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.
Highlights
Molecular diagnostics have played essential roles in life sciences, biosecurity, food safety, and environmental monitoring (Choi et al, 2016; Mumford et al, 2016)
Antibodies are critical to an enzyme-linked immunosorbent assay (ELISA) and provide the basis for the assay’s specificity and sensitivity (Itoh et al, 2002)
It was found that the detection sensitivity for the analytes was different between two matched antibody pairs, suggesting correct antibody pairs are essential to the performance of Cas12a-assisted new immunoassay (CANi)
Summary
Molecular diagnostics have played essential roles in life sciences, biosecurity, food safety, and environmental monitoring (Choi et al, 2016; Mumford et al, 2016). Because of the limited catalytic efficiency of horseradish peroxidase (Acharya et al, 2013), traditional ELISA is not sensitive enough to analyze low-abundant analytes such as hormone (insulin), cancer biomarkers, cytokines, and chemokines, which are in the picogram range in clinical samples at the early stage of the disease. It is well-known that the normal fasting insulin levels in the serum range between 0.17 and 1.34 ng/ml (Carmina et al, 2019).
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