Study on Factors Affecting the Performance of a CRISPR/Cas-Assisted New Immunoassay: Detection of Salivary Insulin as an Example

Xiaoting Lin; Gonglei Wang; Long Ma; Guozhen Liu

doi:10.3389/fbioe.2021.752514

Study on Factors Affecting the Performance of a CRISPR/Cas-Assisted New Immunoassay: Detection of Salivary Insulin as an Example

Front Bioeng Biotechnol. 2021 Nov 11:9:752514. doi: 10.3389/fbioe.2021.752514. eCollection 2021.

Authors

Xiaoting Lin¹, Gonglei Wang², Long Ma³, Guozhen Liu^{1

2}

Affiliations

¹ Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW, Australia.
² School of Life and Health Sciences, The Chinese University of Hong Kong, Shenzhen, China.
³ State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Microbiology, Tianjin Key Laboratory of Industry Microbiology, Ministry of Education, National and Local United Engineering Lab of Metabolic Control Fermentation Technology, China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, China.

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas is now playing a significant role in biosensing applications, especially when the trans-cleavage activity of several Cas effectors is discovered. Taking advantages of both CRISPR/Cas and the enzyme-linked immunosorbent assay (ELISA) in analytical and clinical investigations, CRISPR/Cas-powered ELISA has been successfully designed to detect a spectrum of analytes beyond nucleic acid. Herein, we developed a CRISPR/Cas12a-assisted new immunoassay (CANi) for detection of salivary insulin as an example. Specifically, factors (antibody selection, temperature, and assay time) affecting the CRISPR/Cas-based ELISA system's performance were investigated. It was observed that the concentration of blocking solution, selection of the capture antibody pairs, and the sequences of triggering ssDNA and guiding RNA affected this immunoassay sensitivity. In contrast, the preincubation of CRISPR/Cas12a working solution and pre-mixture of detection antibody with anti-IgG-ssDNA did not show influence on the performance of CANi for the detection of insulin. Under optimized conditions, the sensitivity for detection of salivary insulin was 10 fg/ml with a linear range from 10 fg/ml to 1 ng/ml.

Keywords: CRISPR/Cas; ELISA; biosensing; insulin detection; sensitivity.