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T7 Endonuclease I-mediated voltammetric detection of KRAS mutation coupled with horseradish peroxidase for signal amplification

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Abstract

Rapid and selective sensing of KRAS gene mutation which plays a crucial role in the development of colorectal, pancreatic, and lung cancers is of great significance in the early diagnosis of cancers. In the current study, we developed a simple electrochemical biosensor by differential pulse voltammetry technique for the specific detection of KRAS mutation that uses the mismatch-specific cleavage activity of T7-Endonuclease I (T7EI) coupled with horseradish peroxidase (HRP) to catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate in the presence of hydrogen peroxide (H2O2). In addition, we synthesized the nanocomposite composed of multi-walled carbon nanotube/chitosan-ionic liquid/gold nanoparticles (MWCNT/Chit-IL/AuNPs) on screen-printed carbon electrode surface to increase the electrode surface area and electrochemical signal. In principle, T7E1 enzyme recognized and cleaved the mismatched site formed by the presence of KRAS gene mutation, removing 5′-biotin of capture probes and subsequently reducing the differential pulse voltammetry signal compared to wild-type KRAS gene. With this proposed strategy, a limit of detection of 11.89 fM was achieved with a broad linear relationship from 100 fM to 1 µM and discriminated 0.1% of mutant genes from the wild-type target genes. This confirms that the developed biosensor is a potential platform for the detection of mutations in early disease diagnosis.

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Funding

This work was supported by a grant from the National Research Foundation of Korea (NRF), funded by the Korea government (Ministry of Science and ICT) (No. NRF-2020R1C1C1012275), Korea Institute of Energy Technology Evaluation and Planning (KETEP) and the Ministry of Trade, Industry and Energy (MOTIE, 20194010201900), and by Konkuk University Researcher Fund in 2021.

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Authors and Affiliations

  1. Department of Biological Engineering, College of Engineering, Konkuk University, Seoul, Republic of Korea

    Pinky Chowdhury, Byung Seok Cha, Seokjoon Kim, Eun Sung Lee, Taehwi Yoon, Jisu Woo & Ki Soo Park

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  1. Pinky Chowdhury
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  2. Byung Seok Cha
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  3. Seokjoon Kim
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  5. Taehwi Yoon
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  7. Ki Soo Park
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Contributions

Pinky Chowdhury: conceptualization, methodology, validation, formal analysis, investigation, writing—original draft. Byung Seok Cha: methodology, formal analysis, review. Seokjoon Kim: methodology, formal analysis. Eun Sung Lee: methodology, formal analysis. Taehwi Yoon: formal analysis. Jisu Woo: formal analysis. Ki Soo Park: writing—review and editing, funding acquisition, resources, supervision.

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Correspondence to Ki Soo Park.

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Chowdhury, P., Cha, B.S., Kim, S. et al. T7 Endonuclease I-mediated voltammetric detection of KRAS mutation coupled with horseradish peroxidase for signal amplification. Microchim Acta 189, 75 (2022). https://doi.org/10.1007/s00604-021-05089-1

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