Biotechnology and Bioprocess Engineering 2022; 27(2): 202-212  
Rolling Circle Amplification-based Copper Nanoparticle Synthesis on Cyclic Olefin Copolymer Substrate and Its Application in Aptasensor
Tai-Yong Kim, Min-Cheol Lim, Ji Won Lim, and Min-Ah Woo
Tai-Yong Kim, Min-Cheol Lim, Min-Ah Woo*
Research Group of Food Safety and Distribution, Korea Food Research Institute (KFRI), Wanju, Korea
Tel: +82-63-219-9374; Fax: +82-63-219-9876
E-mail: mawoo@kfri.re.kr
Tai-Yong Kim
Department of Food Science and Technology, Jeonbuk National University, Jeonju, Korea
Ji Won Lim
The 4th R&D Institute, 6th Directorate, Agency for Defense Development, Daejeon, Korea
Received: August 5, 2021; Revised: September 16, 2021; Accepted: September 19, 2021; Published online: April 30, 2022.
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
This study aimed to develop a label-free fluorescent aptasensor for the detection of diazinon (DZN) on a cyclic olefin copolymer (COC) substrate. The aptasensor design was based on rolling circle amplification (RCA) technology and the use of self-assembled copper nanoparticles (CuNPs). A dual-function (DF) probe, capable of binding to circular DNA and an aptamer, was designed and immobilized on a COC-bottom 96-well plate. An aptamer was used for selective recognition of DZN, and the specific site of the aptamer that strongly reacted with DZN was successfully identified using circular dichroism (CD) analysis. In presence of DZN, the aptamer and DZN formed a strong complex, thus providing an opportunity for hybridization of the DF probe and circular DNA, thereby initiating an RCA reaction. Repetitive poly thymine (T) sequence with a length of 30-mer, generated in the RCA reaction, served as a template for the synthesis of fluorescent copper nanoparticles, emitting an orange fluorescence signal (at approximately 620 nm) proportional to the amount of RCA product, within 10 min under UV irradiation. The CuNP fluorescence was imaged and quantified using an image analysis software. A linear correlation of the fluorescence signal was confirmed in the DZN concentration range of 0.1-3 ppm, with a detection limit of 0.15 ppm. Adoption of a label-free detection method, utilizing RCA and fluorescent CuNPs on COC substrates, reduced the need for complex equipment and requirements for DZN analysis, thereby representing a simple and rapid sensing method circumventing the limitations of current complex and labor-intensive methods.
Keywords: diazinon, aptamer, rolling circle amplification, copper nanoparticle, fluorescent sensor, cyclic olefin copolymer


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