KR-102963704-B1 - NANOPARTICLE FOR POLYMERASE CHAIN REACTION

Abstract

The present invention relates to nanoparticles used in a polymerase chain reaction, and specifically, to nanoparticles capable of rapidly increasing the temperature of a polymerase chain reaction solution without interfering with fluorescent signals when carrying out a polymerase chain reaction.

Inventors

• 나건
• 이지민
• 박소연

Assignees

• 가톨릭대학교 산학협력단

Dates

Publication Date

20260512

Application Date

20230418

Priority Date

20220418

Claims (9)

1. Metal nanoparticles exhibiting a surface plasmon resonance effect; and Nanoparticles for polymerase chain reaction comprising hexadecylamine covalently bonded to the above metal nanoparticles.
2. In claim 1, the metal nanoparticles are nanoparticles for polymerase chain reaction selected from the group consisting of gold, silver, and copper.
3. delete
4. delete
5. Nanoparticles for polymerase chain reaction according to claim 1, wherein the covalent bond is a peptide bond.
6. A polymerase chain reaction kit comprising nanoparticles for polymerase chain reaction according to claim 1.
7. In claim 1, the nanoparticles for polymerase chain reaction are nanoparticles for polymerase chain reaction used in real-time polymerase chain reaction.
8. A step of preparing a reaction solution comprising nanoparticles for a polymerase chain reaction according to claim 1 and a nucleic acid to be amplified; and A polymerase chain reaction method comprising the step of applying heat to the above reaction solution.
9. A polymerase chain reaction method according to claim 8, wherein the polymerase chain reaction is a real-time polymerase chain reaction.

Description

Nanoparticles for Polymerase Chain Reaction The present invention relates to nanoparticles used in a polymerase chain reaction, and specifically to nanoparticles capable of rapidly raising the temperature of a solution without interfering with the fluorescent signal when carrying out a polymerase chain reaction. In December 2019, an outbreak of pneumonia of unknown cause began in Wuhan, Hubei Province, China, and a new coronavirus was discovered in patients with this pneumonia. This virus was named severe acute respiratory syndrome coronavirus 2 (SARSCoV-2), and the World Health Organization named the disease caused by this virus Coronavirus Disease 2019 (COVID-19). SARSCoV-2 possesses highly contagious characteristics, spreading infections to approximately 114 countries and causing a global pandemic in less than three months after the virus's identity was confirmed. The viral infection testing methods recommended by the World Health Organization include gene amplification testing and antibody testing. Antibody testing is a method that checks for antibodies against a virus in a person's blood. However, since it takes about 1 to 3 weeks for antibodies to develop after a viral infection, it is difficult to detect early infection, and its sensitivity is low because it requires a large amount of the virus. Gene amplification testing is a method used to confirm viral infection by amplifying the genetic material of a sample, utilizing the Polymerase Chain Reaction (PCR). PCR is a molecular biological technique that replicates and amplifies a desired portion of DNA. Real-time PCR (RT-PCR) is widely used for quantitative analysis because it uses intercalating dyes, allowing the degree of amplification to be verified at every PCR cycle. RT-PCR does not require a separate electrophoresis process to verify amplification products and offers excellent sensitivity as it detects the presence or absence of amplification via fluorescence. Diagnosis via real-time polymerase chain reaction (PCR) is considered the most accurate diagnostic method currently available, with an accuracy rate exceeding 98%. However, PCR has a limitation in that it takes approximately 1.5 to 2 hours to perform the temperature change cycles of about 60–90°C required for gene amplification. Therefore, to meet the increasing demand for testing driven by the rise in viral infections, technology is needed to shorten the PCR execution time. Figure 1 is the result of confirming the shape of copper nanoparticles. Figure 2 shows the results of confirming whether hexadecylamine is bound to copper nanoparticles after reacting copper nanoparticles with hexadecylamine at different ratios: HDA-Cu NP = copper nanoparticles conjugated with hexadecylamine; HDA-Cu NP 1, 2, and 3 = nanoparticles made by reacting copper nanoparticles with 300, 600, and 600 mg of hexadecylamine. Figure 3 shows the results of confirming the concentration of copper ions within the copper nanoparticles after reacting copper nanoparticles with hexadecylamine at different ratios: HDA-Cu NP 1, 2, and 3 = nanoparticles prepared by reacting copper nanoparticles with 300, 600, and 600 mg of hexadecylamine. Figure 4 shows the results of measuring the temperature of a solution while irradiating it with light, containing water, copper nanoparticles, or HDA-Cu NPs. Figure 5 shows the results of measuring the temperature of a solution containing HDA-Cu NPs when light was irradiated or when it was not irradiated. Figure 6 is the result of visually confirming the state of the solution before and after irradiating light onto a solution containing HDA-Cu NPs. Figure 7 shows the results of measuring the fluorescence levels after mixing copper nanoparticles, HDA-Cu NPs, and a solution in which HDA-Cu NPs were separated by irradiation with light, by dispersing each in FITC and an aqueous phase. Figure 8 shows the results of confirming the amplified product after performing a real-time polymerase chain reaction with a reaction solution containing copper nanoparticles or HDA-Cu NPs. Figure 9 shows the results of confirming the fluorescence amount in real time while performing a real-time polymerase chain reaction with a reaction solution containing copper nanoparticles, HDA-Cu NPs, or a solution in which HDA-Cu NPs were separated. Figure 10 shows the results of confirming the amplified product after performing a real-time polymerase chain reaction with a reaction solution containing different templates and HDA-Cu NPs. One or more specific examples are described in more detail below through embodiments. However, these embodiments are intended to illustrate one or more specific examples and the scope of the present invention is not limited to these embodiments. Example: Preparation of copper nanoparticles bound to fatty acids - Copper nanoparticle manufacturing Copper nanoparticles were prepared through a reduction reaction of copper ions using a reducing agent. 100 mg of copper dichloride was dissolved in 7.8