KR20190099658A - Carbon dot-based fluorescentnanosensor and nucleicacid detection method using the same - Google Patents

Abstract

The present invention relates to a composition for detecting a nucleic acid comprising a carbon dot and a nucleic acid detection method using the same. The carbon dot of the present invention has a size of several nanometers to several micrometers and is cationic to combine with a nucleic acid material by electrostatic attraction to form a precipitate. Thus, the nucleic acid detection method of the present invention can be applied to various applications for targeting nucleic acid materials, thereby quickly and simply diagnosing a disease when used in the medical field, and being favorably used in the field of health care, such as health monitoring, pregnancy-related tests. In addition, when used in industry, it is possible to detect contaminants in food and beverage quickly and effectively.

Description

Carbon dot-based fluorescentnanosensor and nucleic acid detection method using the same}

The present invention relates to a carbon quantum dot-based fluorescent nanosensor and a nucleic acid detection method using the same.

Sensing molecular targets is necessary for the industrial, agricultural, environmental and biomedical fields for diagnosing human diseases. In particular, for the rapid diagnosis, early diagnosis and monitoring of treatment response to acute diseases, a simpler and more easily applicable method for detecting a target substance is needed. The high sensitivity and specificity of the detection of nucleic acid material using methods such as real-time polymerase chain reaction (qPCR), sequencing and hybridization assays provide several advantages. However, sensitive detection of a large number of targets requires expensive equipment or tools, and is a limitation as an on-site diagnostic platform in the room or where access to medical facilities is poor.

Various methods of detecting nucleic acid target substances have been studied. Methods based on fluorescent metal sensors that detect fluorescent signals generated by selective binding to byproducts (chemicals) produced during conventional DNA amplification (Tomita, N., et al. Nat. Protoc. 3, 877 (2008) ) Is a complex and indirect way of detecting by-products rather than the target material itself, so its practical application as a diagnostic method is very limited. The method of visually confirming the presence or absence of target DNA based on the hybridization of the fluorescent probe and the target DNA (Mori, Y., et al. BMC Biotechnol. 6, 3 (2006)) requires a large amount of probes. There is a problem of low sensitivity.

Nanomaterials and nanomaterials with unique physicochemical properties can fix a large amount of bioactive molecules, and can be synthesized and manipulated to maximize various detection signals. In the last few decades, research on these nanotechnology has led to breakthroughs in biosensing and molecular diagnostics, and materials such as gold nanoparticles, iron oxide nanoparticles, and quantum dots have been used to detect DNA or RNA.

Especially recently, carbon quantum dot nanoparticles have been proposed as a new kind of carbon-based nanomaterials. Carbon quantum dot nanoparticles have various advantages such as strong photo-luminescence, excellent colloid-stability, and bio-sensing based on properties exhibiting strong fluorescence by aggregation or self-assembly based on crosslink-enhanced emission effect. There are many researches to be utilized in the field of imaging and imaging.

Maiti, S. et al. Detected histones without the use of markers using quaternized carbon quantum dot-DNA nano bio hybrids to detect biomolecules (Maiti, S., et al. Chem). Commun. 49, 8851 (2013)). However, there is a problem that this study is difficult to apply to other molecular targets besides histones. Huang, S. et al. Detected DNA using ethidium bromide and polymer carbon quantum dots. Although this study has shown some utility as an analytical technique, it requires further validation when applied to specific target materials, and there are problems with the toxicity of ethidium bromide reagents (Huang, S., et al. RSC Adv. 5, 44587 (2015)).

In addition, more recently, studies using carbon quantum dots as fluorescent dyes to detect living bacteria and carbon quantum dots as probes to identify Gram-positive bacteria have been reported. However, these studies also have limitations that require high resolution imaging equipment for detection (Hua, XW, et al. Nanoscale 9, 2150 (2017); Yang, J., et al. ACS Appl. Mater. Interfaces 8 , 32170 (2016)).

Therefore, there is a need for research and development of carbon quantum dot-based methods that can detect and analyze nucleic acid materials simply and quickly without high resolution imaging equipment.

The present inventors have made intensive research efforts to develop carbon quantum dot-based nanosensors that can quickly precipitate nucleic acid materials and simply detect and analyze them. As a result, the carbon quantum dot produced by the cationic organic compound of the present invention quickly precipitated the nucleic acid material, and the formed precipitate was completed by elucidating that it can be visually sensed and analyzed by simple UV exposure equipment, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a composition for detecting nucleic acid (nucleic acid) comprising a carbon quantum dot (carbon dot).

Another object of the present invention to provide a nucleic acid (nucleic acid) detection method.

According to one aspect of the invention, the present invention provides a composition for detecting a nucleic acid (nucleic acid) comprising a carbon quantum dot (carbon dot).

In the present invention, "quantum dot" refers to a metal or semiconductor crystal of about 10 nm, usually means a crystal composed of hundreds to thousands of atoms. In general, quantum dots show intermediate properties between a single atom and a bulk material, and feature band-gap inversely proportional to size due to the quantum confinement effect of electrons confined in small spaces. Bars can be applied to various fields such as solar cells, light emitting devices, photocatalysts, and transistors because the energy structure can be adjusted without changing chemical composition.

In the present invention, "carbon quantum dot" may be used in the same sense as "carbon quantum dot" or "carbon dot", and is an amorphous (namorphous) nanostructure based on carbon (C) particles, a diamond-like nanostructure nanodiamond And graphite nanostructures such as graphene, nanotubes and fullerenes. The size of the carbon quantum dots is several nm-several mm, and has the characteristics of a typical quantum dot having a different color depending on the particle size, the fluorescence wavelength varies depending on the particle size. Unlike toxic semiconductor quantum dots such as CdSe, carbon quantum dots are bio-friendly carbon compounds and can be applied to sensors that can be injected into the human body.

The carbon quantum dot is polyethyleneimine, polyamidoamine, ethylenediamine, 2,2-ethylenedioxybisethylamine, 4,7,10-trioxa-1,13-tridecanediamine, polyethylene glycol diamine, Polyenepolyamine, tetraethylenepentamine, urea, chitosan, dimethylaminoethyl methacrylate, cystamine, polylysine, polyarginine, polyornithine, DOTAP, dodecylamine, didecyldimethylammonium, dididodecyldimethylammonium, dimethyldi Tetradecylammonium, tridodecylmethylammonium, dihexadecyldimethylammonium, dimethyldioxadecylammonium, dimethyldioctadecylammonium, dodecylethyldimethylammonium, ethylhexadecyldimethylammonium, decyltrimethylammonium, hexyltrimethylammonium, triethyl At least one cationic polymer reactor selected from the group consisting of hexyl ammonium, tetrabutyl ammonium and trimethyloctadecyl ammonium It can be prepared and, for example, but can be prepared from the polyethyleneimine, and the like.

The preparation can be performed using top-down methods such as chemical ablation, electrochemical carbonization and laser ablation, and wet oxidation, microwave pyrolysis, and high temperature injection. Any method used in the art may be used, such as a bottom-up method such as hot-injection, hydrothermal, and solvent thermal treatment.

The carbon quantum dots may have a diameter of, for example, 1-100 or 1-10 nm, but is not limited thereto. Preferably, when the carbon quantum dots are 1-10 nm, lower toxicity, excellent light emitting ability, and high light stability may be achieved. Can have.

The composition may fluoresce in an aqueous solution or an organic solvent.

The composition may have a zeta potential of 0.1-100 mV, and thus may be positively coupled to anionic nucleic acid (nucleic acid) by electrostatic attraction.

The carbon quantum dots can cause clustering by intermolecular crosslinking with the target nucleic acid. At this time, clustering proceeds until the aggregate is large enough to settle to the bottom of the sample tube, which can be easily visualized under UV exposure, and also visual inspection.

The nucleic acid (nucleic acid) is DNA (deoxyribonucleic acid) or RNA (ribonucleic acid), may be 1-1,000,000,000 nucleotides in length.

The DNA may include, for example, plasmid DNA, genomic DNA, synthetic single-stranded oligodeoxyribonucleotide, synthetic double-stranded oligodeoxyribonucleotide, and the like, and RNA, for example, mRNA, rRNA, tRNA, siRNA, miRNA, synthetic single-stranded oligoribonucleotide and synthetic Double stranded oligoribonucleotide and the like.

In a specific embodiment, the target genes represented by the nucleic acid are mecA, mecR1, aph, NDM-1, KPC, oxa, ure, lrg, cap, spl, KPC, GES, IMP, VIM, KRAS, Stk11, TP53, PTEN, BRCA1 , BRCA2, Akt, Stat, Stat4, JAK3, JAK2, WT1, ERBB2, ERBB3, ERBB4, NF1, NOTCH1, NOTCH3, ATM, ATR, HIF, HIF1 α, HIF3 α, Met, Bcl2, FGFR1, FGFR2, CDKN2α, APC , RB, MEN1, PPAR α, PPAR γ, AR, TSG101, IGF, Igf1, Igf2, Bax, Bcl2, caspase, Kras, Apc, NF1, MTOR, Grm1, Grm5, Grm7, Grm8, mGlurR1, mGlurR5, mGlurR8, PLC , AMPK, MAPK, Raf, ERK, TLR4, BRAF, PI3K, F8, F8C, F9, HEMB, KIR3DL1, NKAT3, NKB1, AMB1I, KIR3DS1, IFNG, CXCLI2, IL2RG, SCIDX1, SCIDX, IMD4, CCR5, SCYAE , TCP228, CXCR2, CXCR3, CXCR4, CCR4, CCR6, CCR7, CX3CR1, CD4, CFTR, HBB, HBA2, HBD, HBA1, LCRB, SCN1A, CHD8, FMR1, VEGF, EGFR, myc, Bcl-2, survivin, SOX2 , Nrgl, Erb4, Cplx1, Tph1, Tph2, GSK3, GSK3α, GSK3 β, El, UBB, PICALM, PSI, SORL1, CR1, Ubal, Uba3, CHIP, UCH, Tau, LRP, CH1P28, Uchl1, Uchl3, APP, Cx3crl, ptpn22, TNF-α, NOD2, IL-1a, IL-1b, IL-6 , IL-10, IL-12, IL-1a, IL-1b IL-13, IL-17a, IL-17b, IL-17c, IL-17d, IL-17f, CT1LA4, Cx3c11J, DJ-1, PINK1, Prp, DJ-1, PINK1, LRRK2, ALAS2, ASB, ANH1, ABCB7, ABC7, ASAT, CDAN1, CDA1, DBA, PKLR, PK1, RIPS19, NT5C3, UMPH1, PSN1, RHAG, RH50A, NRAMP2, SPTB, TBXA2R, P2X1P, 2RX1, HF1, CFH, HUS, MCFD2, Drd2, Drd4, ABAT, BCL7A, BCL7, TALI, TCL5, TAL2, FLT3, NBS1, NBS, ZNEN1A1, TYR, ALDH, ALDH1A1, ADH, FASN, PISN, FASN F5, MDC1C, LAMA2, LAMM, LARGE, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, SGCG, DMDA1, SCG3, SGCA, ADL, DAG2, DMDA2, SGCB, LGMD2B, LGMD2C, LGMD2D, LGMD2E LGMD2G, LGMD2H, LGMD2I, SGCD, SGD, CMD1L, TCAP, CMD1N, TRIM32, HT2A, FKRP, POMT1, CAV3, LGMD1C, SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1, PPP2R1A, PPP2, PPP2 Examples include, but are not limited to, RFP, YFP, tdTomato, mCherry, luciferase, β-actin, GAPDH, and the like.

According to another aspect of the present invention, the present invention comprises the steps of (a) adding a reaction sample containing a nucleic acid to the nucleic acid detection composition for reacting; And (b) provides a method for detecting a nucleic acid (nucleic acid) comprising the step of confirming whether or not the fluorescence of the reactants under UV exposure.

The detection sample of step (a) is DNA (deoxyribonucleic acid) or RNA (ribonucleic acid), and may include 1-1,000,000,000 nucleotides in length.

The DNA may include, for example, plasmid DNA, genomic DNA, synthetic single-stranded oligodeoxyribonucleotide, synthetic double-stranded oligodeoxyribonucleotide, and the like, and RNA, for example, mRNA, rRNA, tRNA, siRNA, miRNA, synthetic single-stranded oligoribonucleotide and synthetic Double stranded oligoribonucleotide and the like.

The reaction of step (b) may be to form a precipitate by a microfuge, incubation, shaking, vortexing or inverting.

More specifically, the microfuge is added for 1-240 minutes, preferably 1-30 minutes at 4-60 ° C., preferably 15-25 ° C., after adding the detection sample containing the nucleic acid to the nucleic acid detection composition. ) Or simply incubation to induce the formation of a precipitate.

The reaction of step (a) may be performed under the condition of pH 1-5. This is because the electrostatic interaction with DNA increases as the primary amine groups of the carbon quantum dots are protonated at an acidic pH (low pH), so that the carbon quantum dots can precipitate the DNA with the highest efficiency at the acidic pH.

The reaction of step (a) may be carried out under NaCl conditions of 0.001-1 M concentration. This is because the electrostatic interaction of carbon quantum dots (cationics) and DNA (anions) by complex formation with counter ions is reduced, so that the lower the salinity to 0.001-1 M concentration, the more efficient the carbon quantum dots can precipitate DNA. Can be.

The step (b) may be to confirm the presence or absence of fluorescence by quantifying the fluorescent image of the reactant, which may be to use naked eyes.

In addition, the step (b) may further use information obtained by using a fluorescence meter or a light analyzer.

According to the detection method of the present invention, the detection signal can be quantitatively represented by analyzing the presence or absence of fluorescence of a fluorescent image obtained by using an experimental analysis or a general digital camera or a simple smartphone camera under UV exposure.

The features and advantages of the present invention are summarized as follows:

(A) The present invention provides a composition for detecting a nucleic acid (nucleic acid) comprising a carbon quantum dot (carbon dot) and a nucleic acid detection method using the same.

(b) The carbon quantum dot of the present invention has a size of several nanometers to several micrometers, which is cationic and can bind to the nucleic acid material by electrostatic attraction to form a precipitate. It can be applied to a variety of applications targeting the material can be quickly and easily diagnose the disease when used in the medical field, it can also be useful in the health care field, such as health monitoring, pregnancy-related tests. In addition, when used in industry, it is possible to detect contaminants in food and beverage quickly and effectively.

(c) In particular, since the analysis can be performed by using a bulky carbon quantum dot instead of the conventional low molecular dyes with the naked eye or by using a simple and inexpensive device, the application to the field diagnostic platform will be high.

1 is a schematic diagram showing a carbon quantum dot-based fluorescent nanosensor and nucleic acid material detection / analysis of the present invention.
Figure 2 is a schematic diagram showing a process for producing a cationic carbon quantum dot of the present invention.
Figure 3 confirms the characteristics of the cationic carbon quantum dot of the present invention, a) spectrophotometric analysis, b) Fourier transform infrared spectroscopy (FT-IR) analysis, c) dynamic light scattering (DLS) analysis, and d) A diagram showing the results of transmission electron microscope analysis.
4 is a schematic diagram showing a nucleic acid substance detection method of the present invention.
Figure 5 confirms the precipitation and detection of nucleic acid material by the cationic carbon quantum dots of the present invention, a / b) fluorescence (photoluminescence) intensity, and c) fluorescence spectrum analysis results.
Figure 6 confirms the physical and chemical properties of the precipitate (carbon quantum dot + nucleic acid material) according to an embodiment of the present invention, a) dynamic light scattering analysis, b) zeta potential measurement, and c) transmission electron microscopy analysis results It is also.
7 is a diagram confirming the precipitation and detection of nucleic acid material of the cationic carbon quantum dots of the present invention according to various solution conditions.
8 is a diagram confirming the precipitation and detection of nucleic acid material of the cationic carbon quantum dots of the present invention according to the conditions of the nucleic acid material.
9 is a diagram confirming the precipitation and detection of nucleic acid material using the image obtained by the smartphone camera.
10 is a schematic diagram showing a method for detecting DNA derived from actual bacteria using the assay method of the present invention.
11 is a diagram showing the results of detecting DNA derived from actual bacteria using the assay method of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Preparation Example 1 Preparation of Fluorescent Carbon Quantum Dots

As a material for precipitating nucleic acid material, in order to produce fluorescent cationic carbon quantum dots, a branched polyethyleneimine polymer having a weight average molecular weight (Mw) of 25,000 was dissolved in 0.1 N hydrogen chloride (HCl) solution, and citric acid (citric acid) was mixed with 1,000 mg. The mixed solution was heated at 800 W microwave for 1 minute to form a carbon quantum dot with a cation around the branched polyethyleneimine, and the solution was sufficiently boiled to remove the solvent. An unreacted reactant was removed by washing with 0.1 N hydrogen chloride solution using an amicon filter. The series of procedures is shown in FIG.

Example 1 Identification of Carbon Quantum Dot Properties of the Invention

The properties of the prepared carbon quantum dots were confirmed through the following experiment.

1-1. Spectrophotometer analysis

As a result of the spectrophotometer analysis, as shown in FIG. 3A, the carbon quantum dots showed the highest value at the wavelength of 450 nm (365 nm excitation wavelength) and showed strong fluorescence. Appeared to be.

1-2. Fourier Transform Infrared Spectrometer (FT-IR) Analysis

As a result of FT-IR analysis, peaks were confirmed at wavelengths of 2821, 2983, and 3360 nm, as shown in FIG. 3B, indicating that hydroxyl groups of primary amine groups and carboxyl groups exist in carbon quantum dots.

1-3. Dynamic light scattering (DLS) analysis

As a result of confirming the particle size of the carbon quantum dots by DLS analysis, it was confirmed that the hydrodynamic diameter of 15.4 nm was shown, as shown in FIG. 3C.

1-4. Transmission electron microscopy analysis

As a result of confirming the core size of the carbon quantum dots by transmission electron microscopic analysis, it was confirmed that the core size of 5.8 ± 1.7 nm, as shown in Figure 3d.

Finally, the composition of the carbon quantum dots was found to be 46.8 wt% carbon, 12.2 wt% nitrogen, 11.2 wt% hydrogen, and the zeta potential measurement (see FIG. 6b) showed that the carbon quantum dots were highly cationic with a surface charge of +6.8 mV. By confirming that, it was confirmed that carbon quantum dots were successfully formed.

Example 2 Confirmation of Precipitation and Detection of Nucleic Acid Material by Carbon Quantum Dots

In order to confirm that the nucleic acid material can be precipitated and detected using the cationic carbon quantum dots prepared in Preparation Example 1, the following experiment was performed. A series of procedures is shown in FIG.

7.4 μg (x 0.5, 1, 3, 5, 7) of carbon quantum dots dispersed in water was added to an aqueous solution containing 0.01 to 1 nmol of synthetic DNA oligonucleotides having a length of 20 to 65 mer, and after about 10 minutes, Gravity was applied by microfuge for 1-2 minutes. After the precipitate was formed, the fluorescence intensity and spectrum of the supernatant were measured by quantifying it with a spectrophotometer, avoiding the precipitate at the bottom of the tube, or by observing and acquiring images with a UV exposure equipment (a standard digital camera mounted on an UV illuminator). .

As a result, as shown in Figure 5a, when the amount of the carbon quantum dot is 7.4 ~ 51.8 μg, the fluorescence intensity was found to be significantly reduced compared to the carbon quantum dot without addition of the nucleic acid material, as shown in Figure 5b, When the amount of quantum dots was 7.4 μg, the precipitate formation ratio (66.7 wt% of the total amount of carbon quantum dots) was highest compared to the fluorescence intensity before adding the nucleic acid material. In addition, as a result of fluorescence spectrum analysis of wavelength band from 380 to 650 nm, as shown in FIG. 5C, the wavelength band in which the maximum value of fluorescence appeared did not change even after precipitation.

Next, the physical and chemical properties of the precipitate (carbon quantum dot + nucleic acid material) according to the experiment was confirmed.

First, as a result of dynamic light scattering analysis, in the case of single-stranded DNA, the size of the precipitate was found to be about 244.5-293.2 nm, which is about 15 times higher than that of the carbon quantum dot without DNA, and the precipitate was 20-, There was no significant difference in 42- and 65-mer lengths. Double-stranded DNA, on the other hand, was found to have a much larger size (1235 nm) due to weak condensation due to charge repulsion between DNA molecules.

Next, as a result of measuring the zeta potential, as shown in FIG. 6B, it was confirmed that in the case of single-stranded DNA, the surface charge also decreased as the length of the DNA decreased. That is, higher zeta potential values are found at longer oligonucleotides (+19.4 mV for 42-mer and +13.4 mV for 65-mer) compared to shorter oligonucleotides (+24.3 mV for 20-mer). It confirmed that it appeared.

In addition, as a result of observing with a transmission electron microscope, as shown in Figure 6c, when the carbon quantum dots are added to the DNA, it was confirmed that large round aggregates of 80-400 nm size is formed.

Example 3. Confirmation of Precipitation and Detection According to Various Solution Conditions

Based on the results of the above examples, in order to determine the binding interaction between carbon quantum dots and nucleic acid material (DNA), the following experiments were carried out.

3-1. Precipitation and detection confirmation according to solution conditions

First, 7.4 μg of carbon quantum dots were prepared by dissolving in various pH conditions (pH 3, 5, 7, 8, and 9) buffers, and 1 nmol of 20-mer synthetic DNA oligonucleotides was added. After about 10 minutes, gravity was applied by centrifugation for 1-2 minutes to form a precipitate. Observations and images were obtained by UV exposure equipment, and the fluorescence intensity of the supernatant was measured to avoid precipitation.

 In addition, NaCl was dissolved in primary distilled water to prepare aqueous NaCl solutions at various concentrations (0, 0.01, 0.02, 0.05, 0.1, 0.15, 0.3, 0.5 and 1 M), 7.4 μg of carbon quantum dots and 20- 1 nmol of mer synthetic DNA oligonucleotide was added. After about 10 minutes, gravity was applied by centrifugation for 1-2 minutes to form a precipitate. Observations and images were obtained by UV exposure equipment, and the fluorescence intensity of the supernatant was measured to avoid precipitation.

As a result, as shown in Figure 7, it was confirmed that as the pH is increased, the fluorescence intensity of the carbon quantum dots is reduced and the amount of precipitate is also reduced. This means that as the primary amine groups of the carbon quantum dots are quantized at acidic pH (low pH), the electrostatic interaction with DNA increases, which means that the carbon quantum dots can precipitate DNA at the highest efficiency at acidic pH. In addition, it was confirmed that as the concentration of salt (NaCl) increases, the rate at which precipitates are formed decreases. This is because the electrostatic interaction of carbon quantum dots (cations) and DNA (anions) by complex formation with counter ions is reduced, which means that the lower the salinity, the more efficient the carbon quantum dots can precipitate DNA.

3-2. Precipitation and detection confirmation according to nucleic acid conditions

Experiments with synthetic oligonucleotides of varying amounts and sizes. The base sequence of the synthetic DNA is shown in Table 1 below, and is selected from the drug resistance gene KPC-2 (GenBank: AY034847), a major gene involved in carbapenem resistance of Gram-negative bacteria K. pneumoniae. It became.

More specifically, 7.4 μg of carbon quantum dots were added to 0.01-1 nmol (0.01, 0.025, 0.05, 0.1, 0.25, 0.5 and 1 nmol) of deoxynucleotide or synthetic single stranded DNA oligonucleotide aqueous solution. At this time, the volume was adjusted to 10 μl. After about 10 minutes, precipitates were formed using a centrifuge, observations and images were obtained with UV exposure equipment, and the fluorescence intensity of the supernatant was measured. In addition, the same experiment was conducted for synthetic double-stranded DNA oligonucleotides of 0.001-0.25 nmol (0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1 and 0.25 nmol).

As a result, as shown in Figure 8, as the length is increased from 20 to 65 mer, it was confirmed that the minimum amount of DNA to form a precipitate is reduced. That is, in the case of 20 mer and 42 mer, it was confirmed that precipitates were formed when DNA of 0.25 nmol or more was added, and in case of 65 mer, when DNA of 0.1 nmol or more was added. In addition, as the amount of DNA increased from 0.01 nmol to 1 nmol, the amount of precipitate formed increased. When comparing single-stranded and double-stranded DNA, double-stranded DNA added less DNA than single-stranded DNA. It was confirmed to form a precipitate.

As a result of the synthesis, it was confirmed that in order for precipitation to occur, longer DNA (eg, 65-mer) is preferable to shorter DNA, and higher concentration (amount) of DNA is more preferable. However, since the target DNA can be detected even at a short length of less than 65-mer and at a low concentration of 0.01-0.25 nmol, the nucleic acid detection method using the carbon quantum dot of the present invention can be usefully used for nucleic acid samples extracted without amplification.

Example 4. Confirmation of Ease of Detection and Analysis

The precipitation result of the DNA target material by the carbon quantum dots of the present invention is a smartphone camera (Samsung Galaxy Note 5) to confirm that the detection and analysis is easy even by the minimum technical components (UV lighting device and smartphone camera) Images were acquired and quantitatively analyzed.

More specifically, when 0.01 to 1 nmol of 20 mer DNA oligonucleotide was added, the digital image of the precipitation result was analyzed on a gray scale, and the cut-off was designated to count the number of pixels showing the intensity above the reference.

As a result, as shown in Figure 9, even when using a smartphone camera it was confirmed that the precipitation effectively occurs when the amount of DNA is 0.25 nmol or more. In addition, when comparing the smartphone image analysis result with the quantitative analysis result of the image obtained with the digital camera (see FIG. 8), it showed a high correlation coefficient of 0.9471, and the trend was similar.

Example 5 Detection and Analysis of Bacterial Nucleic Acid Materials by Carbon Quantum Dots

Finally, based on the results of Examples 1 to 4 above, the DNA derived from the actual bacteria was detected using the assay of the present invention.

More specifically, RNA was extracted from Klebsiella pneumoniae and the target site selected on the gene was amplified by PCR using primers for KPC, which is representative of carbapenem resistance genes during multidrug resistance after reverse transcription process. Finally, the amplified DNA was detected by the above method, and the image was obtained by a smartphone under UV exposure and quantitatively analyzed. At this time, in order to confirm that precipitation was observed only in KPC-positive K. pneumoniae , KPC negative K. pneumoniae , E. coli , Methicillin-resistant Staphylococcus aureus (MRSA) and cDNA were compared. A series of procedures is shown in FIG.

1의 높은 상관관계 계수를 나타내었다. 최종적으로, 상기 분석법으로 3.6 x 10⁶ 개의 박테리아로부터 추출하여 증폭한 DNA를 검출할 수 있음을 확인하였다.As a result, as shown in FIG. 11, it confirmed that precipitation was confirmed only in case of KPC positive K. pneumoniae . In addition, in the quantitative analysis of the smartphone image, it was confirmed that the precipitate was present only in the case of KPC positive K. pneumoniae , even when compared with the image taken with a standard digital camera.

A high correlation coefficient of 1 is shown. Finally, the assay confirmed that the DNA amplified by extracting from 3.6 x 10 ⁶ bacteria was detected.

These results indicate that the assay is a very useful way to quickly and simply detect nucleic acid material of multidrug resistant bacteria.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above are to be understood in all respects as illustrative and not restrictive.

Claims (14)

Nucleic acid (nucleic acid) detection composition comprising a carbon quantum dot (carbon dot).
The method of claim 1, wherein the carbon quantum dots are polyethyleneimine, polyamidoamine, ethylenediamine, 2,2-ethylenedioxybisethylamine, 4,7,10-trioxa-1,13-tridecanediamine , Polyethylene glycol diamine, polyenpolyamine, tetraethylenepentamine, urea, chitosan, dimethylaminoethyl methacrylate, cystamine, polylysine, polyarginine, polyornithine, DOTAP, dodecylamine, didecyldimethylammonium, didido Decyldimethylammonium, dimethylditetradecylammonium, tridodecylmethylammonium, dihexadecyldimethylammonium, dimethyldioxadecylammonium, dimethyldioctadecylammonium, dodecylethyldimethylammonium, ethylhexadecyldimethylammonium, decyltrimethylammonium, At least one amount selected from the group consisting of hexyltrimethylammonium, triethylhexylammonium, tetrabutylammonium and trimethyloctadecylammonium It is prepared from a warm polymer, nucleic acid detection composition.
The composition of claim 1, wherein the carbon quantum dots have a diameter of 1-100 nm.
The composition of claim 1, wherein the composition is fluorescent in an aqueous solution or an organic solvent.
The composition of claim 1, wherein the composition has a zeta potential of 0.1-100 mV.
According to claim 1, wherein the nucleic acid (nucleic acid) is DNA (deoxyribonucleic acid) or RNA (ribonucleic acid), the composition for detecting a nucleic acid.
Nucleic acid detection method comprising the following steps.
(a) reacting the nucleic acid detection composition of claim 1 by adding a sample for detecting the nucleic acid; And
(b) confirming whether the reactants fluoresce under UV exposure.
The method of claim 7, wherein the detecting sample of step (a) is DNA (deoxyribonucleic acid) or RNA (ribonucleic acid).
8. The method of claim 7, wherein the reaction of step (a) is to form a precipitate by a microfuge, incubation, shaking, vortexing or inverting process. That's how.
The method of claim 9, wherein the reaction of step (a) is performed at 4-60 ° C. for 1-240 minutes.
The method of claim 7, wherein the reaction of step (a) is carried out at a condition of pH 1-5.
The method of claim 7, wherein the reaction of step (a) is carried out under NaCl conditions of 0.001-1 M concentration.
The method of claim 7, wherein step (b) confirms the presence or absence of fluorescence by quantifying the fluorescence image of the reactants.
The method of claim 13, wherein step (b) confirms the presence or absence of fluorescence with naked eyes.

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