KR102413053B1 - Nanomaterial complex comprising dopamine-modified black phosphorus nanosheets and gold nanorods, and uses thereof - Google Patents

Abstract

The present invention relates to a nanomaterial composite comprising dopamine-bound black phosphorus nanosheets and gold nanorods, and a use thereof. It enables the detection of genes with high sensitivity. Therefore, the nanomaterial complex of the present invention has the advantage of conveniently diagnosing viral infections by amplifying viral nucleic acids anytime and anywhere without electricity supply, and is expected to be usefully utilized for the diagnosis of various viral infections in the future.

Description

Nanomaterial complex comprising dopamine-modified black phosphorus nanosheets and gold nanorods, and uses thereof

The present invention relates to a nanomaterial composite comprising dopamine-bound black phosphorus nanosheets and gold nanorods, and uses thereof.

In 2019, pneumonia of unknown cause began to occur in the form of viral pneumonia. For the pneumonia, the World Health Organization (WHO) named it Corona virus in 2019 (COVID-19), and the cause of pneumonia caused by severe acute respiratory syndrome is new coronavirus (coronavirus 2, SARS-CoV-2). SARS-CoV-2 is an enveloped virus composed of a 30kb positive single-stranded RNA genome. It consists of 4 major structural proteins (S-spike, E-envelope, M-membrane, N-nucleocapsid) and 16 non-structural proteins ( NSP 1-16). The COVID-19 virus spreads rapidly through respiratory transmission or contact, mainly through droplets, and symptoms are accompanied by high fever, cough, sore throat, and acute pneumonia and acute respiratory distress syndrome in immunocompromised patients. leads to death More than 200 million people have been infected in the 20 months since the COVID-19 virus was identified. Therefore, it is important to quickly and accurately diagnose and investigate the COVID-19 virus, which can prevent the rapid spread of COVID-19 and prevent infection in advance.

The COVID-19 virus diagnostic test developed until recently uses a real-time gene amplification test (RT-PCR) method that enables real-time monitoring based on polymerase chain reaction (PCR), and repeats the reaction cycle. It takes a lot of time for amplification to proceed. In addition, the configuration of diagnostic equipment is complicated because amplification proceeds only when the high temperature is maintained step by step, and a lot of time and skilled technology are required to optimize temperature, time, etc., so a new method for rapid and accurate diagnosis of COVID-19 Diagnostic methods are in demand.

Loop-mediated isothermal amplification (LAMP) uses a primer material capable of specific reaction with a specific region of a target gene to infinitely amplify loop-shaped DNA products and use reagents for fluorescence detection It is a method to check the degree of amplification and the presence or absence of a target gene by performing a precipitation reaction. The LAMP detection method can perform rapid diagnosis as amplification is possible under isothermal conditions through Bst polymerase, thereby solving the disadvantage of PCR detection method, the long time required due to cycle repetition, and the simple configuration of the diagnostic equipment. It is suitable for rapid diagnosis in the field.

Accordingly, the present inventors completed the present invention after studying a method for diagnosing COVID-19.

Republic of Korea Patent Registration No. 10-2173154

In order to solve the above problems and needs, the present inventors discovered a new material applicable to the LAMP diagnostic method, and confirmed that the target gene can be amplified while maintaining high temperature and isothermal conditions. The complex was completed.

Accordingly, an object of the present invention is a black phosphorus nanosheet;

dopamine; and

A nanomaterial composite comprising gold nanorods, comprising:

The black phosphorus nanosheet is one or more dopamine bound,

The gold nanorods are to provide a nanomaterial complex, characterized in that it is bound to dopamine.

Another object of the present invention is to provide beads prepared using a composition comprising the nanomaterial composite according to the present invention.

Another object of the present invention is to (a) modifying the surface of the black phosphorus nanosheet with dopamine; and

(b) to provide a method for preparing a nanomaterial composite according to the present invention, comprising the step of treating the gold nanorods.

Another object of the present invention is a loop-mediated isothermal amplification (LAMP) primer for detection of SARS-CoV-2 virus, comprising at least one selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 6 to provide a set.

Another object of the present invention is to provide a composition for detecting M gene of SARS-CoV-2 virus comprising the primer set according to the present invention.

Another object of the present invention is to isolate RNA from a sample; and

Using the RNA as a template and performing an isothermal amplification reaction using the primer set according to the present invention to amplify the target sequence; to provide an information providing method for diagnosis of coronavirus infection-19, including the.

Another object of the present invention is to provide a kit for ring-mediated isothermal amplification comprising the complex according to the present invention.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

Therefore, the present invention is a black phosphorus nanosheet (black phosphorus nanosheet);

dopamine; and

A nanomaterial composite comprising gold nanorods, comprising:

The black phosphorus nanosheet is one or more dopamine bound,

The gold nanorods provide a nanomaterial complex, characterized in that it is bound to dopamine.

In addition, the present invention provides beads prepared by using the composition comprising the nanomaterial composite according to the present invention.

In one embodiment of the present invention, the gold nanorods may be chemically bonded to a catechol functional group of dopamine, but is not limited thereto.

In another embodiment of the present invention, the gold nanorods may have an aspect ratio of 2:1 to 8:1, but is not limited thereto.

In another embodiment of the present invention, the complex may cause an exothermic reaction at a near-infrared wavelength, but is not limited thereto.

In another embodiment of the present invention, the dopamine may be bound to black phosphorus nanosheets in a polymerized form, but is not limited thereto.

In another embodiment of the present invention, the complex may be maintained in an isothermal state for 20 minutes or more, but is not limited thereto.

In addition, the present invention comprises the steps of (a) modifying the surface of the black phosphorus nanosheet with dopamine; and

(b) provides a method for preparing a nanomaterial composite according to the present invention, comprising the step of treating the gold nanorods.

In one embodiment of the present invention, the black phosphorus nanosheet may be prepared by peeling black phosphorus particles by a sonication method, but is not limited thereto.

In another embodiment of the present invention, dopamine in step (a) may be mixed with black phosphorus nanosheets in a mass ratio of 1:1 to 1:5, but is not limited thereto.

In addition, the present invention provides a loop-mediated isothermal amplification (LAMP) primer set for detection of SARS-CoV-2 virus, comprising at least one selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 6 to provide.

In addition, the present invention provides a composition for detecting the M gene of SARS-CoV-2 virus comprising the primer set according to the present invention.

In addition, the present invention provides a use of the composition comprising the primer set according to the present invention to detect M gene of SARS-CoV-2 virus.

In addition, the present invention comprises the steps of isolating RNA from a sample; and

Using the RNA as a template, and performing an isothermal amplification reaction using the primer set according to the present invention to amplify the target sequence; provides an information providing method for the diagnosis of coronavirus infection-19, including.

In addition, the present invention comprises the steps of isolating RNA from a sample; and

Using the RNA as a template, and performing an isothermal amplification reaction using the primer set according to the present invention to amplify the target sequence; provides a diagnostic method for coronavirus infection-19, including.

In one embodiment of the present invention, the isothermal amplification reaction may be performed at 61 to 71 °C, but is not limited thereto.

In addition, the present invention provides a kit for ring-mediated isothermal amplification comprising the complex or beads according to the present invention.

In one embodiment of the present invention, the kit may further include a primer set according to the present invention, but is not limited thereto.

The nanomaterial complex of the present invention is raised to an optimal temperature that can be amplified by irradiating near-infrared rays, and makes it possible to detect a target gene with high sensitivity. Therefore, the complex of the present invention has the advantage of conveniently diagnosing viral infections by amplifying viral nucleic acids anytime and anywhere, making it possible to detect viruses without huge equipment as it is portable, and can be usefully utilized for diagnosis of various viral infections in the future. is expected to

1 is a diagram showing the mechanism of detection of the M gene of SARS-CoV-2 virus according to the present invention.
2 shows the LAMP reaction optimum temperature result (A) of the primer set of the present invention (Table 1) for the M gene of SARS-CoV-2 virus and raw data (B) of the corresponding bar graph.
3 shows the fluorescence intensity (A) and the fluorescence intensity bar graph (B) according to the reaction time for each target concentration of the primer set LAMP reaction of the present invention with respect to the M gene of SARS-CoV-2 virus DMSO 0%.
4 is a bar graph (B) of the fluorescence intensity (A) and the corresponding fluorescence intensity according to the reaction time for each target concentration of the primer set LAMP reaction of the present invention for the M gene of SARS-CoV-2 virus DMSO 5% (w/w) ) is shown.
5 is a bar graph of fluorescence intensity (A) and corresponding fluorescence intensity according to the reaction time for each target concentration of the primer set LAMP reaction DMSO 7% (w/w) of the present invention for the M gene of SARS-CoV-2 virus (B) ) is shown.
6 shows the fluorescence intensity with respect to the reaction time according to the DMSO concentration of 0%, 5% or 7% (w/w) of the primer set LAMP reaction of the present invention for the M gene of SARS-CoV-2 virus.
7 is an FE-SEM image of the aspect ratio of gold nanorods according to the amount of injected silver nitrate (40 μl, A; 80 μl, B; 160 μl, C). A shows an aspect ratio of 2:1, B shows an aspect ratio of 3:1, and C shows a result of an aspect ratio of 4:1.
8 is a FE-TEM image of the aspect ratio of gold nanorods according to the amount of injected silver nitrate (40 μl, A; 80 μl, B; 160 μl, C). A shows an aspect ratio of 2:1, B shows an aspect ratio of 3:1, and C shows a result of an aspect ratio of 4:1.
9 is a graph illustrating an absorption spectrum according to an aspect ratio of a gold nanorod.
10A to 10F show FE-SEM images of black phosphorus nanosheets transformed into gold nanorods. Specifically, FIG. 10a shows black phosphorus nanosheets alone, and FIGS. 10b to 10f show 0.1 ml (1X), 0.2 ml (2X), 0.4 ml (4X), 0.8 ml (8X), and 1.6 ml (16X) gold nanorods, respectively. It shows an image of black phosphorus nanosheets injected with .
11 is an energy-dispersive X-ray spectroscopic analysis result of black phosphorus nanosheets alone (top) and black phosphorus nanosheets transformed into gold nanorods (bottom).
12 shows absorption spectra of black phosphorus nanosheets alone and black phosphorus nanosheets modified with gold nanorods.
13a is a spectrum of X-ray photoelectron spectroscopy analysis of black phosphorus nanosheets modified with gold nanorods, FIG. 13b is a spectrum of X-ray photoelectron spectroscopy analysis of black phosphorus nanosheets coated with dopamine, and FIG. 13c is X-ray photoelectron spectroscopy of black phosphorus nanosheets The spectrum of the -ray photoelectron spectroscopy analysis is shown.
14A is a graph showing the Raman spectral spectrum of black phosphorus nanosheets, FIG. 14B is a Raman spectrum spectrum of black phosphorus nanosheets transformed into gold nanorods, and FIG. 14C is a graph comparing the two Raman spectral spectra.
15 is a graph showing the FT-IR spectrum of the black phosphorus nanosheets and the black phosphorus nanosheets transformed into gold nanorods.
16a is a graph showing the X-ray diffraction pattern of black phosphorus nanosheets, FIG. 16b is a graph showing the X-ray diffraction pattern of gold nanorods, and FIG. 16c is a graph showing the X-ray diffraction pattern of black phosphorus nanosheets transformed into gold nanorods .
17 is an image of various nanomaterial composites synthesized in the form of hydrogel beads.
18 shows gold nanorods synthesized in the form of beads, black phosphorus nanosheets synthesized in the form of beads, 0.1 ml (1X), 0.2 ml (2X), 0.4 ml (4X), 0.8 ml (8X), and 1.6 ml (16X) ) The temperature change is shown by irradiating near-infrared rays on the black phosphorus nanosheets injected with gold nanorods and the beads that do not contain the material.
19 is a graph showing the results of irradiating near-infrared rays to the black phosphorus nanosheets transformed into gold nanorods in order to confirm the photostability according to the shape of the black phosphorus nanosheets.
20A to 20C show the experimental results of increasing the temperature by irradiating near-infrared rays on black phosphorus nanosheets transformed into gold nanorods synthesized in the form of beads. 20A is a fluorescence intensity according to the reaction time for each target concentration of the primer set LAMP reaction of the present invention DMSO 5% for the M gene of SARS-CoV-2 virus, and FIG. 20B is a bar graph of the corresponding fluorescence intensity. 20c shows a linear relationship between the M gene concentration and fluorescence value of SARS-CoV-2 virus.

The present invention is black phosphorus nanosheet (black phosphorus nanosheet);

dopamine; and

A nanomaterial composite comprising gold nanorods, comprising:

The black phosphorus nanosheet is one or more dopamine bound,

The gold nanorod relates to a nanomaterial complex, characterized in that it is bound to dopamine.

Hereinafter, the present invention will be described in detail.

In the present invention, "Black phosphorus nanosheet (BPNS)" is a sheet-like material with a two-dimensional layer structure composed of only phosphorus element and is made by applying high temperature and high pressure to phosphorus. Black phosphorus has regular wrinkles, so it is easy to control physical properties. In addition, black phosphorus nanosheets and gold nanorods have high light-to-heat conversion efficiency that can be converted into heat when light of a specific wavelength region is received, have excellent biocompatibility, and are easy to manufacture.

In the present invention, the "heuk phosphorus nanosheet" may have one or more dopamine bound to the surface, but is not limited thereto.

In the present invention, “dopamine” is a catecholamine-based organic compound, a hormone or neurotransmitter found in the central nervous system of various animals. The dopamine is not limited thereto, as long as it is bonded to the black phosphorus nanosheet in the form of a single compound of Formula 1 or is bonded to the dopamine-based monomer by surface polymerization, for example, it may be bonded in the form of polydopamine.

[Formula 1]

In the present invention, “gold nanorod (Au nanorod, AuNR)” is widely used as a probe in biomaterial detection because it is easy to modify various surfaces and exhibits excellent optical properties including plasmonic and light-to-heat conversion efficiency.

In the present invention, the gold nanorods may be chemically bound to a catechol functional group of dopamine, but is not limited thereto.

In the present invention, the gold nanorods have an aspect ratio of 0.1 to 10: 1, 0.2 to 10: 1, 0.2 to 9: 1, 0.2 to 8: 1, 0.2 to 7: 1, 0.2 to 6: 1, 0.2 to 5 : 1, 0.2 to 4 : 1, 0.5 to 10 : 1, 0.5 to 9: 1, 0.5 to 6 : 1, 0.5 to 5 : 1, 0.5 to 4 : 1, 1 to 10 : 1, 1 to 9: 1 , 1 to 8: 1, 1 to 7: 1, 1 to 6: 1, 1 to 5: 1, 2 to 10: 1, 2 to 9: 1, 2 to 8: 1, 2 to 7: 1, 2 to 6: 1, 2 to 5: 1, or 2 to 4: 1 may be, but is not limited thereto.

In the present invention, the complex may cause an exothermic reaction at a near-infrared wavelength, but is not limited thereto. In the present invention, the near-infrared wavelength may be a wavelength of 700 to 1200 nm, but is not limited thereto.

In the present invention, the complex may maintain an isothermal state for 20 minutes or more, 25 minutes or more, 30 minutes or more, 35 minutes or more, 40 minutes or more, 45 minutes or more, 50 minutes or more, or 60 minutes or more, but is limited thereto not.

The composite of the present invention is utilized as an efficient portable thermal circulation device by using the light-to-heat conversion properties of black phosphorus nanosheets and gold nanorods. By combining black phosphorus nanosheets with gold nanorods, it is possible to generate heat by receiving light in a specific wavelength region. have.

In addition, the present invention provides bead-shaped particles prepared using the composition comprising the nanomaterial composite according to the present invention.

In the present invention, the beads may have a diameter of 0.5 to 3 mm, but is not limited thereto.

In one embodiment of the present invention, after injecting the complex into sodium alginate and stirring, the mixture was dropped into calcium carbonate using a syringe pump to synthesize beads, and the LAMP reaction was well performed using the prepared beads. was confirmed.

In addition, the present invention comprises the steps of (a) modifying the surface of the black phosphorus nanosheet with dopamine; and

(b) provides a method for preparing a nanomaterial composite according to the present invention, comprising the step of treating the gold nanorods.

In the present invention, the black phosphorus nanosheet may be prepared by peeling black phosphorus particles by a sonication method, but is not limited thereto. The black phosphorus nanosheet may be obtained by peeling bulk black phosphorus with a single layer or a few layers of 2D black phosphorus, but is not limited thereto.

In the present invention, the manufacturing method may further include (c) preparing beads by dropwise adding the composition comprising the nanomaterial composite according to the present invention, but is not limited thereto. The composition including the black phosphorus nanosheet composite may include sodium alginate, calcium alginate, and the like, but is not limited thereto.

The composition may be prepared in the form of beads by dropwise addition to a material that is calcium lactate, calcium chloride, sodium hydroxide, ammonia, sodium carbonate, sodium hydrogen carbonate, magnesium oxide, aluminum hydroxide, calcium carbonate, or a combination thereof, but is limited thereto not.

In the present invention, dopamine in step (a) may be mixed with black phosphorus nanosheets in a mass ratio of 1:1 to 5, 1:1 to 4, 1:1 to 3, 1:1 to 2, or 1:1.5. However, the present invention is not limited thereto.

Meanwhile, the present invention provides a set of loop-mediated isothermal amplification (LAMP) primers for detection of SARS-CoV-2 virus, comprising at least one selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 6 to provide.

The ring-mediated isothermal amplification method uses Bst polymerase, which has the advantage of strand displacement activity, unlike the polymerase chain reaction (PCR) method used as a conventional diagnostic method, so that conjugation and amplification at a constant temperature is not possible. Because it is possible, there is no DNA loss or damage due to a short reaction time of less than 1 hour and maintenance of a constant temperature, and since it only needs to maintain a constant temperature, expensive equipment is not needed, so the field utility is high.

Since the ring-mediated isothermal amplification primer set specific for the M gene of SARS-CoV-2 virus of the present invention is sensitive and specific to the M gene of SARS-CoV-2 virus, it can be effectively distinguished from other viruses and detected. Therefore, it can be usefully used for prevention and diagnosis of COVID-19 virus infection. The primer set of the present invention enables more precise and rapid diagnosis of COVID-19 virus using six newly discovered primers.

The loop-mediated isothermal amplification of the target genetic material according to the present invention may be performed by the following procedure.

First, a sample to be determined whether a target genetic material including a nucleic acid component is included is pretreated. The sample pretreatment method may be, for example, a process of extracting M RNA of SARS-CoV-2 virus using Dynabeads ^TM Intact Virus Enrichment (Introgen, Thermo Fisher Scientific, Waltham, MA, USA).

Prepare an RNA sample extracted from the COVID-19 virus and a mixed reagent that reacts to the target genetic material. For example, 6 primer sets (F3, B3, F2, B2, LF, LB) that bind to the target genetic material, isothermal amplification buffer II (1X), deoxynucleotide solution mixture, Bst DNA polymerase, EvaGreen ^® fluorescence, magnesium sulfate (MgSO ₄ ), Calcein and manganese chloride (MnCl ₂ ) reagents are used, and a primer set consisting of a nucleotide sequence of (F3, B3, F2, B2, LF, LB) is used for the primer set. In the present invention, selection was performed to confirm a primer set capable of specifically amplifying the M gene of SARS-CoV-2 virus. As a result, it was confirmed that the M01 primer set among the candidate primer sequences had the highest detection efficiency for the M gene of SARS-CoV-2 virus.

Thereafter, the mixed reagent and the pretreated sample can be amplified in real time for 60 minutes using a MiniOpticon Real-Time machine. According to one embodiment of the present invention, the isothermal amplification reaction using the primer set consisting of the nucleotide sequence of SEQ ID NOs: 1 to 6 can be carried out at 61°C to 71°C, 63°C to 68°C or 64°C to 67°C. And, the LAMP reaction of the M gene of SARS-CoV-2 virus can be performed at about 65 ℃.

In addition, the isothermal amplification method can be performed under MgSO ₄ conditions at a concentration of 6 mM in the detection of a gene encoding the M protein of SARS-CoV-2 virus using a primer set consisting of the nucleotide sequences of SEQ ID NOs: 1 to 6.

According to one embodiment of the present invention, in order to improve the false-positive signal appearing in the detection of the M gene of SARS-CoV-2 virus, it may be carried out in DMSO conditions of 0% to 7% (w/w basis of DMSO) concentration, Detection of the M gene of SARS-CoV-2 virus can be carried out in the condition of 5% DMSO.

In the present invention, in order to increase the convenience of detecting the M gene of SARS-CoV-2 virus, a detection label may be additionally included. The detection label may be a compound, a biomolecule, or a biomolecule analog, etc. that is mixed with a primer set and can confirm the density, concentration, amount, etc. of the amplification product in a conventional manner. For example, when a reaction such as MnCl ₂ and calcein is used in consideration of detection sensitivity, it is possible to visually identify the M gene of SARS-CoV-2 virus specifically.

In the present invention, the sample may be selected from the group consisting of food, natural products and biological samples.

In the present invention, "natural product" refers to a material produced by an organism, and M of SARS-CoV-2 virus contained in the produced material using the ring-mediated isothermal amplification primer set according to an embodiment of the present invention. genes can be detected.

In the present invention, the biological sample can be used without limitation as long as it is collected from a subject to predict or diagnose an onset, for example, cells or tissues, blood, whole blood, serum, plasma, saliva, cerebrospinal fluid, and various secretions obtained by biopsy, etc. , urine, feces, etc. Preferably, it may be selected from the group consisting of blood, plasma, serum, saliva, nasal fluid, sputum, ascites, vaginal secretion and urine, and preferably blood, plasma, or serum. The sample may be pretreated prior to use for detection or diagnosis. For example, it may include homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like.

In addition, the present invention provides a composition for detecting the M gene of SARS-CoV-2 virus comprising the primer set according to the present invention.

In addition, the present invention comprises the steps of isolating RNA from a sample; and

Using the RNA as a template, and performing an isothermal amplification reaction using the primer set according to the present invention to amplify the target sequence; provides an information providing method for the diagnosis of coronavirus infection-19, including.

In the present invention, the term “detection” is meant to include both measuring and confirming the presence (expression) of a target substance, or measuring and confirming a change in the presence level (expression level) of a target substance. In the same vein, in the present invention, measuring the expression level of the M gene of the SARS-CoV-2 virus is measuring whether or not it is expressed (ie, measuring the presence or absence of expression), or M of the SARS-CoV-2 virus. It means measuring the level of qualitative and quantitative change in a gene. The measurement may be performed without limitation, including both a qualitative method (analysis) and a quantitative method. Types of qualitative and quantitative methods for measuring the presence or absence of the M gene in SARS-CoV-2 virus are well known in the art, and the experimental methods described herein are included therein. A method for comparing the M gene level of specific SARS-CoV-2 virus for each method is well known in the art.

In the present invention, the term “diagnosis” refers to determining the susceptibility of an object to a specific disease or disorder, determining whether an object currently has a specific disease or disorder, or having a specific disease or disorder It is a concept that includes both determining the prognosis of an object, or therametrics (eg, monitoring the condition of an object to provide information on treatment efficacy).

In addition, the term "SARS-CoV-2" or "2019-nCoV" used in the present invention refers to a novel coronavirus, and refers to strains of SARS and MERS as RNA viruses. SARS-CoV-2 shares about 79.7% sequence identity with SARS and about 50% with MERS. However, in contrast to SARS and MERS, the spike glycoprotein of 2019-nCoV forms a structure in which one RBD domain protrudes upward, resulting in 100 to 1,000 times more It shows strong bonding power. This strong binding force makes it easier to penetrate into the cell and acts as a cause to increase the infectious power.

In the present invention, the coronavirus infection may be a coronavirus respiratory infection disease. The viral respiratory infection diseases include cough, sneezing, headache, stuffy nose, sore throat, diarrhea, discoloration of fingers or toes, conjunctivitis, high fever, wheezing, bronchitis, bronchiolitis, pneumonia, asthma, loss of smell and taste, and respiratory failure. symptoms may be present. If the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), fever and respiratory symptoms (cough, sore throat, shortness of breath) are the main symptoms, along with headache, muscle pain, hemoptysis and nausea, Symptoms may include chills, chest pain, and diarrhea. In addition, the coronavirus infection may be coronavirus infection 19 (COVID-19).

In the present invention, the composition for detection may include a polymerase for ring-mediated isothermal amplification, a dNTP mixture, and a reaction buffer. In addition, the kit of the present invention may further include tools or reagents incidentally necessary to perform LAMP and/or user instructions describing optimal reaction conditions.

In addition, the present invention provides a kit for ring-mediated isothermal amplification comprising the complex or beads according to the present invention. In the present invention, the kit may further include a primer set according to the present invention, but is not limited thereto.

Since the present invention can apply various transformations and can have various embodiments, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Hereinafter, the present invention will be described in more detail through one or more embodiments. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

[Example]

Example 1. SARS-CoV-2 Virus M gene Preparation of amplification primers

1-1. Preparation of samples and equipment

The M gene of SARS-CoV-2 virus was extracted using Dynabeads ^TM Intact Virus Enrichment (Introgen, Thermo Fisher Scientific, Waltham, MA, USA), and a specific primer for the M gene of SARS-CoV-2 virus (primer) ) was synthesized by Bioneer (Daejeon, Korea).

Isothermal amplification buffer II (1X), deoxynucleotide solution mixture, Bst DNA polymerase, EvaGreen ^® fluorescence, and magnesium sulfate (MgSO ₄ ) were purchased from New England Biolabs (Ipswich, MA, USA). Evagreen fluorescent DNA staining reagent was purchased from Biofact (Daejeon, Korea), and calcein and manganese chloride (MnCl ₂ ) were purchased from Dojindo (Kumamoto, Japan).

1-2. Primer production

In order to screen the M gene of SARS-CoV-2 virus, the Genbank number and full sequence of the M gene (NC-045512.2) of SARS-CoV-2 virus were obtained from Gene Bank (national center for biotechnology information).

Primers capable of amplifying specifically for the M gene of SARS-CoV-2 virus were designed using the Primer Explorer V5 program on the Eiken website (http://primerexplorer.jp/e/). Primers for specifically amplifying the M gene of SARS-CoV-2 virus using BLAST (Basic Local Alignment Search Tool) were sequenced as shown in Table 1, and all designed primers were synthesized by Bioneer.

serotype genomic location primer
designation LAMP primer sequence (5'→ SEQ ID NO: COVID-19 26588 - 26611 F3 AGT AAT AGG TTT CCT ATT CCT TAC SEQ ID NO: 1 26783 - 26800 B3 AGC CAC ATC AAG CCT ACA SEQ ID NO: 2 26612 - 26635 F2 ATG GAT TTG TCT TCT ACA ATT TGC SEQ ID NO: 3 26763 - 26780 B2 ACA AGC CAT TGC GAT AGC SEQ ID NO: 4 26641 - 26665 LF ATA TAC AAA AAC CTA TTC CTG TTG G SEQ ID NO: 5 26732 - 26754 LB TTA CAG AAT AAA TTG GAT CAC CG SEQ ID NO: 6

Example 2. Establishment of LAMP reaction conditions

The LAMP reaction was carried out by putting 25.0 μl of the reaction solution mixed with the following solution in a PCR tube: 2.5 μl isothermal amplification buffer II (10X), 3.5 μl deoxynucleotide solution mixture (1.4 mM), 1.0 μl inner primer : FIP and BIP, 1.6 μM), outer primer (F3 and B3, 0.2 μM), 1.0 μl loop primer (LF and LB, 0.4 μM), 1.5 μl MgSO ₄ (6 mM), 1.0 μl Bst 3.0 DNA polymerase (320 U/mL), 1.25 μl EvaGreen (1X), 12.5 μl ultrapure purified water, 3 μl purified SARS-CoV-2 virus M gene solution.

As a negative control, ultrapure purified water was used, and the LAMP reaction was performed at a temperature of 61 to 71° C. for 60 minutes using a heating block of Daihan-scientific. The results of adding the M gene of SARS-CoV-2 virus and the amplification results of the negative control were compared.

Example 3. Optimization of LAMP conditions

3-1. SARS-CoV-2 Optimization of LAMP reaction temperature of viral M gene

For optimization of LAMP analysis conditions, the effect of reaction temperature on LAMP reaction was investigated.

Specifically, the LAMP reaction was performed in the method of Example 2 using SEQ ID NOs: 1 to 6, and temperature optimization analysis was performed using CFX manager 3.1 of BIO-RAD.

As a result, as shown in FIG. 2 , the Cq value was the lowest at 65 ° C and the RFU value was high. It was confirmed that 65 ° C was the optimal LAMP reaction temperature for the detection of the M gene of SARS-CoV-2 virus. .

Therefore, thereafter, the LAMP reaction for the detection of the M gene of SARS-CoV-2 virus was analyzed at a temperature of 65 ℃.

3-2. SARS-CoV-2 Optimization of DMSO concentration in the LAMP reaction of the viral M gene

DMSO (Dimethyl Sulfoxide) is known to inhibit secondary structure formation of target DNA during LAMP reaction. The effect of DMSO concentration on LAMP reaction was investigated in order to improve the false-positive result in LAMP reaction for detection of M gene of SARS-CoV-2 virus.

Specifically, the LAMP reaction was performed in the same manner as in Example 3-1 for a concentration of 0%, 5%, or 7% of DMSO at a temperature of 65°C.

As a result, as shown in FIGS. 3 to 5 , it was confirmed that DMSO at a concentration of 5% (w/w) was used in the LAMP reaction of the M gene of SARS-CoV-2 virus. Specifically, when DMSO at a concentration of 5% (w/w) was used, specificity and selectivity increased compared to 0% or 7% DMSO, but it was confirmed that sensitivity decreased and amplification time was prolonged when 7% was used. Conversely, it was confirmed that, when 0% DMSO was used, it was easily amplified even in a false-positive reaction and a low concentration target, resulting in reduced sensitivity.

As a result, as shown in FIG. 6 , as a result of applying to the LAMP reaction for each DMSO concentration, when 0%, 5%, or 7% (w/w) DMSO concentration was used, R ² =0.4754, R ² = As a result of 0.7701 or R ² =0.5004, it was confirmed that 5% DMSO was most effective for the LAMP reaction.

In the following procedure, LAMP analysis was performed for the detection of the M gene of SARS-CoV-2 virus at the optimum concentration of DMSO of 5% (w/w).

Example 4. Preparation of black phosphorus nanosheets modified with gold nanorods synthesized in the form of hydrogel beads

4-1. Preparation of samples and equipment

The black phosphorus sheet was purchased from XFNANO Materials Tech Co., Ltd (Nanjing, China). Gold nanorods and hydrogel raw materials, tetrachloric acid hydrate (HAuCl ₄ ·3H ₂ O) and dopamine hydrochloride It was purchased from Sigma Aldrich (St. Louis, MO, USA). Hexadecyltrimethylammonium bromide (CTAB), silver nitrate (AgNO ₃ ), and sodium borohydride (NaBH ₄ ) were purchased from Daejung (Siheung, Korea). L-Ascorbic acid (L-Ascorbic acid, AA) was purchased from Alfa Aesar (Ward Hill, MA, USA). Tris[hydroxymethyl]aminomethane (tris) was purchased from Bio-Rad Laboratories (Hercules, CA, USA). Hydrochloric acid (HCl) was purchased from Duksan Chemicals (Ansan, Korea), and isopropyl alcohol (Isopropanol) and calcium chloride (CaCl ₂ ) were purchased from Samchun Chemicals (Seoul, Korea). Sodium alginate was purchased from Junsei (Tokyo, Japan). Deionized water was purified at 18.2 MΩ·cm using a Milli-Q system (Millipore, Billerica, MA, USA).

Absorption spectra of gold nanorods and black phosphorus nanosheets transformed into gold nanorods were measured using a UV-vis spectrophotometer (Jasco 670, Jasco, Tokyo, Japan). The FT-IR spectra of gold nanorods and black phosphorus nanosheets transformed into gold nanorods were confirmed by Fourier-transform infrared (FT-IR) (Jasco 6600FV, Jasco, Tokyo, Japan). X-ray diffraction (XRD) patterns were obtained with a D8-Advance instrument (Brucker AXS, Berlin, Germany). The images and sizes of the gold nanorods and the black phosphorus nanosheets modified with the gold nanorods were obtained using a field emission scanning electron microscope (FE-SEM) (SIGMA 300, Carl Zeiss, Cambridge, UK) and a field emission transmission electron microscope (FE-TEM). (JEM-F200, JEOL, Tokyo, Japan) was used. The structure of the black phosphorus nanosheets transformed into gold nanorods was confirmed using X-ray photoelectron spectroscopy (XPS) (K-ALPHA+, Thermofisher Scientific, Waltham, MA, USA).

4-2. Confirmation of Optimization Conditions for Gold Nanorod Synthesis

Gold nanoparticles exhibit strong absorption at about 520 nm due to the SPR phenomenon, whereas gold nanorods simultaneously show an absorption line at 520 nm due to electron vibration in the horizontal axis and a long wavelength absorption line due to electromagnetic vibration in the vertical axis direction. As the aspect ratio of the gold nanorods increases, the UV-Vis absorption line shifts toward longer wavelengths. Accordingly, the longer the gold nanorods in the longitudinal direction (ie, the greater the aspect ratio), the longer the wavelength of light can be absorbed.

In order to develop a kit that can be heated to a temperature of 65 °C in the LAMP reaction for the detection of the M gene of SARS-CoV-2 virus, gold nanorods according to the concentration of AgNO ₃ having absorption in the 800 nm wavelength region were synthesized.

Specifically, for the synthesis of gold nanorods, a conventionally known seeding and growth method was used. By injecting 25 μl of 0.05M HAuCl ₄ and 0.6 mL of 0.01M NaBH ₄ to 4.7 mL of 0.1M CTAB solution and vigorously stirring for 2 minutes in a heating mantle set at 28° C., Au seeds were synthesized. After injecting 0.1mL of 0.05M HAuCl ₄ into 10mL of 0.1M CTAB solution and stirring vigorously for 10 minutes in a heating mantle set at 28℃, 75 μl of 0.1M L-ascorbic acid and 40, 80, 160 μl of 5mM AgNO ₃ was strongly stirred for 5 minutes while injecting into the solution. Then, 0.12 mL of the seed solution was injected into the growth solution, and the mixture was stirred vigorously for 30 minutes in a heating mantle set at 28°C.

The size and shape of the synthesized gold nanorods are shown in FIGS. 7 to 9 .

As shown in FIG. 7 , the gold nanorods are well distributed through the FE-SEM image, and the aspect ratio of the gold nanorods depends on the amount of injected silver nitrate (40 μl, A; 80 μl, B; 160 μl, C). It was confirmed that it increases proportionally.

As shown in FIG. 8 , it was confirmed through the FE-TEM image that the gold nanorods were well distributed, and the aspect ratio of the gold nanorods increased in proportion to the amount of silver nitrate injected (40, 80, 160 μl).

In addition, as shown in FIG. 9 , a red-shifted absorption spectrum of the synthesized gold nanorods according to the amount of injected silver nitrate (40, 80, 160 μl) was confirmed. As the concentration of silver nitrate increased, the aspect ratio of the gold nanorods increased, and as the red shift occurred, it was confirmed that light of a long wavelength was absorbed.

This is because as the aspect ratio increases, that is, as the length of the gold nanorods becomes longer, the absorption peak at a long wavelength causes a red shift, and in general, strong absorption at a wavelength of about 700 ~ 1,200 nm near infrared region depending on the aspect ratio. This is a result that further supports the existence of

4-3. Preparation of black phosphorus nanosheets

A probe sonication reactor was used to make black phosphorus particles into nano-sized sheets. 2mL of 0.2mg/mL concentration of black phosphorus particles was peeled off in a probe sonicator (950W) for 6 hours. Then, a centrifuge was used at 487 x g for 20 minutes to obtain exfoliated black phosphorus nanosheets (repeated 3 times).

The size and shape of the synthesized black phosphorus nanosheets are as shown in FIG. 10A . It was confirmed that the black phosphorus nanosheets had various sizes in the form of thin films, and the average size was 850±77 nm.

4-4. Fabrication of black phosphorus nanosheets modified with gold nanorods

To bind the gold nanorods to the surface of the black phosphorus nanosheets, dopamine was used as a support for the two materials. Dopamine has excellent surface adhesion and polymerizable properties on almost all surfaces regardless of the surface properties in aqueous solution conditions of basic pH. When black phosphorus nanosheets suitable for basic pH conditions (pH 8.5) are reacted with an aqueous dopamine solution, a polydopamine coating layer is formed by oxidation of dopamine catechol. Since the gold nanorods can be fixed through the specific adhesion of catechol functional groups present on the surface of the polymerized dopamine-coated black phosphorus nanosheets, they were used as binding supports.

Specifically, in order to polymerize dopamine on the surface of black phosphorus nanosheets, 2mL of black phosphorus nanosheets, 2mL of 10mM Tris-HCl solution (pH 8.5), and 0.8mL of 0.4mM dopamine were injected while strongly stirring for 24 hours on a stirrer. did Then, centrifugation was performed at 24,979×g for 30 minutes to obtain polymerized dopamine-coated black phosphorus nanosheets (repeated 3 times). In order to bind the gold nanorods to the polymerized dopamine-coated black phosphorus nanosheets, 0.1 to 1.6mL of gold nanorods were injected into 2mL of the polymerized dopamine-coated black phosphorus nanosheets while strongly stirring for 12 hours on a stirrer. Thereafter, centrifugation was performed at 20,595×g for 30 minutes to obtain black phosphorus nanosheets transformed into gold nanorods (repeated 3 times). FE-SEM analysis was performed to verify the binding of black phosphorus nanosheets modified with gold nanorods.

As shown in FIGS. 10b to 10f , during the immobilization of the gold nanorods, it was confirmed that the gold nanorods were bound to the surface of the black phosphorus nanorods in proportion to the amount (0.1 to 1.6mL) of the injected gold nanorods.

In addition, as shown in FIG. 11 , it was confirmed that black phosphorus nanosheets (indicated by P) and gold nanorods (indicated by Au) coexist by energy-dispersive X-ray spectroscopy (EDS). did

In addition, as shown in FIG. 12 , through absorption spectrum analysis, it was confirmed that the absorption spectrum of the gold nanorods was also shown in the absorption spectrum of the black phosphorus nanosheets in which the gold nanorods were deformed, and the gold nanorods were fixed on the surface of the black phosphorus nanosheets. confirmed that there is.

In addition, X-ray photoelectron spectroscopy was performed to analyze the composition of the black phosphorus nanosheets and the black phosphorus nanosheets transformed into gold nanorods and to confirm the chemical bonding state of the materials.

As shown in FIG. 13a , doublet peaks of P2p of black phosphorus nanosheets are observed at 129.5 and 130.2 eV. In addition, the Au4f peaks of the gold nanorods were observed at 85.2 and 88.9 eV. Through this, it was confirmed that the gold nanorods were fixed to the surface of the black phosphorus nanosheets. As shown in FIG. 13b , peaks of carbon, nitrogen, and oxygen of the dopamine-coated black phosphorus nanosheets were observed at 284.7, 399.9 and 532.3 eV. As shown in FIG. 13c , when only black phosphorus nanosheets were present, a peak of PxOy was confirmed, which was confirmed as a result of the surface oxidation layer caused by oxygen in the air.

In addition, Raman spectroscopy was performed to confirm the structural characteristics of the black phosphorus nanosheets and the black phosphorus nanosheets transformed into gold nanorods.

14a to 14c, this was confirmed through phonons according to the vibrational motion of black phosphorus atoms arranged in a constant lattice structure. Intrinsic structural characteristics of black phosphorus nanosheets were confirmed by measuring the A1 _g mode vibrating in a direction perpendicular to the lattice plane of the black phosphorus nanosheet, and B2 _g and A2 _g mode vibrating in a direction parallel to the lattice plane. Through this, it was confirmed that the black phosphorus nanosheets were not deformed in the black phosphorus nanosheets transformed into gold nanorods.

Then, FT-IR spectra were recorded and compared to verify that the gold nanorods were fixed to the surface of the black phosphorus nanosheets.

As shown in FIG. 15 , in the case of black phosphorus nanosheets transformed into gold nanorods, they appear at 1625 cm ⁻¹ due to the C=C bond of dopamine, and 2917 cm due to the carbon chain of CTAB present on the surface of the gold nanorods. A peak at ^-1 was confirmed. This confirmed that the gold nanorods were fixed to the surface of the black phosphorus nanosheets.

In addition, gold nanorods (AuNRs), black phosphorus nanosheets (BP), and black phosphorus nanosheets (BP@AuNRs) transformed into gold nanorods were confirmed by XRD.

As shown in FIGS. 16A to 16C , the XRD peaks of the gold nanorods were (111), (200), (220) and (311), and the XRD peaks of the black phosphorus nanosheets were (020), (021) , (040) and (060). The XRD spectrum indicates the intrinsic properties of the prepared gold nanorods and black phosphorus nanosheets. As for the XRD peaks of the black phosphorus nanosheets transformed into gold nanorods, it was confirmed that both the XRD peaks of the gold nanorods and the black phosphorus nanosheets appeared.

4-5. Production of black phosphorus nanosheets transformed into gold nanorods synthesized in the form of beads

Black phosphorus nanosheets (BP@AuNRs) modified with gold nanorods were synthesized in the form of beads.

Specifically, 0.5 mL of 4-fold concentrated gold nano-rod-modified black phosphorus nanosheets were injected into 1.5 mL of 12 mM sodium alginate, and the mixture was vigorously stirred on a stirrer for 2 hours. Then, it was synthesized by dropping it into 50 mL of 0.1M calcium carbonate using an injection syringe pump at a rate of 0.1 mL/min. Black phosphorus nanosheets transformed into gold nanorods synthesized in the form of beads were placed in an oven at a temperature of 60° C. and dried for 12 hours.

As shown in FIG. 17 , the black phosphorus nanosheets transformed into gold nanorods synthesized in the form of beads had a diameter of 1.78±0.24 mm, and after drying, it was confirmed that they were formed with a size of 0.9±0.1 mm.

Example 5. Heat release experiment based on black phosphorus nanosheets modified with gold nanorods synthesized in the form of hydrogel beads

5-1. Heat release test according to material type

Heat generation was confirmed by irradiating the BP@AuNRs synthesized in the form of beads prepared in Example 4, and it was confirmed whether the LAMP amplification reaction for the M gene of SARS-CoV-2 virus was possible.

Specifically, gold nanorods synthesized in the form of beads, black phosphorus nanosheets, 0.1 ml (1X), 0.2 ml (2X), 0.4 ml (4X), 0.8 ml (8X), and 1.6 ml (16X) gold nanorods were prepared. The injected black phosphorus nano-sheet beads and 10 beads containing no substance were prepared in a 1.5 mL tube containing 0.2 mL of tertiary distilled water, respectively, and irradiated with near infrared rays (808 nm, 0.8 W/cm ² ) for 40 minutes. The temperature change of the tertiary distilled water in the tube was measured. Near-infrared rays (808nm, 0.8W/cm ² ) were irradiated to confirm the heat dissipation efficiency.

As shown in FIG. 18 , it was confirmed that the temperature increased as the irradiation time of near-infrared rays to each bead increased. When the near-infrared irradiation elapsed for 10 minutes, the temperature of the beads containing no material increased to 30°C, and the gold nanorods synthesized in the form of beads were found to be 50°C. The temperature of the black phosphorus nanosheets synthesized in the form of beads was 50°C. The temperature of the black phosphorus nano-sheet beads injected with 2X, 4X, 8X, and 16X gold nano-rods increased to 51, 58, 65, and 69° C., and was then maintained for up to 40 minutes.

5-2. Heat release experiment according to shape

The difference in photostability according to the shape of black phosphorus nanosheets transformed into gold nanorods was confirmed.

As shown in FIG. 19 , the black phosphorus nanosheets transformed into gold nanorods synthesized in the form of hydrogel beads uniformly increased to 65° C. when the set of turning on and off the near-infrared rays at 10-minute intervals was repeated 5 times after 2 weeks of synthesis. was confirmed. When the same process was performed on the black phosphorus nanosheets transformed into nanorods to which the hydrogel beads were not applied, it was confirmed that the temperature did not increase uniformly but decreased. That is, it was confirmed that the form of hydrogel beads plays an important role in the result of maintaining the photostability of black phosphorus nanosheets transformed into gold nanorods.

Therefore, it was confirmed that BP@AuNR in the form of hydrogel beads can be usefully used for LAMP analysis as it has excellent photothermal effect to maintain isothermal conditions at high temperature.

Example 6. Analysis of LAMP based on black phosphorus nanosheets modified with gold nanorods synthesized in the form of hydrogel beads

BP@AuNRs synthesized in the form of beads was prepared in the same manner as in Example 5, and the LAMP reaction was performed in the manner of Example 3.

As a result, as shown in FIGS. 20a and 20b, when BP@AuNRs synthesized in the form of beads was irradiated with near-infrared rays for 20 to 50 minutes, at a concentration of 1.5×10 ⁴ copies/reaction of the M gene of SARS-CoV-2 virus. It was confirmed that an amplified signal was generated. It was confirmed that black phosphorus nanosheets modified with gold nanorods synthesized in the form of beads could be effectively used for the LAMP reaction in that the signal for the LAMP reaction at a concentration of 15 copies/reaction was effectively generated at a reaction time of 50 minutes.

In addition, as shown in FIG. 20c , the signal for the LAMP response increased as the concentration of the M gene of SARS-CoV-2 virus increased (0 to 15,000 copies/reaction). The linear regression equation is as follows. Through this equation, it was confirmed that the detection limit of the M gene of SARS-CoV-2 virus was 7.7 copies/reaction.

Intensity = 0.6169 × [M gene of SARS-CoV-2 virus] + 74.16 (R ² =0.9998)

From the above results, it was confirmed that the M gene of SARS-CoV-2 virus could be detected quickly through LAMP analysis with high sensitivity using the primers of the present invention.

Up to now, the present invention has been looked at focusing on the embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

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Claims (19)

black phosphorus nanosheet;
dopamine; and
A nanomaterial composite comprising gold nanorods, comprising:
The black phosphorus nanosheet is one or more dopamine bound,
The gold nanorods are characterized in that bound to dopamine, nanomaterial composite.
According to claim 1,
The gold nanorods are characterized in that chemically bound to a catechol functional group of dopamine, nanomaterial composite.
According to claim 1,
The gold nanorods have an aspect ratio of 2:1 to 8:1, characterized in that the nanomaterial composite.
The method of claim 1,
The composite is characterized in that the exothermic reaction in the near-infrared wavelength, nanomaterial composite.
According to claim 1,
The dopamine is a nanomaterial composite, characterized in that it is bound to the black phosphorus nanosheet in a polymerized form.
5. The method of claim 4,
The composite is characterized in that the isothermal state is maintained for at least 20 minutes, the nanomaterial composite.
Beads prepared using a composition comprising the nanomaterial composite of claim 1.
(a) modifying the surface of the black phosphorus nanosheet with dopamine; and
(b) A method for preparing the nanomaterial composite of claim 1, comprising the step of treating the gold nanorods.
9. The method of claim 8,
The black phosphorus nanosheet is a production method, characterized in that produced by peeling the black phosphorus particles by a sonication method.
9. The method of claim 8,
In the step (a), dopamine is mixed with black phosphorus nanosheets in a mass ratio of 1:1 to 1:5, a manufacturing method.
delete delete delete delete A kit for ring-mediated isothermal amplification comprising the nanomaterial complex of claim 1 or the beads of claim 7.
16. The method of claim 15,
The kit is characterized in that it further comprises any one or more of the primers of SEQ ID NOs: 1 to 6.
17. The method of claim 16,
The kit is for the detection of the M gene of SARS-CoV-2 virus, the kit.
isolating RNA from the sample; and
Using the RNA as a template, and performing an isothermal amplification reaction using the kit of claim 16 to amplify the target sequence, a method for diagnosing coronavirus infection-19.
19. The method of claim 18,
The isothermal amplification reaction is characterized in that performed at 61 to 71 ℃, information providing method.

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