WO2019088527A2 - Pathogen lysis and nucleic acid extraction method using zinc oxide nanostar - Google Patents

Abstract

The present invention relates to a pathogen lysis and nucleic acid extraction method using a zinc oxide nanostar. A nucleic acid extraction method using a zinc oxide nanostar according to the present invention enables the extraction of nucleic acid through cell lysis of pathogens without using a lysis buffer and allows for the extraction of nucleic acids at high purity and high concentration by preventing nucleic acid degradation and fragmentation attributed to various materials in a lysis buffer, including a high concentration of salts. In addition, zinc oxide nanostars (200 to 900 nm) according to the present invention, which are superior conventional zinc oxide nanoparticles (20 to 50 nm) in terms of cell lysis potential, improve nucleic acid extraction efficiency thanks to their cell lysis potential and allow extraction at room temperature without a heating step, thus finding application as a diagnostic method in the field.

Description

Method of extracting pathogenic bacteria and nucleic acid using zinc oxide nanostar

The present invention relates to a method for extracting pathogenic bacteria and nucleic acids using zinc oxide nanostars.

Nucleic acid is an important analytical tool for identifying disease states, and DNA biomarkers, such as single nucleotide polymorphism (SNP), mutation or DNA methylation, And provides an important clue to providing a great opportunity for prognosis and surveillance as well as diagnosing and monitoring the condition of the disease during the early stages of the disease.

Since nucleic acids such as DNA are present at very low physiological concentrations compared to other components such as proteins (e.g., tens of nanograms of DNA versus a few tens of micrograms of protein per microliter of whole blood), DNA is effectively extracted from clinical samples Preconcentration is very important for subsequent processes such as amplification and detection.

In recent years, the use of high purity purified nucleic acids has been increasing in various fields such as biotechnology, diagnostic medicine, pharmacology, and metabolism. Therefore, efforts are being made to separate nucleic acids more rapidly and neatly from various biological samples.

However, the most significant part of the method for separating nucleic acids to date is a carrier that specifically adsorbs only nucleic acid from various kinds of substances contained in the cell lysis solution such as genomic DNA, plasmid DNA, messenger RNA, protein, Have focused on the research and development of nucleic acid adsorbing materials.

Accordingly, in order to separate nucleic acids more rapidly and purely from various biological samples, it is necessary to develop a technology capable of rapidly separating only desired nucleic acids from cell debris particles, protein denatured aggregates, and various cell degradation materials.

An object of the present invention is to provide a gentle composition for a pathogen including a zinc oxide nanostar, a pathogen ligation method and kit using the same, A composition for extracting nucleic acid including zinc oxide nanostar, a nucleic acid extraction method and kit using the same, and a kit.

In order to achieve the above object, the present invention provides a germicidal composition for a pathogen comprising zinc oxide nanostar.

The present invention also provides a kit for a pathogen comprising the composition.

In addition, the present invention provides a pathogen method comprising contacting a zinc oxide nanostar with a sample containing a pathogen.

The present invention also provides a composition for extracting nucleic acid comprising zinc oxide nanostar.

The present invention also provides a kit for nucleic acid extraction comprising the above composition.

In addition, the present invention provides a method for producing a zinc nanostructure comprising: a first step of adding a zinc oxide nanostar to a nucleic acid sample and reacting the mixture to prepare a mixture; And a second step of extracting the nucleic acid from the mixture.

In addition, the present invention provides a method for producing a zinc nanostructure comprising: a first step of adding a zinc oxide nanostar to a nucleic acid sample and reacting the mixture to prepare a mixture; Adding diatomite modified with a silane compound to the mixture and adding the mixture to a mixture of dimethyl adipimidate (DMA), dimethylpimelimidate (DMP), dimethyl suberimidate (DMS) and dimethyl A second step of preparing a reaction mixture by adding at least one selected from the group consisting of 3,3'-dithiobispropionimidate (DTBP), 3,3'-dithiobispropionimidate (DTBP); And a third step of extracting a nucleic acid from the reaction mixture.

The nucleic acid extraction method using the zinc oxide nanostructure of the present invention can extract the nucleic acid by dissolving the cells of the pathogen without using a lysis buffer and is capable of extracting nucleic acid from a variety of substances including a high concentration of salts contained in the lysis buffer Thereby preventing nucleic acid degradation and fragmentation caused by the nucleic acid degradation. In addition, the zinc oxide nanostar (200 to 900 nm) of the present invention is superior to conventional zinc oxide nanoparticles (20 to 50 nm) in cell solubility to increase nucleic acid extraction efficiency and can be extracted at room temperature without heating It can be used as a field diagnostic method.

1 compares the sizes and shapes of (A) zinc oxide nanostars and (B) zinc oxide nanoparticles.

2 compares the structures of zinc oxide nanostar and zinc oxide nanoparticles using the Raman technique.

FIG. 3 compares the solubility and nucleic acid extraction efficiency of zinc oxide nanostars using brucella bacteria with zinc oxide nanoparticles and a conventional nucleic acid extraction kit (Qiagen).

FIG. 4 compares the solubility and nucleic acid extraction efficiency of zinc oxide nanostars using Brucella, Escherichia coli, Staphylococcus aureus, and Bacillus cereus with a conventional nucleic acid extraction kit (Qiagen).

FIG. 5 confirms that nucleic acid extraction is possible by combining zinc oxide nanostar with a column of a conventional nucleic acid extraction kit (Qiagen).

FIG. 6 shows that the zinc oxide nanostar is combined with diatomaceous earth or the same type 2 functional imidoester to extract nucleic acid.

7 compares the efficiency of nucleic acid extraction of zinc oxide nanostars with zinc oxide nanoparticles according to particle size using HCT116, a colon cancer cell line.

FIG. 8 is a graph comparing (A) DNA extraction and (B) RNA extraction efficiency of a conventional nucleic acid extraction kit (Qiagen), zinc oxide nanostar, and zinc oxide nanoparticles using HCT116 as a colon cancer cell line.

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention have developed an extraction method capable of dissolving a pathogen and extracting a nucleic acid, and the ZnO nanostar (ZnO NS) of the present invention is superior to conventional zinc oxide nanoparticles in dissolving a cell membrane and nucleus of a pathogen The nucleic acid extracting method using the zinc oxide nanostar of the present invention is capable of extracting nucleic acid of high concentration and high concentration without lysis buffer and heating step, The present invention has been accomplished on the basis of the finding that nucleic acid can be extracted and an immediate on-the-spot diagnosis is possible.

The present invention provides a mycelial composition for pathogen comprising zinc oxide nanostar.

The zinc oxide nanostructure refers to a nanoparticle in which a plurality of sharp projections are annularly arranged.

The zinc oxide nanostars have an average particle diameter of 200 to 900 nm, preferably an average particle diameter of 350 to 900 nm, more preferably an average particle diameter of 500 nm, but are not limited thereto Specify.

 The pathogen is a microorganism and the microorganism may be, but is not limited to, a virus, a bacterium, a fungus, a protozoa, a ricketta or a spirotherte.

The present invention also provides a kit for a pathogen comprising the composition.

In addition, the present invention provides a pathogen method comprising contacting a zinc oxide nanostar with a sample containing a pathogen.

The sample containing the pathogenic agent may be used as a sample of a subject suspected of being infected with a pathogen such as feces, urine, tears, saliva, external secretion of skin, external secretion of the respiratory tract, external secretion of the intestinal tract, external secretion of the digestive tract, Spinal fluid, lymph fluid, body fluids, and tissue, but is not limited thereto.

The pathogen is a microorganism and the microorganism may be, but is not limited to, a virus, a bacterium, a fungus, a protozoa, a ricketta or a spirotherte.

The present invention also provides a composition for extracting nucleic acid comprising zinc oxide nanostar.

The zinc oxide nanostructure refers to a nanoparticle in which a plurality of sharp projections are annularly arranged.

The zinc oxide nanostars have an average particle diameter of 200 to 900 nm, preferably an average particle diameter of 350 to 900 nm, more preferably an average particle diameter of 500 nm, but are not limited thereto Specify.

The nucleic acid may be DNA or RNA.

The present invention also provides a kit for nucleic acid extraction comprising the above composition.

In addition, the present invention provides a method for producing a zinc nanostructure comprising: a first step of adding a zinc oxide nanostar to a nucleic acid sample and reacting the mixture to prepare a mixture; And a second step of extracting the nucleic acid from the mixture.

In addition, the present invention provides a method for producing a zinc nanostructure comprising: a first step of adding a zinc oxide nanostar to a nucleic acid sample and reacting the mixture to prepare a mixture; Adding diatomite modified with a silane compound to the mixture and adding the mixture to a mixture of dimethyl adipimidate (DMA), dimethylpimelimidate (DMP), dimethyl suberimidate (DMS) and dimethyl A second step of preparing a reaction mixture by adding at least one selected from the group consisting of 3,3'-dithiobispropionimidate (DTBP), 3,3'-dithiobispropionimidate (DTBP); And a third step of extracting a nucleic acid from the reaction mixture.

The silane compound may be a compound represented by the following general formula (1), but is not limited thereto.

[Chemical Formula 1]

Wherein R ¹ to R ³ may be the same or different and are any one of C 1 to C 4 alkyl or C 1 to C 4 alkoxy, R ⁴ is amino (C 1 to C 10) alkyl, 3- (2 amino (C1 to C4) alkylamino] (C1 to C4) alkyl or 3- [2- (2-amino (C1 to C4) alkylamino) (C1 to C4) alkylamino] (C1 to C4) alkyl.

The silane compound may be at least one selected from the group consisting of (3-aminopropyl) triethoxysilane (APTES), (3-aminopropyl) trimethoxysilane, (1-aminomethyl) triethoxysilane, (2-aminoethyl) triethoxysilane, (4-aminobutyl) triethoxysilane, (5-aminopentyl) triethoxysilane, (6-aminohexyl) triethoxysilane, 3-aminopropyl (diethoxy) methylsilane 3-aminopropyl (diethoxy) methylsilane; APDMS), N- [3- (trimethoxysilyl) propyl] ethylenediamine, N- [3- (trimethoxysilyl ) Propyl] diethylenetriamine, [3- (2-aminoethylamino) propyl] trimethoxysilane ([3- (2-aminoethylamino) propyl] trimethoxysilane; AEAPTMS) and 3 - [(tri (Trimethoxysilyl) propyl] diethylenetriamine; TMPTA), but it is not limited to these.

The present invention also relates to a process for preparing a mixture by adding zinc nitrate hexahydrate and hexadecyltrimethylammonium bromide to water; A second step of heating the mixture at 85 to 95 캜 for 30 to 80 minutes to prepare a reaction mixture; And a third step of adding a solution of ammonium hydroxide to the reaction mixture to prepare a colloidal solution.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example 1: Chemicals and reagents

All reagents were analytically graded and used without purification. Zinc nitrate hexahydrate (Zn NO ₃ * 6H ₂ O, 98%), ammonium hydroxide solution (28% NH ₃ in H ₂ O, 99.99% trace metals), (3-aminopropyl (3-aminopropyl) triethoxysilane (APTES, 98%) was purchased from Sigma-Aldrich (St Louis, MO, USA). Hexadecyltrimethylammonium bromide (CTAB) (C ₁₉ H ₄₂ BrN,> 98%) was purchased from Tokyo Chemical Industry. Commercial zinc oxide nanoparticles (dispersion, 20 wt% in size less than 40 nm in H ₂ O) were used as controls. To design a new nucleic acid extraction system, biomaterials of diatomaceous earth were purchased from Sigma-Aldrich. In addition, Milli-Q water, 99% ethyl alcohol, phosphate buffered saline (PBS, 10x, pH 7.4) and streptavidin conjugated magnetic beads (Dynabeads® MyOne ™ Streptavidin C1) Respectively.

Example 2: Experimental apparatus

The morphology of the sample was analyzed using field-emission scanning electron microscopy (FE-SEM, JEOL JSM-7500F). X-ray diffraction (XRD, Scintag-SDS 2000) was performed at 40 kV voltage and 30 mA current in a continuous scan 2θ mode to analyze the crystal structure of the zinc oxide nanomaterial, (Fourier transform infrared (FT-IR) spectroscopy, Nicolet 6700) was performed to analyze the fluorescence intensity. Raman measurements were performed using Renishaw, a Via Raman microscope system (Renishaw, UK). For the DNA extraction, a commercial QIAamp DNA / RNA mini kit (Spin Colum) was used. A centrifuge (CF-5, 100 to 240 Vas, 50/60 Hz, 8 W), Vortex Mixer (T5AL, 60 Hz, 30 W, 250 V) and MSH- . The Ariamx real-time PCR system (Agilent technologies), the Gene Amp PCR system 9700 (LSK), the Submerge-Mini, the gel documentation system and the Nanodrop 2000 (PeqLab) Respectively.

Example 3: Cells and Pathogens

The cells used in the experiment were HCT116 (ATCC CCL-247), a colon cancer cell line. Brucella ovis (ATCC 25840), Escherichia coli (E coli, ATCC 25922), Staphylococcus aureus Staphylococcus aureus (S. aureus) and Bacillus cereus (B cereus) were used.

Example 4 Synthesis and Morphological Analysis of Zinc Oxide Nanostar

Zno nanostar (Zno NS) crystals were synthesized by hydrothermal method in alkaline medium.

1 M zinc nitrate hexahydrate 1 ml and 1 M CTAB 1 ml were added to the flask containing 98 ml of Milli-Q water in the proper order and stirred (500 rpm) while heating at 90 占 폚 for 50 minutes. Then, under stable stirring conditions, 2 ml of ammonium hydroxide solution was added dropwise to the reaction mixture and stirred for several minutes until a milky colloidal solution was formed.

The reaction temperature, reaction time and stirring speed were controlled for uniform production of zinc oxide nanostars. To control the production of zinc oxide nanostars, the reaction flask of the ice box was immediately taken out and centrifuged, dried at room temperature and washed with Milli-Q water. All syntheses were carried out without any special treatment, and finally the samples were stored in ethanol (99%).

1, (A) zinc oxide nanostar has a particle size of 200 to 600 nm on average and shows a particle shape in which a plurality of sharp projections are annularly arranged, and (B) zinc oxide nanoparticles have an average of 20 ≪ / RTI > to 60 nm and exhibits an irregular spherical particle morphology.

Also, referring to FIG. 2, the structures of zinc oxide nanostars and zinc oxide nanoparticles are different from each other by the Raman technique.

Therefore, the zinc oxide nanostructure of the present invention has a particle size larger than that of the conventional zinc oxide nanoparticles and has a particle shape in which a plurality of sharp projections are annularly arranged to more easily dissolve cell membranes and nuclei of pathogenic bacteria Respectively.

Example 5: Solubility test of zinc oxide nanostar

The commercialization kit was used for comparative analysis of dissolution characteristics of zinc oxide nanostars.

First, in order to dissolve the sample (brucellosis), 1.5 ml of the sample was added with AL buffer, and incubated at 56 ° C for 10 minutes for DNA extraction, followed by incubation at room temperature for 1 minute for RNA extraction. The prepared sample was transferred to a Qiagen column, and washing and elution steps were performed.

On the other hand, in the present invention, conditions for extracting high quality nucleic acid in a large amount by using zinc oxide nanostar instead of commercial AL buffer were optimized, and the extraction method was simplified.

The amount and purity of the extracted nucleic acid was measured with Ariamx real-time PCR system, Gene Amp PCR system 9700, electrophoresis apparatus, electrophoresis gel recording apparatus and Nanodrop 2000.

FIG. 3 and the following Table 1 show the results of using the conventional nucleic acid extraction kit (Qiagen) + lysis buffer, zinc oxide nanoparticles + dissolution buffer unused, zinc oxide nanostar + dissolution buffer unused, conventional nucleic acid extraction kit As a result of comparing the solubility of the nucleic acid extracted under the unused condition of (Qiagen) + lysis buffer, it was confirmed that the nucleic acid extraction of the zinc oxide nanostar of the present invention was the most excellent.

Cq (DELTA R)	Tm Product 1 (-R '(T))	10 ^ 5 Brucella (RT)
23.74	81.5	Qiagen + lysis buffer
25.38	81.5	Zinc oxide nanoparticles (RT)
21.5	81.5	Zinc oxide nanostar (RT)
25.45	81	Qiagen
35.4	83.5	Negative control group

Also, dissolution characteristics of zinc oxide nanostar were comparatively analyzed using Brucella, Escherichia coli, Staphylococcus aureus and Bacillus cereus as in the above method.

As a result, referring to FIG. 4, it was confirmed that the zinc oxide nanostar of the present invention was superior to the conventional nucleic acid extraction kit in solubility and nucleic acid extraction in all four kinds of bacteria.

Further, FIG. 5 is an analysis of whether the zinc oxide nanostar of the present invention is capable of nucleic acid extraction by grafting into a conventional nucleic acid extraction kit (Qiagen) column. As a result of comparing nucleic acid extraction efficiencies with different conditions as shown in Table 2 below, it was confirmed that zinc oxide nanostar could efficiently extract nucleic acid even at room temperature without heating.

	A1	A2	B1	B2	C1	C2
Nucleic acid extract composition	Qiagen	Qiagen	Qiagen + zinc oxide nanostar	Qiagen + zinc oxide nanostar	Qiagen	Qiagen
Dissolution buffer	use	use	unused	unused	unused	unused
Temperature (℃)	56	Room temperature	56	Room temperature	56	Room temperature
Time (minutes)	10	10	10	10	10	10

Example 6: Validation of solubility of functionalized diatomite and optical zinc oxide nanostar for single tube nucleic acid extraction

To further analyze the dissolution characteristics of zinc oxide nanostars, a single tube nucleic acid extraction system was used, which was performed with reference to a previous paper (Biosensors and Bioelectronics 99 (2018) 443-449.).

Briefly, (1) the diatomite pore structure can accommodate a variety of molecules, and the high density silanol groups on the walls are beneficial in linking with functional amine groups, so the washed diatomite is functionalized with APTES. The detailed experimental procedure is as follows.

i) 2 ml of APTES was added dropwise to 100 ml of a 95% ethanol solution and stirred at 400 rpm for 3 minutes at room temperature.

ii) 500 mg of washed diatomite was dispersed in the solution at 600 rpm for 4 hours.

iii) After the reforming, the precipitate was washed twice with ethanol to remove the free silanol.

iv) centrifuged to obtain diatomaceous earth functionalized with APTES and dried overnight at room temperature under vacuum.

v) Finally, diatomite functionalized with APTES was dispersed in distilled water at a concentration of 50 mg / ml.

The characteristics of APTES - functionalized diatomite and pure diatomite were analyzed by field emission scanning electron microscopy, Fourier transform infrared spectroscopy and Raman microscopy system.

(2) Based on the modification, nucleic acid was extracted by adding diatomaceous earth functionalized with APTES and an imidoester crosslinking agent (Dimethyl suberimidate. 2 HCl, DMS) to a 5 ml tube containing the prepared biological sample. The detailed experimental procedure is as follows.

i) 100 μl of the sample solution was added to a 1.5 ml tube containing 10 μl of protease K and 10 μl of a lysis buffer (M-SDS lysis buffer or zinc oxide nanostar solution), mixed using a pipette, Min. ≪ / RTI > 10 [mu] l of DNase was added for RNA extraction.

ii) After adding 2 mg / ml of diatomaceous earth-APTES, 100 占 of a 100 mg / ml DMS solution was added.

iii) After mixing, the cells were incubated at 56 ° C for 10 minutes for M-SDS lysis or mixed with a pipette for zinc oxide nanostar dissolution and cultured at room temperature for 2 minutes.

iv) After incubation, the supernatant was removed and the precipitate was washed twice with 200 μl of PBS.

v) and finally, the addition of elution buffer _{(pH ~ 10.6 NaHCO 3) 60} ㎕ and incubated for 1 min at room temperature.

vi) After centrifugation, the supernatant was transferred to a 1.5-ml tube and the extracted DNA or RNA was stored at -20 ° C.

The amount and purity of the extracted nucleic acid was measured with Ariamx real-time PCR system, Gene Amp PCR system 9700, electrophoresis apparatus, electrophoresis gel recording apparatus and Nanodrop 2000.

6 shows that the zinc oxide nanostar of the present invention is a diatomaceous earth functionalized with APTES and a homobifunctional imidoester [DMA (Dimethyl adipimidate), Dimethyl pimelimidate (DMP) (DMS) or dimethyl 3,3'-dithiobispropionimidate (DTBP)], as shown in Table 1 below.

The zinc oxide nanostar of the present invention was grafted together with diatomite and DMS functionalized with APTES and the nucleic acid extraction efficiency was compared according to the conditions as shown in Table 3. As a result, Extraction was possible. In addition, since it does not use a lysis buffer, it can be applied to the detection technique through the final PCR by giving less damage to the nucleic acid.

	Qiagen	A1	A2	A3	B1	B2	B3
Dissolution buffer	use	use	Zinc oxide nanostar	unused	use	Zinc oxide nanostar	unused
Temperature (℃)	56	56	56	56	Room temperature	Room temperature	Room temperature
Time (minutes)	10	10	10	10	2	2	2

In addition, referring to FIG. 7 and Table 4, the extraction efficiency of the nucleic acid according to the particle size was compared, and the extraction efficiency of the nucleic acid of the zinc oxide nanostar having the particle size of 350 to 900 nm was the best, Zinc oxide nanostar with particle size of.

Cq (DELTA R)	Tm Product 1 (-R '(T))	HCT 116 10 ^ 4 / ml 100 [mu] l (60 [mu] l DNA)
26.63	85	~ 20 nm zinc oxide nanoparticles
26.2	85	~ 100 nm zinc oxide nanostar
25.75	90	~ 350 nm zinc oxide nanostar
25.21	85.5	~ 500 nm zinc oxide nanostar
25.25	85.5	~ 900 nm zinc oxide nanostar
27.21	85	~ 1000 nm zinc oxide nanostar
30.78	85	Negative control group

Example 7: Nucleic acid extraction method using zinc oxide nanostar

7-1. Synthesis of zinc oxide nanostar

Zinc oxide nanostar was synthesized by hydrothermal method on alkaline medium.

i) 1 ml of 1 M zinc nitrate hexahydrate and 1 ml of 1 M CTAB were added to a flask containing 98 ml of Milli-Q water in the proper order and stirred (500 rpm) while heating at 90 占 폚 for 50 minutes.

ii) Subsequently, under stable stirring conditions, 2 ml of ammonium hydroxide solution was added dropwise to the reaction mixture and stirred for several minutes until a milky colloidal solution was formed.

The reaction temperature, reaction time and stirring speed were controlled for uniform production of zinc oxide nanostars.

iii) To control the production of zinc oxide nanostar, the reaction flask of the ice box was immediately taken out, centrifuged, dried at room temperature and washed with Milli-Q water. All syntheses were carried out without any special treatment, and finally the samples were stored in ethanol (99%).

7-2. Nucleic acid extraction method using zinc oxide nanostar and Qiagen kit

i) The zinc oxide nanostar solution was added to a 1.5 ml tube containing 100 占 퐇 of brucellosis (CFU: 10 ^ 5 / ml), and gently mixed with a pipette within 10 minutes at room temperature.

ii) Next, the prepared sample was transferred to a Qiagen column, washed, and then the DNA was eluted with 60 Q of Qiagen elution buffer.

7-3. Nucleic acid extraction method using zinc oxide nanostar, diatomaceous earth-APTES and imidoesters

i) 100 μl of Brucella strain was added to a 1.5 ml tube containing 10 μl of protease K and 10 μl of a lysis buffer (M-SDS lysis buffer or zinc oxide nanostar solution), mixed using a pipette, Min. ≪ / RTI > 10 [mu] l of DNase was added for RNA extraction.

ii) Thereafter, diatomite-APTES 2 mg / ml (50 mg / ml, 40 [mu] l) was added and then 100 [mu] l of a 100 mg / ml DMS solution was added. After mixing, the cells were incubated at 56 ° C for 10 minutes for M-SDS lysis, or mixed with a pipette for zinc oxide nanostar dissolution and cultured at room temperature for 2 minutes.

iii) After incubation, the supernatant was removed and the precipitate was washed twice with 200 [mu] l PBS.

iv) and finally, the addition of an elution buffer solution _{(pH ~ 10.6 NaHCO 3) 60} ㎕ and incubated for 1 min at room temperature.

7-4. Real-time PCR for DNA extraction efficiency analysis

Was performed in a 96 well plate (96 Well Plate) using 5 [mu] l of template (NA).

i) 20 μl of master mix was dispensed into each well of a 96-well plate using a multi-channel pipet.

ii) Add 5 μl of template (NA) to the master mix and mix well with a pipette.

iii) The optical strip lid was placed in the well, being careful not to cross-contaminate and stain the surface of the lid.

iv) The 96-well plate was centrifuged at 2000 rpm for 1 minute.

v) Real-time PCR was performed under the following conditions by placing a 96-well plate in the correct orientation on a qPCR machine.

   Step 1: 95 ° C, 10 min

   Step 2: 95 캜, 10 seconds; 60 캜, 20 seconds; 72 ° C, 20 seconds (40 to 45 cycles)

   Step 3: 72 ° C, 10 min

vi) Data analysis was performed.

Example 8: Solubility test of zinc oxide nanostar using eukaryotic cells

In order to verify the usefulness of zinc oxide nanostars in eukaryotic cells, HCT116 cells, a colon cancer cell line, were serially diluted to a concentration of 1 to 10 ⁴ cells / 100 μl, and then zinc oxide nanostar, zinc oxide nano- And extraction efficiency of DNA and RNA was analyzed using a conventional nucleic acid extraction kit (Qiagen).

As a result, referring to FIG. 8, it was confirmed that DNA and RNA were detected up to 1 cell / ml when the zinc oxide nanostructure of the present invention was used under optimized conditions. The detection limits of zinc oxide nanostars were 100 times higher for DNA and 10 times higher for RNA than conventional nucleic acid extraction kits.

In addition, comparing the cell solubility of zinc oxide nanostars and zinc oxide nanoparticles, it was confirmed that zinc oxide nanostars were able to detect DNA and RNA 1 to 1.5 cycles earlier than zinc oxide nanoparticles (20 nm) .

In addition, zinc oxide nanostar extracts nucleic acids at a higher concentration and purity than conventional nucleic acid extraction kits, and even in a small number of cells, high concentrations and high purity (3.5 ± 2.3 ng / μl and 1.73 ± 0.26 at 10 ¹ cells / ).

Zinc oxide nanostars enable rapid cell lysis at room temperature without the use of lysis buffers in various types of cells, making them useful as diagnostic systems in clinical settings.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims (17)

1. A composition for a pathogen comprising a zinc oxide nanostar.
2. The antifungal composition according to claim 1, wherein the zinc oxide nanostar has an average particle diameter of 200 to 900 nm.
3. The antifungal composition according to claim 1, wherein the pathogen is a microorganism.
4. [Claim 5] The composition according to claim 3, wherein the microorganism is a virus, a bacterium, a fungus, a protozoa, a ricketta or a spiroheta.
5. 8. A kit for a pathogen, comprising the composition of any one of claims 1 to 4.
6. Contacting the zinc oxide nanostar with a sample containing the pathogen.
7. 7. The method according to claim 6, wherein the sample containing the pathogen is selected from the group consisting of feces, urine, tears, saliva, external secretion of the skin, external secretion of the respiratory tract, external secretion of the intestinal tract, Wherein the method is any one selected from the group consisting of plasma, serum, blood, spinal fluid, lymph fluid, body fluids, and tissues.
8. [Claim 7] The method according to claim 6, wherein the pathogen is a microorganism.
9. 9. The method according to claim 8, wherein the microorganism is a virus, a bacterium, a fungus, an insect, a ricketta or a spiroheta.
10. A composition for extracting nucleic acid comprising zinc oxide nanostar.
11. [Claim 11] The composition for extracting nucleic acid according to claim 10, wherein the zinc oxide nanostar has an average particle diameter of 200 to 900 nm.
12. 11. The nucleic acid extracting composition according to claim 10, wherein the nucleic acid is DNA or RNA.
13. A kit for nucleic acid extraction comprising the composition of any one of claims 10 to 12.
14. A first step of adding a zinc oxide nanostar to a nucleic acid sample and reacting to prepare a mixture; And

    And a second step of extracting nucleic acid from the mixture.
15. A first step of adding a zinc oxide nanostar to a nucleic acid sample and reacting to prepare a mixture;

    Adding diatomite modified with a silane compound to the mixture and adding the mixture to a mixture of dimethyl adipimidate (DMA), dimethylpimelimidate (DMP), dimethyl suberimidate (DMS) and dimethyl A second step of preparing a reaction mixture by adding at least one selected from the group consisting of 3,3'-dithiobispropionimidate (DTBP), 3,3'-dithiobispropionimidate (DTBP); And

    And a third step of extracting nucleic acid from the reaction mixture.
16. The nucleic acid extracting method according to claim 15, wherein the silane compound is a compound represented by the following formula (1)

    [Chemical Formula 1]

    

    Wherein R ¹ to R ³ may be the same or different and are any one of C 1 to C 4 alkyl or C 1 to C 4 alkoxy, R ⁴ is amino (C 1 to C 10) alkyl, 3- (2 amino (C1 to C4) alkylamino] (C1 to C4) alkyl or 3- [2- (2-amino (C1 to C4) alkylamino) (C1 to C4) alkylamino] (C1 to C4) alkyl.
17. The method of claim 16, wherein the silane compound is selected from the group consisting of (3-aminopropyl) triethoxysilane (APTES), (3-aminopropyl) trimethoxysilane, (1-aminomethyl) triethoxysilane, (2-aminoethyl) triethoxysilane, (4-aminobutyl) triethoxysilane (( 4-aminobutyl) triethoxysilane, (5-aminopentyl) triethoxysilane, (6-aminohexyl) triethoxysilane, 3-aminopropyl 3-aminopropyl (diethoxy) methylsilane (APDMS), N- [3- (trimethoxysilyl) propyl] ethylenediamine, N- [3 Propyl] diethylenetriamine), [3- (2-aminoethylamino) propyl] trimethoxysilane ([3- (2- aminoethylamino) propyl] trimethoxysilane; AEAPTMS) and 3 - [(trimethoxysilyl) propyl] diethylenetriamine (3 - [(trimethoxysilyl) propyl] diethylenetriamine].

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