Fabrication of ultrasensitive electrochemical biosensor for dengue fever viral RNA Based on CRISPR/Cpf1 reaction

Abstract

Dengue virus (DENV) causes several dengue-related diseases, such as Dengue Fever (DF), Dengue Hemorrhagic Fever (DHF), and Dengue Shock Syndrome (DSS). Despite its high fatality, there are few reports for the vaccine and adequate treatment. To increase the survival rate, therefore, it is highly demanded to early diagnosis and highly sensitive detection of the Dengue viral infection for the rapid initial treatment. In this study, a highly sensitive electrochemical biosensor is developed using a clustered regularly interspaced short palindromic repeats (CRISPR) system with methylene blue (MB)-conjugated Au nanoparticles (MB-AuNPs) for the detection of DENV. The CRISPR/CRISPR from the Prevotella and Francisella 1 (CRISPR/Cpf1) system, which consisted of CRISPR RNA (crRNA) and Cpf1 enzymatic protein, recognizes a target sequence and cleaved a nonspecific single-strand DNA (ssDNA) randomly with targeted DNA. Using this biological reaction, Dengue viral RNA was measured by using MB-AuNPs to enhance the electrochemical signal with a highly sensitive manner. The developed electrochemical biosensor exhibited the ultrasensitive detection of DENV-4, as low as 100 fM, without any RNA amplification methods.