanostructure may include a cationic polymer. The type of the cationic polymer is not limited. A specific example of the cationic polymer is polyethyleneimine (PEI), which may be a cationic branched polymer polyethyleneimine.

[0050] In addition, nanowires labeled with avidin-based proteins can be mixed with a biotin-conjugated PEI solution to further bind cationic branched polyethyleneimine (PEI) to the nanowires through the biotin-avidin protein interaction. As a result, nanoparticles are embedded in a nanostructure (PEI / mPpy NW) with a high density and irregular distribution, with the cationic polymer polyethyleneimine bound to the surface.

[0051] Such nanowires can successfully capture genomic DNA and cfDNA at high efficiency and low concentrations. In particular, nanowire features such as a large surface area for binding to target molecules such as DNA and enhanced mobility for promoting interaction with DNA enable efficient and effective capture of target cfDNA.

[0052] In this case, the target cfDNA refers to the cfDNA of interest to be detected. As used herein, cfDNA is double-stranded. In this case, a portion of the cfDNA may be unwound. The cfDNA may also be derived from genes in prostate cancer cells. Specifically, cfDNA may contain a nucleic acid sequence that is overexpressed in prostate cancer cells. The nucleic acid sequence that is overexpressed in cancer cells refers to a nucleic acid sequence that is expressed at an appropriate level in normal cells but is overexpressed in specific cancer cells.

[0053] Specifically, the degree or cutoff of overexpression of the nucleic acid sequence in cancer cells may be an OD value of 0.007 or more when measuring optical density using a marker. More specifically, the degree or cutoff of overexpression of the nucleic acid sequence in cancer cells may be an OD value determined by the maximum sensitivity and specificity of a receiver operating characteristic curve after measuring absorbance using a marker. In this case, the wavelength of irradiation for measuring absorbance may be appropriately determined depending on the marker. The cfDNA may be DNA with unwinding of the double strand. Furthermore, the cfDNA may be appropriately determined depending on the purpose.

[0054] Furthermore, the cfDNA derived from the prostate cancer cells may be characterized by: i) having a lower Tm value than cfDNA having a double helix structure derived from normal cells; or ii) being denatured under conditions under which cfDNA having a double helix structure derived from normal cells is not denatured.

[0055] Furthermore, the cfDNA can be bound to a probe of about 15 mer to about 30 mer that can complementarily bind to the cfDNA under any one of the following conditions: i) leaving the sample at room temperature for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes; iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C to about 75°C for about 30 seconds to about 30 minutes; v) heating at about 25°C to about 40°C for about 10 minutes to about 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; vii) treating with DNase for about 1 minute to about 30 minutes; and viii) treating with chemicals (e.g., sodium hydroxide, DMSO, surfactants, etc.).

[0056] The genes overexpressed in prostate cancer cells include KLK3 (NCBI Gene ID: 354), FOLH1 (NCBI Gene ID: 2346), ACPP (NCBI Gene ID: 55), PCA3 (NCBI Gene ID: 50652), PDE4D7 (NCBI Gene ID: 5144), SFMBT2 (NCBI Gene ID: 57713), EFEMP1 (NCBI Gene ID:2202), RETN(NCBI Gene ID:56729), ACADL(NCBI Gene ID:33), AGR2(NCBI Gene ID:10551), COL1A1(NCBI Gene ID:1277), FAM13C(NCBI Gene ID:220965), GPX8(NCBI Gene ID:493869), GRHL2(NCBI Gene ID:79977), HNF1A(NCBI Gene ID:6927), HOXB13(NCBI Gene ID:10481), KLK2(NCBI Gene ID:3817), MYBPC1(NCBI Gene ID:4604), NR0B1(NCBI Gene ID:190), PITX2(NCBI Gene ID:5308), SFRP4(NCBI Gene ID:6424), SLCO1B3(NCBI Gene ID:28234), TMEFF2(NCBI Gene ID:23671), CPT1A(NCBI Gene ID:1374), IFNG(NCBI Gene ID:3458), CD274(NCBI Gene ID:29126), FOLR1(NCBI Gene ID:2348), EPCAM(NCBI Gene ID:4072), OGT(NCBI Gene ID:8473), TMPRSS2-ERG, and combinations thereof.

[0057] In one embodiment, the genes specific to prostate cancer are KLK3, FOLH1, PCA3, PDE4D7, SFMBT2, EFEMP1, RETN, ACADL, AGR2, COL1A1, FAM13C, GPX8, GRHL2, HNF1A, HOXB13, KLK2, MYBPC1, NR0B1, PITX2, SFRP4, SLCO1B3, TMEFF2, and TMPRSS2-ERG genes. Prostate cancer can be diagnosed by additionally detecting one or more additional marker genes selected from the group consisting of ACPP, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, and EPCAM. Although the additional marker genes are not specific to prostate cancer, their use in combination with the genes specific to prostate cancer can significantly improve the sensitivity and specificity of the prostate cancer diagnostic method.

[0058] As used herein, the term "KLK3" refers to a gene encoding kallikrein-3, gamma-seminoprotein, or prostate-specific antigen (PSA). PSA is a protease synthesized in prostate epithelial cells and is rarely expressed in tissues other than the prostate. It is a useful tumor marker used in the screening of prostate cancer. PSA is also useful not only for screening prostate cancer but also for assessing recurrence after surgery.

[0059] As used herein, the term "FOLH1" refers to a gene encoding prostate-specific membrane antigen (PSMA). PSMA is highly expressed in the prostate, and its expression in prostate cancer cells is known to be approximately 8 to 12 times higher than in normal prostate cells. PSMA is used as a tumor marker for diagnosing prostate cancer.

[0060] As used herein, the term "ACPP" refers to a gene encoding prostatic acid phosphatase (PAP). PAP is an enzyme produced in the prostate. PAP expression is increased in men suffering from prostate cancer or prostate disease, and it is used as an indicator of prostate cancer or prostate disease.

[0061] As used herein, the term "PCA3" refers to a gene expressed in the form of non-coding RNA in human prostate tissue. PCA3 is expressed only in human prostate tissue and is highly overexpressed in prostate cancer cells. Thus, PCA3 is used as a tumor marker for prostate cancer.

[0062] The term "probe" as used herein refers to DNA or RNA for detecting cfDNA. The probe may have a specific sequence that allows it to bind complementarily to cfDNA. A probe having a sequence complementary to cfDNA refers to a probe having a nucleic acid sequence that can bind complementarily to the target double-stranded cfDNA present in plasma that is to be detected. In this case, the probe may be bound to biotin. The probe may bind to a marker bound to a biotin-binding protein.

[0063] As used herein, the term "marker" refers to a substance used to detect probes bound to cfDNA. The marker may be a nanoparticle, a fluorescent dye, a fluorescent protein, or an enzyme. Specifically, the marker may be any one selected from the group consisting of quantum dots, HRP, and fluorescent proteins. In one specific example, the marker may be, but is not limited to, green fluorescent protein (GFP), blue fluorescent protein (BFP), cyan fluorescent protein (BFP), yellow fluorescent protein (YFP), or horse radish peroxidase (HRP).

[0064] The marker may be conjugated with a biotin-binding protein. The biotin-binding protein may be an avidin-based protein such as avidin, streptavidin, traptavidin, or neutravidin, but any protein that can specifically bind to biotin may be used without limitation. In one specific example, the marker may be conjugated with streptavidin.

[0065] The term "avidin" as used herein refers to a homotetrameric protein produced in the oviducts of birds, reptiles, and amphibians, distributed in egg white, and capable of binding to biotin with high affinity. Although its function in nature has not yet been elucidated, it is presumed to inhibit bacterial growth by binding to biotin, which is essential for bacterial growth.

[0066] As used herein, the term "streptavidin" refers to a tetrameric biotin-binding protein with a molecular weight of approximately 60 kDa isolated from Streptomyces avidinii. Although it has very low homology to avidin, its structure is very similar. Like avidin, streptavidin possesses antibacterial activity and a very high binding affinity for biotin. Unlike avidin, streptavidin does not contain carbohydrates, exhibits an acidic isoelectric point (pI = 5), and has significantly lower solubility than avidin. Commercially available streptavidin, such as Thermo Scientific Pierce Streptavidin, is a recombinant form of streptavidin with a molecular weight of approximately 53 kDa and a near-neutral isoelectric point (pI = 6.8-7.5). The lack of glycosylation and low pI of streptavidin result in a lower level of nonspecific binding (especially lectin binding) compared to avidin. These properties of streptavidin make it an ideal reagent of choice for many detection systems.

[0067] As used herein, the term "traptavidin" refers to a streptavidin variant or mutein, a protein that exhibits approximately 10-fold slower biotin dissociation rate, increased mechanical strength, and improved thermal stability. Traptavidin also specifically binds to biotin.

[0068] The term "neutravidin" used in the present invention, also known as "deglycosylated avidin," was developed to overcome the major drawbacks of natural avidin and streptavidin. As the name suggests, neutravidin is produced by deglycosylating avidin. It is a protein that maintains high biotin-binding ability while retaining a reduced molecular weight (approximately 60 kDa) compared to avidin. Deglycosylation of avidin reduces lectin binding to undetectable levels and lowers its isoelectric point (pI = approximately 6.3), effectively eliminating the main cause of nonspecific binding to avidin. Because the lysine residues remain available, neutravidin can be easily derivatized or conjugated, like streptavidin. Furthermore, its high biotin-binding ability and low nonspecific binding make it an ideal biotin-binding protein with a wide range of applications.

[0069] As used herein, the term "detecting a marker" refers to detecting a marker bound to a probe through a biotin-avidin reaction. The detection of the marker can be measured by a color change, a change in UV absorbance, the presence or absence of bioluminescence, a change in a fluorescent reaction, or an electrochemical change. Specifically, the method for detecting the marker varies depending on the marker used. For example, when HRP is used as a marker, the marker can be detected by observing the color reaction resulting from the reaction of hydrogen peroxide with a substrate. When the marker is a fluorescent protein such as GFP, the presence or absence of the marker can be detected by observing the light emitted after irradiation with light of a specific wavelength. When the marker is luciferase, the presence of the marker can be detected by adding a substrate such as luciferin and measuring the bioluminescence generated using a bioluminometer.

[0070] The diagnostic method of the present invention may further include a step of denaturing cfDNA. In this case, the denaturation step may be performed so as to selectively denature only cfDNA derived from prostate cancer, while leaving normal double-stranded cfDNA undenatured. For this purpose, the denaturation step may be performed at about 50°C to about 100°C for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is typically about 0.1 seconds to about 8 minutes. Alternatively, denaturation may be performed for about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds, or about 90 seconds. In one example, the denaturation step of cfDNA may be performed before step c).

[0071] Specifically, prior to step c), the method may further include denaturing the sample or the cfDNA bound to the positively charged substance under one of the following conditions: (i) leaving the sample at room temperature for about 1 to 10 minutes; (ii) heating at about 90°C to about 95°C for 1 second to 1 minute; (iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; (iv) heating at about 60°C to about 75°C for about 1 to 30 minutes; (v) heating at about 25°C to about 40°C for about 5 to 60 minutes; (vi) treating with protease for about 1 to 10 minutes; and (vii) treating with DNase I for about 1 to 10 minutes. This denaturation step selectively denatures only the cfDNA derived from cancer cells, while not denaturing double-stranded cfDNA derived from normal cells, thereby further facilitating binding to the probe. The denaturation conditions i) to vii) can be performed after obtaining the sample. The denaturation conditions i) to vii) can be performed after obtaining the cfDNA bound to the positively charged substance. The temperature, protease, and DNase treatment times for the denaturation conditions i) to vii) can be adjusted as appropriate, as long as they do not denature stable cfDNA.

[0072] Prostate cancer diagnostic kit Another aspect of the present invention is to provide a diagnostic kit for prostate cancer, comprising: a biotin-conjugated probe that binds complementarily to a gene specifically expressed in prostate cancer; a positively charged substance; a marker bound to an avidin-based protein; and instructions.

[0073] In this case, the gene specifically expressed in prostate cancer may be any one or more selected from the group consisting of KLK3, FOLH1, PCA3, PDE4D7, SFMBT2, EFEMP1, RETN, ACADL, AGR2, COL1A1, FAM13C, GPX8, GRHL2, HNF1A, HOXB13, KLK2, MYBPC1, NR0B1, PITX2, SFRP4, SLCO1B3, TMEFF2, TMPRSS2-ERG, and combinations thereof.

[0074] The instructions may also describe that the kit configuration can diagnose prostate cancer by the following protocol: a) isolating cfDNA from a biological sample isolated from an individual using a positively charged substance included in the kit; b) sequentially or simultaneously mixing the isolated cfDNA with a biotin-conjugated probe included in the kit and a marker included in the kit; c) removing probes and markers that do not bind to cfDNA; and d) detecting signals from the markers.

[0075] The antibody may additionally contain a biotin-conjugated probe that binds complementarily to at least one gene selected from the group consisting of ACPP, CPT1A, IFNG, CD274, FOLR1, EPCAM, OGT, and combinations thereof.

[0076] The probe, positively charged substance, and marker are as described above.

[0077] Prostate cancer diagnostic device Another aspect of the present invention is to provide an apparatus for diagnosing prostate cancer by detecting genes derived from prostate cancer cells from a sample without amplification, comprising: a) a mixing section that mixes a biological sample isolated from an individual containing cfDNA with a positively charged substance; b) an acquisition section that removes the sample from which the positively charged substance to which cfDNA is bound has been removed; c) a reaction section that sequentially or simultaneously adds a biotin-binding probe capable of complementarily binding to a gene specifically expressed in prostate cancer, and nanoparticles containing streptavidin and a marker, to the positively charged substance to which cfDNA is bound; d) a detection section that detects the marker; and e) an information processing section that determines, based on the presence or absence of detection of the marker, that cfDNA derived from prostate cancer and having a sequence complementary to the probe is present in the sample.

[0078] In this case, the gene specifically expressed in prostate cancer may be any one or more selected from the group consisting of KLK3, FOLH1, PCA3, PDE4D7, SFMBT2, EFEMP1, RETN, ACADL, AGR2, COL1A1, FAM13C, GPX8, GRHL2, HNF1A, HOXB13, KLK2, MYBPC1, NR0B1, PITX2, SFRP4, SLCO1B3, TMEFF2, TMPRSS2-ERG, and combinations thereof.

[0079] The antibody may additionally contain a biotin-conjugated probe that binds complementarily to at least one gene selected from the group consisting of ACPP, CPT1A, IFNG, CD274, FOLR1, EPCAM, OGT, and combinations thereof.

[0080] <Lung cancer> How lung cancer is diagnosed One aspect of the present invention provides a method for diagnosing lung cancer by detecting genes derived from lung cancer cells in the sample without amplification, the method comprising: (a) mixing a biological sample isolated from an individual containing cell-free DNA (cfDNA) with a positively charged substance; (b) separating the positively charged substance bound to the cfDNA; (c) sequentially or simultaneously mixing a probe and a marker having a sequence complementary to the cfDNA with the mixture; (d) removing the probe and marker that do not bind to the cfDNA; and (e) detecting the marker, wherein the probe having a sequence complementary to the cfDNA complementarily binds to a gene known to be a lung cancer biomarker. In this case, the gene known to be a lung cancer biomarker may be a gene encoding a protein overexpressed in lung cancer.

[0081] Specifically, the probe having a sequence complementary to the cfDNA may be one that binds complementarily to at least one gene selected from the group consisting of ENO2, SART3, KRT19, PLAT, EGFR, ALK, ROS1, RET, ERBB2, PI3K, S100P, MMP11, CDCA7, S100A2, ETV4, TOP2A, UBE2C, and combinations thereof.

[0082] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in the sample can be separated and / or concentrated using various methods. In one embodiment, a nitrocellulose membrane, which has a strong affinity for nucleic acids, can be used. In another embodiment, a positively charged substance can be used to capture negatively charged cfDNA. The positively charged substance can be, but is not limited to, nanoparticles, nanowires, a mesh structure, or a positively charged filter. A specific example of the "positively charged substance" can be a positively charged nanostructure or a positively charged membrane.

[0083] An example of the nanostructure may include a cationic polymer. The type of the cationic polymer is not limited. A specific example of the cationic polymer is polyethyleneimine (PEI), which may be a cationic branched polymer polyethyleneimine.

[0084] In addition, by mixing streptavidin-labeled nanowires with a biotin-conjugated PEI solution, cationic branched polyethyleneimine (PEI) can be additionally bound to the nanowires through the biotin-streptavidin interaction, resulting in nanoparticles embedded in a nanostructure (PEI / mPpy NW) with a high density and irregular distribution, with the cationic polymer polyethyleneimine bound to the surface.

[0085] Such nanowires can successfully capture genomic DNA and cfDNA at high efficiency and low concentrations. In particular, nanowire features such as a large surface area for binding to target molecules such as DNA and enhanced mobility for promoting interaction with DNA enable efficient and effective capture of target cfDNA.

[0086] In this context, the target cfDNA refers to the cfDNA of interest to be detected. As used herein, cfDNA is double-stranded. In this context, the double strand of the cfDNA may be partially unwound. The cfDNA may also be derived from genes in lung cancer cells. Specifically, cfDNA may contain a nucleic acid sequence overexpressed in lung cancer cells. The nucleic acid sequence overexpressed in cancer cells refers to a nucleic acid sequence that exhibits an appropriate expression level in normal cells but is overexpressed in specific cancer cells. Specifically, the degree or cutoff for the nucleic acid sequence overexpressed in cancer cells may be a value of about 0.010 or greater when measuring optical density using a marker. More specifically, the degree or cutoff for the nucleic acid sequence overexpressed in cancer cells may be a value of about 0.012 or about 0.015 or greater when measuring optical density using a marker. In this context, the wavelength of irradiation for measuring the absorbance may be determined appropriately depending on the marker. The cfDNA may be DNA in which the double strand is unwound (unwinding of DNA). Furthermore, the cfDNA can be appropriately determined depending on the purpose.

[0087] Furthermore, the cfDNA derived from the lung cancer cells may be characterized by: i) having a lower Tm value than cfDNA having a double helix structure derived from normal cells; or ii) being denatured under conditions under which cfDNA having a double helix structure derived from normal cells is not denatured.

[0088] Furthermore, the cfDNA can be bound to a probe of approximately 15 mer to approximately 30 mer that can complementarily bind to the cfDNA under any one of the following conditions: i) leaving it at room temperature for approximately 1 minute to approximately 120 minutes; ii) heating at approximately 90°C to approximately 95°C for approximately 1 second to approximately 3 minutes; iii) heating at approximately 75°C to approximately 90°C for approximately 1 second to approximately 5 minutes; iv) heating at approximately 60°C to approximately 75°C for approximately 30 seconds to approximately 30 minutes; v) heating at approximately 25°C to approximately 40°C for 10 minutes to 120 minutes; vi) treating with protease for approximately 1 minute to approximately 30 minutes; and vii) treating with DNase for approximately 1 minute to approximately 30 minutes.

[0089] The genes overexpressed in lung cancer cells include ENO2 (NCBI Gene ID: 2026), SART3 (NCBI Gene ID: 9733), ACPP (NCBI Gene ID: 55), KRT19 (NCBI Gene ID: 3880), PLAT (NCBI Gene ID: 5327), EGFR (NCBI Gene ID: 1956), KRAS (NCBI Gene ID:3845), ALK (NCBI Gene ID:238), ROS1 (NCBI Gene ID:6098), RET (NCBI Gene ID:5979), ERBB2 (NCBI Gene ID:2064), PI3K (NCBI Gene ID:5291), S100P (NCBI Gene ID:6286), MMP11 (NCBI Gene ID:4320), CDCA7 (NCBI Gene ID:83879), S100A2(NCBI Gene ID: 6273), ETV4 (NCBI Gene ID: 2118), TOP2A (NCBI Gene ID: 7153), UBE2C (NCBI Gene ID: 11065), CPT1A (NCBI Gene ID: 1374), IFNG (NCBI Gene ID: 3458), CD274 (NCBI Gene ID: 29126), FOLR1 (NCBI Gene ID: 2348), EPCAM (NCBI Gene ID: 4072), OGT (NCBI Gene ID: 8473), and combinations thereof.

[0090] In one specific example, genes that are specifically present in lung cancer are SART3, PLAT, ALK, ROS1, PI3K, S100P, CDCA7, S100A2, and ETV4 genes, and additionally, lung cancer can be diagnosed by detecting the ENO2, ACPP, KRT19, EGFR, KRAS, RET, ERBB2, MMP11, TOP2A, UBE2C, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, or EPCAM gene.

[0091] As used herein, the term "ENO2" refers to a gene encoding an enzyme known as gamma-enolase, enolase 2, or NSE (neuron-specific enolase). NSE is used as a tumor marker for small cell lung cancer, neuroblastoma, and medullary thyroid cancer.

[0092] As used herein, the term "SART3" refers to a gene encoding squamous cell carcinoma antigen (SCCA). SCCA is found to be positive in the blood of patients with many types of squamous cell carcinoma, including not only cervical squamous cell carcinoma but also vulvar cancer, vaginal cancer, esophageal cancer, tongue cancer, and pharyngeal cancer, and is therefore used as a tumor marker.

[0093] As used herein, the term "KRT19" refers to a gene encoding a protein known as Cyfra21-1, CK-19 (cytokeratin-19), or K19 (keratin-19). Cyfra21-1 is known to be associated with cancers derived from epithelial cells, such as lung cancer and head and neck cancer. Furthermore, Cyfra21-1 has been reported to have higher blood levels in patients with pneumonia or lung disease than in normal individuals, and is therefore used as a tumor marker.

[0094] As used herein, the term "PLAT" refers to the gene encoding TPA (Tissue plasminogen activator), a protein involved in the breakdown of blood mochi.

[0095] As used herein, the term "probe" refers to DNA or RNA for detecting cfDNA. The probe may have a specific sequence that allows it to bind complementarily to cfDNA. A probe having a sequence complementary to cfDNA refers to a probe having a nucleic acid sequence that can bind complementarily to the target double-stranded cfDNA present in plasma that is to be detected. In this case, the probe may be bound to biotin. The probe may bind to a marker bound to a biotin-binding protein.

[0096] As used herein, the term "marker" refers to a substance used to detect probes bound to cfDNA. The marker may be a nanoparticle, a fluorescent dye, a fluorescent protein, or an enzyme. Specifically, the marker may be any one selected from the group consisting of quantum dots, HRP, and fluorescent proteins. In one specific example, the marker may be, but is not limited to, green fluorescent protein (GFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), or horse radish peroxidase (HRP).

[0097] The marker may be conjugated with a biotin-binding protein. The biotin-binding protein may be an avidin-based protein such as streptavidin, traptavidin, or neutravidin, but any protein that can specifically bind to biotin may be used without limitation. In one specific example, the marker may be conjugated with streptavidin.

[0098] As used herein, the term "streptavidin" refers to a tetrameric biotin-binding protein with a molecular weight of 60 kDa isolated from Streptomyces avidinii. Although it has very low homology to avidin, its structure is very similar. Like avidin, streptavidin possesses antibacterial activity and a very high binding affinity for biotin. Unlike avidin, streptavidin does not contain carbohydrates, exhibits an acidic isoelectric point (pI = approximately 5), and has significantly lower solubility than avidin. Commercially available streptavidin, such as Thermo Scientific Pierce Streptavidin, is a recombinant form of streptavidin with a molecular weight of approximately 53 kDa and a near-neutral isoelectric point (pI = approximately 6.8 to approximately 7.5). The lack of glycosylation and low pI of streptavidin result in a lower level of nonspecific binding (especially lectin binding) compared to avidin. These properties of streptavidin make it an ideal reagent of choice for many detection systems.

[0099] As used herein, the term "traptavidin" refers to a streptavidin variant or mutein, a protein that exhibits approximately 10-fold slower biotin dissociation rate, increased mechanical strength, and improved thermal stability. Traptavidin also specifically binds to biotin.

[0100] The term "neutravidin" used in the present invention, also known as "deglycosylated avidin," was created to avoid the major drawbacks of natural avidin and streptavidin. As the name suggests, neutravidin is produced by deglycosylating avidin. It is a protein that maintains high biotin-binding ability while having a reduced molecular weight (60 kDa) compared to avidin. Deglycosylation of avidin reduces lectin binding to undetectable levels and lowers its isoelectric point (pI = approximately 6.3), effectively eliminating the main cause of nonspecific binding to avidin. Because the lysine residues remain available, it can be easily derivatized or conjugated, like streptavidin. Furthermore, its high biotin-binding ability and low nonspecific binding make it an ideal biotin-binding protein, making it suitable for a variety of uses.

[0101] As used herein, the term "detecting a marker" refers to detecting a marker bound to a probe through a biotin-avidin reaction. The detection of the marker can be measured by a color change, a change in UV absorbance, the presence or absence of bioluminescence, a change in a fluorescent reaction, or an electrochemical change. Specifically, the method for detecting the marker varies depending on the marker used. For example, when HRP is used as a marker, the marker can be detected by observing the color reaction resulting from the reaction of hydrogen peroxide with a substrate. When the marker is a fluorescent protein such as GFP, the presence or absence of the marker can be detected by observing the light emitted after irradiation with light of a specific wavelength. When the marker is luciferase, the presence of the marker can be detected by adding a substrate such as luciferin and measuring the bioluminescence generated using a bioluminometer.

[0102] The diagnostic method of the present invention may further include a step of denaturing cfDNA. In this case, the denaturation step can be performed so as to selectively denature only lung cancer-derived cfDNA while leaving normal double-stranded cfDNA undenatured. For this purpose, the denaturation step can be performed at about 50°C to about 100°C for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is typically about 0.1 seconds to about 8 minutes. Alternatively, denaturation can be performed for about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds, or about 90 seconds. In one example, the denaturation step of cfDNA can be performed before step c).

[0103] Specifically, prior to step c), the method may further include denaturing the sample or the cfDNA bound to the positively charged substance under one of the following conditions: (i) leaving the sample at room temperature for about 1 to about 10 minutes; (ii) heating at about 90°C to about 95°C for about 1 second to about 1 minute; (iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; (iv) heating at about 60°C to about 75°C for about 1 to about 30 minutes; (v) heating at about 25°C to about 40°C for about 5 minutes to about 60 minutes; (vi) treating with protease for about 1 to about 10 minutes; and (vii) treating with DNase I for about 1 to about 10 minutes. This denaturation step selectively denatures only the cfDNA derived from cancer cells, while not denaturing double-stranded cfDNA derived from normal cells, thereby further facilitating binding to the probe. The denaturation conditions i) to vii) can be performed after obtaining the sample. The denaturation conditions i) to vii) can be performed after obtaining the cfDNA bound to the positively charged substance. The temperature, protease, and DNase treatment times for the denaturation conditions i) to vii) can be adjusted as appropriate, as long as they do not denature stable cfDNA.

[0104] Lung cancer diagnostic kit Another aspect of the present invention is to provide a diagnostic kit for lung cancer, comprising: a biotin-conjugated probe that binds complementarily to a gene specifically expressed in lung cancer; a positively charged substance; a marker bound to an avidin-based protein; and instructions.

[0105] In this case, the gene specifically expressed in lung cancer may be any one or more selected from the group consisting of SART3, PLAT, ALK, ROS1, PI3K, S100P, CDCA7, S100A2, ETV4, and combinations thereof.

[0106] The instructions may also describe that the kit configuration can diagnose lung cancer by the following protocol: a) isolating cfDNA from a biological sample isolated from an individual using a positively charged substance included in the kit; b) sequentially or simultaneously mixing the isolated cfDNA with a biotin-conjugated probe included in the kit and a marker included in the kit; c) removing probes and markers that do not bind to cfDNA; and d) detecting signals from the markers.

[0107] The kit may further include a biotin-conjugated probe that complementarily binds to at least one gene selected from the group consisting of ENO2, ACPP, KRT19, EGFR, KRAS, RET, ERBB2, MMP11, TOP2A, UBE2C, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, or EPCAM, and combinations thereof.

[0108] The probe, positively charged substance, and marker are as described above.

[0109] Lung cancer diagnostic device Another aspect of the present invention is to provide an apparatus for diagnosing lung cancer by detecting genes derived from lung cancer cells from a sample without amplification, the apparatus comprising: a) a mixing unit that mixes a biological sample isolated from an individual containing cfDNA with a positively charged substance; b) a harvesting unit that removes the sample from which the positively charged substance to which cfDNA is bound has been removed; c) a reaction unit that sequentially or simultaneously adds a biotin-binding probe capable of complementarily binding to a gene specifically expressed in lung cancer, and nanoparticles containing streptavidin and a marker, to the positively charged substance to which cfDNA is bound; d) a detection unit that detects the marker; and e) an information processing unit that determines, based on the presence or absence of detection of the marker, that cfDNA derived from lung cancer and having a sequence complementary to the probe is present in the sample.

[0110] In this case, the gene specifically expressed in lung cancer may be any one or more selected from the group consisting of SART3, PLAT, ALK, ROS1, PI3K, S100P, CDCA7, S100A2, ETV4, and combinations thereof.

[0111] The kit may further include a biotin-conjugated probe that complementarily binds to at least one gene selected from the group consisting of ENO2, ACPP, KRT19, EGFR, KRAS, RET, ERBB2, MMP11, TOP2A, UBE2C, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, or EPCAM, and combinations thereof.

[0112] <Thyroid cancer> How thyroid cancer is diagnosed One aspect of the present invention provides a method for diagnosing thyroid cancer by detecting genes derived from thyroid cancer cells in the sample without amplification, the method comprising the steps of: a) mixing a biological sample isolated from an individual containing cell-free DNA (cfDNA) with a positively charged substance; b) separating the positively charged substance bound to the cfDNA; c) sequentially or simultaneously mixing a probe and a marker having a sequence complementary to the cfDNA with the mixture; d) removing the probe and marker that do not bind to the cfDNA; and e) detecting the marker, wherein the probe having a sequence complementary to the cfDNA complementarily binds to a gene known to be a biomarker for thyroid cancer. The gene known to be a biomarker for thyroid cancer may be a gene encoding a protein overexpressed in thyroid cancer.

[0113] Specifically, the probe having a sequence complementary to the cfDNA may bind complementarily to at least one gene selected from the group consisting of TG, CALCA, APOC1, HIG2, and combinations thereof.

[0114] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in the sample can be separated and / or concentrated using various methods. As a specific example, a nitrocellulose membrane, which has a strong affinity for nucleic acids, can be used. As another specific example, a positively charged substance can be used to capture negatively charged cfDNA. The positively charged substance can be, but is not limited to, nanoparticles, nanowires, a mesh structure, or a positively charged filter. A specific example of the "positively charged substance" can be a positively charged nanostructure or a positively charged membrane.

[0115] An example of the nanostructure may include a cationic polymer. The type of the cationic polymer is not limited. A specific example of the cationic polymer is polyethyleneimine (PEI), which may be a cationic branched polymer polyethyleneimine.

[0116] In addition, by mixing streptavidin-labeled nanowires with a biotin-conjugated PEI solution, cationic branched polyethyleneimine (PEI) can be additionally bound to the nanowires through the biotin-streptavidin interaction, resulting in nanoparticles embedded in a nanostructure (PEI / mPpy NW) with a high density and irregular distribution, with the cationic polymer polyethyleneimine bound to the surface.

[0117] Such nanowires can successfully capture genomic DNA and cfDNA at high efficiency and low concentrations. In particular, nanowire features such as a large surface area for binding to target molecules such as DNA and enhanced mobility for promoting interaction with DNA enable efficient and effective capture of target cfDNA.

[0118] In this case, the target cfDNA refers to the cfDNA of interest to be detected. As used herein, cfDNA is double-stranded. In this case, a portion of the cfDNA may be unwound. The cfDNA may also be derived from genes in thyroid cancer cells. Specifically, cfDNA may contain a nucleic acid sequence that is overexpressed in thyroid cancer cells. The nucleic acid sequence that is overexpressed in cancer cells refers to a nucleic acid sequence that is expressed at an appropriate level in normal cells but is overexpressed in specific cancer cells.

[0119] Specifically, the degree or cutoff of overexpression of the nucleic acid sequence in cancer cells may be when the OD value is 0.010 or more when measuring optical density using a marker. More specifically, the degree or cutoff of overexpression of the nucleic acid sequence in cancer cells may be when the OD value is 0.012 or 0.015 or more when measuring optical density using a marker. In this case, the wavelength of irradiation for measuring the absorbance may be appropriately determined depending on the marker. The cfDNA may be DNA in which the double strand is unwound (unwinding of DNA). Furthermore, the cfDNA may be appropriately determined depending on the purpose.

[0120] Furthermore, the cfDNA derived from the thyroid cancer cells may be characterized by: i) having a lower Tm value than cfDNA having a double helix structure derived from normal cells; or ii) being denatured under conditions under which cfDNA having a double helix structure derived from normal cells is not denatured.

[0121] Furthermore, the cfDNA can be bound to a probe of about 15 mer to about 30 mer that can complementarily bind to the cfDNA under any one of the following conditions: i) leaving the sample at room temperature for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes; iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C to about 75°C for about 30 seconds to about 30 minutes; v) heating at about 25°C to about 40°C for about 10 minutes to about 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; vii) treating with DNase for about 1 minute to about 30 minutes; and viii) treating with chemicals (e.g., sodium hydroxide, DMSO, surfactants, etc.).

[0122] The gene overexpressed in thyroid cancer cells may be any one selected from the group consisting of ACPP (NCBI Gene ID: 55), ENO2 (NCBI Gene ID: 2026), TG (NCBI Gene ID: 7038), CALCA (NCBI Gene ID: 796), APOC1 (NCBI Gene ID: 341), HIG2 (NCBI Gene ID: 29923), TYRO3 (NCBI Gene ID: 7301), CPT1A (NCBI Gene ID: 1374), IFNG (NCBI Gene ID: 3458), CD274 (NCBI Gene ID: 29126), FOLR1 (NCBI Gene ID: 2348), EPCAM (NCBI Gene ID: 4072), and combinations thereof.

[0123] In one specific example, genes that are specifically present in thyroid cancer are the TG, CALCA, APOC1, and HIG2 genes, and additionally, the ENO2, ACPP, TYRO3, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, or EPCAM genes can be detected to diagnose thyroid cancer.

[0124] As used herein, the term "TG" refers to a gene encoding thyroglobulin (TG). Thyroglobulin is produced exclusively in the thyroid gland in the human body, and when thyroid cancer develops or metastasizes, the level of thyroglobulin in the blood increases. The level of thyroglobulin in the blood is used as a thyroid cancer marker.

[0125] As used herein, the term "CALCA" refers to the gene encoding calcitonin gene-related peptide.

[0126] As used herein, the term "probe" refers to DNA or RNA for detecting cfDNA. The probe may have a specific sequence that allows it to bind complementarily to cfDNA. A probe having a sequence complementary to cfDNA refers to a probe having a nucleic acid sequence that can bind complementarily to the target double-stranded cfDNA present in plasma that is to be detected. In this case, the probe may be bound to biotin. The probe may bind to a marker bound to a biotin-binding protein.

[0127] As used herein, the term "marker" refers to a substance used to detect probes bound to cfDNA. The marker may be a nanoparticle, a fluorescent dye, a fluorescent protein, or an enzyme. Specifically, the marker may be any one selected from the group consisting of quantum dots, HRP, and fluorescent proteins. In one specific example, the marker may be, but is not limited to, green fluorescent protein (GFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), or horse radish peroxidase (HRP).

[0128] The marker may be conjugated with a biotin-binding protein. The biotin-binding protein may be an avidin-based protein such as streptavidin, traptavidin, or neutravidin, but any protein that can specifically bind to biotin may be used without limitation. In one specific example, the marker may be conjugated with streptavidin.

[0129] As used herein, the term "streptavidin" refers to a tetrameric biotin-binding protein with a molecular weight of 60 kDa isolated from Streptomyces avidinii. Although it has very low homology to avidin, its structure is very similar. Like avidin, streptavidin possesses antibacterial activity and a very high binding affinity for biotin. Unlike avidin, streptavidin does not contain carbohydrates, exhibits an acidic isoelectric point (pI = 5), and has significantly lower solubility than avidin. Commercially available streptavidin, such as Thermo Scientific Pierce Streptavidin, is a recombinant form of streptavidin with a molecular weight of 53 kDa and a near-neutral isoelectric point (pI = 6.8-7.5). The lack of glycosylation and low pI of streptavidin result in a lower level of nonspecific binding (especially lectin binding) compared to avidin. These properties of streptavidin make it an ideal reagent of choice for many detection systems.

[0130] As used herein, the term "traptavidin" refers to a streptavidin variant or mutein, a protein that exhibits approximately 10-fold slower biotin dissociation rate, increased mechanical strength, and improved thermal stability. Traptavidin also specifically binds to biotin.

[0131] The term "neutravidin" used in the present invention, also known as "deglycosylated avidin," was created to avoid the major drawbacks of natural avidin and streptavidin. As the name suggests, neutravidin is produced by deglycosylating avidin. It is a protein that maintains high biotin-binding ability while having a reduced molecular weight (60 kDa) compared to avidin. Deglycosylation of avidin reduces lectin binding to undetectable levels and lowers its isoelectric point (pI = 6.3), effectively eliminating the main cause of nonspecific binding to avidin. Because the lysine residues remain available, neutravidin can be easily derivatized or conjugated, like streptavidin. Furthermore, its high biotin-binding ability and low nonspecific binding make it an ideal biotin-binding protein with a wide range of applications.

[0132] As used herein, the term "detecting a marker" refers to detecting a marker bound to a probe through a biotin-avidin reaction. The detection of the marker can be measured by a color change, a change in UV absorbance, the presence or absence of bioluminescence, a change in a fluorescent reaction, or an electrochemical change. Specifically, the method for detecting the marker varies depending on the marker used. For example, when HRP is used as a marker, the marker can be detected by observing the color reaction resulting from the reaction of hydrogen peroxide with a substrate. When the marker is a fluorescent protein such as GFP, the presence or absence of the marker can be detected by observing the light emitted after irradiation with light of a specific wavelength. When the marker is luciferase, the presence of the marker can be detected by adding a substrate such as luciferin and measuring the bioluminescence generated using a bioluminometer.

[0133] The diagnostic method of the present invention may further include a step of denaturing cfDNA. In this case, the denaturation step does not denature normal double-stranded cfDNA. Therefore, the denaturation step may be performed at about 50°C to about 100°C for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is usually about 0.1 seconds to about 8 minutes. Denaturation may be performed for about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds, or about 90 seconds. In one example, the step of denaturing cfDNA may be performed before step c).

[0134] Specifically, prior to step c), the method may further include denaturing the sample or the cfDNA bound to the positively charged substance under one of the following conditions: (i) leaving the sample at room temperature for about 1 to 10 minutes; (ii) heating at about 90°C to about 95°C for 1 second to 1 minute; (iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; (iv) heating at about 60°C to about 75°C for about 1 to 30 minutes; (v) heating at about 25°C to about 40°C for about 5 to 60 minutes; (vi) treating with protease for about 1 to 10 minutes; and (vii) treating with DNase I for about 1 to 10 minutes. This denaturation step selectively denatures only the cfDNA derived from cancer cells, while not denaturing double-stranded cfDNA derived from normal cells, thereby further facilitating binding to the probe. The denaturation conditions i) to vii) can be performed after obtaining the sample. The denaturation conditions i) to vii) can be performed after obtaining the cfDNA bound to the positively charged substance. The temperature, protease, and DNase treatment times for the denaturation conditions i) to vii) can be adjusted as appropriate as long as they do not denature stable cfDNA.

[0135] Thyroid cancer diagnostic kit Another aspect of the present invention is to provide a diagnostic kit for thyroid cancer, comprising: a biotin-conjugated probe that binds complementarily to a gene specifically expressed in thyroid cancer; a positively charged substance; a marker bound to an avidin-based protein; and instructions.

[0136] In this case, the gene specifically expressed in thyroid cancer may be any one or more selected from the group consisting of TG, CALCA, APOC1, HIG2, and combinations thereof.

[0137] The instructions may also describe that the kit configuration can diagnose thyroid cancer by the following protocol: a) isolating cfDNA from a biological sample isolated from an individual using a positively charged substance included in the kit; b) sequentially or simultaneously mixing the isolated cfDNA with a biotin-conjugated probe included in the kit and a marker included in the kit; c) removing probes and markers that do not bind to cfDNA; and d) detecting signals from the markers.

[0138] It may also additionally contain a biotin-conjugated probe that complementarily binds to at least one gene selected from the group consisting of ENO2, ACPP, TYRO3, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof.

[0139] The probe, positively charged substance, and marker are as described above.

[0140] Thyroid cancer diagnostic device Another aspect of the present invention is to provide an apparatus for diagnosing thyroid cancer by detecting genes derived from thyroid cancer cells from a sample without amplification, the apparatus comprising: a) a mixing unit that mixes a biological sample isolated from an individual containing cfDNA with a positively charged substance; b) a harvesting unit that removes the sample from which the positively charged substance to which cfDNA is bound has been removed; c) a reaction unit that sequentially or simultaneously adds a biotin-binding probe capable of complementarily binding to a gene specifically expressed in thyroid cancer, and nanoparticles containing streptavidin and a marker, to the positively charged substance to which cfDNA is bound; d) a detection unit that detects the marker; and e) an information processing unit that determines, based on the presence or absence of detection of the marker, that cfDNA derived from thyroid cancer and having a sequence complementary to the probe is present in the sample.

[0141] In this case, the gene specifically expressed in thyroid cancer may be any one or more selected from the group consisting of TG, CALCA, APOC1, HIG2, and combinations thereof.

[0142] It may also additionally contain a biotin-conjugated probe that complementarily binds to at least one gene selected from the group consisting of ENO2, ACPP, TYRO3, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof.

[0143] <Bladder cancer> How Bladder Cancer is Diagnosed

[0144] One aspect of the present invention provides a method for diagnosing bladder cancer by detecting genes derived from bladder cancer cells in the sample without amplification, the method comprising the steps of: a) mixing a biological sample isolated from an individual containing cell-free DNA (cfDNA) with a positively charged substance; b) separating the positively charged substance bound to the cfDNA; c) sequentially or simultaneously mixing a probe and a marker having a sequence complementary to the cfDNA with the mixture; d) removing the probe and marker that do not bind to the cfDNA; and e) detecting the marker, wherein the probe having a sequence complementary to the cfDNA complementarily binds to a gene known to be a bladder cancer biomarker. The gene known to be a bladder cancer biomarker may be a gene encoding a protein overexpressed in bladder cancer.

[0145] Specifically, the probe having a sequence complementary to the cfDNA may be one that binds complementarily to at least one gene selected from the group consisting of OGT, FGFR3, TP53, NUMA1, COCH, CELSR3, HMOX1, KIF1A, MGC17624, MTAP, PFKFB4, S100A8, RSPH9, FOXM1, FANCB, FANCC, FANCD2, RUSC1-AS1, CACNA1B, IMP-1, PDE3A, POU3F4, SOX3, DMC1, PLXDC2, ZNF312, SYCP2L, HOXA9, ISL1, ALDH1A3, and combinations thereof.

[0146] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in the sample can be separated and / or concentrated using various methods. As a specific example, a nitrocellulose membrane, which has a strong affinity for nucleic acids, can be used. As another specific example, a positively charged substance can be used to capture negatively charged cfDNA. The positively charged substance can be, but is not limited to, nanoparticles, nanowires, a mesh structure, or a positively charged filter. A specific example of the "positively charged substance" can be a positively charged nanostructure or a positively charged membrane.

[0147] An example of the nanostructure may include a cationic polymer. The type of the cationic polymer is not limited. A specific example of the cationic polymer is polyethyleneimine (PEI), which may be a cationic branched polymer polyethyleneimine.

[0148] In addition, by mixing streptavidin-labeled nanowires with a biotin-conjugated PEI solution, cationic branched polyethyleneimine (PEI) can be additionally bound to the nanowires through the biotin-streptavidin interaction, resulting in nanoparticles embedded in a nanostructure (PEI / mPpy NW) with a high density and irregular distribution, with the cationic polymer polyethyleneimine bound to the surface.

[0149] Such nanowires can successfully capture genomic DNA and cfDNA at high efficiency and low concentrations. In particular, nanowire features such as a large surface area for binding to target molecules such as DNA and enhanced mobility for promoting interaction with DNA enable efficient and effective capture of target cfDNA.

[0150] In this case, the target cfDNA refers to the cfDNA of interest to be detected. As used herein, cfDNA is double-stranded. In this case, a portion of the cfDNA may be unwound. The cfDNA may also be derived from genes in bladder cancer cells. Specifically, cfDNA may contain a nucleic acid sequence that is overexpressed in bladder cancer cells. The nucleic acid sequence that is overexpressed in cancer cells refers to a nucleic acid sequence that is expressed at an appropriate level in normal cells but is overexpressed in specific cancer cells.

[0151] Specifically, the degree or cutoff of overexpression of the nucleic acid sequence in cancer cells may be when the OD value is 0.010 or more when measuring optical density using a marker. More specifically, the degree or cutoff of overexpression of the nucleic acid sequence in cancer cells may be when the OD value is 0.012 or 0.015 or more when measuring optical density using a marker. In this case, the wavelength of irradiation for measuring the absorbance may be appropriately determined depending on the marker. The cfDNA may be DNA in which the double strand is unwound (unwinding of DNA). Furthermore, the cfDNA may be appropriately determined depending on the purpose.

[0152] Furthermore, the cfDNA derived from the bladder cancer cells may be characterized by: i) having a lower Tm value than cfDNA having a double helix structure derived from normal cells; or ii) being denatured under conditions under which cfDNA having a double helix structure derived from normal cells is not denatured.

[0153] Furthermore, the cfDNA can be bound to a probe of about 15 mer to about 30 mer that can complementarily bind to the cfDNA under any one of the following conditions: i) leaving the sample at room temperature for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes; iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C to about 75°C for about 30 seconds to about 30 minutes; v) heating at about 25°C to about 40°C for about 10 minutes to about 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; vii) treating with DNase for about 1 minute to about 30 minutes; and viii) treating with chemicals (e.g., sodium hydroxide, DMSO, surfactants, etc.).

[0154] The genes overexpressed in bladder cancer cells include OGT (NCBI Gene ID: 8473), FGFR3 (NCBI Gene ID: 2261), TP53 (NCBI Gene ID: 7157), NUMA1 (NCBI Gene ID: 4926), KRT19 (NCBI Gene ID: 3880), COCH (NCBI Gene ID: 1690), and CELSR3 (NCBI Gene ID:1951), HMOX1(NCBI Gene ID:3162), KIF1A(NCBI Gene ID:547), MGC17624(NCBI Gene ID:404550), MTAP(NCBI Gene ID:4507), PFKFB4(NCBI Gene ID:5210), S100A8(NCBI Gene ID:6279), RSPH9(NCBI Gene ID:221421), CCNB1(NCBI Gene ID:891), FOXM1(NCBI Gene ID:2305), FANCB(NCBI Gene ID:2187), FANCC(NCBI Gene ID:2176), FANCD2(NCBI Gene ID:2177), RUSC1-AS1(NCBI Gene ID:284618), CACNA1B(NCBI Gene ID:774), IMP-1(NCBI Gene ID:10642), PDE3A(NCBI Gene ID:5139), POU3F4(NCBI Gene ID:5456), SOX3(NCBI Gene ID:6658), DMC1(NCBI Gene ID:11144), PLXDC2(NCBI Gene ID:84898), ZNF312(NCBI Gene ID:55079), SYCP2L(NCBI Gene ID:221711), HOXA9(NCBI Gene ID: 3205), ISL1 (NCBI Gene ID: 3670), ALDH1A3 (NCBI Gene ID: 220), CPT1A (NCBI Gene ID: 1374), IFNG (NCBI Gene ID: 3458), CD274 (NCBI Gene ID: 29126), FOLR1 (NCBI Gene ID: 2348), EPCAM (NCBI Gene ID: 4072), and combinations thereof.

[0155] In one specific example, genes that are specifically present in bladder cancer are OGT, FGFR3, TP53, NUMA1, COCH, CELSR3, HMOX1, KIF1A, MGC17624, MTAP, PFKFB4, S100A8, RSPH9, FOXM1, FANCB, FANCC, FANCD2, RUSC1-AS1, CACNA1B, IMP-1, PDE3A, POU3F4, SOX3, DMC1, PLXDC2, ZNF312, SYCP2L, HOXA9, ISL1, and ALDH1A3 genes. Additionally, bladder cancer can be diagnosed by detecting the KRT19, CCNB1, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, or EPCAM genes.

[0156] As used herein, the term "OGT" refers to the gene encoding O-GlcNAc transferase.

[0157] As used herein, the term "FGFR1" refers to the gene encoding fibroblast growth factor receptor 1.

[0158] As used herein, the term "NUMA1" refers to a gene encoding NMP22 (nuclear matrix protein-22). NMP22 is found in higher than normal levels in urine samples from patients with certain types of cancer, including bladder cancer, and is widely used as a bladder cancer marker.

[0159] As used herein, the term "probe" refers to DNA or RNA for detecting cfDNA. The probe may have a specific sequence that allows it to bind complementarily to cfDNA. A probe having a sequence complementary to cfDNA refers to a probe having a nucleic acid sequence that can bind complementarily to the target double-stranded cfDNA present in plasma that is to be detected. In this case, the probe may be bound to biotin. The probe may bind to a marker bound to a biotin-binding protein.

[0160] As used herein, the term "marker" refers to a substance used to detect probes bound to cfDNA. The marker may be a nanoparticle, a fluorescent dye, a fluorescent protein, or an enzyme. Specifically, the marker may be any one selected from the group consisting of quantum dots, HRP, and fluorescent proteins. In one specific example, the marker may be, but is not limited to, green fluorescent protein (GFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), or horse radish peroxidase (HRP).

[0161] The marker may be conjugated with a biotin-binding protein. The biotin-binding protein may be an avidin-based protein such as streptavidin, traptavidin, or neutravidin, but any protein that can specifically bind to biotin may be used without limitation. In one specific example, the marker may be conjugated with streptavidin.

[0162] As used herein, the term "streptavidin" refers to a tetrameric biotin-binding protein with a molecular weight of 60 kDa isolated from Streptomyces avidinii. Although it has very low homology to avidin, its structure is very similar. Like avidin, streptavidin possesses antibacterial activity and a very high binding affinity for biotin. However, unlike avidin, streptavidin does not contain carbohydrates, exhibits an acidic isoelectric point (pI = 5), and has significantly lower solubility than avidin. Commercially available streptavidin, such as Thermo Scientific Pierce Streptavidin, is a recombinant form of streptavidin with a molecular weight of 53 kDa and a near-neutral isoelectric point (pI = 6.8-7.5). The lack of glycosylation and low pI of streptavidin result in a lower level of nonspecific binding (especially lectin binding) compared to avidin. These properties of streptavidin make it an ideal reagent of choice for many detection systems.

[0163] As used herein, the term "traptavidin" refers to a streptavidin variant or mutein, a protein that exhibits approximately 10-fold slower biotin dissociation rate, increased mechanical strength, and improved thermal stability. Traptavidin also specifically binds to biotin.

[0164] The term "neutravidin" used in the present invention, also known as "deglycosylated avidin," was developed to overcome the major drawbacks of natural avidin and streptavidin. As the name suggests, neutravidin is produced by deglycosylating avidin. It is a protein that maintains high biotin-binding ability while having a reduced molecular weight (60 kDa) compared to avidin. Deglycosylation of avidin reduces lectin binding to undetectable levels and lowers its isoelectric point (pI = 6.3), effectively eliminating the main cause of nonspecific binding to avidin. Because the lysine residues remain available, neutravidin can be easily derivatized or conjugated, like streptavidin. Furthermore, its high biotin-binding ability and low nonspecific binding make it an ideal biotin-binding protein with a wide range of applications.

[0165] As used herein, the term "detecting a marker" refers to detecting a marker bound to a probe through a biotin-avidin reaction. The detection of the marker can be measured by a color change, a change in UV absorbance, the presence or absence of bioluminescence, a change in a fluorescent reaction, or an electrochemical change. Specifically, the method for detecting the marker varies depending on the marker used. For example, when HRP is used as a marker, the marker can be detected by observing the color reaction resulting from the reaction of hydrogen peroxide with a substrate. When the marker is a fluorescent protein such as GFP, the presence or absence of the marker can be detected by observing the light emitted after irradiation with light of a specific wavelength. When the marker is luciferase, the presence of the marker can be detected by adding a substrate such as luciferin and measuring the bioluminescence generated using a bioluminometer.

[0166] The diagnostic method of the present invention may further include a step of denaturing cfDNA. In this case, the denaturation step may be performed so as to selectively denature only bladder cancer-derived cfDNA while leaving normal double-stranded cfDNA undenatured. For this purpose, the denaturation step may be performed at about 50°C to about 100°C for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is typically about 0.1 seconds to about 8 minutes. Alternatively, denaturation may be performed for about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds, or about 90 seconds. In one example, the denaturation step of cfDNA may be performed before step c).

[0167] Specifically, prior to step c), the method may further include denaturing the sample or the cfDNA bound to the positively charged substance under one of the following conditions: (i) leaving the sample at room temperature for about 1 to 10 minutes; (ii) heating at about 90°C to about 95°C for 1 second to 1 minute; (iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; (iv) heating at about 60°C to about 75°C for about 1 to 30 minutes; (v) heating at about 25°C to about 40°C for about 5 to 60 minutes; (vi) treating with protease for about 1 to 10 minutes; and (vii) treating with DNase I for about 1 to 10 minutes. This denaturation step selectively denatures only the cfDNA derived from cancer cells, while not denaturing double-stranded cfDNA derived from normal cells, thereby further facilitating binding to the probe. The denaturation conditions i) to vii) can be performed after obtaining the sample. The denaturation conditions i) to vii) can be performed after obtaining the cfDNA bound to the positively charged substance. The temperature, protease, and DNase treatment times for the denaturation conditions i) to vii) can be adjusted as appropriate, as long as they do not denature stable cfDNA.

[0168] Bladder cancer diagnostic kit Another aspect of the present invention is to provide a diagnostic kit for bladder cancer, comprising: a biotin-conjugated probe that binds complementarily to a gene that is specifically expressed in bladder cancer; a positively charged substance; a marker bound to an avidin-based protein; and instructions.

[0169] In this case, the gene specifically expressed in bladder cancer may be any one or more selected from the group consisting of OGT, FGFR3, TP53, NUMA1, COCH, CELSR3, HMOX1, KIF1A, MGC17624, MTAP, PFKFB4, S100A8, RSPH9, FOXM1, FANCB, FANCC, FANCD2, RUSC1-AS1, CACNA1B, IMP-1, PDE3A, POU3F4, SOX3, DMC1, PLXDC2, ZNF312, SYCP2L, HOXA9, ISL1, ALDH1A3, and combinations thereof.

[0170] The instructions may also describe that the kit configuration can diagnose bladder cancer by the following protocol: a) isolating cfDNA from a biological sample isolated from an individual using a positively charged substance included in the kit; b) sequentially or simultaneously mixing the isolated cfDNA with a biotin-conjugated probe included in the kit and a marker included in the kit; c) removing probes and markers that do not bind to cfDNA; and d) detecting signals from the markers.

[0171] It may also additionally contain a biotin-conjugated probe that complementarily binds to at least one gene selected from the group consisting of KRT19, CCNB1, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof.

[0172] The probe, positively charged substance, and marker are as described above.

[0173] Bladder cancer diagnostic device Another aspect of the present invention is to provide an apparatus for diagnosing bladder cancer by detecting genes derived from bladder cancer cells from a sample without amplification, comprising: a) a mixing section that mixes a biological sample isolated from an individual containing cfDNA with a positively charged substance; b) a harvesting section that removes the sample from which the positively charged substance to which cfDNA is bound has been removed; c) a reaction section that sequentially or simultaneously adds a biotin-binding probe capable of complementarily binding to a gene specifically expressed in bladder cancer, and nanoparticles containing streptavidin and a marker, to the positively charged substance to which cfDNA is bound; d) a detection section that detects the marker; and e) an information processing section that determines, based on the presence or absence of detection of the marker, that cfDNA derived from bladder cancer and having a sequence complementary to the probe is present in the sample.

[0174] In this case, the gene specifically expressed in bladder cancer may be any one or more selected from the group consisting of OGT, FGFR3, TP53, NUMA1, COCH, CELSR3, HMOX1, KIF1A, MGC17624, MTAP, PFKFB4, S100A8, RSPH9, FOXM1, FANCB, FANCC, FANCD2, RUSC1-AS1, CACNA1B, IMP-1, PDE3A, POU3F4, SOX3, DMC1, PLXDC2, ZNF312, SYCP2L, HOXA9, ISL1, ALDH1A3, and combinations thereof.

[0175] It may also additionally contain a biotin-conjugated probe that complementarily binds to at least one gene selected from the group consisting of KRT19, CCNB1, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof.

[0176] <Breast cancer> How breast cancer is diagnosed One aspect of the present invention provides a method for diagnosing breast cancer by detecting genes derived from breast cancer cells in the sample without amplification, the method comprising the steps of: a) mixing a biological sample isolated from an individual containing cell-free DNA (cfDNA) with a positively charged substance; b) separating the positively charged substance bound to the cfDNA; c) sequentially or simultaneously mixing a probe and a marker having a sequence complementary to the cfDNA with the mixture; d) removing the probe and marker that do not bind to the cfDNA; and e) detecting the marker, wherein the probe having a sequence complementary to the cfDNA complementarily binds to a gene known to be a breast cancer biomarker. In this case, the gene known to be a breast cancer biomarker may be a gene encoding a protein overexpressed in breast cancer.

[0177] Specifically, the probe having a sequence complementary to the cfDNA may be one that binds complementarily to at least one gene selected from the group consisting of MEST, NR1D1, BIRC5, RACGAP1, DHCR7, STC2, AZGP1, RBBP8, IL6ST, MGP, TRBC1, MMP11, COL10A1, C10orf64, COL11A1, POTEG, FSIP1, HER2, and combinations thereof.

[0178] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in the sample can be separated and / or concentrated using various methods. As a specific example, a nitrocellulose membrane, which has a strong affinity for nucleic acids, can be used. As another specific example, a positively charged substance can be used to capture negatively charged cfDNA. The positively charged substance can be, but is not limited to, nanoparticles, nanowires, a mesh structure, or a positively charged filter. A specific example of the "positively charged substance" can be a positively charged nanostructure or a positively charged membrane.

[0179] An example of the nanostructure may include a cationic polymer. The type of the cationic polymer is not limited. A specific example of the cationic polymer is polyethyleneimine (PEI), which may be a cationic branched polymer polyethyleneimine.

[0180] In addition, by mixing streptavidin-labeled nanowires with a biotin-conjugated PEI solution, cationic branched polyethyleneimine (PEI) can be additionally bound to the nanowires through the biotin-streptavidin interaction, resulting in nanoparticles embedded in a nanostructure (PEI / mPpy NW) with a high density and irregular distribution, with the cationic polymer polyethyleneimine bound to the surface.

[0181] Such nanowires can successfully capture genomic DNA and cfDNA at high efficiency and low concentrations. In particular, nanowire features such as a large surface area for binding to target molecules such as DNA and enhanced mobility for promoting interaction with DNA enable efficient and effective capture of target cfDNA.

[0182] In this case, the target cfDNA refers to the cfDNA of interest to be detected. As used herein, cfDNA is double-stranded. In this case, a portion of the cfDNA may be unwound. The cfDNA may also be derived from genes in breast cancer cells. Specifically, cfDNA may contain a nucleic acid sequence that is overexpressed in breast cancer cells. The nucleic acid sequence that is overexpressed in cancer cells refers to a nucleic acid sequence that is expressed at an appropriate level in normal cells but is overexpressed in specific cancer cells.

[0183] Specifically, the degree or cutoff of overexpression of the nucleic acid sequence in cancer cells may be when the OD value is 0.010 or more when measuring optical density using a marker. More specifically, the degree or cutoff of overexpression of the nucleic acid sequence in cancer cells may be when the OD value is 0.012 or 0.015 or more when measuring optical density using a marker. In this case, the wavelength of irradiation for measuring the absorbance may be appropriately determined depending on the marker. The cfDNA may be DNA in which the double strand is unwound (unwinding of DNA). Furthermore, the cfDNA may be appropriately determined depending on the purpose.

[0184] Furthermore, the cfDNA derived from the breast cancer cells may be characterized by: i) having a lower Tm value than cfDNA having a double helix structure derived from normal cells; or ii) being denatured under conditions under which cfDNA having a double helix structure derived from normal cells is not denatured.

[0185] Furthermore, the cfDNA can be bound to a probe of about 15 mer to about 30 mer that can complementarily bind to the cfDNA under any one of the following conditions: i) leaving the sample at room temperature for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes; iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C to about 75°C for about 30 seconds to about 30 minutes; v) heating at about 25°C to about 40°C for about 10 minutes to about 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; vii) treating with DNase for about 1 minute to about 30 minutes; and viii) treating with chemicals (e.g., sodium hydroxide, DMSO, surfactants, etc.).

[0186] The genes overexpressed in breast cancer cells are MUC1 (NCBI Gene ID: 4582), ACPP (NCBI Gene ID: 55), MEST (NCBI Gene ID: 4232), TYRO3 (NCBI Gene ID: 7301), NR1D1 (NCBI Gene ID: 9572), UBE2C (NCBI Gene ID: 11065), BIRC5 (NCBI Gene ID:332), RACGAP1(NCBI Gene ID:29127), DHCR7(NCBI Gene ID:1717), STC2(NCBI Gene ID:8614), AZGP1(NCBI Gene ID:563), RBBP8(NCBI Gene ID:5932), IL6ST(NCBI Gene ID:3572), MGP(NCBI Gene ID:4256), TRBC1(NCBI Gene ID:28639), MMP11(NCBI Gene ID: 4320), COL10A1 (NCBI Gene ID: 1300), C10orf64 (NCBI Gene ID: 57705), COL11A1 (NCBI Gene ID: 1301), POTEG (NCBI Gene ID: 404785), FSIP1 (NCBI Gene ID: 161835), HER2 (NCBI Gene ID: 2064), CPT1A (NCBI Gene ID: 1374), IFNG (NCBI Gene ID: 3458), CD274 (NCBI Gene ID: 29126), FOLR1 (NCBI Gene ID: 2348), EPCAM (NCBI Gene ID: 4072), and combinations thereof.

[0187] In one specific example, genes that are specifically present in breast cancer are MEST, NR1D1, BIRC5, RACGAP1, DHCR7, STC2, AZGP1, RBBP8, IL6ST, MGP, TRBC1, MMP11, COL10A1, C10orf64, COL11A1, POTEG, FSIP1, and HER2 genes, and additionally, MUC1, ACPP, TYRO3, UBE2C, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, or EPCAM genes can be detected to diagnose breast cancer.

[0188] As used herein, the term "MUC1" refers to a breast cancer-associated gene encoding proteins including CA 15-3 (Carcinoma Antigen 15-3) and CA 27-29. CA15-3 has been shown to increase the likelihood of early recurrence of breast cancer and is used as a breast cancer marker.

[0189] As used herein, the term "probe" refers to DNA or RNA for detecting cfDNA. The probe may have a specific sequence that allows it to bind complementarily to cfDNA. A probe having a sequence complementary to cfDNA refers to a probe having a nucleic acid sequence that can bind complementarily to the target double-stranded cfDNA present in plasma that is to be detected. In this case, the probe may be bound to biotin. The probe may bind to a marker bound to a biotin-binding protein.

[0190] As used herein, the term "marker" refers to a substance used to detect probes bound to cfDNA. The marker may be a nanoparticle, a fluorescent dye, a fluorescent protein, or an enzyme. Specifically, the marker may be any one selected from the group consisting of quantum dots, HRP, and fluorescent proteins. In one specific example, the marker may be, but is not limited to, green fluorescent protein (GFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), or horse radish peroxidase (HRP).

[0191] The marker may be conjugated with a biotin-binding protein. The biotin-binding protein may be an avidin-based protein such as streptavidin, traptavidin, or neutravidin, but any protein that can specifically bind to biotin may be used without limitation. In one specific example, the marker may be conjugated with streptavidin.

[0192] As used herein, the term "streptavidin" refers to a tetrameric biotin-binding protein with a molecular weight of 60 kDa isolated from Streptomyces avidinii. Although it has very low homology to avidin, its structure is very similar. Like avidin, streptavidin possesses antibacterial activity and a very high binding affinity for biotin. Unlike avidin, streptavidin does not contain carbohydrates, exhibits an acidic isoelectric point (pI = 5), and has significantly lower solubility than avidin. Commercially available streptavidin, such as Thermo Scientific Pierce Streptavidin, is a recombinant form of streptavidin with a molecular weight of 53 kDa and a near-neutral isoelectric point (pI = 6.8-7.5). The lack of glycosylation and low pI of streptavidin result in a lower level of nonspecific binding (especially lectin binding) compared to avidin. These properties of streptavidin make it an ideal reagent of choice for many detection systems.

[0193] As used herein, the term "traptavidin" refers to a streptavidin variant or mutein, a protein that exhibits approximately 10-fold slower biotin dissociation rate, increased mechanical strength, and improved thermal stability. Traptavidin also specifically binds to biotin.

[0194] The term "neutravidin" used in the present invention, also known as "deglycosylated avidin," was created to avoid the major drawbacks of natural avidin and streptavidin. As the name suggests, neutravidin is produced by deglycosylating avidin. It is a protein that maintains high biotin-binding ability while having a reduced molecular weight (60 kDa) compared to avidin. Deglycosylation of avidin reduces lectin binding to undetectable levels and lowers its isoelectric point (pI = 6.3), effectively eliminating the main cause of nonspecific binding to avidin. Because the lysine residues remain available, neutravidin can be easily derivatized or conjugated, like streptavidin. Furthermore, its high biotin-binding ability and low nonspecific binding make it an ideal biotin-binding protein with a wide range of applications.

[0195] As used herein, the term "detecting a marker" refers to detecting a marker bound to a probe through a biotin-avidin reaction. The detection of the marker can be measured by a color change, a change in UV absorbance, the presence or absence of bioluminescence, a change in a fluorescent reaction, or an electrochemical change. Specifically, the method for detecting the marker varies depending on the marker used. For example, when HRP is used as a marker, the marker can be detected by observing the color reaction resulting from the reaction of hydrogen peroxide with a substrate. Furthermore, when the marker is a fluorescent protein such as GFP, the presence or absence of the marker can be detected by observing the light emitted after irradiation with light of a specific wavelength. Furthermore, when the marker is luciferase, the presence of the marker can be detected by adding a substrate such as luciferin and measuring the bioluminescence generated using a bioluminometer.

[0196] The diagnostic method of the present invention may further include a step of denaturing cfDNA. In this case, the denaturation step can be performed so as to selectively denature only breast cancer-derived cfDNA while leaving normal double-stranded cfDNA undenatured. For this purpose, the denaturation step can be performed at about 50°C to about 100°C for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is typically about 0.1 seconds to about 8 minutes. Alternatively, denaturation can be performed for about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds, or about 90 seconds. In one example, the denaturation step of cfDNA can be performed before step c).

[0197] Specifically, prior to step c), the method may further include denaturing the sample or the cfDNA bound to the positively charged substance under one of the following conditions: (i) leaving the sample at room temperature for about 1 to 10 minutes; (ii) heating at about 90°C to about 95°C for 1 second to 1 minute; (iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; (iv) heating at about 60°C to about 75°C for about 1 to 30 minutes; (v) heating at about 25°C to about 40°C for about 5 to 60 minutes; (vi) treating with protease for about 1 to 10 minutes; and (vii) treating with DNase I for about 1 to 10 minutes. This denaturation step selectively denatures only the cfDNA derived from cancer cells, while not denaturing double-stranded cfDNA derived from normal cells, thereby further facilitating binding to the probe. The denaturation conditions i) to vii) can be performed after obtaining the sample. The denaturation conditions i) to vii) can be performed after obtaining the cfDNA bound to the positively charged substance. The temperature, protease, and DNase treatment times for the denaturation conditions i) to vii) can be adjusted as appropriate, as long as they do not denature stable cfDNA.

[0198] Breast cancer diagnostic kit Another aspect of the present invention is to provide a diagnostic kit for breast cancer, comprising: a biotin-conjugated probe that binds complementarily to a gene specifically expressed in breast cancer; a positively charged substance; a marker bound to an avidin-based protein; and instructions.

[0199] In this case, the gene specifically expressed in breast cancer may be any one or more selected from the group consisting of MEST, NR1D1, BIRC5, RACGAP1, DHCR7, STC2, AZGP1, RBBP8, IL6ST, MGP, TRBC1, MMP11, COL10A1, C10orf64, COL11A1, POTEG, FSIP1, HER2, and combinations thereof.

[0200] The instructions may also describe that the kit configuration can diagnose breast cancer by the following protocol: a) isolating cfDNA from a biological sample isolated from an individual using a positively charged substance included in the kit; b) sequentially or simultaneously mixing the isolated cfDNA with a biotin-conjugated probe included in the kit and a marker included in the kit; c) removing probes and markers that do not bind to cfDNA; and d) detecting signals from the markers.

[0201] It may also additionally contain a biotin-conjugated probe that complementarily binds to at least one gene selected from the group consisting of MUC1, ACPP, TYRO3, UBE2C, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof.

[0202] The probe, positively charged substance, and marker are as described above.

[0203] Breast cancer diagnostic equipment Another aspect of the present invention is to provide an apparatus for diagnosing breast cancer by detecting genes derived from breast cancer cells from a sample without amplification, comprising: a) a mixing unit that mixes a biological sample isolated from an individual containing cfDNA with a positively charged substance; b) a harvesting unit that removes the sample from which the positively charged substance to which cfDNA is bound has been removed; c) a reaction unit that sequentially or simultaneously adds a biotin-binding probe capable of complementarily binding to a gene specifically expressed in breast cancer, and nanoparticles containing streptavidin and a marker, to the positively charged substance to which cfDNA is bound; d) a detection unit that detects the marker; and e) an information processing unit that determines, based on the presence or absence of detection of the marker, that cfDNA derived from breast cancer and having a sequence complementary to the probe is present in the sample.

[0204] In this case, the gene that is complementarily expressed in breast cancer may be any one or more selected from the group consisting of MEST, NR1D1, BIRC5, RACGAP1, DHCR7, STC2, AZGP1, RBBP8, IL6ST, MGP, TRBC1, MMP11, COL10A1, C10orf64, COL11A1, POTEG, FSIP1, HER2, and combinations thereof.

[0205] It may also additionally contain a biotin-conjugated probe that complementarily binds to at least one gene selected from the group consisting of MUC1, ACPP, TYRO3, UBE2C, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof.

[0206] <Colon cancer> How colon cancer is diagnosed One aspect of the present invention provides a method for diagnosing colorectal cancer by detecting genes derived from colorectal cancer cells in a sample without amplification, the method comprising the steps of: a) mixing a biological sample isolated from an individual containing cell-free DNA (cfDNA) with a positively charged substance; b) separating the positively charged substance bound to the cfDNA; c) sequentially or simultaneously mixing a probe and a marker having a sequence complementary to the cfDNA with the mixture; d) removing the probe and marker that do not bind to the cfDNA; and e) detecting the marker, wherein the probe having a sequence complementary to the cfDNA complementarily binds to a gene known to be a colorectal cancer biomarker. In this case, the gene known to be a colorectal cancer biomarker may be a gene encoding a protein overexpressed in colorectal cancer.

[0207] Specifically, the probes having a sequence complementary to the cfDNA include NCKAP1, AUNIP, NOTUM, KRT5, TUBB, COL6A1, JUP, CDX2, MELTF, EFEMP2, DEFA5, CHEK1, MAD2L1, ENC1, CSE1L, RAD51AP1, ERICH3, SLC7A11, KRT23, PLAU, CDCA1, KLK6, DPEP1, CDH3, ANLN, CXCL1, CTHRC1, LCN2, HS6ST2, EGFL6, CXCL3, CA9, The gene may be complementary to at least one gene selected from the group consisting of PROX1, SPP1, CST1, CXCL2, TSTA3, RRM2, MMP3, MMP7, MMP10, CXCL5, SERPINB5, TEAD4, BUB1, CDC2, CLDN2, HSPH1, LY6G6D, PRC1, PUS1, SQLE, TTK, ECT2, RNF183, FBXO39, TEX38, TTLL2, PRR7, CANP, KIAA010, and combinations thereof.

[0208] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in the sample can be separated and / or concentrated using various methods. As a specific example, a nitrocellulose membrane, which has a strong affinity for nucleic acids, can be used. As another specific example, a positively charged substance can be used to capture negatively charged cfDNA. The positively charged substance can be, but is not limited to, nanoparticles, nanowires, a mesh structure, or a positively charged filter. A specific example of the "positively charged substance" can be a positively charged nanostructure or a positively charged membrane.

[0209] An example of the nanostructure may include a cationic polymer. The type of the cationic polymer is not limited. A specific example of the cationic polymer is polyethyleneimine (PEI), which may be a cationic branched polymer polyethyleneimine.

[0210] In addition, by mixing streptavidin-labeled nanowires with a biotin-conjugated PEI solution, cationic branched polyethyleneimine (PEI) can be additionally bound to the nanowires through the biotin-streptavidin interaction, resulting in nanoparticles embedded in a nanostructure (PEI / mPpy NW) with a high density and irregular distribution, with the cationic polymer polyethyleneimine bound to the surface.

[0211] Such nanowires can successfully capture genomic DNA and cfDNA at high efficiency and low concentrations. In particular, nanowire features such as a large surface area for binding to target molecules such as DNA and enhanced mobility for promoting interaction with DNA enable efficient and effective capture of target cfDNA.

[0212] In this case, the target cfDNA refers to the cfDNA of interest to be detected. As used herein, cfDNA is double-stranded. In this case, a portion of the cfDNA may be unwound. The cfDNA may also be derived from genes of colorectal cancer cells. Specifically, cfDNA may contain a nucleic acid sequence that is overexpressed in colorectal cancer cells. The nucleic acid sequence that is overexpressed in cancer cells refers to a nucleic acid sequence that is expressed at an appropriate level in normal cells but is overexpressed in specific cancer cells.

[0213] Specifically, the degree or cutoff of overexpression of the nucleic acid sequence in cancer cells may be when the OD value is 0.010 or more when measuring optical density using a marker. More specifically, the degree or cutoff of overexpression of the nucleic acid sequence in cancer cells may be when the OD value is 0.012 or 0.015 or more when measuring optical density using a marker. In this case, the wavelength of irradiation for measuring the absorbance may be appropriately determined depending on the marker. The cfDNA may be DNA in which the double strand is unwound (unwinding of DNA). Furthermore, the cfDNA may be appropriately determined depending on the purpose.

[0214] Furthermore, the cfDNA derived from the colon cancer cells may be characterized by: i) having a lower Tm value than cfDNA having a double helix structure derived from normal cells; or ii) being denatured under conditions under which cfDNA having a double helix structure derived from normal cells is not denatured.

[0215] Furthermore, the cfDNA can be bound to a probe of about 15 mer to about 30 mer that can complementarily bind to the cfDNA under any one of the following conditions: i) leaving the sample at room temperature for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes; iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C to about 75°C for about 30 seconds to about 30 minutes; v) heating at about 25°C to about 40°C for about 10 minutes to about 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; vii) treating with DNase for about 1 minute to about 30 minutes; and viii) treating with chemicals (e.g., sodium hydroxide, DMSO, surfactants, etc.).

[0216] Additional genes were ACPP (NCBI Gene ID:55), FLU3 (NCBI Gene ID:837968), TYRO3 (NCBI Gene ID:7301), and NCKAP1 (NCBI Gene ID:10787), AUNIP(NCBI Gene ID:79000), NOTUM(NCBI Gene ID:147111), KRT5(NCBI Gene ID:3852), TUBB(NCBI Gene ID:203068), COL6A1(NCBI Gene ID:1291), JUP(NCBI Gene ID:1291). ID:3728), COTL1(NCBI Gene ID:23406), CK7(NCBI Gene ID:3855), CK20(NCBI Gene ID:54474), CDX2(NCBI Gene ID:1045), MUC2(NCBI Gene ID:4583), MELTF(NCBI Gene ID:1045). ID:4241), SDC2(NCBI Gene ID:6383), EFEMP2(NCBI Gene ID:30008), DEFA5(NCBI Gene ID:1670), ASB9(NCBI Gene ID:140462),CHEK1(NCBI Gene ID:1111), MAD2L1(NCBI Gene). ID:4085, ENC1(NCBI Gene ID:8507), CSE1L(NCBI Gene ID:1434), RAD51AP1(NCBI Gene ID:10635), ERICH3(NCBI Gene ID:127524), SLC7A11(NCBI Gene ID:23657), KRT23(NCBI Gene ID:23657). ID:25984), PLAU (NCBI Gene ID:5328), CCNB1 (NCBI Gene ID:891), MELK (NCBI Gene ID:9833), CDCA1 (NCBI Gene ID:83540), KLK6 (NCBI Gene ID:5653), CKS2 (NCBI Gene ID:5653). ID:1164), IFITM1(NCBI Gene ID:8519), DPEP1(NCBI Gene ID:1800), CDH3(NCBI Gene ID:1001), ANLN(NCBI Gene ID:54443), CXCL1(NCBI Gene).ID:2919)、CTHRC1(NCBI Gene ID:115908)、CEACAM6(NCBI Gene ID:4680)、LCN2(NCBI Gene ID:3934)、HS6ST2(NCBI Gene ID:90161)、EGFL6(NCBI Gene ID:25975)、CXCL3(NCBI Gene ID:2921)、CA9(NCBI Gene ID:768)、ATAD2(NCBI Gene ID:29028)、PROX1(NCBI Gene ID:5629)、SPP1(NCBI Gene ID:6696)、CST1(NCBI Gene ID:1469)、CXCL2(NCBI Gene ID:2920)、TSTA3(NCBI Gene ID:7264)、RRM2(NCBI Gene ID:6241)、MMP3(NCBI Gene ID:4314)、MMP7(NCBI Gene ID:4316)、MMP10(NCBI Gene ID:4319)、CXCL5(NCBI Gene ID:6374)、SERPINB5(NCBI Gene ID:5268)、TEAD4(NCBI Gene ID:7004)、BUB1(NCBI Gene ID:699)、CDC2(NCBI Gene ID:983)、CLDN2(NCBI Gene ID:9075)、HSPH1(NCBI Gene ID:10808)、LY6G6D(NCBI Gene ID:58530)、PRC1(NCBI Gene ID:9055)、PUS1(NCBI Gene ID:80324)、SQLE(NCBI Gene ID:6713)、TOP2A(NCBI Gene ID:7153)、TTK(NCBI Gene ID:7272)、DSCC1(NCBI Gene ID:79075)、ECT2(NCBI Gene ID:1894)、RNF183(NCBI Gene ID:138065)、FBXO39(NCBI Gene ID:162517)、TEX38(NCBI Gene ID:374973)、TTLL2(NCBI Gene ID:83887)、PRR7(NCBI Gene ID:80758)、CANP(NCBI Gene ID:823)、KIAA0101(NCBI GeneID: 9768), CPT1A (NCBI Gene ID: 1374), IFNG (NCBI Gene ID: 3458), CD274 (NCBI Gene ID: 29126), FOLR1 (NCBI Gene ID: 2348), EPCAM (NCBI Gene ID: 4072), KRAS (NCBI Gene ID: 3845), and combinations thereof.

[0217] As a specific example, genes that are specifically present in colorectal cancer include NCKAP1, AUNIP, NOTUM, KRT5, TUBB, COL6A1, JUP, CDX2, MELTF, EFEMP2, DEFA5, CHEK1, MAD2L1, ENC1, CSE1L, RAD51AP1, ERICH3, SLC7A11, KRT23, PLAU, CDCA1, KLK6, DPEP1, CDH3, ANLN, CXCL1, CTHRC1, LCN2, HS6ST2, EGFL6, CXCL3, CA9, PROX1, SPP1, CST1, CXCL2, TSTA3, RRM2, MMP3, MMP7, MMP10, CXCL5, and SERPINB5. , TEAD4, BUB1, CDC2, CLDN2, HSPH1, LY6G6D, PRC1, PUS1, SQLE, TTK, ECT2, RNF183, FBXO39, TEX38, TTLL2, PRR7, CANP, KIAA010 genes, and additionally, ACPP, FLU3, TYRO3, COTL1, CK7, CK20, MUC2, SDC2, ASB9, CCNB1, MELK, CKS2, IFITM1, CEACAM6, ATAD2, TOP2A, CPT1A, DSCC1, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1 or EPCAM genes can be detected to diagnose colorectal cancer.

[0218] As used herein, the term "FLU3" refers to a gene encoding a protein including CA19-9 (Carcinoma Antigen 19-9).

[0219] As used herein, the term "probe" refers to DNA or RNA for detecting cfDNA. The probe may have a specific sequence that allows it to bind complementarily to cfDNA. A probe having a sequence complementary to cfDNA refers to a probe having a nucleic acid sequence that can bind complementarily to the target double-stranded cfDNA present in plasma that is to be detected. In this case, the probe may be bound to biotin. The probe may bind to a marker bound to a biotin-binding protein.

[0220] As used herein, the term "marker" refers to a substance used to detect probes bound to cfDNA. The marker may be a nanoparticle, a fluorescent dye, a fluorescent protein, or an enzyme. Specifically, the marker may be any one selected from the group consisting of quantum dots, HRP, and fluorescent proteins. In one specific example, the marker may be, but is not limited to, green fluorescent protein (GFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), or horse radish peroxidase (HRP).

[0221] The marker may be conjugated with a biotin-binding protein. The biotin-binding protein may be an avidin-based protein such as streptavidin, traptavidin, or neutravidin, but any protein that can specifically bind to biotin may be used without limitation. In one specific example, the marker may be conjugated with streptavidin.

[0222] As used herein, the term "streptavidin" refers to a tetrameric biotin-binding protein with a molecular weight of 60 kDa isolated from Streptomyces avidinii. Although it has very low homology to avidin, its structure is very similar. Like avidin, streptavidin possesses antibacterial activity and a very high binding affinity for biotin. Unlike avidin, streptavidin does not contain carbohydrates, exhibits an acidic isoelectric point (pI = 5), and has significantly lower solubility than avidin. Commercially available streptavidin, such as Thermo Scientific Pierce Streptavidin, is a recombinant form of streptavidin with a molecular weight of 53 kDa and a near-neutral isoelectric point (pI = 6.8-7.5). The lack of glycosylation and low pI of streptavidin result in a lower level of nonspecific binding (especially lectin binding) compared to avidin. These properties of streptavidin make it an ideal reagent of choice for many detection systems.

[0223] As used herein, the term "traptavidin" refers to a streptavidin variant or mutein, a protein that exhibits approximately 10-fold slower biotin dissociation rate, increased mechanical strength, and improved thermal stability. Traptavidin also specifically binds to biotin.

[0224] The term "neutravidin" used in the present invention, also known as "deglycosylated avidin," was created to avoid the major drawbacks of natural avidin and streptavidin. As the name suggests, neutravidin is produced by deglycosylating avidin. It is a protein that maintains high biotin-binding ability while having a reduced molecular weight (60 kDa) compared to avidin. Deglycosylation of avidin reduces lectin binding to undetectable levels and lowers its isoelectric point (pI = 6.3), effectively eliminating the main cause of nonspecific binding to avidin. Because the lysine residues remain available, neutravidin can be easily derivatized or conjugated, like streptavidin. Furthermore, its high biotin-binding ability and low nonspecific binding make it an ideal biotin-binding protein with a wide range of applications.

[0225] As used herein, the term "detecting a marker" refers to detecting a marker bound to a probe through a biotin-avidin reaction. The detection of the marker can be measured by a color change, a change in UV absorbance, the presence or absence of bioluminescence, a change in a fluorescent reaction, or an electrochemical change. Specifically, the method for detecting the marker varies depending on the marker used. For example, when HRP is used as a marker, the marker can be detected by observing the color reaction resulting from the reaction of hydrogen peroxide with a substrate. Furthermore, when the marker is a fluorescent protein such as GFP, the presence or absence of the marker can be detected by observing the light emitted after irradiation with light of a specific wavelength. Furthermore, when the marker is luciferase, the presence of the marker can be detected by adding a substrate such as luciferin and measuring the bioluminescence generated using a bioluminometer.

[0226] The diagnostic method of the present invention may further include a step of denaturing cfDNA. In this case, the denaturation step can be performed so as to selectively denature only cfDNA derived from colorectal cancer, while leaving normal double-stranded cfDNA undenatured. For this purpose, the denaturation step can be performed at about 50°C to about 100°C for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is typically about 0.1 seconds to about 8 minutes. Alternatively, denaturation can be performed for about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds, or about 90 seconds. In one example, the denaturation step of cfDNA can be performed before step c).

[0227] Specifically, prior to step c), the method may further include denaturing the sample or the cfDNA bound to the positively charged substance under one of the following conditions: (i) leaving the sample at room temperature for about 1 to 10 minutes; (ii) heating at about 90°C to about 95°C for 1 second to 1 minute; (iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; (iv) heating at about 60°C to about 75°C for about 1 to 30 minutes; (v) heating at about 25°C to about 40°C for about 5 to 60 minutes; (vi) treating with protease for about 1 to 10 minutes; and (vii) treating with DNase I for about 1 to 10 minutes. This denaturation step selectively denatures only the cfDNA derived from cancer cells, while not denaturing double-stranded cfDNA derived from normal cells, thereby further facilitating binding to the probe. The denaturation conditions i) to vii) can be performed after obtaining the sample. The denaturation conditions i) to vii) can be performed after obtaining the cfDNA bound to the positively charged substance. The temperature, protease, and DNase treatment times for the denaturation conditions i) to vii) can be adjusted as appropriate, as long as they do not denature stable cfDNA.

[0228] Colon cancer diagnostic kit Another aspect of the present invention is to provide a diagnostic kit for colon cancer, comprising: a biotin-conjugated probe that binds complementarily to a gene specifically expressed in colon cancer; a positively charged substance; a marker bound to an avidin-based protein; and instructions.

[0229] In this case, the genes specifically expressed in colon cancer include NCKAP1, AUNIP, NOTUM, KRT5, TUBB, COL6A1, JUP, CDX2, MELTF, EFEMP2, DEFA5, CHEK1, MAD2L1, ENC1, CSE1L, RAD51AP1, ERICH3, SLC7A11, KRT23, PLAU, CDCA1, KLK6, DPEP1, CDH3, ANLN, CXCL1, CTHRC1, LCN2, HS6ST2, EGFL6, and CXCL 3, CA9, PROX1, SPP1, CST1, CXCL2, TSTA3, RRM2, MMP3, MMP7, MMP10, CXCL5, SERPINB5, TEAD4, BUB1, CDC2, CLDN2, HSPH1, LY6G6D, PRC1, PUS1, SQLE, TTK, ECT2, RNF183, FBXO39, TEX38, TTLL2, PRR7, CANP, KIAA0101, and combinations thereof.

[0230] The instructions may also describe that the kit configuration can diagnose colorectal cancer by the following protocol: a) isolating cfDNA from a biological sample isolated from an individual using a positively charged substance included in the kit; b) sequentially or simultaneously mixing the isolated cfDNA with a biotin-conjugated probe included in the kit and a marker included in the kit; c) removing probes and markers that do not bind to cfDNA; and d) detecting signals from the markers.

[0231] The kit may further comprise a biotin-conjugated probe that complementarily binds to at least one gene selected from the group consisting of ACPP, FLU3, TYRO3, COTL1, CK7, CK20, MUC2, SDC2, ASB9, CCNB1, MELK, CKS2, IFITM1, CEACAM6, ATAD2, TOP2A, CPT1A, DSCC1, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof.

[0232] The probe, positively charged substance, and marker are as described above.

[0233] Colon cancer diagnostic device Another aspect of the present invention is to provide an apparatus for diagnosing colorectal cancer by detecting genes derived from colorectal cancer cells from a sample without amplification, the apparatus comprising: a) a mixing unit that mixes a biological sample isolated from an individual containing cfDNA with a positively charged substance; b) a harvesting unit that removes the sample from which the positively charged substance to which cfDNA is bound has been removed; c) a reaction unit that sequentially or simultaneously adds a biotin-binding probe capable of complementarily binding to a gene specifically expressed in colorectal cancer, and nanoparticles containing streptavidin and a marker, to the positively charged substance to which cfDNA is bound; d) a detection unit that detects the marker; and e) an information processing unit that determines, based on the presence or absence of detection of the marker, that cfDNA derived from colorectal cancer and having a sequence complementary to the probe is present in the sample.

[0234] In this case, the genes specifically expressed in colon cancer include NCKAP1, AUNIP, NOTUM, KRT5, TUBB, COL6A1, JUP, CDX2, MELTF, EFEMP2, DEFA5, CHEK1, MAD2L1, ENC1, CSE1L, RAD51AP1, ERICH3, SLC7A11, KRT23, PLAU, CDCA1, KLK6, DPEP1, CDH3, ANLN, CXCL1, CTHRC1, LCN2, HS6ST2, EGFL6, and CXCL 3, CA9, PROX1, SPP1, CST1, CXCL2, TSTA3, RRM2, MMP3, MMP7, MMP10, CXCL5, SERPINB5, TEAD4, BUB1, CDC2, CLDN2, HSPH1, LY6G6D, PRC1, PUS1, SQLE, TTK, ECT2, RNF183, FBXO39, TEX38, TTLL2, PRR7, CANP, KIAA0101, and combinations thereof.

[0235] The kit may further comprise a biotin-conjugated probe that complementarily binds to at least one gene selected from the group consisting of ACPP, FLU3, TYRO3, COTL1, CK7, CK20, MUC2, SDC2, ASB9, CCNB1, MELK, CKS2, IFITM1, CEACAM6, ATAD2, TOP2A, CPT1A, DSCC1, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof.

[0236] <Bile duct cancer> How bile duct cancer is diagnosed One aspect of the present invention provides a method for diagnosing cholangiocarcinoma by detecting genes derived from cholangiocarcinoma cells in the sample without amplification, the method comprising the steps of: a) mixing a biological sample isolated from an individual containing cell-free DNA (cfDNA) with a positively charged substance; b) separating the positively charged substance bound to the cfDNA; c) sequentially or simultaneously mixing the mixture with a probe and a marker having a sequence complementary to the cfDNA; d) removing the probe and marker that do not bind to the cfDNA; and e) detecting the marker, wherein the probe having a sequence complementary to the cfDNA complementarily binds to a gene known to be a biomarker for cholangiocarcinoma. The gene known to be a biomarker for cholangiocarcinoma may be a gene encoding a protein overexpressed in cholangiocarcinoma.

[0237] Specifically, the probe having a sequence complementary to the cfDNA may bind complementarily to at least one gene selected from the group consisting of MUC16, ASH1L, DOCK70, and combinations thereof.

[0238] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in the sample can be separated and / or concentrated using various methods. As a specific example, a nitrocellulose membrane, which has a strong affinity for nucleic acids, can be used. As another specific example, a positively charged substance can be used to capture negatively charged cfDNA. The positively charged substance can be, but is not limited to, nanoparticles, nanowires, a mesh structure, or a positively charged filter. A specific example of the "positively charged substance" can be a positively charged nanostructure or a positively charged membrane.

[0239] An example of the nanostructure may include a cationic polymer. The type of the cationic polymer is not limited. A specific example of the cationic polymer is polyethyleneimine (PEI), which may be a cationic branched polymer polyethyleneimine.

[0240] In addition, by mixing streptavidin-labeled nanowires with a biotin-conjugated PEI solution, cationic branched polyethyleneimine (PEI) can be additionally bound to the nanowires through the biotin-streptavidin interaction, resulting in nanoparticles embedded in a nanostructure (PEI / mPpy NW) with a high density and irregular distribution, with the cationic polymer polyethyleneimine bound to the surface.

[0241] Such nanowires can successfully capture genomic DNA and cfDNA at high efficiency and low concentrations. In particular, nanowire features such as a large surface area for binding to target molecules such as DNA and enhanced mobility for promoting interaction with DNA enable efficient and effective capture of target cfDNA.

[0242] In this case, the target cfDNA refers to the cfDNA of interest to be detected. As used herein, cfDNA is double-stranded. In this case, a portion of the cfDNA may be unwound. The cfDNA may also be derived from genes in cholangiocarcinoma cells. Specifically, cfDNA may contain a nucleic acid sequence that is overexpressed in cholangiocarcinoma cells. The nucleic acid sequence that is overexpressed in cancer cells refers to a nucleic acid sequence that is expressed at an appropriate level in normal cells but is overexpressed in specific cancer cells.

[0243] Specifically, the degree or cutoff of overexpression of the nucleic acid sequence in cancer cells may be when the OD value is 0.010 or more when measuring optical density using a marker. More specifically, the degree or cutoff of overexpression of the nucleic acid sequence in cancer cells may be when the OD value is 0.012 or 0.015 or more when measuring optical density using a marker. In this case, the wavelength of irradiation for measuring the absorbance may be appropriately determined depending on the marker. The cfDNA may be DNA in which the double strand is unwound (unwinding of DNA). Furthermore, the cfDNA may be appropriately determined depending on the purpose.

[0244] Furthermore, the cfDNA derived from the bile duct cancer cells may be characterized by: i) having a lower Tm value than cfDNA having a double helix structure derived from normal cells; or ii) being denatured under conditions under which cfDNA having a double helix structure derived from normal cells is not denatured.

[0245] Furthermore, the cfDNA can be bound to a probe of about 15 mer to about 30 mer that can complementarily bind to the cfDNA under any one of the following conditions: i) leaving the sample at room temperature for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes; iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C to about 75°C for about 30 seconds to about 30 minutes; v) heating at about 25°C to about 40°C for about 10 minutes to about 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; vii) treating with DNase for about 1 minute to about 30 minutes; and viii) treating with chemicals (e.g., sodium hydroxide, DMSO, surfactants, etc.).

[0246] The gene overexpressed in the cholangiocarcinoma cells may be any one selected from the group consisting of ACPP (NCBI Gene ID: 55), FLU3 (NCBI Gene ID: 837968), MUC16 (NCBI Gene ID: 94025), ASH1L (NCBI Gene ID: 55870), DOCK7 (NCBI Gene ID: 85440), CPT1A (NCBI Gene ID: 1374), IFNG (NCBI Gene ID: 3458), CD274 (NCBI Gene ID: 29126), FOLR1 (NCBI Gene ID: 2348), EPCAM (NCBI Gene ID: 4072), CA125 (NCBI Gene ID: 94025), CEACAM5 (NCBI Gene ID: 1048), and combinations thereof.

[0247] In one specific example, genes that are specifically present in bile duct cancer are MUC16, ASH1L, and DOCK70 genes, and additionally, ACPP, FLU3, CPT1A, DSCC1, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, or EPCAM genes can be detected to diagnose bile duct cancer.

[0248] As used herein, the term "MUC16" refers to the gene encoding CA-125 (Carcinoma Antigen 125), which is used as a tumor marker or biomarker that is positive in the blood of some patients with certain types of cancer.

[0249] As used herein, the term "probe" refers to DNA or RNA for detecting cfDNA. The probe may have a specific sequence that allows it to bind complementarily to cfDNA. A probe having a sequence complementary to cfDNA refers to a probe having a nucleic acid sequence that can bind complementarily to the target double-stranded cfDNA present in plasma that is to be detected. In this case, the probe may be bound to biotin. The probe may bind to a marker bound to a biotin-binding protein.

[0250] As used herein, the term "marker" refers to a substance used to detect probes bound to cfDNA. The marker may be a nanoparticle, a fluorescent dye, a fluorescent protein, or an enzyme. Specifically, the marker may be any one selected from the group consisting of quantum dots, HRP, and fluorescent proteins. In one specific example, the marker may be, but is not limited to, green fluorescent protein (GFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), or horse radish peroxidase (HRP).

[0251] The marker may be conjugated with a biotin-binding protein. The biotin-binding protein may be an avidin-based protein such as streptavidin, traptavidin, or neutravidin, but any protein that can specifically bind to biotin may be used without limitation. In one specific example, the marker may be conjugated with streptavidin.

[0252] As used herein, the term "streptavidin" refers to a tetrameric biotin-binding protein with a molecular weight of 60 kDa isolated from Streptomyces avidinii. Although it has very low homology to avidin, its structure is very similar. Like avidin, streptavidin possesses antibacterial activity and a very high binding affinity for biotin. Unlike avidin, streptavidin does not contain carbohydrates, exhibits an acidic isoelectric point (pI = 5), and has significantly lower solubility than avidin. Commercially available streptavidin, such as Thermo Scientific Pierce Streptavidin, is a recombinant form of streptavidin with a molecular weight of 53 kDa and a near-neutral isoelectric point (pI = 6.8-7.5). The lack of glycosylation and low pI of streptavidin result in a lower level of nonspecific binding (especially lectin binding) compared to avidin. These properties of streptavidin make it an ideal reagent of choice for many detection systems.

[0253] As used herein, the term "traptavidin" refers to a streptavidin variant or mutein, a protein that exhibits approximately 10-fold slower biotin dissociation rate, increased mechanical strength, and improved thermal stability. Traptavidin also specifically binds to biotin.

[0254] The term "neutravidin" used in the present invention, also known as "deglycosylated avidin," was created to avoid the major drawbacks of natural avidin and streptavidin. As the name suggests, neutravidin is produced by deglycosylating avidin. It is a protein that maintains high biotin-binding ability while having a reduced molecular weight (60 kDa) compared to avidin. Deglycosylation of avidin reduces lectin binding to undetectable levels and lowers its isoelectric point (pI = 6.3), effectively eliminating the main cause of nonspecific binding to avidin. Because the lysine residues remain available, neutravidin can be easily derivatized or conjugated, like streptavidin. Furthermore, its high biotin-binding ability and low nonspecific binding make it an ideal biotin-binding protein with a wide range of applications.

[0255] As used herein, the term "detecting a marker" refers to detecting a marker bound to a probe through a biotin-avidin reaction. The detection of the marker can be measured by a color change, a change in UV absorbance, the presence or absence of bioluminescence, a change in a fluorescent reaction, or an electrochemical change. Specifically, the method for detecting the marker varies depending on the marker used. For example, when HRP is used as a marker, the marker can be detected by observing the color reaction resulting from the reaction of hydrogen peroxide with a substrate. Furthermore, when the marker is a fluorescent protein such as GFP, the presence or absence of the marker can be detected by observing the light emitted after irradiation with light of a specific wavelength. Furthermore, when the marker is luciferase, the presence of the marker can be detected by adding a substrate such as luciferin and measuring the bioluminescence generated using a bioluminometer.

[0256] The diagnostic method of the present invention may further include a step of denaturing cfDNA. In this case, the denaturation step may be performed so as to selectively denature only cfDNA derived from bile duct cancer, while leaving normal double-stranded cfDNA undenatured. For this purpose, the denaturation step may be performed at about 50°C to about 100°C for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is typically about 0.1 seconds to about 8 minutes. Alternatively, denaturation may be performed for about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds, or about 90 seconds. In one example, the denaturation step of cfDNA may be performed before step c).

[0257] Specifically, prior to step c), the method may further include denaturing the sample or the cfDNA bound to the positively charged substance under one of the following conditions: (i) leaving the sample at room temperature for about 1 to 10 minutes; (ii) heating at about 90°C to about 95°C for 1 second to 1 minute; (iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; (iv) heating at about 60°C to about 75°C for about 1 to 30 minutes; (v) heating at about 25°C to about 40°C for about 5 to 60 minutes; (vi) treating with protease for about 1 to 10 minutes; and (vii) treating with DNase I for about 1 to 10 minutes. This denaturation step selectively denatures only the cfDNA derived from cancer cells, while not denaturing double-stranded cfDNA derived from normal cells, thereby further facilitating binding to the probe. The denaturation conditions i) to vii) can be performed after obtaining the sample. The denaturation conditions i) to vii) can be performed after obtaining the cfDNA bound to the positively charged substance. The temperature, protease, and DNase treatment times for the denaturation conditions i) to vii) can be adjusted as appropriate, as long as they do not denature stable cfDNA.

[0258] Bile duct cancer diagnostic kit Another aspect of the present invention is to provide a diagnostic kit for bile duct cancer, comprising: a biotin-conjugated probe that binds complementarily to a gene specifically expressed in bile duct cancer; a positively charged substance; a marker bound to an avidin-based protein; and instructions.

[0259] In this case, the gene specifically expressed in bile duct cancer may be any one or more selected from the group consisting of MUC16, ASH1L, DOCK7, and combinations thereof.

[0260] The instructions may also describe that the kit configuration can diagnose bile duct cancer by the following protocol: a) isolating cfDNA from a biological sample isolated from an individual using a positively charged substance included in the kit; b) sequentially or simultaneously mixing the isolated cfDNA with a biotin-conjugated probe included in the kit and a marker included in the kit; c) removing probes and markers that do not bind to cfDNA; and d) detecting signals from the markers.

[0261] It may also additionally contain a biotin-conjugated probe that complementarily binds to at least one gene selected from the group consisting of ACPP, FLU3, CPT1A, DSCC1, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof.

[0262] The probe, positively charged substance, and marker are as described above.

[0263] Bile duct cancer diagnostic device Another aspect of the present invention is to provide an apparatus for diagnosing cholangiocarcinoma by detecting genes derived from cholangiocarcinoma cells from a sample without amplification, the apparatus comprising: a) a mixing section that mixes a biological sample isolated from an individual containing cfDNA with a positively charged substance; b) a harvesting section that removes the sample from which the positively charged substance to which cfDNA is bound has been removed; c) a reaction section that sequentially or simultaneously adds a biotin-binding probe capable of complementarily binding to a gene specifically expressed in cholangiocarcinoma, and nanoparticles containing streptavidin and a marker, to the positively charged substance to which cfDNA is bound; d) a detection section that detects the marker; and e) an information processing section that determines, based on the presence or absence of detection of the marker, that cfDNA derived from cholangiocarcinoma is present in the sample, the sequence of which is complementary to the probe.

[0264] In this case, the gene specifically expressed in bile duct cancer may be any one or more selected from the group consisting of ACPP, FLU3, CPT1A, DSCC1, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof.

[0265] It may also additionally contain a biotin-conjugated probe that specifically binds to at least one gene selected from the group consisting of ACPP, FLU3, CPT1A, DSCC1, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof.

[0266] <Stomach cancer> How stomach cancer is diagnosed One aspect of the present invention provides a method for diagnosing gastric cancer by detecting genes derived from gastric cancer cells in the sample without amplification, the method comprising the steps of: a) mixing a biological sample isolated from an individual containing cell-free DNA (cfDNA) with a positively charged substance; b) separating the positively charged substance bound to the cfDNA; c) sequentially or simultaneously mixing a probe and a marker having a sequence complementary to the cfDNA with the mixture; d) removing the probe and marker that do not bind to the cfDNA; and e) detecting the marker, wherein the probe having a sequence complementary to the cfDNA complementarily binds to a gene known to be a gastric cancer biomarker. In this case, the gene known to be a gastric cancer biomarker may be a gene encoding a protein overexpressed in gastric cancer.

[0267] Specifically, the probe having a sequence complementary to the cfDNA may be one that binds complementarily to at least one gene selected from the group consisting of CGB, PARP1, FOXO3A, MED30, CCNE1, MYC, TFF1, FABP1, LAMP5, MATN3, CLIP4, NOX4, ADRA2C, CSK, FZD9, GALR1, GRM6, INSR, LPHN1, LYN, MRGPRX3, ADCY3, HDAC2, CFL1, NRP2, ANXA10, TFF2, CDCA5, NUSAP1, and combinations thereof.

[0268] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in the sample can be separated and / or concentrated using various methods. As a specific example, a nitrocellulose membrane, which has a strong affinity for nucleic acids, can be used. As another specific example, a positively charged substance can be used to capture negatively charged cfDNA. The positively charged substance can be, but is not limited to, nanoparticles, nanowires, a mesh structure, or a positively charged filter. A specific example of the "positively charged substance" can be a positively charged nanostructure or a positively charged membrane.

[0269] An example of the nanostructure may include a cationic polymer. The type of the cationic polymer is not limited. A specific example of the cationic polymer is polyethyleneimine (PEI), which may be a cationic branched polymer polyethyleneimine.

[0270] In addition, by mixing streptavidin-labeled nanowires with a biotin-conjugated PEI solution, cationic branched polyethyleneimine (PEI) can be additionally bound to the nanowires through the biotin-streptavidin interaction, resulting in nanoparticles embedded in a nanostructure (PEI / mPpy NW) with a high density and irregular distribution, with the cationic polymer polyethyleneimine bound to the surface.

[0271] Such nanowires can successfully capture genomic DNA and cfDNA at high efficiency and low concentrations. In particular, nanowire features such as a large surface area for binding to target molecules such as DNA and enhanced mobility for promoting interaction with DNA enable efficient and effective capture of target cfDNA.

[0272] In this case, the target cfDNA refers to the cfDNA of interest to be detected. As used herein, cfDNA is double-stranded. In this case, a portion of the cfDNA may be unwound. The cfDNA may also be derived from a gene in a gastric cancer cell. Specifically, the cfDNA may contain a nucleic acid sequence that is overexpressed in gastric cancer cells. The nucleic acid sequence that is overexpressed in cancer cells refers to a nucleic acid sequence that is expressed at an appropriate level in normal cells but is overexpressed in specific cancer cells.

[0273] Specifically, the degree or cutoff of overexpression of the nucleic acid sequence in cancer cells may be when the OD value is 0.010 or more when measuring optical density using a marker. More specifically, the degree or cutoff of overexpression of the nucleic acid sequence in cancer cells may be when the OD value is 0.012 or 0.015 or more when measuring optical density using a marker. In this case, the wavelength of irradiation for measuring the absorbance may be appropriately determined depending on the marker. The cfDNA may be DNA in which the double strand is unwound (unwinding of DNA). Furthermore, the cfDNA may be appropriately determined depending on the purpose.

[0274] Furthermore, the cfDNA derived from the gastric cancer cells may be characterized by: i) having a lower Tm value than cfDNA having a double helix structure derived from normal cells; or ii) being denatured under conditions under which cfDNA having a double helix structure derived from normal cells is not denatured.

[0275] Furthermore, the cfDNA can be bound to a probe of about 15 mer to about 30 mer that can complementarily bind to the cfDNA under any one of the following conditions: i) leaving the sample at room temperature for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes; iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C to about 75°C for about 30 seconds to about 30 minutes; v) heating at about 25°C to about 40°C for about 10 minutes to about 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; vii) treating with DNase for about 1 minute to about 30 minutes; and viii) treating with chemicals (e.g., sodium hydroxide, DMSO, surfactants, etc.).

[0276] Additional genes were ACPP (NCBI Gene ID:55), FLU3 (Gene ID:837968), CGB (NCBI Gene ID:1082), and KRT19 (NCBI Gene ID:3880), PARP1(NCBI Gene ID:142), FOXO3A(NCBI Gene ID:2309), MED30(NCBI Gene ID:90390), ERBB2(NCBI Gene ID:2064), CCNE1(NCBI Gene ID:898), MYC(NCBI Gene ID:898). ID:4609), EGFR(NCBI Gene ID:1956), KRAS(NCBI Gene ID:3845), TFF1(NCBI Gene ID:7031), FABP1(NCBI Gene ID:2168), CK20(NCBI Gene ID:54474), MUC2(NCBI Gene ID:4583), SDC2(NCBI). Gene ID:6383, LAMP5(NCBI Gene ID:24141), MATN3(NCBI Gene ID:4148), CLIP4(NCBI Gene ID:79745), NOX4(NCBI Gene ID:50507), ADRA2C(NCBI Gene ID:152), CSK(NCBI Gene). ID:1445, FZD9 (NCBI Gene ID:8326), GALR1 (NCBI Gene ID:2587), GRM6 (NCBI Gene ID:2916), INSR (NCBI Gene ID:3643), LPHN1 (NCBI Gene ID:22859), LYN (NCBI Gene ID:22859). ID:4067), MRGPRX3(NCBI Gene ID:117195), ADCY3(NCBI Gene ID:109), HDAC2(NCBI Gene ID:3066), CFL1(NCBI Gene ID:1072), COTL1(NCBI Gene ID:23406), NRP2(NCBI Gene). ID:8828), ANXA10(NCBI Gene ID:11199), TFF2(NCBI Gene ID:7032), CDCA5(NCBI Gene ID:113130), ATAD2(NCBI Gene ID:29028), ASB9(NCBI Gene).ID:140462), MMP1(NCBI Gene ID:4312), CEACAM6(NCBI Gene ID:4680), DSCC1(NCBI Gene ID:79075), CKS2(NCBI Gene ID:1164), CST1(NCBI Gene ID:1469), IFITM1(NCBI Gene ID:8519), NUSAP1(NCBI Gene ID:51203), MELK(NCBI Gene ID:9833), LGALS3BP(NCBI Gene ID:3959), CPT1A(NCBI Gene ID:1374), IFNG(NCBI Gene ID:3458), CD274(NCBI Gene ID:29126), FOLR1(NCBI Gene ID:2348), EPCAM(NCBI Gene ID:4072), CEACAM5(NCBI Gene ID: 1048) and combinations thereof.

[0277] As a specific example, genes that are specifically present in gastric cancer are CGB, PARP1, FOXO3A, MED30, CCNE1, MYC, TFF1, FABP1, LAMP5, MATN3, CLIP4, NOX4, ADRA2C, CSK, FZD9, GALR1, GRM6, INSR, LPHN1, LYN, MRGPRX3, ADCY3, HDAC2, CFL1, NRP2, ANXA10, TFF2, CDCA5, and NUSAP1 genes; Additionally, ACPP, FLU3, KRT19, ERBB2, EGFR, KRAS, DSCC1, CK20, MUC2, SDC2, COTL1, ATAD2, ASB9, MMP1, CEACAM6, DSCC1, CKS2, CST1, IFITM1, MELK, LGALS3BP, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, or EPCAM gene can be detected to diagnose gastric cancer.

[0278] As used herein, the term "CGB" refers to the gene that encodes the hormone hCG (human chorionic gonadotropin). Some cancers may also produce the hormone hCG. Therefore, elevated levels of hCG measured when a patient is not pregnant may be indicative of a cancer diagnosis.

[0279] As used herein, the term "probe" refers to DNA or RNA for detecting cfDNA. The probe may have a specific sequence that allows it to bind complementarily to cfDNA. A probe having a sequence complementary to cfDNA refers to a probe having a nucleic acid sequence that can bind complementarily to the target double-stranded cfDNA present in plasma that is to be detected. In this case, the probe may be bound to biotin. The probe may bind to a marker bound to a biotin-binding protein.

[0280] As used herein, the term "marker" refers to a substance used to detect probes bound to cfDNA. The marker may be a nanoparticle, a fluorescent dye, a fluorescent protein, or an enzyme. Specifically, the marker may be any one selected from the group consisting of quantum dots, HRP, and fluorescent proteins. In one specific example, the marker may be, but is not limited to, green fluorescent protein (GFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), or horse radish peroxidase (HRP).

[0281] The marker may be conjugated with a biotin-binding protein. The biotin-binding protein may be an avidin-based protein such as streptavidin, traptavidin, or neutravidin, but any protein that can specifically bind to biotin may be used without limitation. In one specific example, the marker may be conjugated with streptavidin.

[0282] As used herein, the term "streptavidin" refers to a tetrameric biotin-binding protein with a molecular weight of 60 kDa isolated from Streptomyces avidinii. Although it has very low homology to avidin, its structure is very similar. Like avidin, streptavidin possesses antibacterial activity and a very high binding affinity for biotin. Unlike avidin, streptavidin does not contain carbohydrates, exhibits an acidic isoelectric point (pI = 5), and has significantly lower solubility than avidin. Commercially available streptavidin, such as Thermo Scientific Pierce Streptavidin, is a recombinant form of streptavidin with a molecular weight of 53 kDa and a near-neutral isoelectric point (pI = 6.8-7.5). The lack of glycosylation and low pI of streptavidin result in a lower level of nonspecific binding (especially lectin binding) compared to avidin. These properties of streptavidin make it an ideal reagent of choice for many detection systems.

[0283] As used herein, the term "traptavidin" refers to a streptavidin variant or mutein, a protein that exhibits approximately 10-fold slower biotin dissociation rate, increased mechanical strength, and improved thermal stability. Traptavidin also specifically binds to biotin.

[0284] The term "neutravidin" used in the present invention, also known as "deglycosylated avidin," was created to avoid the major drawbacks of natural avidin and streptavidin. As the name suggests, neutravidin is produced by deglycosylating avidin. It is a protein that maintains high biotin-binding ability while having a reduced molecular weight (60 kDa) compared to avidin. Deglycosylation of avidin reduces lectin binding to undetectable levels and lowers its isoelectric point (pI = 6.3), effectively eliminating the main cause of nonspecific binding to avidin. Because the lysine residues remain available, neutravidin can be easily derivatized or conjugated, like streptavidin. Furthermore, its high biotin-binding ability and low nonspecific binding make it an ideal biotin-binding protein with a wide range of applications.

[0285] As used herein, the term "detecting a marker" refers to detecting a marker bound to a probe through a biotin-avidin reaction. The detection of the marker can be measured by a color change, a change in UV absorbance, the presence or absence of bioluminescence, a change in a fluorescent reaction, or an electrochemical change. Specifically, the method for detecting the marker varies depending on the marker used. For example, when HRP is used as a marker, the marker can be detected by observing the color reaction resulting from the reaction of hydrogen peroxide with a substrate. Furthermore, when the marker is a fluorescent protein such as GFP, the presence or absence of the marker can be detected by observing the light emitted after irradiation with light of a specific wavelength. Furthermore, when the marker is luciferase, the presence of the marker can be detected by adding a substrate such as luciferin and measuring the bioluminescence generated using a bioluminometer.

[0286] The diagnostic method of the present invention may further include a step of denaturing cfDNA. In this case, the denaturation step may be performed so as to selectively denature only cfDNA derived from gastric cancer, while leaving normal double-stranded cfDNA undenatured. For this purpose, the denaturation step may be performed at about 50°C to about 100°C for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is typically about 0.1 seconds to about 8 minutes. Alternatively, denaturation may be performed for about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds, or about 90 seconds. In one example, the denaturation step of cfDNA may be performed before step c).

[0287] Specifically, prior to step c), the method may further include denaturing the sample or the cfDNA bound to the positively charged substance under one of the following conditions: (i) leaving the sample at room temperature for about 1 to 10 minutes; (ii) heating at about 90°C to about 95°C for 1 second to 1 minute; (iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; (iv) heating at about 60°C to about 75°C for about 1 to 30 minutes; (v) heating at about 25°C to about 40°C for about 5 to 60 minutes; (vi) treating with protease for about 1 to 10 minutes; and (vii) treating with DNase I for about 1 to 10 minutes. This denaturation step selectively denatures only the cfDNA derived from cancer cells, while not denaturing double-stranded cfDNA derived from normal cells, thereby further facilitating binding to the probe. The denaturation conditions i) to vii) can be performed after obtaining the sample. The denaturation conditions i) to vii) can be performed after obtaining the cfDNA bound to the positively charged substance. The temperature, protease, and DNase treatment times for the denaturation conditions i) to vii) can be adjusted as appropriate, as long as they do not denature stable cfDNA.

[0288] Stomach cancer diagnostic kit Another aspect of the present invention is to provide a diagnostic kit for gastric cancer, comprising: a biotin-conjugated probe that binds complementarily to a gene specifically expressed in gastric cancer; a positively charged substance; a marker bound to an avidin-based protein; and instructions.

[0289] In this case, the gene specifically expressed in gastric cancer may be any one or more selected from the group consisting of CGB, PARP1, FOXO3A, MED30, CCNE1, MYC, TFF1, FABP1, LAMP5, MATN3, CLIP4, NOX4, ADRA2C, CSK, FZD9, GALR1, GRM6, INSR, LPHN1, LYN, MRGPRX3, ADCY3, HDAC2, CFL1, NRP2, ANXA10, TFF2, CDCA5, NUSAP1, and combinations thereof.

[0290] The instructions may also describe that the kit is capable of diagnosing gastric cancer by the following protocol: a) isolating cfDNA from a biological sample isolated from an individual using a positively charged substance included in the kit; b) sequentially or simultaneously mixing the isolated cfDNA with a biotin-conjugated probe included in the kit and a marker included in the kit; c) removing probes and markers that do not bind to cfDNA; and d) detecting signals from the markers.

[0291] The kit may further comprise a biotin-conjugated probe that complementarily binds to at least one gene selected from the group consisting of ACPP, FLU3, KRT19, ERBB2, EGFR, KRAS, DSCC1, CK20, MUC2, SDC2, COTL1, ATAD2, ASB9, MMP1, CEACAM6, DSCC1, CKS2, CST1, IFITM1, MELK, LGALS3BP, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof.

[0292] The probe, positively charged substance, and marker are as described above.

[0293] Gastric cancer diagnostic device Another aspect of the present invention is to provide an apparatus for diagnosing gastric cancer by detecting genes derived from gastric cancer cells from a sample without amplification, the apparatus comprising: a) a mixing unit that mixes a biological sample isolated from an individual containing cfDNA with a positively charged substance; b) a harvesting unit that removes the sample from which the positively charged substance to which cfDNA is bound has been removed; c) a reaction unit that sequentially or simultaneously adds a biotin-binding probe capable of complementarily binding to a gene specifically expressed in gastric cancer, and nanoparticles containing streptavidin and a marker, to the positively charged substance to which cfDNA is bound; d) a detection unit that detects the marker; and e) an information processing unit that determines, based on the presence or absence of detection of the marker, that cfDNA derived from gastric cancer and having a sequence complementary to the probe is present in the sample.

[0294] In this case, the gene specifically expressed in gastric cancer may be any one or more selected from the group consisting of CGB, PARP1, FOXO3A, MED30, CCNE1, MYC, TFF1, FABP1, LAMP5, MATN3, CLIP4, NOX4, ADRA2C, CSK, FZD9, GALR1, GRM6, INSR, LPHN1, LYN, MRGPRX3, ADCY3, HDAC2, CFL1, NRP2, ANXA10, TFF2, CDCA5, NUSAP1, and combinations thereof.

[0295] The kit may further comprise a biotin-conjugated probe that complementarily binds to at least one gene selected from the group consisting of ACPP, FLU3, KRT19, ERBB2, EGFR, KRAS, DSCC1, CK20, MUC2, SDC2, COTL1, ATAD2, ASB9, MMP1, CEACAM6, DSCC1, CKS2, CST1, IFITM1, MELK, LGALS3BP, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof.

[0296] <Pancreatic cancer> How pancreatic cancer is diagnosed One aspect of the present invention provides a method for diagnosing pancreatic cancer by detecting genes derived from pancreatic cancer cells in a sample without amplification, the method comprising the steps of: a) mixing a biological sample isolated from an individual containing cell-free DNA (cfDNA) with a positively charged substance; b) separating the positively charged substance bound to the cfDNA; c) sequentially or simultaneously mixing a probe and a marker having a sequence complementary to the cfDNA with the mixture; d) removing the probe and marker that do not bind to the cfDNA; and e) detecting the marker, wherein the probe having a sequence complementary to the cfDNA complementarily binds to a gene known to be a pancreatic cancer biomarker. In this case, the gene known to be a pancreatic cancer biomarker may be a gene encoding a protein overexpressed in pancreatic cancer.

[0297] Specifically, the probe having a sequence complementary to the cfDNA may bind complementarily to at least one gene selected from the group consisting of SMAD4, APC, GNAS, and combinations thereof.

[0298] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in the sample can be separated and / or concentrated using various methods. As a specific example, a nitrocellulose membrane, which has a strong affinity for nucleic acids, can be used. As another specific example, a positively charged substance can be used to capture negatively charged cfDNA. The positively charged substance can be, but is not limited to, nanoparticles, nanowires, a mesh structure, or a positively charged filter. A specific example of the "positively charged substance" can be a positively charged nanostructure or a positively charged membrane.

[0299] An example of the nanostructure may include a cationic polymer. The type of the cationic polymer is not limited. A specific example of the cationic polymer is polyethyleneimine (PEI), which may be a cationic branched polymer polyethyleneimine.

[0300] In addition, by mixing streptavidin-labeled nanowires with a biotin-conjugated PEI solution, cationic branched polyethyleneimine (PEI) can be additionally bound to the nanowires through the biotin-streptavidin interaction, resulting in nanoparticles embedded in a nanostructure (PEI / mPpy NW) with a high density and irregular distribution, with the cationic polymer polyethyleneimine bound to the surface.

[0301] Such nanowires can successfully capture genomic DNA and cfDNA at high efficiency and low concentrations. In particular, nanowire features such as a large surface area for binding to target molecules such as DNA and enhanced mobility for promoting interaction with DNA enable efficient and effective capture of target cfDNA.

[0302] In this case, the target cfDNA refers to the cfDNA of interest to be detected. As used herein, cfDNA is double-stranded. In this case, a portion of the cfDNA may be unwound. The cfDNA may also be derived from genes in pancreatic cancer cells. Specifically, the cfDNA may contain a nucleic acid sequence that is overexpressed in pancreatic cancer cells. The nucleic acid sequence that is overexpressed in cancer cells refers to a nucleic acid sequence that is expressed at an appropriate level in normal cells but is overexpressed in specific cancer cells.

[0303] Specifically, the degree or cutoff of overexpression of the nucleic acid sequence in cancer cells may be when the OD value is 0.010 or more when measuring optical density using a marker. More specifically, the degree or cutoff of overexpression of the nucleic acid sequence in cancer cells may be when the OD value is 0.012 or 0.015 or more when measuring optical density using a marker. In this case, the wavelength of irradiation for measuring the absorbance may be appropriately determined depending on the marker. The cfDNA may be DNA in which the double strand is unwound (unwinding of DNA). Furthermore, the cfDNA may be appropriately determined depending on the purpose.

[0304] Furthermore, the cfDNA derived from the pancreatic cancer cells may be characterized by: i) having a lower Tm value than cfDNA having a double helix structure derived from normal cells; or ii) being denatured under conditions under which cfDNA having a double helix structure derived from normal cells is not denatured.

[0305] Furthermore, the cfDNA can be bound to a probe of about 15 mer to about 30 mer that can complementarily bind to the cfDNA under any one of the following conditions: i) leaving the sample at room temperature for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes; iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C to about 75°C for about 30 seconds to about 30 minutes; v) heating at about 25°C to about 40°C for about 10 minutes to about 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; vii) treating with DNase for about 1 minute to about 30 minutes; and viii) treating with chemicals (e.g., sodium hydroxide, DMSO, surfactants, etc.).

[0306] The gene overexpressed in pancreatic cancer cells may be any one selected from the group consisting of KRAS (NCBI Gene ID: 3845), SMADA4 (NCBI Gene ID: 4089), APC (NCBI Gene ID: 324), GNAS (NCBI Gene ID: 2788), MUC1 (NCBI Gene ID: 4582), CEACAM5 (NCBI Gene ID: 1048), CEACAM1 (NCBI Gene ID: 634), MUC16 (NCBI Gene ID: 94025), and combinations thereof.

[0307] In one specific example, genes that are specifically present in pancreatic cancer are the SMAD4, APC, and GNAS genes, and pancreatic cancer can additionally be diagnosed by detecting the KRAS, MUC1, MSLN, CEACAM1, CEACAM5, or MUC16 genes.

[0308] As used herein, the term "probe" refers to DNA or RNA for detecting cfDNA. The probe may have a specific sequence that allows it to bind complementarily to cfDNA. A probe having a sequence complementary to cfDNA refers to a probe having a nucleic acid sequence that can bind complementarily to the target double-stranded cfDNA present in plasma that is to be detected. In this case, the probe may be bound to biotin. The probe may bind to a marker bound to a biotin-binding protein.

[0309] As used herein, the term "marker" refers to a substance used to detect probes bound to cfDNA. The marker may be a nanoparticle, a fluorescent dye, a fluorescent protein, or an enzyme. Specifically, the marker may be any one selected from the group consisting of quantum dots, HRP, and fluorescent proteins. In one specific example, the marker may be, but is not limited to, green fluorescent protein (GFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), or horse radish peroxidase (HRP).

[0310] The marker may be conjugated with a biotin-binding protein. The biotin-binding protein may be an avidin-based protein such as streptavidin, traptavidin, or neutravidin, but any protein that can specifically bind to biotin may be used without limitation. In one specific example, the marker may be conjugated with streptavidin.

[0311] As used herein, the term "streptavidin" refers to a tetrameric biotin-binding protein with a molecular weight of 60 kDa isolated from Streptomyces avidinii. Although it has very low homology to avidin, its structure is very similar. Like avidin, streptavidin possesses antibacterial activity and a very high binding affinity for biotin. Unlike avidin, streptavidin does not contain carbohydrates, exhibits an acidic isoelectric point (pI = 5), and has significantly lower solubility than avidin. Commercially available streptavidin, such as Thermo Scientific Pierce Streptavidin, is a recombinant form of streptavidin with a molecular weight of 53 kDa and a near-neutral isoelectric point (pI = 6.8-7.5). The lack of glycosylation and low pI of streptavidin result in a lower level of nonspecific binding (especially lectin binding) compared to avidin. These properties of streptavidin make it an ideal reagent of choice for many detection systems.

[0312] As used herein, the term "traptavidin" refers to a streptavidin variant or mutein, a protein that exhibits approximately 10-fold slower biotin dissociation rate, increased mechanical strength, and improved thermal stability. Traptavidin also specifically binds to biotin.

[0313] The term "neutravidin" used in the present invention, also known as "deglycosylated avidin," was created to avoid the major drawbacks of natural avidin and streptavidin. As the name suggests, neutravidin is produced by deglycosylating avidin. It is a protein that maintains high biotin-binding ability while having a reduced molecular weight (60 kDa) compared to avidin. Deglycosylation of avidin reduces lectin binding to undetectable levels and lowers its isoelectric point (pI = 6.3), effectively eliminating the main cause of nonspecific binding to avidin. Because the lysine residues remain available, neutravidin can be easily derivatized or conjugated, like streptavidin. Furthermore, its high biotin-binding ability and low nonspecific binding make it an ideal biotin-binding protein with a wide range of applications.

[0314] As used herein, the term "detecting a marker" refers to detecting a marker bound to a probe through a biotin-avidin reaction. The detection of the marker can be measured by a color change, a change in UV absorbance, the presence or absence of bioluminescence, a change in a fluorescent reaction, or an electrochemical change. Specifically, the method for detecting the marker varies depending on the marker used. For example, when HRP is used as a marker, the marker can be detected by observing the color reaction resulting from the reaction of hydrogen peroxide with a substrate. Furthermore, when the marker is a fluorescent protein such as GFP, the presence or absence of the marker can be detected by observing the light emitted after irradiation with light of a specific wavelength. Furthermore, when the marker is luciferase, the presence of the marker can be detected by adding a substrate such as luciferin and measuring the bioluminescence generated using a bioluminometer.

[0315] The diagnostic method of the present invention may further include a step of denaturing cfDNA. In this case, the denaturation step can be performed so as to selectively denature only cfDNA derived from pancreatic cancer, while leaving normal double-stranded cfDNA undenatured. For this purpose, the denaturation step can be performed at about 50°C to about 100°C for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is typically about 0.1 seconds to about 8 minutes. Alternatively, denaturation can be performed for about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds, or about 90 seconds. In one example, the step of denaturing cfDNA can be performed before step c).

[0316] Specifically, prior to step c), the method may further include denaturing the sample or the cfDNA bound to the positively charged substance under one of the following conditions: (i) leaving the sample at room temperature for about 1 to 10 minutes; (ii) heating at about 90°C to about 95°C for 1 second to 1 minute; (iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; (iv) heating at about 60°C to about 75°C for about 1 to 30 minutes; (v) heating at about 25°C to about 40°C for about 5 to 60 minutes; (vi) treating with protease for about 1 to 10 minutes; and (vii) treating with DNase I for about 1 to 10 minutes. This denaturation step selectively denatures only the cfDNA derived from cancer cells, while not denaturing double-stranded cfDNA derived from normal cells, thereby further facilitating binding to the probe. The denaturation conditions i) to vii) can be performed after obtaining the sample. The denaturation conditions i) to vii) can be performed after obtaining the cfDNA bound to the positively charged substance. The temperature, protease, and DNase treatment times for the denaturation conditions i) to vii) can be adjusted as appropriate, as long as they do not denature stable cfDNA.

[0317] Pancreatic cancer diagnostic kit Another aspect of the present invention is to provide a diagnostic kit for pancreatic cancer, comprising: a biotin-conjugated probe that binds complementarily to a gene specifically expressed in pancreatic cancer; a positively charged substance; a marker bound to an avidin-based protein; and instructions.

[0318] In this case, the gene specifically expressed in pancreatic cancer may be any one or more selected from the group consisting of SMAD4, APC, GNAS, and combinations thereof.

[0319] The instructions may also describe that the kit configuration can diagnose pancreatic cancer by the following protocol: a) isolating cfDNA from a biological sample isolated from an individual using a positively charged substance included in the kit; b) sequentially or simultaneously mixing the isolated cfDNA with a biotin-conjugated probe included in the kit and a marker included in the kit; c) removing probes and markers that do not bind to cfDNA; and d) detecting signals from the markers.

[0320] The kit may additionally contain a biotin-conjugated probe that binds complementarily to at least one gene selected from the group consisting of KRAS, MUC1, MSLN, CEACAM1, CEACAM5, or MUC16, and combinations thereof.

[0321] The probe, positively charged substance, and marker are as described above.

[0322] Pancreatic cancer diagnostic device Another aspect of the present invention is to provide an apparatus for diagnosing pancreatic cancer by detecting genes derived from pancreatic cancer cells from a sample without amplification, the apparatus comprising: a) a mixing unit that mixes a biological sample isolated from an individual containing cfDNA with a positively charged substance; b) a harvesting unit that removes the sample from which the positively charged substance to which cfDNA is bound has been removed; c) a reaction unit that sequentially or simultaneously adds a biotin-binding probe capable of complementarily binding to a gene specifically expressed in pancreatic cancer, and nanoparticles containing streptavidin and a marker, to the positively charged substance to which cfDNA is bound; d) a detection unit that detects the marker; and e) an information processing unit that determines the presence of cfDNA derived from pancreatic cancer in the sample, having a sequence complementary to the probe, based on the presence or absence of detection of the marker.

[0323] In this case, the gene specifically expressed in pancreatic cancer may be any one or more selected from the group consisting of SMAD4, APC, GNAS, and combinations thereof.

[0324] The kit may additionally contain a biotin-conjugated probe that binds complementarily to at least one gene selected from the group consisting of KRAS, MUC1, MSLN, CEACAM1, CEACAM5, or MUC16, and combinations thereof.

[0325] <Early diagnosis and prognosis prediction> Cancer diagnosis methods One aspect of the present invention provides a method for early diagnosis or prognosis prediction of cancer by detecting genes derived from cancer cells from the sample without amplification, the method comprising the steps of: a) mixing a biological sample isolated from an individual containing cell-free DNA (hereinafter referred to as cfDNA) with a positively charged substance; b) separating the positively charged substance bound to the cfDNA; c) sequentially or simultaneously mixing a probe and a marker having a sequence complementary to the cfDNA with the mixture; d) removing the probe and marker that do not bind to the cfDNA; and e) detecting the marker, wherein the probe having a sequence complementary to the cfDNA complementarily binds to a gene known to be a cancer biomarker.

[0326] Specifically, the probe having a sequence complementary to the cfDNA may complementarily bind to at least one gene selected from the group consisting of CPT1A, IFNG, IFNGR1, CD279, CD274, and combinations thereof, and preferably a combination of two or more genes selected from the group.

[0327] The cancer may be any one selected from the group consisting of lung cancer, colon cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymus cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, lymphoma, acute leukemia, multiple myeloma, and combinations thereof.

[0328] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in the sample can be separated and / or concentrated using various methods. As a specific example, a nitrocellulose membrane, which has a strong affinity for nucleic acids, can be used. As another specific example, a positively charged substance can be used to capture negatively charged cfDNA. The positively charged substance can be, but is not limited to, nanoparticles, nanowires, a mesh structure, or a positively charged filter. A specific example of the "positively charged substance" can be a positively charged nanostructure or a positively charged membrane.

[0329] An example of the nanostructure may include a cationic polymer. The type of the cationic polymer is not limited. A specific example of the cationic polymer is polyethyleneimine (PEI), which may be a cationic branched polymer polyethyleneimine.

[0330] In addition, by mixing streptavidin-labeled nanowires with a biotin-conjugated PEI solution, cationic branched polyethyleneimine (PEI) can be additionally bound to the nanowires through the biotin-streptavidin interaction, resulting in nanoparticles embedded in a nanostructure (PEI / mPpy NW) with a high density and irregular distribution, with the cationic polymer polyethyleneimine bound to the surface.

[0331] Such nanowires can successfully capture genomic DNA and cfDNA at high efficiency and low concentrations. In particular, nanowire features such as a large surface area for binding to target molecules such as DNA and enhanced mobility for promoting interaction with DNA enable efficient and effective capture of target cfDNA.

[0332] In this case, the target cfDNA refers to the cfDNA of interest to be detected. As used herein, cfDNA is double-stranded. In this case, a portion of the cfDNA may be unwound. The cfDNA may also be derived from genes in cancer cells. Specifically, cfDNA may contain a nucleic acid sequence that is overexpressed in cancer cells. The nucleic acid sequence that is overexpressed in cancer cells refers to a nucleic acid sequence that is expressed at an appropriate level in normal cells but is overexpressed in specific cancer cells.

[0333] Specifically, the degree or cutoff of overexpression of the nucleic acid sequence in cancer cells may be when the OD value is 0.010 or more when measuring optical density using a marker. More specifically, the degree or cutoff of overexpression of the nucleic acid sequence in cancer cells may be when the OD value is 0.012 or 0.015 or more when measuring optical density using a marker. In this case, the wavelength of irradiation for measuring the absorbance may be appropriately determined depending on the marker. The cfDNA may be DNA in which the double strand is unwound (unwinding of DNA). Furthermore, the cfDNA may be appropriately determined depending on the purpose.

[0334] Furthermore, the cfDNA derived from the cancer cells may be characterized by: i) having a lower Tm value than cfDNA having a double helix structure derived from normal cells; or ii) being denatured under conditions under which cfDNA having a double helix structure derived from normal cells is not denatured.

[0335] Furthermore, the cfDNA can be bound to a probe of about 15 mer to about 30 mer that can complementarily bind to the cfDNA under any one of the following conditions: i) leaving the sample at room temperature for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes; iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C to about 75°C for about 30 seconds to about 30 minutes; v) heating at about 25°C to about 40°C for about 10 minutes to about 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; vii) treating with DNase for about 1 minute to about 30 minutes; and viii) treating with chemicals (e.g., sodium hydroxide, DMSO, surfactants, etc.).

[0336] The gene overexpressed in the cancer cells may be any one selected from the group consisting of FNG (NCBI Gene ID: 3458), IFNGR1 (NCBI Gene ID: 3459), CD279 (NCBI Gene ID: 5133), CD274 (NCBI Gene ID: 29126), and combinations thereof. Specifically, when the combination of two genes overexpressed in the cancer cells is IFNG / IFNGR1, IFNG / CD274, or IFNG / CD279, when the combination of three genes overexpressed in the cancer cells is IFNG / IFNGR1 / CD274, IFNG / CD274 / CD279, or IFNGR1 / CD274 / CD279, and when the combination of four genes overexpressed in the cancer cells is IFNG / IFNGR1 / CD274 / CD279. When two or more genes that are overexpressed in the cancer cells are analyzed, the reliability of the analysis results can be improved.

[0337] As used herein, the term "IFNG" refers to the gene encoding interferon gamma.

[0338] As used herein, the term "IFNGR1" refers to the gene encoding interferon gamma receptor 1.

[0339] As used herein, the term "CD274" refers to the gene encoding PD-L1 (Programmed death-ligand 1).

[0340] As used herein, the term "probe" refers to DNA or RNA for detecting cfDNA. The probe may have a specific sequence that allows it to bind complementarily to cfDNA. A probe having a sequence complementary to cfDNA refers to a probe having a nucleic acid sequence that can bind complementarily to the target double-stranded cfDNA present in plasma that is to be detected. In this case, the probe may be bound to biotin. The probe may bind to a marker bound to a biotin-binding protein.

[0341] As used herein, the term "marker" refers to a substance used to detect probes bound to cfDNA. The marker may be a nanoparticle, a fluorescent dye, a fluorescent protein, or an enzyme. Specifically, the marker may be any one selected from the group consisting of quantum dots, HRP, and fluorescent proteins. In one specific example, the marker may be, but is not limited to, green fluorescent protein (GFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), or horse radish peroxidase (HRP).

[0342] The marker may be conjugated with a biotin-binding protein. The biotin-binding protein may be an avidin-based protein such as streptavidin, traptavidin, or neutravidin, but any protein that can specifically bind to biotin may be used without limitation. In one specific example, the marker may be conjugated with streptavidin.

[0343] As used herein, the term "streptavidin" refers to a tetrameric biotin-binding protein with a molecular weight of 60 kDa isolated from Streptomyces avidinii. Although it has very low homology to avidin, its structure is very similar. Like avidin, streptavidin possesses antibacterial activity and a very high binding affinity for biotin. Unlike avidin, streptavidin does not contain carbohydrates, exhibits an acidic isoelectric point (pI = 5), and has significantly lower solubility than avidin. Commercially available streptavidin, such as Thermo Scientific Pierce Streptavidin, is a recombinant form of streptavidin with a molecular weight of 53 kDa and a near-neutral isoelectric point (pI = 6.8-7.5). The lack of glycosylation and low pI of streptavidin result in a lower level of nonspecific binding (especially lectin binding) compared to avidin. These properties of streptavidin make it an ideal reagent of choice for many detection systems.

[0344] As used herein, the term "traptavidin" refers to a streptavidin variant or mutein, a protein that exhibits approximately 10-fold slower biotin dissociation rate, increased mechanical strength, and improved thermal stability. Traptavidin also specifically binds to biotin.

[0345] The term "neutravidin" used in the present invention, also known as "deglycosylated avidin," was created to avoid the major drawbacks of natural avidin and streptavidin. As the name suggests, neutravidin is produced by deglycosylating avidin. It is a protein that maintains high biotin-binding ability while having a reduced molecular weight (60 kDa) compared to avidin. Deglycosylation of avidin reduces lectin binding to undetectable levels and lowers its isoelectric point (pI = 6.3), effectively eliminating the main cause of nonspecific binding to avidin. Because the lysine residues remain available, neutravidin can be easily derivatized or conjugated, like streptavidin. Furthermore, its high biotin-binding ability and low nonspecific binding make it an ideal biotin-binding protein with a wide range of applications.

[0346] As used herein, the term "detecting a marker" refers to detecting a marker bound to a probe through a biotin-avidin reaction. The detection of the marker can be measured by a color change, a change in UV absorbance, the presence or absence of bioluminescence, a change in a fluorescent reaction, or an electrochemical change. Specifically, the method for detecting the marker varies depending on the marker used. For example, when HRP is used as a marker, the marker can be detected by observing the color reaction resulting from the reaction of hydrogen peroxide with a substrate. Furthermore, when the marker is a fluorescent protein such as GFP, the presence or absence of the marker can be detected by observing the light emitted after irradiation with light of a specific wavelength. Furthermore, when the marker is luciferase, the presence of the marker can be detected by adding a substrate such as luciferin and measuring the bioluminescence generated using a bioluminometer.

[0347] The diagnostic method of the present invention may further include a step of denaturing cfDNA. In this case, the denaturation step can be performed so as to selectively denature only cancer-derived cfDNA while leaving normal double-stranded cfDNA undenatured. For this purpose, the denaturation step can be performed at about 50°C to about 100°C for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is typically about 0.1 seconds to about 8 minutes. Alternatively, denaturation can be performed for about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds, or about 90 seconds. In one example, the denaturation step of cfDNA can be performed before step c).

[0348] Specifically, prior to step c), the method may further include denaturing the sample or the cfDNA bound to the positively charged substance under one of the following conditions: (i) leaving the sample at room temperature for about 1 to 10 minutes; (ii) heating at about 90°C to about 95°C for 1 second to 1 minute; (iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; (iv) heating at about 60°C to about 75°C for about 1 to 30 minutes; (v) heating at about 25°C to about 40°C for about 5 to 60 minutes; (vi) treating with protease for about 1 to 10 minutes; and (vii) treating with DNase I for about 1 to 10 minutes. This denaturation step selectively denatures only the cfDNA derived from cancer cells, while not denaturing double-stranded cfDNA derived from normal cells, thereby further facilitating binding to the probe. The denaturation conditions i) to vii) can be performed after obtaining the sample. The denaturation conditions i) to vii) can be performed after obtaining the cfDNA bound to the positively charged substance. The temperature, protease, and DNase treatment times for the denaturation conditions i) to vii) can be adjusted as appropriate, as long as they do not denature stable cfDNA.

[0349] Cancer diagnostic kits Another aspect of the present invention is to provide a kit for early diagnosis or prognosis prediction of cancer, comprising: a biotin-conjugated probe that binds complementarily to a gene that is specifically expressed in cancer; a positively charged substance; a marker bound to an avidin-based protein; and instructions.

[0350] In this case, the gene specifically expressed in cancer may be any one or more selected from the group consisting of CPT1A, IFNG, IFNGR1, CD279, CD274, and combinations thereof.

[0351] The instructions may also describe that the kit is capable of diagnosing cancer early or predicting cancer prognosis by the following protocol: a) isolating cfDNA from a biological sample isolated from an individual using a positively charged substance included in the kit; b) sequentially or simultaneously mixing the isolated cfDNA with a biotin-conjugated probe included in the kit and a marker included in the kit; c) removing probes and markers that do not bind to cfDNA; and d) detecting signals from the markers.

[0352] The probe, positively charged substance, and marker are as described above.

[0353] Cancer diagnostic equipment Another aspect of the present invention is to provide an apparatus for detecting genes derived from cancer cells from a sample, without amplification, for early diagnosis or prognosis of cancer, comprising: a) a mixing unit that mixes a biological sample isolated from an individual containing cfDNA with a positively charged substance; b) a harvesting unit that removes the sample from which the positively charged substance to which cfDNA is bound has been removed; c) a reaction unit that sequentially or simultaneously adds a biotin-binding probe capable of complementarily binding to a gene specifically expressed in the cancer, and nanoparticles containing streptavidin and a marker, to the positively charged substance to which cfDNA is bound; d) a detection unit that detects the marker; and e) an information processing unit that determines, based on the presence or absence of detection of the marker, that cfDNA derived from the cancer is present in the sample, the sample having a sequence complementary to the probe.

[0354] In this case, the gene specifically expressed in cancer may be any one or more selected from the group consisting of CPT1A, IFNG, IFNGR1, CD279, CD274, and combinations thereof.

[0355] <Cancer> Method for confirming cancer, cancer metastasis, and cancer resistance to drugs One aspect of the present invention provides a method for determining the presence or absence of cancer, the presence or absence of cancer metastasis, and / or the presence or absence of cancer resistance by detecting genes derived from cancer cells from the sample without amplification, the method comprising the steps of: a) mixing a biological sample isolated from an individual containing cell-free DNA (hereinafter referred to as cfDNA) with a positively charged substance; b) separating the positively charged substance bound to the cfDNA; c) sequentially or simultaneously mixing a probe and a marker having a sequence complementary to the cfDNA with the mixture; d) removing the probe and marker that do not bind to the cfDNA; and e) detecting the marker, wherein the cfDNA is derived from cancer cells, and the probe having a sequence complementary to the cfDNA complementarily binds to a gene known as a biomarker for the presence or absence of cancer, a cancer metastasis indicator, or resistance, thereby providing a method for determining the state of cancer metastasis or the presence or absence of resistance. In this regard, genes known as biomarkers for cancer, the presence or absence of cancer metastasis, or resistance are genes that contain single nucleotide polymorphisms (snp) in cancer (see Examples 6 and 7).

[0356] Specifically, the probe having a sequence complementary to the cfDNA may bind complementarily to at least one gene selected from the group consisting of CPT1A, IFNG, IFNGR1, CD279, CD274G, and combinations thereof.

[0357] The probe having a sequence complementary to the cfDNA may bind complementarily to a gene overexpressed in cancer cells, a gene specifically present in cancer, a gene associated with metastasis, or a gene associated with drug resistance.

[0358] In one specific example, the gene overexpressed in the cancer cells may be any one selected from the group consisting of CPT1A, IFNG, IFNGR1, CD279, CD274, NSE, SCC, CEA, cyfra21-1, TPA, NMP22, OGT, Thyroglobulin (TG), Calcitonin (CALCA), BRAF V600E, TERT C228T / C250T, AFP, β-HCG (CGB), CA19-9, PSA, PSMA, PAP, PCA3, TMPRSS2-ERG, CA125, HIF-1a, VEGF, CA15-3, HER2, SCC (SART3), TOP2A, MCM2, p16INK4a (CDKN2A), Ki-67 (MKI167), HE4 (WEDC2), and combinations thereof.

[0359] In one specific example, the genes specifically present in cancer are CPT1A, IFNG, IFNGR1, CD279, and CD274 genes. Additionally, the type of cancer can be diagnosed by detecting the NSE, SCC, CEA, cyfra21-1, TPA, NMP22, OGT, thyroglobulin (TG), calcitonin (CALCA), BRAF V600E, TERT C228T / C250T, AFP, β-HCG (CGB), CA19-9, PSA, PSMA, PAP, PCA3, TMPRSS2-ERG, CA125, HIF-1a, VEGF, CA15-3, HER2, SCC (SART3), TOP2A, MCM2, p16INK4a (CDKN2A), Ki-67 (MKI167), or HE4 (WEDC2) genes.

[0360] In one specific example, the genes associated with cancer metastasis are "proliferation" and "invasion" related genes, such as Ki67, STK15, Survivin, Cyclin B1, MYBL2, Stromelysin3, and Cathepsin L2.

[0361] In one embodiment, the gene associated with drug resistance can be determined depending on the drug and cancer type. In one embodiment, the gene associated with acquired resistance to EGFR-TKI can be any one selected from the group consisting of EGFR T790M, PI3K, BRAF, MAPK1, HER2, KRAS, NRAS, RB deletion, p53 deletion, PTEN, and NFkB.

[0362] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in the sample can be separated and / or concentrated using various methods. As a specific example, a nitrocellulose membrane, which has a strong affinity for nucleic acids, can be used. As another specific example, a positively charged substance can be used to capture negatively charged cfDNA. The positively charged substance can be, but is not limited to, nanoparticles, nanowires, a mesh structure, or a positively charged filter. A specific example of the "positively charged substance" can be a positively charged nanostructure or a positively charged membrane.

[0363] An example of the nanostructure may include a cationic polymer. The type of the cationic polymer is not limited. A specific example of the cationic polymer is polyethyleneimine (PEI), which may be a cationic branched polymer polyethyleneimine.

[0364] In addition, by mixing streptavidin-labeled nanowires with a biotin-conjugated PEI solution, cationic branched polyethyleneimine (PEI) can be additionally bound to the nanowires through the biotin-streptavidin interaction, resulting in nanoparticles embedded in a nanostructure (PEI / mPpy NW) with a high density and irregular distribution, with the cationic polymer polyethyleneimine bound to the surface.

[0365] Such nanowires can successfully capture genomic DNA and cfDNA at high efficiency and low concentrations. In particular, nanowire features such as a large surface area for binding to target molecules such as DNA and enhanced mobility for promoting interaction with DNA enable efficient and effective capture of target cfDNA.

[0366] In this case, the target cfDNA refers to the cfDNA of interest to be detected. As used herein, cfDNA is double-stranded. In this case, a portion of the cfDNA may be unwound. The cfDNA may also be derived from genes in cancer cells. Specifically, cfDNA may contain a nucleic acid sequence that is overexpressed in cancer cells. The nucleic acid sequence that is overexpressed in cancer cells refers to a nucleic acid sequence that is expressed at an appropriate level in normal cells but is overexpressed in specific cancer cells.

[0367] Specifically, the degree or cutoff of overexpression of the nucleic acid sequence in cancer cells may be when the OD value is 0.010 or more when measuring optical density using a marker. More specifically, the degree or cutoff of overexpression of the nucleic acid sequence in cancer cells may be when the OD value is 0.012 or 0.015 or more when measuring optical density using a marker. In this case, the wavelength of irradiation for measuring the absorbance may be appropriately determined depending on the marker. The cfDNA may be DNA in which the double strand is unwound (unwinding of DNA). Furthermore, the cfDNA may be appropriately determined depending on the purpose.

[0368] Furthermore, the cfDNA derived from the cancer cells may be characterized by: i) having a lower Tm value than cfDNA having a double helix structure derived from normal cells; or ii) being denatured under conditions under which cfDNA having a double helix structure derived from normal cells is not denatured.

[0369] Furthermore, the cfDNA can be bound to a probe of about 15 mer to about 30 mer that can complementarily bind to the cfDNA under any one of the following conditions: i) leaving the sample at room temperature for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes; iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C to about 75°C for about 30 seconds to about 30 minutes; v) heating at about 25°C to about 40°C for about 10 minutes to about 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; vii) treating with DNase for about 1 minute to about 30 minutes; and viii) treating with chemicals (e.g., sodium hydroxide, DMSO, surfactants, etc.).

[0370] As used herein, the term "probe" refers to DNA or RNA for detecting cfDNA. The probe may have a specific sequence that allows it to bind complementarily to cfDNA. A probe having a sequence complementary to cfDNA refers to a probe having a nucleic acid sequence that can bind complementarily to the target double-stranded cfDNA present in plasma that is to be detected. In this case, the probe may be bound to biotin. The probe may bind to a marker bound to a biotin-binding protein.

[0371] As used herein, the term "marker" refers to a substance used to detect probes bound to cfDNA. The marker may be a nanoparticle, a fluorescent dye, a fluorescent protein, or an enzyme. Specifically, the marker may be any one selected from the group consisting of quantum dots, HRP, and fluorescent proteins. In one specific example, the marker may be, but is not limited to, green fluorescent protein (GFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), or horse radish peroxidase (HRP).

[0372] The marker may be conjugated with a biotin-binding protein. The biotin-binding protein may be an avidin-based protein such as streptavidin, traptavidin, or neutravidin, but any protein that can specifically bind to biotin may be used without limitation. In one specific example, the marker may be conjugated with streptavidin.

[0373] As used herein, the term "streptavidin" refers to a tetrameric biotin-binding protein with a molecular weight of 60 kDa isolated from Streptomyces avidinii. Although it has very low homology to avidin, its structure is very similar. Like avidin, streptavidin possesses antibacterial activity and a very high binding affinity for biotin. Unlike avidin, streptavidin does not contain carbohydrates, exhibits an acidic isoelectric point (pI = 5), and has significantly lower solubility than avidin. Commercially available streptavidin, such as Thermo Scientific Pierce Streptavidin, is a recombinant form of streptavidin with a molecular weight of 53 kDa and a near-neutral isoelectric point (pI = 6.8-7.5). The lack of glycosylation and low pI of streptavidin result in a lower level of nonspecific binding (especially lectin binding) compared to avidin. These properties of streptavidin make it an ideal reagent of choice for many detection systems.

[0374] As used herein, the term "traptavidin" refers to a streptavidin variant or mutein, a protein that exhibits approximately 10-fold slower biotin dissociation rate, increased mechanical strength, and improved thermal stability. Traptavidin also specifically binds to biotin.

[0375] The term "neutravidin" used in the present invention, also known as "deglycosylated avidin," was created to avoid the major drawbacks of natural avidin and streptavidin. As the name suggests, neutravidin is produced by deglycosylating avidin. It is a protein that maintains high biotin-binding ability while having a reduced molecular weight (60 kDa) compared to avidin. Deglycosylation of avidin reduces lectin binding to undetectable levels and lowers its isoelectric point (pI = 6.3), effectively eliminating the main cause of nonspecific binding to avidin. Because the lysine residues remain available, neutravidin can be easily derivatized or conjugated, like streptavidin. Furthermore, its high biotin-binding ability and low nonspecific binding make it an ideal biotin-binding protein with a wide range of applications.

[0376] As used herein, the term "detecting a marker" refers to detecting a marker bound to a probe through a biotin-avidin reaction. The detection of the marker can be measured by a color change, a change in UV absorbance, the presence or absence of bioluminescence, a change in a fluorescent reaction, or an electrochemical change. Specifically, the method for detecting the marker varies depending on the marker used. For example, when HRP is used as a marker, the marker can be detected by observing the color reaction resulting from the reaction of hydrogen peroxide with a substrate. Furthermore, when the marker is a fluorescent protein such as GFP, the presence or absence of the marker can be detected by observing the light emitted after irradiation with light of a specific wavelength. Furthermore, when the marker is luciferase, the presence of the marker can be detected by adding a substrate such as luciferin and measuring the bioluminescence generated using a bioluminometer.

[0377] The diagnostic method of the present invention may further include a step of denaturing cfDNA. In this case, the denaturation step can be performed so as to selectively denature only cancer-derived cfDNA while leaving normal double-stranded cfDNA undenatured. For this purpose, the denaturation step can be performed at about 50°C to about 100°C for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is typically about 0.1 seconds to about 8 minutes. Alternatively, denaturation can be performed for about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds, or about 90 seconds. In one example, the denaturation step of cfDNA can be performed before step c).

[0378] Specifically, prior to step c), the method may further include denaturing the sample or the cfDNA bound to the positively charged substance under one of the following conditions: (i) leaving the sample at room temperature for about 1 to 10 minutes; (ii) heating at about 90°C to about 95°C for 1 second to 1 minute; (iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; (iv) heating at about 60°C to about 75°C for about 1 to 30 minutes; (v) heating at about 25°C to about 40°C for about 5 to 60 minutes; (vi) treating with protease for about 1 to 10 minutes; and (vii) treating with DNase I for about 1 to 10 minutes. This denaturation step selectively denatures only the cfDNA derived from cancer cells, while not denaturing double-stranded cfDNA derived from normal cells, thereby further facilitating binding to the probe. The denaturation conditions i) to vii) can be performed after obtaining the sample. The denaturation conditions i) to vii) can be performed after obtaining the cfDNA bound to the positively charged substance. The temperature, protease, and DNase treatment times for the denaturation conditions i) to vii) can be adjusted as appropriate, as long as they do not denature stable cfDNA.

[0379] Cancer diagnostic kits Another aspect of the present invention is to provide a cancer diagnostic kit comprising: a biotin-conjugated probe that binds complementarily to a gene that is specifically expressed in cancer; a positively charged substance; a marker bound to an avidin-based protein; and instructions.

[0380] In this case, the gene specifically expressed in cancer may be any one or more selected from the group consisting of CPT1A, IFNG, IFNGR1, CD279, CD274G, and combinations thereof.

[0381] The instructions may also describe that the kit configuration can diagnose cancer by the following protocol: a) isolating cfDNA from a biological sample isolated from an individual using a positively charged substance included in the kit; b) sequentially or simultaneously mixing the isolated cfDNA with a biotin-conjugated probe included in the kit and a marker included in the kit; c) removing probes and markers that do not bind to cfDNA; and d) detecting signals from the markers.

[0382] The kit may also contain a biotin-conjugated probe that complementarily binds to at least one gene selected from the group consisting of NSE, SCC, CEA, cyfra21-1, TPA, NMP22, OGT, Thyroglobulin (TG), Calcitonin (CALCA), BRAF V600E, TERT C228T / C250T, AFP, β-HCG (CGB), CA19-9, PSA, PSMA, PAP, PCA3, TMPRSS2-ERG, CA125, HIF-1a, VEGF, CA15-3, HER2, SCC (SART3), TOP2A, MCM2, p16INK4a (CDKN2A), Ki-67 (MKI167), HE4 (WEDC2), and combinations thereof.

[0383] The probe, positively charged substance, and marker are as described above.

[0384] Cancer diagnostic equipment Another aspect of the present invention is to provide an apparatus for diagnosing cancer by detecting genes derived from cancer cells from a sample without amplification, the apparatus comprising: a) a mixing unit that mixes a biological sample isolated from an individual containing cfDNA with a positively charged substance; b) an acquisition unit that removes the sample from which the positively charged substance to which cfDNA is bound has been removed; c) a reaction unit that sequentially or simultaneously adds a biotin-binding probe capable of complementarily binding to a gene specifically expressed in the cancer, and nanoparticles containing streptavidin and a marker, to the positively charged substance to which cfDNA is bound; d) a detection unit that detects the marker; and e) an information processing unit that determines, based on the presence or absence of detection of the marker, that cfDNA derived from the cancer is present in the sample, the sample having a sequence complementary to the probe.

[0385] In this case, the gene specifically expressed in cancer may be any one or more selected from the group consisting of CPT1A, IFNG, IFNGR1, CD279, CD274, and combinations thereof.

[0386] The kit may also contain a biotin-conjugated probe that complementarily binds to at least one gene selected from the group consisting of NSE, SCC, CEA, cyfra21-1, TPA, NMP22, OGT, Thyroglobulin (TG), Calcitonin (CALCA), BRAF V600E, TERT C228T / C250T, AFP, β-HCG (CGB), CA19-9, PSA, PSMA, PAP, PCA3, TMPRSS2-ERG, CA125, HIF-1a, VEGF, CA15-3, HER2, SCC (SART3), TOP2A, MCM2, p16INK4a (CDKN2A), Ki-67 (MKI167), HE4 (WEDC2), and combinations thereof. [Example]

[0387] The present invention will be described in more detail with reference to the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0388] Experimental Method 1: cfDNA Detection Method Derived from Tumor-Specific Genes Step 1: Sample preparation and nanowire addition Immediately after receiving the patient's plasma, urine, saliva, or sputum, the sample was centrifuged at 3,000 x g for 10 minutes at 4°C. The patient's plasma, urine, saliva, or sputum was diluted with DPBS at a certain ratio. For plasma, 1 μl to 30 μl of plasma was mixed with 150 μl of DW and placed in a spin column (Type G or Type Q). PEI / Ppy nanowires (150 μl) were added and mixed in a thermomixer at 1,200 rpm at room temperature for 20 minutes.

[0389] Stage 2: Vacuum / Washing / Temperature Denaturation The spin column was attached to a vacuum suction device and suctioned at 550 mBar. 400 μl of 1x DPBS was added and suction was applied again. The same process was repeated once more. Only the nanowire-DNA complex obtained through step 2 was filtered into the spin column. If temperature denaturation was required, the suctioned spin column was placed in a heating block preheated to 95°C, incubated at 95°C for 1 minute, and then immediately removed. Samples for which the temperature denaturation step was not required were not subjected to this process.

[0390] We demonstrated that cfDNA was separated using a spin column using nanowires without magnetic nanoparticles (Figure 85). The top photo is an SEM image of the spin column before centrifugation, and the bottom photo is an SEM image of the spin column with separated cfDNA after centrifugation.

[0391] Step 3: Adding probes and HRP / STR NPs The appropriate probe (200 μl) and HRP / STR NPs solution (200 μl) were added to the spin column. The mixture was mixed at room temperature for 30 minutes at 850-1,000 rpm using a thermomixer. The spin column was then attached to a vacuum device and suctioned. 400 μl of 1x DPBS was added, and suction was applied again. The same process was repeated once more.

[0392] Step 4: TMB reaction for gene mutation detection After replacing the collection tube with a new one, 200 μl of sodium acetate buffer (0.2 M, pH 7.0) and 50 μl of H2O2 (0.1 M) were added to the spin column using a syringe pump, followed by incubation for 3 minutes. After incubation, the spin column was centrifuged at 3,500 rpm to 5,000 rpm for 30 seconds. 200 μl of the solution collected in the collection tube was transferred to a 96-well plate, and the absorbance was measured in the wavelength range of 500 nm to 850 nm using a UV / VIS spectrophotometer.

[0393] Overview of detection methods The detection steps of the present invention are diagrammed in Figures 84a to 84g. Figure 84a is a schematic diagram of a method for collecting cfDNA from a patient's body fluid using polypyrrole nanowires (PEI / Ppy NWs) with polyethyleneimine (PEI) attached to their surface, followed by analysis of cancer cell-derived genes within 60 minutes through a reaction with probes that complementarily bind to the target cfDNA and HRP / streptavidin nanoparticles (HRP / st-tagged NPs). Figure 84b is a diagram illustrating a method for detecting cancer cell-derived cfDNA using nanowires, probes, and HRP / streptavidin nanoparticles. Figure 84c illustrates a process for detecting cancer cell-derived genes using a spin column with nanowires. In one embodiment of the present invention, a lysis buffer treatment step may be additionally included. Figure 84d is a timeline diagram illustrating a method for detecting cancer cell-derived cfDNA from samples such as blood, cerebrospinal fluid, or pleural effusion. Figure 84e shows a timeline of a method for detecting cancer cell-derived cfDNA from samples such as urine. Figure 84f shows a diagram illustrating the differences in denaturation conditions depending on the state of cfDNA obtained from blood. Figure 84g shows a diagram illustrating the differences in denaturation conditions depending on the state of cfDNA obtained from urine, saliva, and sputum.

[0394] Production Example 1. Production of nanowires surface-treated with cationic polymers As shown in Figure 1a, nanowires with a cationic polymer, polyethyleneimine (PEI), attached to the surface were fabricated. One side of an anodic aluminum oxide (AAO) was coated with 5 × 10 -3The AAO molds were coated with a gold (Au) layer (approximately 150 nm thick) at 50 mA and 100 mbar for 600 seconds. All electrochemical experiments were performed using a potentiostat / galvanostat (BioLogic SP-150) equipped with a platinum wire counter electrode and a Ag / AgCl (3.0 M NaCl type) reference electrode.

[0395] To fabricate nanowires surface-treated with a cationic polymer (PEI / Ppy NWs), electrochemical deposition was performed on the pores of the AAO template with 0.01 M poly(4-styrene sulfonic acid) and 0.01 M pyrrole solution containing 1 mg / ml biotin by applying chronoamperometry at 1.0 V (vs. Ag / AgCl) for 7 min.

[0396] The resulting AAO template was washed multiple times with distilled water, immersed in 2 M sodium hydroxide (NaOH) solution for 3 hours, and then sonicated (Bioruptor UCD-200, Diagenode) to obtain free-standing polypyrrole nanowires (PPy NWs) doped with biotin molecules. The nanowires were then treated with 30 mM N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and 6 mM N-hydroxysuccinimide (NHS) to activate the carboxylic acid (-COOH) groups. PEI solution was then added and reacted at room temperature for 1 hour. The resulting nanostructures (PEI / PPy NWs) with polyethyleneimine attached to the surface were obtained by rinsing with water. The resulting nanostructures (PEI / PPy NWs) were dispersed in deionized water and stored at room temperature until use.

[0397] By this fabrication method, after the AAO template was selectively dissolved, each polypyrrole (Ppy) nanowire was released from the AAO template, and cationic branched polyethyleneimine (PEI, 25 kDa) was additionally conjugated to the nanowire through biotin-streptavidin interaction.

[0398] Production Example 2: Production of nanowires surface-treated with cationic polymers By substantially the same method as in Preparation Example 1, a nanostructure (PL / Ppy NW) in which polylysine was bonded to the surface instead of polyethyleneimine was obtained.

[0399] Preparation Example 3. Preparation of polypyrrole nanoparticles labeled with HRP and streptavidin To prepare HRP and streptavidin-conjugated nanoparticles, 0.5 g of polyvinylpyrrolidone (PVP) was added to 12.5 ml of triple-distilled water and stirred for 30 minutes. 65 μl of pyrrole was then added and stirred for an additional 10 minutes. 0.5 ml of a 0.75 g / ml FeCl3 solution was then added and allowed to react for 3 hours. 20 ml of a hyaluronic acid aqueous solution (400 mg / 20 ml) was then added and stirred for 3 hours to prepare polypyrrole-hyaluronic acid nanoparticles (Ppy-HA-NPs).

[0400] The NPs were dialyzed against triple-distilled water for two days using a 50,000 MWCO membrane. Large particle aggregates were removed by centrifugation at 1,200 rpm for 3 minutes and then freeze-dried. 200 μg of the Ppy-HA-NPs prepared above was placed in 1 ml of triple-distilled water, and a 100 mM EDC / 50 mM NHS solution was added and reacted for 45 minutes to activate the carboxyl groups of the hyaluronic acid. The NPs were washed twice, with centrifugation at 15,000 rpm for 10 minutes and the supernatant removed. 1 mg of HRP and 1 mg of streptavidin were added to the Ppy-HA-NPs and mixed at 4°C. The NPs were then centrifuged at 15,000 rpm for 10 minutes, with the supernatant removed, and then stored in triple-distilled water. The morphology of the HRP and streptavidin-conjugated nanoparticles (HRP / st-tagged NPs) was observed using a scanning electron microscope (Figure 1b).

[0401] Example 4: Probe fabrication Probes were prepared to detect cfDNA with unstable double helix structures. The probes were prepared differently depending on the type of cancer cfDNA to be detected. Biotin was conjugated to the probes. Specific nucleic acid sequences of the probes are listed in Tables 1, 5, 9, and 11-28, depending on the type of cancer to be diagnosed.

[0402] I. Confirmation of accuracy of gene detection derived from cancer cell lines Example 1. Detecting expressed genes in cancer cell lines Example 1.1. Confirmation of PD-L1 detection accuracy The accuracy of PD-L1 detection was confirmed using known PD-L1-positive cancer cell lines, MDA-MB-231, HCC827, H1975, PC9, and H460, as well as known PD-L1-negative cancer cell lines, A549, MDA-MB-468, HeLa, and MCF7, obtained from the ATCC and the Korea Cell Line Bank. PD-L1-positive and PD-L1-negative cancer cell lines were classified based on the PD-L1 mRNA levels of each cancer cell line provided by the Cancer Cell Line Encyclopedia (CCLE).

[0403] Specifically, genomic DNA was extracted from each PD-L1-positive and PD-L1-negative cancer cell line and then sonicated to prepare fDNA (fragmented DNA). 50 ng / μl of fDNA was added to PBS, and the nanowires prepared in Preparation Example 1 were added and reacted for 20 minutes for isolation. After denaturation at 95°C for 1 minute, a biotinylated PD-L1 probe was added and reacted for an additional 20 minutes. The probes used are listed in Table 1 below.

[0404] [Table 1]

[0405] Colorimetric detection was confirmed by adding 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2 to sodium acetate buffer. DNA-based PD-L1 expression (ΔOD) was analyzed for each cancer cell line. Two replicate experiments showed clear PD-L1 DNA expression (ΔOD; cutoff OD > 0.012) in the fDNA-only samples of MDA-MB-231, HCC827, H1975, PC9, and H460 cancer cell lines. On the other hand, two replicate experiments showed no PD-L1 DNA expression in the A549, MDA-MB-461, HeLa, and MCF7 cancer cell lines (Figures 4 and 5). The PD-L1 DNA expression results for each cancer cell line were also compared with the PD-L1 (CD274) mRNA expression results for each cancer cell line obtained from the Cancer Cell Line Encyclopedia (CCLE). The PD-L1 (CD274) mRNA levels for each cancer cell line are shown in Table 2 below.

[0406] [Table 2]

[0407] The results showed high mRNA expression in MDA-MB-231, HCC827, H1975, PC9, and H460 cancer cell lines, whereas A549, MDA-MB-461, HeLa, and MCF7 cancer cell lines showed almost no mRNA expression.

[0408] Example 1.2. Confirmation of EpCAM detection accuracy The accuracy of EpCAM detection was confirmed using EpCAM-positive cancer cell lines MDA-MB468, HCC827, MCF7, H1975, and MDA-MB-231 obtained from the ATCC and the Korean Cell Line Bank, and EpCAM-negative cancer cell lines A549, H460, and Hela. EpCAM-positive and EpCAM-negative cancer cell lines were classified based on the EpCAM mRNA levels of each cancer cell line provided by the Cancer Cell Line Encyclopedia (CCLE).

[0409] Specifically, genomic DNA was extracted from EpCAM-positive and EpCAM-negative cancer cell lines and sonicated to prepare fDNA. 50 ng / μL of fDNA was added to PBS, and the nanowires prepared in Preparation Example 1 were added and reacted for 20 minutes before separation. After denaturing at 95°C for 1 minute, a biotinylated EpCAM probe was added and reacted for an additional 20 minutes. The probes used are listed in Table 3 below.

[0410] [Table 3]

[0411] For colorimetric detection, a solution containing TMB and H2O2 in sodium acetate buffer was prepared to confirm the detection results. Analysis of DNA-based EpCAM expression (ΔOD) for each cancer cell line revealed clear EpCAM DNA expression (ΔOD; cutoff OD > 0.012) in fDNA alone for the PC9, MDA-MB468, HCC827, MCF7, H1975, and MDA-MB-231 cancer cell lines in two replicate experiments. On the other hand, no EpCAM DNA expression was observed in the H460 and HeLa cancer cell lines in two replicate experiments (Figures 6 and 7). The EpCAM DNA expression results for each cancer cell line were also compared with the EpCAM mRNA expression results for each cancer cell line obtained from the CCLE (cancer cell line encyclopedia). The EpCAM mRNA values ​​for each cancer cell line are shown in Table 4 below.

[0412] [Table 4]

[0413] The results showed high mRNA expression in PC9, MDA-MB468, HCC827, MCF7, H1975, and MDA-MB-231 cancer cell lines, whereas H460 and Hela cancer cell lines showed almost no mRNA expression.

[0414] Example 1.3. Confirmation of FOLR1 detection accuracy The accuracy of FOLR1 detection was confirmed using known FOLR1-positive cancer cell lines, HeLa, MDA-MB-468, HCC827, MCF7, and MDA-MB-231, and known FOLR1-negative cancer cell lines, H460, PC9, H1975, and A549, obtained from ATCC and the Korea Cell Line Bank. FOLR1-positive and FOLR1-negative cancer cell lines were classified based on the FOLR1 mRNA levels of each cancer cell line provided by the Cancer Cell Line Encyclopedia (CCLE).

[0415] Specifically, genomic DNA was extracted from FOLR1-positive and FOLR1-negative cancer cell lines and sonicated to prepare fDNA. 50 ng / μL of fDNA was added to PBS, and the nanowires prepared in Preparation Example 1 were added and reacted for 20 minutes before separation. After denaturing at 95°C for 1 minute, a biotinylated FOLR1 probe was added and reacted for an additional 20 minutes. The probes used are listed in Table 5 below.

[0416] [Table 5]

[0417] For colorimetric detection, a solution containing TMB and H2O2 in sodium acetate buffer was prepared to confirm the detection results. Analysis of DNA-based FOLR1 expression (ΔOD) for each cancer cell line revealed clear FOLR1 DNA expression (ΔOD; cutoff OD > 0.012) in fDNA alone for HeLa, MDA-MB468, HCC827, MCF7, and MDA-MB-231 cancer cell lines in two replicate experiments. On the other hand, no FOLR1 DNA expression was observed in H460, PC9, H1975, and A549 cancer cells in two replicate experiments (Figures 8 and 9). The FOLR1 DNA expression results for each cancer cell line were also compared with the FOLR1 mRNA results for each cancer cell line obtained from the CCLE (cancer cell line encyclopedia). The FOLR1 mRNA values ​​for each cancer cell line are shown in Table 6 below.

[0418] [Table 6]

[0419] The results showed high mRNA expression in HeLa, MDA-MB-468, HCC827, MCF7, and MDA-MB-231 cancer cell lines, whereas H460, PC9, H1975, and A549 cancer cell lines showed almost no mRNA expression.

[0420] Example 1.4. Confirmation of EGFR detection accuracy The accuracy of EGFR detection was confirmed using known EGFR-positive cancer cell lines, HeLa, PC9, A549, H1975, H460, MDA-MB-468, MCF7, and MDA-MB-231, as well as known EGFR-negative cancer cell line MCF7, obtained from ATCC and the Korea Cell Line Bank. EGFR-positive and EGFR-negative cancer cell lines were classified based on the EGFR mRNA levels of each cancer cell line provided by the Cancer Cell Line Encyclopedia (CCLE).

[0421] Specifically, genomic DNA was extracted from EGFR-positive and EGFR-negative cancer cell lines and sonicated to prepare fDNA. 50 ng / μL of fDNA was added to PBS, and the nanowires prepared in Preparation Example 1 were added and reacted for 20 minutes before separation. After denaturation at 95°C for 1 minute, a biotinylated EGFR probe was added and reacted for an additional 20 minutes. The probes used are listed in Table 7 below.

[0422] [Table 7]

[0423] The detection results were confirmed by adding TMB and H2O2 to sodium acetate buffer for colorimetric detection. Analysis of DNA-based EGFR expression (ΔOD) for each cancer cell line revealed clear EGFR DNA expression (ΔOD; cutoff OD > 0.012) in fDNA alone for HCC827, PC9, MDA-MB468, MCF7, and MDA-MB-231 cancer cell lines in two replicate experiments. On the other hand, no EGFR DNA expression was observed in the MCF7 cancer cell line in two replicate experiments (Figures 10 and 11). The EGFR DNA expression results for each cancer cell line were also compared with the EGFR mRNA expression results from CCLE. The EGFR mRNA values ​​for each cancer cell line are shown in Table 8 below.

[0424] [Table 8]

[0425] The results showed high mRNA expression in HeLa, H460, PC9, H1975, MDA-MB-468, MCF7, and MDA-MB-231 cancer cell lines, whereas the MCF7 cancer cell line showed almost no mRNA expression.

[0426] Example 1.5. Confirmation of ERBB2 (HER2) detection accuracy The accuracy of ERBB2 detection was confirmed using ERBB2-positive cancer cell lines MCF7, PC9, A549, H1975, H460, MDA-MB468, MCF7, and MDA-MB-231 obtained from the ATCC and the Korea Cell Line Bank, and the ERBB2-negative cancer cell line H460. ERBB2-positive and ERBB2-negative cancer cell lines were classified based on the ERBB2 mRNA levels of each cancer cell line provided by the Cancer Cell Line Encyclopedia (CCLE).

[0427] Specifically, genomic DNA was extracted from ERBB2-positive and ERBB2-negative cancer cell lines and sonicated to prepare fDNA. 50 ng / μL of fDNA was added to PBS, and the nanowires prepared in Preparation Example 1 were added and reacted for 20 minutes before separation. After denaturation at 95°C for 1 minute, a biotinylated ERBB2 probe was added and reacted for an additional 20 minutes. The probes used are listed in Table 9 below.

[0428] [Table 9]

[0429] For colorimetric detection, a solution containing TMB and H2O2 in sodium acetate buffer was prepared to confirm the detection results. Analysis of DNA-based ERBB2 expression (ΔOD) for each cancer cell line revealed clear ERBB2 DNA expression (ΔOD; cutoff OD > 0.010) in fDNA alone for the MCF7, PC9, A549, H1975, MDA-MB468, MCF7, and MDA-MB-231 cancer cell lines in two replicate experiments. On the other hand, no ERBB2 DNA expression was observed in the H460 cancer cell line in two replicate experiments (Figures 12 and 13). Furthermore, the ERBB2 DNA expression results for each cancer cell line were compared with the ERBB2 mRNA expression results obtained from CCLE. The EGFR mRNA levels for each cancer cell line are shown in Table 10.

[0430] [Table 10]

[0431] As a result, the experiment showed high mRNA expression in MCF7, PC9, A549, H1975, MDA-MB468, MCF7, H460 and MDA-MB-231 cancer cell lines.

[0432] Example 1.6. Confirmation of OGT (O-linked β-N-acetylglucosamine transferase) detection accuracy The accuracy of OGT detection was confirmed using OGT-positive cancer cell lines (UMUC3, KU19-19, 253J, J82, T24, MBT2) and OGT-negative cancer cell lines (RT4, MDCK, HBL EpC, and Jurkat) obtained from ATCC and the Korea Cell Line Bank. OGT-positive and OGT-negative cancer cell lines were classified based on the OGT mRNA levels of each cancer cell line provided by the Cancer Cell Line Encyclopedia (CCLE).

[0433] Specifically, genomic DNA was extracted from OGT-positive and OGT-negative cancer cell lines and sonicated to prepare fDNA. 50 ng / μL of fDNA was added to PBS, and the nanowires prepared in Preparation Example 1 were added and reacted for 20 minutes before separation. After denaturation at 95°C for 1 minute, a biotinylated OGT probe was added and reacted for an additional 20 minutes. The probes used are listed in Table 11 below.

[0434] [Table 11]

[0435] For colorimetric detection, a solution containing TMB and H2O2 in sodium acetate buffer was prepared and processed to confirm the detection results. After detecting fDNA extracted from OGT-positive and OGT-negative cancer cell lines using the nanowires prepared in Example 1, DNA-based OGT expression (ΔOD) was analyzed. In two replicate experiments, clear OGT DNA expression (ΔOD; cutoff OD > 0.010) was observed only in the fDNA of UMUC3, KU19-19, 253J, J82, T24, and MBT2 cancer cell lines. On the other hand, no OGT DNA expression was observed in either the RT4 positive bladder cancer cell line, the MDCK and HBL_EpC normal bladder cancer cell lines, or Jurkat T-lymphocyte cells (Figures 14 and 15).

[0436] II. Detection of tumor markers derived from cancer cell lines Example 2. Detection of cancer cell line-derived biomarkers Example 2.1. CEA detection CEA was detected using known CEA-positive cancer cell lines LoVo, MKN45, and SW1116, and known CEA-negative cancer cell lines HCT8, HCT15, HeLa, and MDA-MB-231 obtained from the ATCC and the Korea Cell Line Bank. CEA-positive and CEA-negative cancer cell lines were classified based on the CEA mRNA levels of each cancer cell line provided by the Cancer Cell Line Encyclopedia (CCLE).

[0437] Specifically, genomic DNA was extracted from CEA-positive and CEA-negative cancer cell lines and sonicated to prepare fDNA. 50 ng / μL of fDNA was added to PBS, and the nanowires were added and reacted for 20 minutes to separate the DNA. After denaturing at 95°C for 1 minute, a biotinylated CEA probe was added and reacted for another 20 minutes. The probes used are listed in Table 12 below.

[0438] [Table 12]

[0439] For colorimetric detection, a solution containing TMB and H2O2 in sodium acetate buffer was prepared to confirm the detection results. Analysis of DNA-based CEA expression (ΔOD) for each cancer cell line revealed clear CEA DNA expression (ΔOD; cutoff OD > 0.010) in the fDNA of the LoVo, MKN45, and SW1116 cancer cell lines in three replicate experiments. On the other hand, the HCT8, HCT15, HeLa, and MDA-MB-231 cancer cell lines showed no CEA DNA expression in all three replicate experiments (Figures 16-18).

[0440] Example 2.2. PSA detection PSA was detected using the LNE and LNCaP cancer cell lines, known as PSA-positive cancer cell lines, and the PC3, DU145, and MCF7 cancer cell lines, known as PSA-negative cancer cell lines, obtained from the ATCC and the Korea Cell Line Bank. PSA-positive and PSA-negative cancer cell lines were classified based on the PSA mRNA levels of each cancer cell line provided by the CCLE (Cancer Cell Line Encyclopedia).

[0441] Specifically, genomic DNA was extracted from PSA-positive and PSA-negative cancer cell lines and sonicated to prepare fDNA. 50 ng / μL of fDNA was added to PBS, and the nanowires prepared in Preparation Example 1 were added and reacted for 20 minutes before separation. After denaturation at 95°C for 1 minute, a biotinylated PSA probe was added and reacted for an additional 20 minutes. The probes used are listed in Table 13 below.

[0442] [Table 13]

[0443] For colorimetric detection, a solution containing TMB and H2O2 in sodium acetate buffer was prepared to confirm the detection results. Analysis of DNA-based PSA expression (ΔOD) for each cancer cell line revealed clear PSA DNA expression (ΔOD; cutoff OD > 0.010) in the fDNA of LNE and LNCaP cancer cell lines in two replicate experiments. On the other hand, no PSA DNA expression was observed in the PC3, DU145, and MCF7 cancer cell lines in both replicate experiments (Figures 19 and 20).

[0444] Example 2.3. CA19-9 Detection CA19-9 was detected using the CA19-9-positive cancer cell lines Capan1, Capn2, and AsPC1, as well as the CA19-9-negative cancer cell lines MIA-PaCa2 and Panc1, obtained from the ATCC and the Korea Cell Line Bank. CA19-9-positive and CA19-9-negative cancer cell lines were classified based on the CA19-9 mRNA levels of each cancer cell line provided by the CCLE (Cancer Cell Line Encyclopedia).

[0445] Specifically, genomic DNA was extracted from CA19-9-positive and CA19-9-negative cancer cell lines and sonicated to prepare fDNA. 50 ng / μL of fDNA was added to PBS, and the nanowires were added and reacted for 20 minutes to separate the DNA. After denaturing at 95°C for 1 minute, a biotinylated CA19-9 probe was added and reacted for another 20 minutes. The probes used are listed in Table 14 below.

[0446] [Table 14]

[0447] For colorimetric detection, a solution containing TMB and H2O2 in sodium acetate buffer was added to confirm the detection results. Analysis of DNA-based CA19-9 expression (ΔOD) for each cancer cell line revealed clear CA19-9 DNA expression (ΔOD; cutoff OD > 0.010) in the fDNA of Capan1, Capn2, and AsPC1 cancer cell lines in two replicate experiments. On the other hand, the MIA-PaCa2 and Panc1 cancer cell lines showed no CA19-9 DNA expression in both replicate experiments (Figures 21 and 22).

[0448] Example 2.4. CA125 detection CA125 was detected using the A549 cancer cell line, known to be a CA125-positive cancer cell line, and the A431 cancer cell line, known to be a CA125-negative cancer cell line, obtained from the ATCC and the Korea Cell Line Bank. CA125-positive and CA125-negative cancer cell lines were classified based on the CA125 mRNA levels of each cancer cell line provided by the CCLE (Cancer Cell Line Encyclopedia).

[0449] Specifically, genomic DNA was extracted from CA125-positive and CA125-negative cancer cell lines and sonicated to prepare fDNA. 50 ng / μL of fDNA was added to PBS, and the nanowires were added and reacted for 20 minutes to separate the DNA. After denaturing at 95°C for 1 minute, a biotinylated CA125 probe was added and reacted for another 20 minutes. The probes used are listed in Table 15 below.

[0450] [Table 15]

[0451] For colorimetric detection, a solution containing TMB and H2O2 in sodium acetate buffer was prepared to confirm the detection results. Analysis of DNA-based CA125 expression (ΔOD) for each cancer cell line revealed clear CA125 DNA expression (ΔOD; cutoff OD > 0.010) in the fDNA of the A549 cancer cell line in two replicate experiments. On the other hand, the A431 cancer cell line showed no CA125 DNA expression in either of the two replicate experiments (Figures 23 and 24).

[0452] Example 2.5. AFP detection AFP was detected using known AFP-positive cancer cell lines, Huh7, HepG2, Hep3B, and PLC, and known AFP-negative cancer cell lines, SNU475, SNU387, SNU423, SNU449, SK Hep1, and HeLa, obtained from the ATCC and the Korea Cell Line Bank. AFP-positive and AFP-negative cancer cell lines were classified based on the AFP mRNA levels of each cancer cell line provided by the CCLE (Cancer Cell Line Encyclopedia).

[0453] Specifically, genomic DNA was extracted from AFP-positive and AFP-negative cancer cell lines and sonicated to prepare fDNA. 50 ng / μL of fDNA was added to PBS, and the nanowires were added and reacted for 20 minutes to separate the DNA. After denaturing at 95°C for 1 minute, a biotinylated AFP probe was added and reacted for another 20 minutes. The probes used are listed in Table 16 below.

[0454] [Table 16]

[0455] The detection results were confirmed by processing a solution containing TMB and H2O2 in sodium acetate buffer for colorimetric detection. Analysis of DNA-based AFP expression (ΔOD) for each cancer cell line revealed clear AFP DNA expression (ΔOD; cutoff OD > 0.010) in the fDNA of Huh7, HepG2, Hep3B, and PLC cancer cell lines in two replicate experiments. On the other hand, SNU475, SNU387, SNU423, SNU449, SK Hep1, and HeLa cancer cell lines showed no AFP DNA expression in both replicate experiments (Figures 25 and 26).

[0456] III. Confirmation of biomarker detection using plasma or urine of cancer patients Example 3. Detection of biomarkers derived from cancer patients Example 3.1. Confirmation of tumor marker detection using plasma from prostate cancer patients Prostate cancer tumor markers PSA, PSMA, PAP, and PCA3 were detected in plasma obtained from normal subjects or prostate cancer patients. The prostate cancer markers PSA, PSMA, PAP, and PAC3 are highly expressed in prostate cancer patients, and are currently used to differentiate prostate cancer by checking the levels of prostate cancer antigens (PSA, PSMA, PAP, and PAC3) through cancer tissue and blood tests.

[0457] Specifically, plasma was collected from normal subjects or prostate cancer patients, and nanowires were added and reacted for 20 minutes to isolate circulating tumor DNA (ctDNA). The ctDNA then attached to the nanowires to form complexes. Experiments were conducted on the ctDNA / nanowire complexes in two ways: i) at 27°C without temperature denaturation, and ii) after denaturation at 95°C for 1 minute. Biotinylated probes and HRP- and streptavidin-tagged polypyrrole nanoparticles (HRP / st-tagged NPs) were then added and reacted for an additional 20 minutes. The probes used are listed in Table 17 below.

[0458] [Table 17]

[0459] For colorimetric detection, a solution containing TMB and H2O2 in sodium acetate buffer was added to confirm the expression of PSA, PSMA, PAP, and PAC3. Analysis of PSA, PSMA, PAP, and PAC3 ctDNA expression (ΔOD) revealed clear PSA, PSMA, PAP, and PAC3 ctDNA expression (ΔOD; cutoff OD > 0.015) in prostate cancer patients without or with temperature denaturation. On the other hand, in normal humans, neither without nor with temperature denaturation demonstrated PSA, PSMA, PAP, nor PAC3 ctDNA expression (ΔOD; cutoff OD > 0.015) (Figures 27-32).

[0460] Example 3.2. Confirmation of tumor marker detection using plasma from lung cancer patients Lung cancer tumor markers NSE, SCC, CEA, Cyfra21-1, and TPA were detected in plasma obtained from normal subjects or lung cancer patients. The lung cancer markers NSE, SCC, CEA, Cyfra21-1, and TPA are highly expressed in lung cancer patients, and are currently used to differentiate lung cancer by checking the levels of lung cancer antigens (NSE, SCC, CEA, Cyfra21-1, TPA) through cancer tissue and blood tests.

[0461] Specifically, plasma was collected from normal subjects or lung cancer patients, and nanowires were added and reacted for 20 minutes to isolate circulating tumor DNA (ctDNA). The ctDNA then attached to the nanowires to form complexes. Experiments were conducted on the ctDNA and nanowire complexes in two ways: i) at 27°C without temperature denaturation, and ii) after denaturation at 95°C for 1 minute. Biotinylated probes and HRP- and streptavidin-tagged polypyrrole nanoparticles (HRP / st-tagged NPs) were then added and reacted for an additional 20 minutes. The probes used are listed in Table 18 below.

[0462] [Table 18]

[0463] For colorimetric detection, we added TMB and H2O2 to sodium acetate buffer to confirm the expression of NSE, SCC, CEA, Cyfra21-1, and TPA. Analysis of ctDNA-based NSE, SCC, CEA, Cyfra21-1, and TPA ctDNA expression (ΔOD) showed clear NSE, SCC, CEA, Cyfra21-1, and TPA ctDNA expression (ΔOD; cutoff OD > 0.010) in two lung cancer patients without or with temperature denaturation. On the other hand, normal human ctDNA expression (ΔOD; cutoff OD > 0.010) was not observed in either without or with temperature denaturation (Figures 33-35).

[0464] Example 3.3. Confirmation of tumor marker detection using plasma from thyroid cancer patients Thyroid cancer tumor markers CEA, NSE, TG, and CALCA were detected in plasma obtained from normal subjects or thyroid cancer patients. Thyroid cancer markers CEA, NSE, TG, and CALCA are highly expressed in thyroid cancer patients, and are currently used to differentiate thyroid cancer by checking the levels of thyroid cancer antigens (CEA, NSE, TG, CALCA) through cancer tissue and blood tests.

[0465] Specifically, plasma was collected from normal subjects or thyroid cancer patients, and nanowires were added and reacted for 20 minutes to isolate circulating tumor DNA (ctDNA). The ctDNA then attached to the nanowires to form complexes. Experiments were conducted on the ctDNA and nanowire complexes in two ways: (i) at 27°C without temperature denaturation, and (ii) after denaturation at 95°C for 1 minute. Biotinylated probes and HRP- and streptavidin-tagged polypyrrole nanoparticles (HRP / st-tagged NPs) were then added and reacted for an additional 20 minutes. The probes used are listed in Table 19 below.

[0466] [Table 19]

[0467] For colorimetric detection, we added TMB and H2O2 to sodium acetate buffer to confirm the expression of CEA, NSE, TG, and CALCA. Analysis of ctDNA-based CEA, NSE, TG, and CALCA ctDNA expression (ΔOD) showed clear CEA, NSE, TG, and CALCA ctDNA expression (ΔOD; cutoff OD > 0.010) in thyroid cancer patients without or after temperature denaturation. On the other hand, normal human ctDNA expression (ΔOD; cutoff OD > 0.010) was not observed, either without or after temperature denaturation (Figures 36-40).

[0468] Example 3.4. Confirmation of tumor marker detection using urine from bladder cancer patients Bladder cancer tumor markers OGT, FGFR3, TP53, NMP22, and Cyfra21-1 were detected in urine collected from normal subjects or bladder cancer patients. As bladder cancer markers, OGT, FGFR3, TP53, NMP22, and Cyfra21-1 show high levels in the urine of bladder cancer patients, and can be used to differentiate bladder cancer.

[0469] Specifically, urine was collected from normal subjects or bladder cancer patients, and nanowires were added and reacted for 20 minutes to isolate circulating tumor DNA (ctDNA). The ctDNA then attached to the nanowires to form complexes. Experiments were conducted on the ctDNA and nanowire complexes in two ways: i) at 27°C without temperature denaturation, and ii) after denaturation at 95°C for 1 minute. Biotinylated probes and HRP- and streptavidin-tagged polypyrrole nanoparticles (HRP / st-tagged NPs) were then added and reacted for an additional 20 minutes. The probes used are listed in Table 20 below.

[0470] [Table 20]

[0471] For colorimetric detection, we added TMB and H2O2 to sodium acetate buffer to confirm the expression of OGT, FGFR3, TP53, NMP22, and Cyfra21-1. Analysis of ctDNA-based OGT, FGFR3, TP53, NMP22, and Cyfra21-1 ctDNA expression (ΔOD) showed clear OGT, FGFR3, TP53, NMP22, and Cyfra21-1 ctDNA expression (ΔOD; cutoff OD > 0.010) in bladder cancer patients without or with temperature denaturation. On the other hand, normal subjects and cystitis patients showed no OGT, FGFR3, TP53, NMP22, or Cyfra21-1 ctDNA expression (ΔOD; cutoff OD > 0.010) even without or with temperature denaturation (Figures 41-46).

[0472] Example 3.5. Confirmation of tumor marker detection using plasma from breast cancer patients Breast cancer tumor markers CA27-29, CA15-3, and CEA were detected in plasma obtained from normal subjects or breast cancer patients. CA27-29, CA15-3, and CEA are highly expressed in breast cancer patients, and are currently used to differentiate breast cancer by checking the levels of breast cancer antigens (CA27-29, CA15-3, CEA) in cancer tissue and blood tests.

[0473] Specifically, plasma was collected from normal subjects or breast cancer patients, and nanowires were added and reacted for 20 minutes to isolate circulating tumor DNA (ctDNA). The ctDNA then attached to the nanowires to form complexes. Experiments were conducted on the ctDNA and nanowire complexes in two ways: i) at 27°C without temperature denaturation, and ii) after denaturation at 95°C for 1 minute. Biotinylated probes and HRP- and streptavidin-tagged polypyrrole nanoparticles (HRP / st-tagged NPs) were then added and reacted for an additional 20 minutes. The probes used are listed in Table 21 below.

[0474] [Table 21]

[0475] For colorimetric detection, we added TMB and H2O2 to sodium acetate buffer to confirm the expression of CA27-29, CA15-3, and CEA. Analysis of ctDNA-based CA27-29, CA15-3, and CEA ctDNA expression (ΔOD) showed clear CA27-29, CA15-3, and CEA ctDNA expression (ΔOD; cutoff OD > 0.010) in breast cancer patients without or with temperature denaturation. On the other hand, in normal humans, no expression of CA27-29, CA15-3, or CEA ctDNA (ΔOD; cutoff OD > 0.010) was observed, even without or with temperature denaturation (Figures 47-50).

[0476] Example 3.6. Confirmation of tumor marker detection using plasma from colorectal cancer patients Colon cancer tumor markers CEA and CA19-9 were detected in plasma obtained from normal individuals or colon cancer patients. CEA and CA19-9 are colon cancer markers that are highly expressed in colon cancer patients, and are currently used to differentiate colon cancer by checking the levels of colon cancer antigens (CEA, CA19-9) in cancer tissue and blood tests.

[0477] Specifically, plasma was collected from normal subjects or colon cancer patients, and nanowires were added and reacted for 20 minutes to isolate circulating tumor DNA (ctDNA). The ctDNA then attached to the nanowires to form complexes. Experiments were conducted on the ctDNA / nanowire complexes in two ways: i) at 27°C without temperature denaturation, and ii) after denaturation at 95°C for 1 minute. Biotinylated probes and HRP- and streptavidin-tagged polypyrrole nanoparticles (HRP / st-tagged NPs) were then added and reacted for an additional 20 minutes. The probes used are listed in Table 22 below.

[0478] [Table 22]

[0479] For colorimetric detection, we added TMB and H2O2 to sodium acetate buffer to confirm the expression of CEA and CA19-9. Analysis of ctDNA-based CEA and CA19-9 ctDNA expression (ΔOD) showed clear CEA and CA19-9 ctDNA expression (ΔOD; cutoff OD > 0.010) in colorectal cancer patients without or with temperature denaturation. On the other hand, normal human samples showed no CEA or CA19-9 ctDNA expression (ΔOD; cutoff OD > 0.010) in either without or with temperature denaturation (Figures 51-55).

[0480] Example 3.7. Confirmation of tumor marker detection using plasma from bile duct cancer patients CEA, CA19-9, and CA125, tumor markers for cholangiocarcinoma, were detected in plasma obtained from normal subjects or patients with cholangiocarcinoma. CA19-9, CA125, and CEA, which are cholangiocarcinoma markers, are highly expressed in patients with cholangiocarcinoma, and checking the levels of cholangiocarcinoma antigens (CA19-9, CA125, CEA) through blood tests can be used to differentiate cholangiocarcinoma.

[0481] Specifically, plasma was collected from normal subjects or bile duct cancer patients, and nanowires were added and reacted for 20 minutes to isolate circulating tumor DNA (ctDNA). The ctDNA then attached to the nanowires to form complexes. Experiments were conducted on the ctDNA / nanowire complexes in two ways: i) at 27°C without temperature denaturation, and ii) after denaturation at 95°C for 1 minute. Biotinylated probes and HRP- and streptavidin-tagged polypyrrole nanoparticles (HRP / st-tagged NPs) were then added and reacted for an additional 20 minutes. The probes used are listed in Table 23 below.

[0482] [Table 23]

[0483] For colorimetric detection, we added TMB and H2O2 to sodium acetate buffer to confirm the expression of CEA, CA19-9, and CA125. Analysis of ctDNA-based CEA, CA19-9, and CA125 ctDNA expression (ΔOD) showed clear CEA, CA19-9, and CA125 ctDNA expression (ΔOD; cutoff OD > 0.010) in cholangiocarcinoma patients without or with temperature denaturation. On the other hand, normal human samples showed no CEA, CA19-9, or CA125 ctDNA expression (ΔOD; cutoff OD > 0.010) even without or with temperature denaturation (Figures 56-58).

[0484] Example 3.8. Confirmation of tumor marker detection using plasma from gastric cancer patients Gastric cancer tumor markers CEA, CA19-9, CGB, and Cyfra21-1 were detected in plasma obtained from normal subjects or gastric cancer patients. The gastric cancer markers CEA, CA19-9, CGB, and Cyfra21-1 are highly expressed in gastric cancer patients, and checking the levels of gastric cancer antigens (CEA, CA19-9, CGB, Cyfra21-1) through blood tests can be used to differentiate gastric cancer.

[0485] Specifically, plasma was collected from normal subjects or gastric cancer patients, and nanowires were added and reacted for 20 minutes to isolate circulating tumor DNA (ctDNA). The ctDNA then attached to the nanowires to form complexes. Experiments were conducted on the ctDNA and nanowire complexes in two ways: (i) at 27°C without temperature denaturation, and (ii) after denaturation at 95°C for 1 minute. Biotinylated probes and HRP- and streptavidin-tagged polypyrrole nanoparticles (HRP / st-tagged NPs) were then added and reacted for an additional 20 minutes. The probes used are listed in Table 24 below.

[0486] [Table 24]

[0487] For colorimetric detection, a solution containing TMB and H2O2 in sodium acetate buffer was added to confirm the expression of CEA, CA19-9, CGB, and Cyfra21-1. Analysis of ctDNA-based CEA, CA19-9, CGB, and Cyfra21-1 ctDNA expression (ΔOD) showed clear CEA, CA19-9, CGB, and Cyfra21-1 ctDNA expression (ΔOD; cutoff OD > 0.010) in gastric cancer patients without or with temperature denaturation. On the other hand, normal human samples showed no CEA, CA19-9, CGB, or Cyfra21-1 ctDNA expression (ΔOD; cutoff OD > 0.010) in either without or with temperature denaturation (Figures 59-61).

[0488] Example 3.9. Confirmation of tumor marker detection using plasma from pancreatic cancer patients Pancreatic cancer tumor markers CA19-9, CA125, and CEA were detected in plasma obtained from normal subjects or pancreatic cancer patients.

[0489] Specifically, plasma was collected from normal subjects or pancreatic cancer patients, and nanowires were added and reacted for 20 minutes to isolate circulating tumor DNA (ctDNA). The ctDNA then attached to the nanowires to form complexes. Experiments were conducted on the ctDNA / nanowire complexes in two ways: i) at 27°C without temperature denaturation, and ii) after denaturation at 95°C for 1 minute. Biotinylated probes and HRP- and streptavidin-tagged polypyrrole nanoparticles (HRP / st-tagged NPs) were then added and reacted for an additional 20 minutes. The probes used are listed in Table 25 below.

[0490] [Table 25]

[0491] For colorimetric detection, a solution containing TMB and H2O2 in sodium acetate buffer was processed to confirm the expression of CEA, CA19-9, and CA125. Analysis of ctDNA-based CEA, CA19-9, and CA125 ctDNA expression (ΔOD) showed clear CEA, CA19-9, and CA125 ctDNA expression (ΔOD; cutoff OD > 0.010) in pancreatic cancer patients without temperature denaturation. On the other hand, in normal humans, no CEA, CA19-9, or CA125 ctDNA expression (ΔOD; cutoff OD > 0.010) was observed without temperature denaturation (Figures 62-69).

[0492] IV. Evaluation of tumor marker detection for early diagnosis and prognosis of cancer Example 4. Detection of biomarkers derived from cancer patients Example 4.1. Detection of CPT1A in plasma or urine of cancer patients CPT1A (Carnitine palmitoyltransferase 1A) was detected in plasma and urine obtained from normal subjects or lung cancer patients. CPTA1 was also detected in urine obtained from bladder cancer patients. It is known that CPT1A expression is significantly higher in cancer tissues than in normal tissues, making it a useful diagnostic target for cancer.

[0493] Specifically, after collecting the collected plasma or urine, nanowires were added and reacted for 20 minutes to isolate circulating tumor DNA (ctDNA). The ctDNA then attached to the nanowires to form a complex. Experiments were conducted on the ctDNA and nanowire complexes in two ways: i) at 27°C without temperature denaturation, and ii) after denaturation at 95°C for 1 minute. Then, biotinylated probes and HRP- and streptavidin-tagged polypyrrole nanoparticles (HRP / st-tagged NPs) were added and reacted for an additional 20 minutes. The probes used are listed in Table 26 below.

[0494] [Table 26]

[0495] CPT1A expression was confirmed by processing a solution containing TMB and H2O2 in sodium acetate buffer for colorimetric detection. Analysis of ctDNA-based CPT1A ctDNA expression (ΔOD) showed clear CPT1A ctDNA expression (ΔOD; cutoff OD > 0.010) in lung cancer or bladder cancer patients without or after temperature denaturation. On the other hand, normal human samples showed no CPT1A ctDNA expression (ΔOD; cutoff OD > 0.010) in either the absence or the presence of temperature denaturation (Figures 70-73).

[0496] Example 4.2. Confirmation of IFN-γ, IFN-γ receptor, and PD-L1 expression using cancer cell lines The accuracy of IFN-γ, IFN-γ receptor, and PD-L1 detection was evaluated using known PD-L1-positive cancer cell lines, MDA-MB-231, HCC827, H1975, PC9, and H460, and known PD-L1-negative cancer cell lines, A549, MDA-MB-461, HeLa, and MCF7, obtained from ATCC and the Korea Cell Line Bank. PD-L1-positive and PD-L1-negative cancer cell lines were classified based on the PD-L1 mRNA levels of each cancer cell line provided by CCLE (Cancer Cell Line Encyclopedia). 1 x 10 cells were plated in a 100 mm dish. 6 After culturing the same cells, one dish was supplemented with 10 nmol of IFN-γ, while the other dish was left untreated with IFN-γ. After culturing for one day, genomic DNA was extracted from the PD-L1-positive and PD-L1-negative cancer cell lines.

[0497] The fDNA was then sonicated to produce fDNA of less than 200 bp. 50 ng / μL of fDNA was added to PBS, and the nanowires were added and reacted for 20 minutes to separate the fDNA. After denaturing at 95°C for 1 minute, biotinylated probes and streptavidin-tagged polypyrrole nanoparticles (HRP / st-tagged NPs) were added and reacted for an additional 20 minutes. The probes used are listed in Table 27 below.

[0498] [Table 27]

[0499] For colorimetric detection, we added TMB and H2O2 to sodium acetate buffer to confirm the detection results for IFN-γ, IFN-γ receptor, and PD-L1. First, we analyzed DNA-based PD-L1 expression (ΔOD) in cancer cell lines without IFN-γ. In all three experiments, only the MDA-MB-231, HCC827, H1975, PC9, and H460 cancer cell lines showed clear PD-L1 DNA expression (cutoff: OD > 0.6) by fDNA analysis. On the other hand, the A549, MDA-MB-461, HeLa, and MCF7 cancer cell lines showed no PD-L1 DNA expression in all three experiments (Figures 74-76). However, when IFN-γ was added, analysis of DNA-based PD-L1 expression (ΔOD) in cancer cell lines showed clear fDNA-based PD-L1 DNA expression (cutoff: OD>0.6) in all three experiments, not only in PD-L1-positive cancer cell lines MDA-MB-231, HCC827, H1975, PC9, and H460, but also in PD-L1-negative cancer cell lines, namely A549, MDA-MB-461, HeLa, and MCF7 (Figure 77). Furthermore, IFN-γ and IFN-γ receptors were detected through fDNA analysis of PD-L1-positive cancer cell lines MDA-MB-231, HCC827, H1975, PC9, and H460, as well as PD-L1-negative cancer cell lines A549, MDA-MB-461, HeLa, and MCF7. Results showed that no IFN-γ was detected in any of the three experiments, regardless of whether IFN-γ was added or not. However, IFN-γ receptors were detected in all cell lines (Figures 78 and 79).

[0500] Furthermore, PD-L1, IFN-γ, and IFN-γ receptors were analyzed through fDNA analysis of PD-L1-positive cancer cell lines MDA-MB-231, HCC827, H1975, PC9, and H460, and PD-L1-negative cancer cell lines A549, MDA-MB-461, HeLa, and MCF7 in three replicate experiments with and without IFN-γ. Graphs show that when IFN-γ was added, high PD-L1 expression was observed in the PD-L1-negative cancer cell lines, whereas no IFN-γ was detected, regardless of whether IFN-γ was added or not, and IFN-γ receptors were detected in all cell lines (Figures 80 to 83).

[0501] Graphs analyzing DNA-based PD-L1 expression (ΔOD) of cancer cell lines before and after the addition of IFN-γ to PD-L1-positive and -negative cancer cell lines are shown (Figures 80 to 83).

[0502] V. Confirmation of the detectability of cancer-associated cfDNA by collection method Example 5. Confirmation of biomarker detection by sample collection method cfDNA was detected from lung cancer patients and normal subjects using the same method as described above. However, venous blood was collected using a hypodermic needle and capillary blood was collected using a lancet. The nanowires and probes used were those prepared in Preparation Examples 1 and 3. The DNA expression levels of cancer-related biomarkers, such as AKL Fusion and PIK3CA, were measured in blood collected from lung cancer patients using a hypodermic needle (Figure 86). The DNA expression levels of cancer-related biomarkers, such as AKL Fusion and PIK3CA, were also measured in blood collected from lung cancer patients using a lancet (Figure 87). The DNA expression levels of cancer-related biomarkers, such as AKL Fusion and PIK3CA, were also measured in blood collected from normal subjects using a hypodermic needle (Figure 88). The DNA expression levels of cancer-related biomarkers, such as AKL Fusion and PIK3CA, were also measured in blood collected from normal subjects using a lancet (Figure 89). These results confirmed that cancer-related biomarkers could be detected in both venous and capillary blood.

[0503] VI. Confirmation of detectability of lung cancer-related cfDNA Example 6. Confirmation of mutation biomarker detection in lung cancer cell lines The presence or absence of mutations (snp) occurring in cancer-specific genes can determine whether resistance to a particular treatment exists or will occur, thereby providing useful information for finding appropriate treatment methods.

[0504] To confirm whether mutations could be detected in lung cancer cell lines, we analyzed the EML4-ALK gene. First, we confirmed the expression level by RT-PCR. The expression level of EML4-ALK was confirmed by RT-PCR in cfDNA from EML4-ALK variant 3a / b positive cell lines (H2228) and EML4-ALK negative cell lines (A549, H1993, PC9, RT4) (Figure 90). Furthermore, the expression level of EML4-ALK was confirmed by Western blot in cfDNA from EML4-ALK variant 3a / b positive cell lines (H2228) and EML4-ALK negative cell lines (A549, H1993, PC9, RT4) (Figure 91). In addition, the expression level of EML4-ALK in cfDNA from EML4-ALK variant 3a / b positive cell (H2228) and EML4-ALK negative cell (A549, H1993, PC9, RT4) cancer cell lines was confirmed using RT-PCR and Western blot (Figure 92).

[0505] EML4-ALK was then detected using the fDNA-based method described herein. The detection method was the same as described above. The results confirmed that EML4-ALK could also be detected using the fDNA detection method. The DNA expression levels of EML4-ALK fusion var. 1 or EML4-ALK fusion var. 3 were measured in cfDNA from EML4-ALK variant 3a / b-positive cells (H2228) and EML4-ALK-negative cells (A549, H1993, PC9, RT4) cancer cell lines (Figure 93). The DNA expression levels of EML4-ALK fusion var. 1 or EML4-ALK fusion var. 3 were also measured in cfDNA from EML4-ALK variant 3a / b-positive cells (H2228) and EML4-ALK-negative cells (A549, H1993, PC9, RT4) cancer cell lines (Figure 94).

[0506] [Table 28]

[0507] Example 7. Validation of mutation biomarker detection in lung cancer patients: drug resistance Using the same method as described above, various mutations were detected in lung cancer patients. The probes used to detect the cfDNA of lung cancer patients were nucleic acid sequences complementary to the lung cancer biomarker genes described above. Patient information and analyzed genes are as shown in the figure.

[0508] Specifically, we measured the DNA expression levels of cancer-related biomarkers, such as EML4-ALK fusion var.3, KRAS, SYP, NCAM1, and NKX2-1, in blood collected from small cell lung cancer patients (Figure 95). As a result, EML4-ALK fusion was found in both cancer tissue and blood ctDNA, and ...

Claims

1. a) mixing a biological sample isolated from an individual containing cell-free DNA (hereinafter referred to as cfDNA) with a positively charged substance; b) mixing a probe and a marker into the mixture and capturing the cfDNA with a positively charged substance; and c) detecting a marker that binds to cfDNA; a method for providing information for diagnosing cancer by detecting genes overexpressed in cancer cells from the sample without amplification, Here, the positively charged substance is a nanowire having a positively charged surface, the probe has a sequence complementary to cfDNA and a material capable of binding to the marker; the marker further comprises a substance capable of binding to the probe; The probe binds to a gene known as a cancer biomarker by complementarity, The cfDNA is a cfDNA having a stable double helix structure, and The method provides information for cancer diagnosis, and does not involve PCR or nucleic acid amplification.

2. The method for providing information for cancer diagnosis according to claim 1 , wherein the cancer is prostate cancer, lung cancer, thyroid cancer, bladder cancer, breast cancer, colon cancer, biliary tract cancer, stomach cancer, or pancreatic cancer.

3. 2. The method for providing information for cancer diagnosis according to claim 1, wherein step (b) is a step of sequentially or simultaneously mixing a probe and a marker into the mixture.

4. The probe is At least one gene selected from the group consisting of KLK3, FOLH1, PCA3, PDE4D7, SFMBT2, EFEMP1, RETN, ACADL, AGR2, COL1A1, FAM13C, GPX8, GRHL2, HNF1A, HOXB13, KLK2, MYBPC1, NR0B1, PITX2, SFRP4, SLCO1B3, TMEFF2, TMPRSS2-ERG, ACPP, CPT1A, IFNG, CD274, FOLR1, EPCAM, OGT, and combinations thereof. at least one gene selected from the group consisting of SART3, PLAT, ALK, ROS1, PI3K, S100P, CDCA7, S100A2, ETV4, ENO2, ACPP, KRT19, EGFR, KRAS, RET, ERBB2, MMP11, TOP2A, UBE2C, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof; at least one gene selected from the group consisting of TG, CALCA, APOC1, HIG2, ENO2, ACPP, TYRO3, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof; at least one gene selected from the group consisting of OGT, FGFR3, TP53, NUMA1, COCH, CELSR3, HMOX1, KIF1A, MGC17624, MTAP, PFKFB4, S100A8, RSPH9, FOXM1, FANCB, FANCC, FANCD2, RUSC1-AS1CACNA1B, IMP-1, PDE3A, POU3F4, SOX3, DMC1, PLXDC2, ZNF312, SYCP2L, HOXA9, ISL1, ALDH1A3, KRT19, CCNB1, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof; at least one gene selected from the group consisting of MEST, NR1D1, BIRC5, RACGAP1, DHCR7, STC2, AZGP1, RBBP8, IL6ST, MGP, TRBC1, MMP11, COL10A1, C10orf64, COL11A1, POTEG, FSIP1, HER2, MUC1, ACPP, TYRO3, UBE2C, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof; NCKAP1, AUNIP, NOTUM, KRT5, TUBB, COL6A1, JUP, CDX2, MELTF, EFEMP2, DEFA5, CHEK1, MAD2L1, ENC1, CSE1L, RD51AP1, ERICH3, SLC7A11, KRT23 , Plau, CDCA1, KLK6, DPEP1, CDH3, Anln, CXCL1, CTHRC1, LCN2, HS6ST2, EGFL6, CXCL3, CA9, Prox1, SPL1, CST1, CXCL2, TSTA3, RRM2, MMP3, MMP7, MMP10, CXCL5, SERPINB5, TEAD4, BUB1, CDC2, CLDN2, HSPH1, LY6G6D, PRC1, PUS1, SQLE, TTK , ECT2, RNF183, FBXO39, TEX38, TTLL2, PRR7, CANP, KIAA0101, ACPP, FLU3, TYRO3, COTOR1, at least one gene selected from the group consisting of CK7, CK20, MUC2, SDC2, ASB9, CCNB1, MELK, CKS2, IFITM1, CEACAM6, ATAD2, TOP2A, CPT1A, DSCC1, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof; at least one gene selected from the group consisting of MUC16, ASH1L, DOCK7, ACPP, FLU3, CPT1A, DSCC1, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM, and a combination thereof; CGB, PARP1, FOXO3A, MED30, CCNE1, MYC, TFF1, FABP1, LAMP5, MATN3, CLIP4, NOX4, ADRA2C, CSK, FZD9, GALR1, GR M6, INSR, LPHN1, LYN, MRGPRX3, ADCY3, HDAC2, CFL1, NRP2, ANXA10, TFF2, CDCA5, NUSAP1, ACPP, FLU3, KRT19, ERB at least one gene selected from the group consisting of B2, EGFR, KRAS, DSCC1, CK20, MUC2, SDC2, COTL1, ATAD2, ASB9, MMP1, CEALAM6, DSCC1, CKS2, CST1, IFITM1, MELK, LGALS3BP, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof; or At least one gene selected from the group consisting of SMAD4, APC, GNAS, KRAS, MUC1, MSLN, CEACAM1, CEACAM5, MUC16, and combinations thereof The method for providing information for cancer diagnosis according to claim 1 , wherein the nucleic acid sequence complementarily binds to the following:

5. 2. The method for providing information for cancer diagnosis according to claim 1, wherein the sample is any one selected from the group consisting of urine, cerebrospinal fluid, plasma, blood, pleural effusion, ascites, saliva, sputum, and body fluids.

6. The method for providing information for cancer diagnosis according to claim 1, wherein the sequence of the probe complementary to the cfDNA is a 15-mer to 30-mer nucleotide sequence.

7. 2. The method for providing information for cancer diagnosis according to claim 1, wherein the marker is any one selected from the group consisting of quantum dots, horseradish peroxidase (HRP), fluorescent proteins, fluorophores, alkaline phosphatase, and luciferase.

8. 2. The method for providing information for cancer diagnosis according to claim 1, wherein the marker is a nanoparticle containing one or more selected from the group consisting of a conductive polymer, hyaluronic acid, avidin, or streptavidin, and one or more selected from the group consisting of horseradish peroxidase (HRP) or a fluorescent protein, and wherein the marker is detected in step c) by a change in color, ultraviolet absorbance, fluorescence, or electrochemical change.

9. a) mixing a biological sample isolated from an individual containing cell-free DNA (hereinafter referred to as cfDNA) with a positively charged substance; b) adding a probe and a marker to the mixture and capturing the cfDNA with a positively charged substance; and c) a method for providing information for early diagnosis of cancer or predicting the prognosis of cancer by detecting genes overexpressed in cancer cells from the sample without amplification, the method comprising the step of detecting the marker, Here, the positively charged substance is a nanowire having a positively charged surface, the probe has a sequence complementary to cfDNA and a material capable of binding to the marker; the marker further comprises a substance capable of binding to the probe; The probe binds to a gene known as a cancer biomarker by complementarity, The cfDNA is a cfDNA having a stable double helix structure, and The method does not involve PCR or nucleic acid amplification.

10. The probes having a sequence complementary to the cfDNA include CPT1A, IFNG, IFNGR1, CD279, CD274, NSE, SCC, CEA, cyfra21-1, TPA, NMP22, OGT, Thyroglobulin (TG), Calcitonin (CALCA), BRAF V600E, TERT C228T / C250T, AFP, β-HCG (CGB), CA19-9, PSA, PSMA, PAP, PCA3, TMPRSS2-ERG, CA125, HIF-1a, VEGF, CA15-3, HER2, SCC (SART3), TOP2A, The method for providing information for early diagnosis of cancer or predicting the prognosis of cancer according to claim 9, wherein the gene complementarily binds to at least one gene selected from the group consisting of MCM2, p16INK4a (CDKN2A), Ki-67 (MKI167), HE4 (WEDC2), and combinations thereof.

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