Nowadays, isothermal amplification-based biomolecular detection have gathered extensive interests due to its easy applicability toward various point-of-care testing (POCT) devices. It is a promising strategy which can successfully replace conventional polymerase chain reaction (PCR)-based assays. In this study, we utilized fluorescent copper nanoparticles (CuNPs) as our signaling materials for isothermal amplification-based assays due to their easy, simple, eco-friendly, and rapid synthesis compared with other fluorescent nanomaterials. In chapter 2, we devised a rapid and ultrasensitive microRNA (miRNA) detection strategy by employing target-assisted isothermal exponential amplification (TAIEA) combined with poly (thymine)-templated fluorescent CuNPs as signaling probes. The target miRNA hybridizes to a unimolecular template DNA and works as a primer for extension reaction to generate double-stranded product which contains two nicking endonuclease recognition sites. By simultaneous nicking and displacement reactions, exponential amplification generates poly (thymine) strands as final products, which are directly utilized for the synthesis of fluorescent CuNPs. As a result, our target miRNA was successfully detected as low as 0.27 fM around 1 hour of total analysis time. In chapter 3 and 4, we developed a novel rolling circle amplification (RCA) method coupled with poly (thymine)-templated fluorescent CuNPs for rapid and ultrasensitive target DNA detection and enzymatic assay. At first, ligation reaction is performed to generate circular template for initiating RCA reaction. Then, long and single-stranded DNA is generated by using phi 29 DNA polymerase and consuming dNTPs. Resultant product contains many repetitive poly (thymine) sequences which can be further utilized as templates for the synthesis of fluorescent CuNPs. Therefore, amplified fluorescence signal is obtained from resultant RCA product for ultrasensitive assay. As a result, our target DNA and enzyme were successfully detected around 2 hours of total analysis time as low as 0.49 aM and 0.01 U/mL, respectively.
등온증폭반응은 기존의 중합효소 연쇄 반응을 대체할 수 있는 유망한 기술로써, 최근 이를 이용한 여러 생체물질을 검출하는 기술이 개발되고 있다. 등온증폭반응의 다양한 신호 검출 방법들 가운데 형광을 이용한 신호 검출 방법은 간편함으로 큰 주목을 받고 있다. 이들 중 DNA를 주형으로 합성한 형광 구리나노입자는 생성과정이 매우 간단하고 수 분이라는 매우 짧은 반응시간을 통해 손쉽게 합성할 수 있다는 장점이 있다. 따라서 본 연구에서는 DNA를 주형으로 합성한 형광 구리나노입자를 이용하여 다양한 생체물질을 등온증폭반응을 통해 검출하는 기술 개발에 초점을 맞추었다. 제 2장에서는 표적 핵산의 도움을 통한 등온증폭방법과 DNA를 주형으로 합성한 형광 구리나노입자를 통해 표적 마이크로 RNA를 빠르고 초고감도로 검출하는 방법을 개발하였으며, 제 3장과 4장에서는 회전환증폭방법과 DNA를 주형으로 합성한 형광 구리나노입자를 통해 표적 DNA와 효소를 빠르고 초고감도로 검출하는 방법을 개발하였다.
