OA18785A - Quencher containing water soluble polymerconjugated nanomaterial and use thereof. - Google Patents
Quencher containing water soluble polymerconjugated nanomaterial and use thereof. Download PDFInfo
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- OA18785A OA18785A OA1201800123 OA18785A OA 18785 A OA18785 A OA 18785A OA 1201800123 OA1201800123 OA 1201800123 OA 18785 A OA18785 A OA 18785A
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- nucleic acid
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- 239000002086 nanomaterial Substances 0.000 title claims abstract description 47
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title description 4
- 239000000463 material Substances 0.000 claims abstract description 84
- 239000000523 sample Substances 0.000 claims abstract description 73
- 239000000203 mixture Substances 0.000 claims abstract description 72
- 229920003169 water-soluble polymer Polymers 0.000 claims abstract description 21
- 150000007523 nucleic acids Chemical class 0.000 claims description 36
- 108020004707 nucleic acids Proteins 0.000 claims description 27
- 229920002224 Peptide nucleic acid Polymers 0.000 claims description 24
- OKTJSMMVPCPJKN-UHFFFAOYSA-N carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 19
- 239000002202 Polyethylene glycol Substances 0.000 claims description 16
- 229920001223 polyethylene glycol Polymers 0.000 claims description 16
- 229910021389 graphene Inorganic materials 0.000 claims description 14
- 229920002873 Polyethylenimine Polymers 0.000 claims description 11
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- 108090000623 proteins and genes Proteins 0.000 claims description 6
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- 239000000126 substance Substances 0.000 claims description 5
- OMDQUFIYNPYJFM-XKDAHURESA-N (2R,3R,4S,5R,6S)-2-(hydroxymethyl)-6-[[(2R,3S,4R,5S,6R)-4,5,6-trihydroxy-3-[(2S,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]methoxy]oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@H](O)[C@H](O)O1 OMDQUFIYNPYJFM-XKDAHURESA-N 0.000 claims description 4
- 229920001661 Chitosan Polymers 0.000 claims description 4
- 229920000926 Galactomannan Polymers 0.000 claims description 4
- PYWVYCXTNDRMGF-UHFFFAOYSA-N Rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims description 4
- MPLHNVLQVRSVEE-UHFFFAOYSA-N Texas Red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 claims description 4
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- 229920000615 alginic acid Polymers 0.000 claims description 4
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 4
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- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 claims description 2
- 229920001817 Agar Polymers 0.000 claims description 2
- XJKJWTWGDGIQRH-BFIDDRIFSA-N Alginic acid Chemical compound O1[C@@H](C(O)=O)[C@@H](OC)[C@H](O)[C@H](O)[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](C)[C@@H](O)[C@H]1O XJKJWTWGDGIQRH-BFIDDRIFSA-N 0.000 claims description 2
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- 230000001808 coupling Effects 0.000 claims description 2
- 238000010168 coupling process Methods 0.000 claims description 2
- 238000005859 coupling reaction Methods 0.000 claims description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 2
- 229920002674 hyaluronan Polymers 0.000 claims description 2
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- MAKUBRYLFHZREJ-JWBQXVCJSA-M sodium;(2S,3S,4R,5R,6R)-3-[(2S,3R,5S,6R)-3-acetamido-5-hydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4,5,6-trihydroxyoxane-2-carboxylate Chemical compound [Na+].CC(=O)N[C@@H]1C[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](C([O-])=O)O[C@@H](O)[C@H](O)[C@H]1O MAKUBRYLFHZREJ-JWBQXVCJSA-M 0.000 claims description 2
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims description 2
- WHGYBXFWUBPSRW-FOUAGVGXSA-N β-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims description 2
- 230000021615 conjugation Effects 0.000 claims 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 claims 1
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- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 6
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- OBYNJKLOYWCXEP-UHFFFAOYSA-N 2-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9-yl]-4-isothiocyanatobenzoate Chemical compound C=12C=CC(=[N+](C)C)C=C2OC2=CC(N(C)C)=CC=C2C=1C1=CC(N=C=S)=CC=C1C([O-])=O OBYNJKLOYWCXEP-UHFFFAOYSA-N 0.000 description 2
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- VZJVWSHVAAUDKD-UHFFFAOYSA-N Potassium permanganate Chemical compound [K+].[O-][Mn](=O)(=O)=O VZJVWSHVAAUDKD-UHFFFAOYSA-N 0.000 description 2
- 238000001069 Raman spectroscopy Methods 0.000 description 2
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- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
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Abstract
The present invention relates to a quencher containing water-soluble polymer-conjugated nanomaterial and a use thereof. The quencher containing water-soluble polymer-conjugated nanomaterial effectively quenches the fluorescence of a fluorescent material-conjugated probe. In addition, a composition including the quencher and a fluorescent material-conjugated probe can detect a target material existing at a low concentration, and thus can be favorably used as a composition or kit for providing information necessary for the detection of a biomaterial or the diagnosis of a disease.
Description
[DESCRIPTION]
[invention Titlel
QUENCHER CONTAINING WATER-SOLUBLE POLVMERCONJUGATED NANOMATERIAL AND USE THEREOF
[Technical Field]
The present invention relates to a quencher containing a water-soluble polymer-conjugated nanomaterial and a use thereof.
[Background Art]
Methods for detecting a spécifie nucleic acid (DNA or RNA) or protein are critical techniques in the field of scientific research. As a spécifie nucleic acid or protein can be detected, researchers were able to détermine which genetic or biological marker is a marker indicating the health condition of a human. According to such methods for detecting a nucleic acid or protein, modification of a pathogenic gene present in a sample or the expression of a spécifie gene present in a sample may be found.
However, an organic or inorganic polymer, such as graphene oxide, having a planar structure filled with a two-dimensional lattice and dérivatives thereof can transfer électrons. Such électron transfer is caused by physical properties such as the crystalline and lattice structures of the polymer. Particularly, the fluorescent signal of an organic fluorescent dye may be quenched by fluorescence résonance energy transfer (FRET).
Therefore, a change in the fluorescent signal of a fluorescent dye-conjugated nucleic acid polymer may be observed using the physical properties of a material such as graphene oxide, and thus a biomaterial such as a nucleic acid or a protein may be detected (Korean Patent Publication No. 10-1496671). In addition, a method for detecting the activity of a fluorescence analysis-based enzyme using such a material was developed (Korean Patent Publication No. 10-1554173). Recently, techniques using such a material hâve been used to observe a gene or protein of a pathogen present in a sample, and therefore hâve become significant in the initial research on a disease and an illness.
However, while such a détection method using graphene oxide is simple, it can be greatly influenced by an amount of a target material included in a sample, and since the size distribution of nucleic acid polymers used in détection is not uniform, an error range is wide. In addition, in an in vitro environment for in vitro diagnosis, the polymers are instable, and thus reproducibility is reduced.
[Disclosure]
[Technical Problem]
It is an object of the present invention to provide a quencher containing water-soluble polymer-conjugated nanomaterial and a composition including the quencher and a fluorescent material-conjugated probe. In addition, it is another object of the present invention to provide a method for providing information necessary for the diagnosis of a disease using the composition.
[Technical Solution]
To achieve the objects, the présent invention provides a quencher containing a water-soluble polymer-conjugated nanomaterial.
In addition, the présent invention provides a composition including the quencher and a fluorescent material-conjugated probe.
In addition, the présent invention provides a method for providing information necessary for the diagnosis of a disease, the method including: preparing a mixture by mixing the composition with an isolated sample; measuring a fluorescence level of the mixture; and comparing the resulting level with a fluorescence level of a normal control sample.
In addition, the présent invention provides a kit including the quencher and a fluorescent material-conjugated probe.
[Advantageous Effectsl
A quencher containing a water-soluble polymer-conjugated nanomaterial of the présent invention effectively quenches the fluorescence emitted from a fluorescent material-conjugated probe. In addition, the water-soluble polymerconjugated nanomaterial is stably bound with the fluorescent material-conjugated probe. Moreover, in the presence of a target material, as the probe is bound to the target material, the probe can be easily released from the water-soluble polymerconjugated nanomaterial, and thus the target material can be effectively detected. Therefore, a composition including the quencher and the fluorescent materialconjugated probe can also detect a target material présent at a low concentration. For this reason, the composition can be effectively used as a composition or kit for providing information necessary for the détection of a biomaterial or the diagnosis of a disease.
[Description of Drawings]
FIG. I is a schematic diagram illustrating a method for preparing a watersoluble polymer-conjugated nanomaterial and a method for detecting a biomaterial using the material prepared thereby.
FIG. 2 is an atomic force microscope (AFM) image of a graphene oxide nanocolloid (GON) prepared in an example.
FIG. 3 includes scanning-transmission électron microscope (STEM; a) and AFM (b) images of two-dimensional nanomaterial, manganèse dioxide, prepared in an example.
FIG. 4 is an AFM image of nano graphene oxide (NGO) prepared in an example.
FIG. 5 includes an AFM image (a) and a graph representing Raman spectra (b) of GON which is surface-modified with dextran (DReGON).
FIG. 6 is an AFM image of NGO which is surface-modified with polyethylene glycol (PEG-NGO; a), or GON which is surface-modified with PEG and polyethyleneimine (PEI) (PEG-PEI-GON; b).
FIG. 7 is a graph representing a fluorescent signal emitted from a mixture of DReGON and PNA-US5-2 (a) or PNA-DENV (b) as a probe, to confirm whether a détection composition is formed or not.
FIG. 8 is a graph representing a fluorescent signal emitted from a mixture of PEG-NGO and PNA-Sa (a) or PNA-Pa (b) as a probe, to confirm whether a détection composition is formed or not.
FIG. 9 is a graph representing a fluorescent signal emitted from a mixture of PEG-PEI-GON and PNA-TS as a probe, to confirm whether a détection composition is formed or not.
FIG. 10 includes diagrams for comparing the fluorescence stability of GON (a) with that of DReGON (b).
FIG. 11 is a graph for confirming the target material détection capability of DReGON and GON .
FIG. 12 is a graph for confirming the détection capability of PEG-NGO when miR-21 (a) or miR-223 (b) is added as a target material at various concentrations.
FIG. 13 is a graph for confirming the détection capability of PEG-PEI-GON when miR-TS is added as a target material at various concentrations.
FIG. 14 includes images for confirming that no fluorescent signal is detected when a probe is not added to various cancer cell lines.
FIG. 15 includes images for confirming the cancer cell détection capability of DReGON mixed with PNA484 or PNA31 as a probe in various cancer cell lines.
FIG. 16 includes graphs for confirming the blood cell détection capability of DReGON mixed with PNA21, PNA223, Let-7a or a mixture thereof (scrambled) as a probe.
FIG. 17 is a graph of fluorescence levels shown after various concentrations of a fluorescence-labeled nucleic acid sequence specifically binding to Pseudomonas aeruginosa (a) or Staphylococcus aureus (b) are mixed with PEG-NGO, and mixed with Pseudomonas aeruginosa or Staphylococcus aureus.
FIG. 18 is a graph of fluorescence levels shown after a nucleic acid sequence specifically binding to human cytomégalovirus (a) or dengue virus (b) is mixed with DReGON, and mixed with human cytomégalovirus or dengue virus.
[Modes of the Invention]
Hereinafter, the présent invention will be described in detail.
In one aspect, the présent invention provides a quencher containing a watersoluble polymer-conjugated nanomaterial.
The term “water-soluble polymer” used herein refers to a resin or polymer that can be dissolved in water or dispersed as fine particles in water. The watersoluble polymer may be a natural polymer, semi-synthetic polymer or synthetic polymer. The water-soluble polymer that can be used in the présent invention may hâve a molecular weight of l to 20 kDa, 5 to 15 kDa or 8 to 12 kDa. In one embodiment, the molecular weight of the water-soluble polymer may be 10 kDa.
The water-soluble polymer may be selected from the group consisting of chitosan and a dérivative thereof, a chitosan sait, dextran and a dérivative thereof, hyaluronic acid and a dérivative thereof, a hyaluronate, pectin and a dérivative thereof, pectin sait, an alginate and a dérivative thereof, alginic acid, agar, a galactomannan and a dérivative thereof, a galactomannan sait, xanthan and a dérivative thereof, a xanthan sait, β-cyclodextrin and a dérivative thereof, a βcyclodextrinate, polyethylene glycol (PEG), polyethyleneimine (PEI) and a combination thereof. In an embodiment, the water-soluble polymer may be selected from the group consisting of dextran, polyethylene glycol, polyethyleneimine and a combination thereof.
The term “nanomaterial” used herein refers to a nano-scale material. Due to a small size, the nanomaterial may easily pass through a cell membrane. The nanomaterial may be in a sheet or particle shape. The sheet may consist of a single layer or multiple layers. In addition, the sheet conformation may include a fiat or curved surface, and may be present in various forms. In one embodiment, the nanomaterial may be a two-dimensional single-layer sheet-type nanomaterial. In addition, the particle shape may include various forms such as a sphere, an oval, a rod, and a polygon.
A particle size of the nanomaterial may be approximately 10 to 500, 10 to 200, 10 to 150, 10 to 100, 10 to 50, 20 to 200, 20 to 150, 20 to 100, 20 to 50, 30 to 200, 30 to 150, 30 to 100, 30 to 50, 50 to 200, 50 to 150, 50 to 100, 50 to 80, 60 to 200, 60 to 100, 60 to 80, 80 to 200, 80 to 150, 80 to 100, 90 to 200, 90 to 150, or 90 to 100 nm, but the present invention îs not limited thereto. In one embodiment of the present invention, the particle size of the nanomaterial is preferably 50 to 80, 90 to 200, 90 to 150 or 80 to 100 nm. Here, the particle size is an average of experimental values measured using dynamic light scattering or sizes shown in AFM or STEM images, referring to a value obtained under the assumption that the nanomaterial is spherical or circular.
The nanomaterial may be prepared as a carbon nanomaterial or manganèse dioxide. Here, the carbon nanomaterial may be selected from the group consisting of NGO and a dérivative thereof, reduced graphene oxide and a dérivative thereof, GON and a combination thereof. In one embodiment, the nanomaterial may be NGO, GON or manganèse dioxide.
The term “quencher” used herein refers to a material that absorbs fluorescence energy of a material emitting fluorescence by absorbing light or a wavelength. A quenching effect occurs due to the interaction between a fluorescent material and a nanomaterial. The quenching effect usually occurs when the fluorescent material is located a short distance, for example, approximately 10 nm or less, from the nanomaterial. Here, the fluorescent material serves as an energy donor, and the nanomaterial serves as an energy acceptor.
The quencher may be prepared by modifying the surface of a nanomaterial by a water-soluble polymer. Here, the water-soluble polymer and the nanomaterial may be conjugated by a Chemical or physical bond. The Chemical bond may be an amide bond, an ester bond or an ether bond, but the present invention is not limited thereto. In addition, the Chemical bond may be achieved through a crosslinker. In one embodiment, the water-soluble polymer and the nanomaterial may be conjugated by EDC coupling. In addition, the physical bond may be an electrostatic attraction, a hydrogen bond, or a Van deer Waals bond, but the present invention is not limited thereto. In addition, such a nanomaterial which is surface-modified with a watersoluble polymer may be improved in dispersibility, stability and biocompatibility.
In addition, in another aspect, the present invention provides a composition including the quencher and a fluorescent material-conjugated probe.
The term “probe” used herein refers to a material capable of specifically binding to a target material. The probe may be any one selected from the group consisting of an antibody, a nucleic acid, a peptide, a protein and a combination thereof. In addition, any material that is known as a material with high affinity to a target material can be used. In an embodiment, the antibody may specifically bind to an epitope of a target protein to detect the target material. In addition, when the nucleic acid has a complementary sequence to the sequence of a nucleic acid of the target material, the nucleic acid may bind to a target base sequence to detect the target material. In addition, the peptide may specifically bind to a receptor or ligand expressed on a cell surface to detect the target material.
The nucleic acid may be any one selected from the group consisting of DNA, RNA, mRNA, miRNA, non-coding RNA, double hélix RNA, double hélix DNA, a DNA-based enzyme, a deoxyribozyme, an aptamer, a peptide nucleic acid (PNA), a locked nucleic acid (LNA) and a combination thereof.
Here, while the nucleic acid may consist of 10 to 50, 10 to 30, 12 to 28, 15 to 25, or 18 to 22 bases, if the nucleic acid is capable of complementary binding to a target nucleic acid sequence, there is no lîmit to the number of bases. In one embodiment, the nucleic acid may consist of 15 to 22 bases. In one embodiment of the présent invention, the nucleic acid may be any one selected from the group consisting of SEQ ID NOs: 1 to 20.
A fluorescent material binds to the probe according to the présent invention. The fluorescent material is présent in a quenched state by absorption of fluorescence energy by a water-soluble polymer-conjugated nanomaterial, and when the probe is released from the nanomaterial due to spécifie binding to the target material, fluorescence is emitted. The fluorescent material may bind to one end or the middle of the probe. When the probe is a nucleic acid, the fluorescent material may be located at the 5’ or 3’ position of the nucleic acid or in the nucleic acid. When the probe is a peptide, the fluorescent material may bind to the N- or C-terminal of the peptide, or in the peptide. The fluorescent material may bind to the probe directly or through a crosslinker.
The fluorescent material may be selected from the group consisting of fluorescein, fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, fluorescein isothiocyanate (FITC), Oregon green, an Alexa Fluor dye, carboxyfluorescein (FAM), 6-carboxy-4’,5’-dichloro-2’,7’dimethoxyfluorescein (JOE), carboxy-X-rhodamine (ROX), 6-carboxy-2',4,4’,5’,7,7'hexachlorofluorescein (HEX), Texas red (sulforhodamine 101 acid chloride), 6carboxy-2',4,7’,7-tetrachlorofluorescein (TET), tetramethylrhodamine-isothiocyanate (TRITC), carboxytetramethylrhodamine (TAMRA), a cyanine-based dye, a thiodicarbocyanine dye, and a combination thereof. The cyanine-based dye may be selected from the group consisting of Cy3, Cy5, Cy5.5, Cy7 and a combination thereof.
The quencher may detect one or more targets. To detect one or more targets, the nanomaterial may include two or more different probes. Here, each probe may include a different fluorescent material. Here, each probe may bind a different target material so as to detect a different target material.
In addition, the composition may be used to provide information necessary for the détection of a biomaterial or the diagnosis of a disease. The disease may be cancer, an infections disease, an inflammatory disease, or a genetic disease, and in one embodiment, the disease is preferably cancer or an inflammatory disease.
The cancer may be selected from the group consisting of breast cancer, lung cancer, liver cancer, pancreatic cancer, stomach cancer, colorectal cancer, bone cancer, skin cancer, brain tumors, sarcoma, eye cancer, bone marrow cancer, blood cancer and a combination thereof. Meanwhile, the infections disease may be caused by an infection of any one selected from the group consisting of bacteria, fungi, viruses, parasites and a combination thereof. Here, the bacteria may be enterobacteria or enterococci. Examples of the bacteria include Pseudomonas aeruginosa, Staphylococcus aureus and Acinetobacter baumannii. In one embodiment of the present invention, the bacteria are preferably Pseudomonas aeruginosa or Staphylococcus aureus.
Examples of the viruses may include a double-stranded DNA virus, a singlestranded DNA virus, a double-stranded RNA virus, a positive-sense single-stranded RNA virus, a negative-sense single-stranded RNA virus, a single-stranded RNA retrovirus and a double-stranded DNA retrovirus. In one embodiment of the present invention, the virus may be selected from the group consisting of cytomégalovirus, Dengue virus and a combination thereof.
In still another aspect, the present invention provides a method for providing information necessary for the diagnosis of a disease, the method including: mixing the composition including the quencher and the fluorescent material-conjugated probe with an isolated sample; measuring a fluorescence level of the mixture; and comparing the resulting level with a fluorescence level of a normal control sample.
The quencher and the fluorescent material-conjugated probe, which are included in the composition, are as described above. In addition, the sample may be a sample isolated and discharged from a diagnosis target, and may be cells, a cell culture medium, tissue, saliva, urine, feces, semen, blood, plasma or sérum. In addition, a normal control sample refers to a sample isolated and discharged from a normal person without a disease.
The method according to the present invention may be used to detect a biomaterial such as a nucleic acid or protein, or to diagnose a disease as described above. Here, the fluorescence may be determined by measuring light emitted while being released by specifically contacting or binding a fluorescent material quenched by a nanomaterial with a target material. To measure the fluorescence level, flow cytometry, fluorescence-activated cell sorting (FACS), or a method for analyzing a fluorescent signal or an image may be used.
In yet another aspect, the present invention provides a kit including the quencher and the fluorescent material-conjugated probe. The quencher and the fluorescent material-conjugated probe, which are included in the composition, are as described above. The kit may be used to detect a biomaterial such as a nucleic acid or protein, or to diagnose a disease as described above.
Hereinafter, the present invention will be fiilly described with reference to the following examples. However, the following examples are merely provided to exemplify the present invention, and the present invention is not limited by the examples.
I. Préparation of two-dimensional nanomaterial
Example 1. Préparation of graphene oxide nanocolloid (GON) g of IGSzOg and 4 g of P4O10 were added to 50 ml of H2SO4 and dissolved while stirring. 2 g of graphite nanofiber was added to the resulting solution, and heated at 90 deg C for 16 hours. The heated mixture was cooled to room température, 250 ml of distilled water was added thereto, followed by filtration with a paper filter (Whatman-GE Healthcare, USA). The purified mixture was washed with distilled water twice or more and dried in air.
1.5 g of the dry powder-type graphite nanofiber was added to 250 ml of H2SO4, 10 g of KMnO4 was slowly added thereto, and reacted while stîrring. Here, a reaction température did not exceed 10 deg C. The reaction products were warmed up in 35 deg C distilled water for further reaction while stîrring for 6 hours. After the reaction, 1,000 ml of distilled water was added, and here, the température was maintained at 55 deg C or less. 50 ml of H2O2 was added to the mixture to allow a reaction, and the resulting mixture was centrifuged at 10,000 rpm for 30 minutes, thereby obtaining a pellet. The obtained pellet was washed with 3.4% (w/w) HCl and acetone three times or more using a centrifuge.
The acetone contained in the finally-obtained brownish supematant was removed under vacuum, and distilled water was added to the remaining solution to adjust the final concentration to 1 mg/ml, followed by vortexting for complété suspension. The collected product was purified and neutralized using a 10,000 Da dialysis membrane, and the final product was lyophilized to obtain powder-type GON. The obtained GON was observed using AFM, and the resuit is shown in FIG.
2. As shown in FIG. 2, GON with a size of 100 nm or less and a thickness of 2 nm or less was obtained.
Example 2. Préparation of manganèse dioxide (MnO2)
Distilled water was added to a solution of 32 ml of sodium dodecyl sulfate (SDS) and 1.6 ml of H2SO4 to adjust the final volume to 300 ml. The resulting solution was heated at 95 deg C for 15 minutes, and then 3.2 ml of a KMnC>4 solution was rapidly added thereto. Afterward, heating was continued for 60 minutes, and a dark brown manganèse dioxide sheet was obtained.
Triple distilled water and an alcohol were added at a volume ratio of 1:1 to the obtained manganèse dioxide sheet, and centrifuged at 12,000 rpm, thereby obtaining a pellet. In addition, centrifugation was performed twice under the same conditions to allow the obtained product to be resuspended in triple distilled water, thereby obtaining the final product. The obtained MnO2 was observed by STEM and AFM, and the resuit is shown in FIG. 3. As shown in FIG. 3b, MnOi with a size of 200 nm or less and a thickness of 2 nm or less was obtained.
Example 3. Préparation of nano graphene oxide (NGO)
0.5 g of Na2NO3 was added to 23 ml of H2SO4 and dissolved while stirring. 0.5 g of graphite nanofiber was added to the resulting solution, and then 3 g of KMnC>4 was slowly added thereto while stirring to allow a reaction. Here, a reaction température did not exceed 10 deg C. The reaction products were warmed up in 35 deg C distilled water for further reaction while stirring for 1 hour, and then were further reacted at 90 deg C for 30 minutes. After the reaction, 1 ml of distilled water was added, and the température was maîntained at 55 deg C or less. 3 ml of H2Ot was added to the reaction products to allow a reaction, cooled to room température, and then 250 ml of distilled water added thereto. After the resulting solution was filtered using a paper filter, a filtrate obtained thereby was washed with distilled water twice or more and dried in air.
To préparé graphene oxide (GO), 50 ml of 9M NaOH was added to 10 mg/ml of a graphene oxide solution, followed by tip-sonication for 90 minutes. The graphene oxide was purified and neutralized using a 3,800 Da dialysis membrane, and the final product was lyophilized to obtain powder-type NGO. The obtained NGO was observed using AFM, and the resuit is shown in FIG. 4. As shown in FIG. 4, NGO with a size of 200 nm or less and a thickness of 1.5 nm or less was obtained.
II. Préparation of two-dimensional nanomaterial surface-modified with polymer
Exaniple 4. Préparation of GON surface-modified with dextran (DReGON)
The surface of the GON obtained in Example 1 was modified with dextran. Specifically, 50 mg of the GON was suspended in 50 ml of distilled water, and a 0.1%(w/w) dextran aqueous solution was added thereto. The mixture was subjected to ultrasonication for 30 minutes, and then reacted with 25 μΙ of an aqueous ammonia solution at 95 deg C for 3 hours while stirring. The reaction product was washed with distilled water, isolated by centrifugation at 10,000 rpm for 30 minutes, and lyophilized, thereby obtaining a final product DReGON.
Raman analysis was performed using the obtained DReGON. Specifically, DReGON was put on a Silicon wafer, and then the Silicon wafer was mounted in a Raman spectrometer (LabRAM HR UV/vis/NIR), followed by spectrum analysis by irradiation of 514 nm CW laser.
The resuit of observing the obtained DReGON by AFM is shown in FIG. 5a, and the resuit of Raman spectrum analysis is shown in FIG. 5b. As shown in FIG. 5a, DReGON with a size of 100 nm or less and a thickness of 7 nm or less was obtained. Meanwhile, as shown in FIG. 5b, D and G peaks were observed at 1370 cm'1 and 1600 cm'1, respectively, and the ID/IG ratio was 0.85.
Example 5. Préparation of NGO surface-modified with polyethylene glycol (PEG)
The surface of the NGO obtained in Example 3 was modified with PEG. Specifically, 5 mg of NGO was mixed with the same amount of PEG (10 kDa), followed by bath-sonication. After adding 5 mg of 1 -ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC), the bath-sonication of the mixture was further performed for 5 minutes. After being stirred for 6 hours, the mixture was purified and neutralized using a 10,000 Da dialysis membrane, and the final product was lyophilized, thereby obtaining powder-type NGO that was surface-modified with PEG (PEG-NGO).
The obtained PEG-NGO was observed by AFM, and the resuit is shown in FIG. 6a. As shown in FIG. 6a, the PEG-NGO with a size of 200 nm or less and a thickness of 1 nm or less was obtained.
Example 6. Préparation of GON surface-modified with polyethylene glycol and polyethyleneimide (PEI)
The surface of the GON obtained in Example 1 was modified with PEG and PEI. Specifically, 20 mg of PEG (10 kDa) was added to 10 ml of a 2 mg/ml GON solution, followed by bath-sonication for 5 minutes. By adding 20 mg of EDC, the bath-sonication of the mixture was further performed for 5 minutes. The bath sonication was performed for 5 minutes each after the addition of 10 mg of PEI and EDC to the reaction product, followed by stirring at room température for 6 hours to allow uniform reactions. Afterward, the mixture was purified and neutralized using a 12,000 Da dialysis membrane, and the final product was lyophilized, thereby obtaining powder-type GON that was surface-modified with PEG and PEI (PEGPEI-GON).
The obtained PEG-PEI-GON was observed by AFM, and the resuit is shown in FIG. 6b. As shown in FIG. 6b, the PEG-PEI-GON with a size of 100 nm or less and a thickness of 10 nm or less was obtained.
ΙΠ. Confirmation of préparation of composition of two-dimensional nanomaterial surface-modified with polymer for detecting target material
Experimental Example 1. Confirmation of préparation of composition of two-dimensional nanomaterial surface-modified with polymer for detecting target material
Experimental Example 1-1. Confirmation of préparation of DReGON composition for detecting target material-(l)
To confirm whether the DReGON prepared in Example 4 can be used as a composition for detecting a target material, an experiment was carried out as follows. Specifically, a peptide nucleic acid (PNA) probe for detecting a target material was prepared by requesting Panagene (Korea) to label Cy5 at the 5’-end, and include two units, as linkers, in which two carbon atoms are bound to one oxygen atom between Cy5 and the probe sequence. 1 μΜ of the PNA probe was added to 20 μΐ of nuclease-free water, and completely dissolved by being heated at 80 deg C for approximately 3 minutes. 20 μί of the dissolved PNA probe was mixed with the DReGON prepared in Example 4, and reacted at room température for 30 minutes. Four, eight, twelve or twenty-four hours after the reaction, a fluorescence signal was measured using a fluorescence reader at Ex/Em=647/670 nm. Here, only a PNA probe was used as a control.
As a resuit, provided that the fluorescence signal of the PNA probe is 100%, when the PNA probe is reacted with a two-dimensional nanomaterial that is surfacemodified with a polymer, the fluorescence signal is reduced to less than 5%, and therefore it was confîrmed that the material forms a détection composition.
Experimental Example 1-2. Confirmation of préparation of DReGON composition for detecting target material-(2)
To confinn whether the DReGON prepared în Example 4 can be used as a composition for detecting a target material, an experiment was performed under the same conditions and in the same manner as in Experimental Example 1-1. Here, as a PNA probe, PNA-US5-2 or PNA-DENV listed in Table 2 was used. 10 pmol of the PNA probe was mixed with each of 0, 0,2, 0.4, 0.6, 0.8, 1.0, 1,2, 1.4 or 1.6 pg of DReGON. As a resuit, a value of the measured fluorescence signal is shown in FIG. 7, As shown in FIG. 7, a réduction in the fluorescence signal is dépendent on a concentration of the added PNA probe, and therefore it was confîrmed that the material forms a détection composition.
Experimental Example 1-3. Confirmation of préparation of PEG-NGO composition for detecting target material
To confirm whether the PEG-NGO prepared in Example 5 can be used as a composition for detecting a target material, an experiment was performed under the same conditions and in the same manner as in Experimental Example l - I. Here, 10 pmol of a PNA probe was mixed with each of 0, 0.1, 0.2, 0.5 or l .0 pg of PEG-NGO. As the PNA probe, one in which a Cy5 fluorescent dye is bound to PNA-Sa or PNAPa, listed in Table l below, was used. As a resuit, a value of the measured fluorescence signal is shown in FIG. 8. As shown in FIG. 8, a réduction in the fluorescence signal is dépendent on a concentration of the added PNA probe, and therefore it was confirmed that the material forms a détection composition.
Experimental Example 1-4. Confirmation of préparation of PEG-PEIGON composition for detecting target material
To confirm whether the PEG-PEI-GON prepared in Example 6 can be used as a composition for detecting a target material, an experiment was performed under the same conditions and in the same manner as in Experimental Example 1-1. 10 pmol of a PNA probe was mixed with each of 0, 0.1, 0.2, 0.5 or 1.0 pg of PEG-PEINGO. As the PNA probe, one in which an F1TC fluorescent dye is bound to PNATS listed in Table 1 below was used. As a resuit, a value of the measured fluorescence signal is shown in FIG. 9. As shown in FIG. 9, a réduction in the fluorescence signal is dépendent on a concentration of the added PNA probe, and therefore it was confirmed that the material forms a détection composition.
Experimental Example 2. Confirmation of stability of DReGON particles
To confirm the stability of the DReGON partivles prepared in Example 4, an experiment was carried out as follows. Specifically, 0.1 mg/ml of a DReGON solution was well suspended în a PBS solution containing sérum, and maintained for
0, 4, 8, 12 or 24 hours at room température, followed by measurement of absorbance over time. As a control, the GON prepared in Example l was used. As a resuit, as shown in FIG. 10, DReGON (b) showed more stable absorption spectra in a physiologically active environment for a long period of time than those of GON (a), and such stable dispersibility was retained for 24 hours or longer.
IV. Confirmation of target material detectability of two-dimensional nanomaterial surface-modified with polymer
Experimental Example 3. Confirmation of ability to detect cancer cellspecific nucleic acid sequence
Experimental Example 3-1. Confirmation of detectability of DReGON
To measure a détection limit of the DReGON prepared in Example 4, an experiment was carried out as follows. First, PNA21 and DReGON were mixed as probes and reacted under the same conditions and in the same manner as in Experimental Example 1-1. Thirty minutes after the reaction, a target material including a cancer cell-specific sequence, that is, miR-21, was added to the reaction product to hâve a concentration of 0, 0.001, 0.01, 0.1, 1, 10, 100 or 1,000 nM, and reacted at room température for 2 hours. Fluorescence signais were measured using a fluorescence reader at Ex/Em=647/670 nm. The sequences of the PNA probe and the target materials used in the experiment are listed in Tables 1 and 2 below.
[Table 1]
| Target disease | Probe | Sequence (5’—>3') | SEQ ID NO: |
| Cancer | PNA21 | TCAACATCAGTCTGATAAGCTA | SEQ ID NO: 1 |
| PNA31 | AGCTATGCCAGCATCTTGCCT | SEQ ID NO: 2 |
| PNA223 | ATTTGACAAACTGAC | SEQIDNO: 3 | |
| PNA484 | GGAGGGGACTGAGCCTG | SEQ ID NO: 4 | |
| Let-7a | AACTATACAACCTACTACCTCA | SEQ ID NO: 5 | |
| PNA-TS | CTGCCCCAAAATGCCT | SEQ ID NO: 6 | |
| Bacterial disease | PNA-Pa | GCGGCATGGCTGGATC | SEQ ID NO: 7 |
| PNA-Sa | ACAGAGTTTTACGATC | SEQ ID NO: 8 | |
| Viral disease | PNA-US5-2 | AGACATCGTCACACCTATCATA | SEQ ID NO: 9 |
| PNA-DENV | GCGTTTCAGCATATTGA | SEQ IDNO: 10 |
[Table 2]
| Target disease | Target material | Sequence (5'—>3') | SEQ ID NO: |
| Cancer | mtR-21 | UAGCUUAUCAGACUGAUGUUGA | SEQ ID NO: 11 |
| niiR-31 | AGGCAAGAUGCUGGCAUAGCU | SEQ IDNO: 12 | |
| miR-223 | GUCAGUUUGUCAAAU | SEQ IDNO: 13 | |
| miR-484 | CAGGCUCAGUCCCCUCC | SEQ ID NO: 14 | |
| Let-7a | UGAGGUAGUAGGUUGUAUAGUU | SEQIDNO: 15 | |
| miR-TS | AGGCAUUUUGGGGCAG | SEQ ID NO: 16 | |
| Bacterial disease | miR-Pa | GAUCCAGCCAUGCCGC | SEQ ID NO: 17 |
| miR-Sa | GAUCGUAAAACUCUGU | SEQ ID NO: 18 | |
| Viral disease | niiR-US5-2 | UAUGAUAGGUGUGACGAUGUCU | SEQ ID NO: 19 |
| miR-DENV | UCAAUAUGCUGAAACGC | SEQ ID NO: 20 |
Results derived by substituting changes in fluorescence before and after the addition of a target material into Equation l below are shown in FIG. 11. Here, as a control, the GON prepared in Example 1 was used.
[Equation 1]
SD
Détection Limit = 3.3(-y-) *SD: Standard déviation, S: Slope of calibration line
As shown in FIG. 11, GON detected up to 230 pM of the target material, but DReGON detected up to 10 pM of the target material. From the resuit, it was confirmed that the two-dimensional nanomaterial that was surface-modified with a polymer according to the présent invention can also detect a target nucleic acid présent at a low concentration in a sample.
Experimental Example 3-2. Confirmation of detectability of PEG-NGO
The ability of PEG-NGO to detect a cancer cell-specific nucleic acid sequence was confirmed under the same conditions and in the same manner as in Experimental Example 3-l. Here, 10 pmol of the PEG-NGO prepared in Example 5, instead of DReGON, was used, and PNA21 and PNA233 as probes and miR-2l and miR-233 as target materials were used at 0, 0.002, 0.02, 0.2, 2, 20, 200 or 2,000 nM. In addition, after the target material was added, a reaction was performed for 4 hours, a fluorescence change was measured at intervals of 20 minutes, and the resuit is shown in FIG. 12. As shown in FIG. 12, as the concentration of the target material was increased, the fluorescence signal became stronger, and fluorescence was restored to different levels according to the concentration and type of a probe.
Experimental Exaniple 3-3. Confirmation of detectability of PEG-PE1GON
The ability of PEG-PEI-NGO to detect a cancer cell-specific nucleic acid sequence was confirmed under the same conditions and in the same manner as in
Experimental Example 3-1. Here, 10 pmol of the PEG-PEI-NGO prepared in Example 6, instead of DReGON, was used, and PNA-TS as a probe and miR-TS as a target material were used at 100, 200, 300 or 500 nM. In addition, after the target material was added, a reaction was performed for 4 hours, a fluorescence change was measured at întervals of 20 minutes, and the resuit is shown in FIG. 13. As shown in FIG. 13, as the concentration of the target material was increased, the fluorescence signal became stronger, and fluorescence was restored in a different level according to the concentration and type of a probe.
Experimental Exaniple 3-4. Confirmation of detectability of DReGON in cancer cell Unes
The ability of DReGON to detect a cancer cell-specific nucleic acid sequence was confirmed using various types of cancer cell lines. First, MCF-7, HeLa and SW620 cell lines were prepared by being cultured in a DMEM or RPMI medium. However, PNA484 or PNA31 as a probe was mixed with DReGON under the same conditions and in the same manner as in Experimental Example 1-1 to allow a reaction. The prepared cells were seeded into a 12-well plate to hâve a density of IxlO5 cells per well, and after 24 hours, a reaction product of DReGON, and PNA484 or PNA31 was added to the cells at a concentration of 80 pmol.
After 14 hours, images of fluorescence signais of cells observed using a fluorescence microscope are shown in FIG. 15. Here, as a control, images taken without addition of a probe to cells are shown in FIG. 14. As shown in FIG. 15, détection of the target material by DReGON was identified through a fluorescence signal of a fluorescent material-conjugated probe.
Experimental Example 3-5. Confirmation of detectability of DReGON in blood cells
To confirm the detectability of DReGON for a spécifie base sequence présent in blood cells, an experiment was carried out as follows. Specifically, blood cells were collected from 10 ml of healthy human blood by a known method, and cultured in an RPMI medium. The cultured cells were fixed and 200 nM of a PNA probe to which 3.0 pg of DReGON and a Cy5 fluorescent dye were bound was added. Here, as a PNA probe, PNA21, PNA223, Let-7a or a mixture thereof (scrambled) was used. As a control, an untreated cell group was used.
After 4 hours, the intensity of fluorescence restored in the blood cells, detected by flow cytometry, is shown in FIG. 16. As shown in FIG. 16, it was confirmed that a target base sequence expressed in the blood cells is detected by the PNA probe.
Experimental Example 4. Confirmation of ability of PEG-NGO to detect bacteria-specific nucleic acid sequence
The ability of PEG-NGO to detect a Pseudomonas aeruginosa or Staphylococcus aurez/s-specific nucleic acid sequence was confirmed under the same conditions and in the same manner as in Experimental Example 3-l. Here, 0.2 pg of the PEG-NGO prepared in Example 5, instead of DReGON, and 10 pmol of PNAPa or PNA-Sa were added, and then 0, 10, 20, 40, 70 or 100 nM of Pseudomonas aeruginosa or Staphylococcus aureus AS-DNA was added thereto as a target material. As a resuit, the measured fluorescence change is shown in FIG. 17. As shown in FIG. 17, the intensity of fluorescence was increased depending on the concentration of the added Pseudonionas aeniginosa or Staphylococcus aureus ASDNA.
Experimental Example 5. Confirmation of ability DReGON to detect virus-specific nucleic acid sequence
The ability of DReGON to detect a human cytomégalovirus (HCMV) or dengue virus (DENV)-specific nucleic acid sequence was confirmed under the same conditions and in the same manner as in Experimental Example 3-l. Here, 0.5 pg of DReGON, and 20 pmol of PNA-US5-2 or PNA-DENV were added, and 20 pmol of miR-US5-2 or miR-DENV was added thereto as a target material. As a resuit, fluorescence changes measured over reaction time are shown in FIG. 18. As shown in FIG. 18, the intensity of fluorescence for a HCMV or DENV-specific nucleic acid sequence added depending on a reaction time was increased.
Consequently, from the results, it can be demonstrated that, unlike a microarray or RT-PCR, the composition of the present invention can easily detect a target sequence, and can be used to indirectly confirm the concentration of the target sequence.
Sequence Listing Free Text
SEQ ID NO: l : tcaacatcag tctgataagc ta
SEQ ID NO: 2: agctatgcca gcatcttgcc t
SEQ ID NO: 3: atttgacaaa ctgac
SEQ ID NO: 4: ggaggggact gagcctg
SEQ ID NO: 5: aactatacaa cctactacct ca
SEQ ID NO: 6: ctgccccaaa atgcct
SEQ ID NO: 7: gcggcatggc tggatc
SEQ ID NO: 8: acagagtttt acgatc
SEQ ID NO: 9: agacatcgtc acacctatca ta
SEQ ID NO: 10: gcgtttcagc atattga
SEQ ID NO: 11 : uagcuuauca gacugauguu ga
SEQ ID NO: 12: aggcaagaug cuggcauagc u
SEQ ID NO: 13: gucaguuugu caaau
SEQ ID NO: 14: caggcucagu ccccucc
SEQ ID NO: 15: ugagguagua gguuguauaguu
SEQ ID NO: 16: aggcauuuug gggcag
SEQ ID NO: 17: gauccagcca ugccgc
SEQ ID NO; 18: gaucguaaaa cucugu
SEQ ID NO: 19: uaugauaggu gugacgaugu eu
SEQ ID NO: 20: ucaauaugcu gaaacgc
Claims (10)
- [CLAIMS] [Claim l]A composition comprising a water-soluble polymer-conjugated nanomaterial and a fluorescent material-conjugated probe, wherein the water-soluble polymer is any one selected from the group consisting of chitosan and a dérivative thereof, a chitosan sait, dextran and a dérivative thereof, hyaluronic acid and a dérivative thereof, a hyaluronate, pectin and a dérivative thereof, a pectin sait, an alginate and a dérivative thereof, alginic acid, agar, a galactomannan and a dérivative thereof, a galactomannan sait, xanthan and a dérivative thereof, a xanthan sait, β-cyclodextrin and a dérivative thereof, a β-cyclodextrinate, polyethylene glycol (PEG), polyethyleneimine (PEI) and a combination thereof.wherein the nanomaterial has a particle size of 0.01 to l pm, and is any one selected from the group consisting of nano graphene oxide (NGO) and a dérivative thereof, reduced graphene oxide and a dérivative thereof, a graphene oxide nanocolloid (GON) and a combination thereof.
- [Claim 2]The composition of claim l, wherein the nanomaterial has a particle or sheet form.
- [Claim 3]The composition of claim l, wherein the conjugation is achieved by a Chemical or physical bond.
- [Claim 4]The composition of claim 3, wherein the Chemical bond is EDC coupling.
- [Claim 5]The composition of claim 3, wherein the physical bond is a hydrogen bond.
- [Claim 6]The composition of claim 1, wherein the probe is any one selected from the group consisting of an antibody, a nucleic acid, a peptide, a protein and a combination thereof.
- [Claim 7]The composition of claim 6, wherein the nucleic acid consists of 10 to 50 bases.
- [Claim 8]The composition of claim 6, wherein the nucleic acid is any one selected from the group consisting of DNA, RNA, mRNA, miRNA, non-coding RNA, double hélix RNA, double hélix DNA, a DNA-based enzyme, a deoxyribozyme, an aptamer, a peptide nucleic acid (PNA), a locked nucleic acid (LNA) and a combination thereof.
- [Claim 9]The composition of claim 6, wherein the nucleic acid is any one selected from the group consisting of SEQ ID NOs: 1 to 10.
- [Claim 10]The composition of claim 1, wherein the fluorescent material is selected from the group consisting of fluorescein, fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, fluorescein isothiocyanate (FITC), Oregon green, an Alex Fluor dye, carboxyfluorescein (FAM), 6-carboxy-4’,5’-dichloro-2’,7’-dimethoxyfluorescein (JOE), carboxy-X-rhodamine (ROX), 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein (HEX), Texas red (sulforhodamine 101 acid chloride), 6-carboxy-2',4,7',7-tetrachlorofluorescein (TET),
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2015-0139174 | 2015-10-02 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| OA18785A true OA18785A (en) | 2019-06-28 |
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