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

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 (...

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Autores principales:	Xiaoting Lin, Gonglei Wang, Long Ma, Guozhen Liu
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	biosensing CRISPR/Cas ELISA sensitivity insulin detection Biotechnology TP248.13-248.65
Acceso en línea:	https://doaj.org/article/2a62b3911a59413ab049641ab75ba058
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spelling	oai:doaj.org-article:2a62b3911a59413ab049641ab75ba0582021-11-11T08:59:04ZStudy on Factors Affecting the Performance of a CRISPR/Cas-Assisted New Immunoassay: Detection of Salivary Insulin as an Example2296-418510.3389/fbioe.2021.752514https://doaj.org/article/2a62b3911a59413ab049641ab75ba0582021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fbioe.2021.752514/fullhttps://doaj.org/toc/2296-4185The 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.Xiaoting LinGonglei WangLong MaGuozhen LiuGuozhen LiuFrontiers Media S.A.articlebiosensingCRISPR/CasELISAsensitivityinsulin detectionBiotechnologyTP248.13-248.65ENFrontiers in Bioengineering and Biotechnology, Vol 9 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	biosensing CRISPR/Cas ELISA sensitivity insulin detection Biotechnology TP248.13-248.65
spellingShingle	biosensing CRISPR/Cas ELISA sensitivity insulin detection Biotechnology TP248.13-248.65 Xiaoting Lin Gonglei Wang Long Ma Guozhen Liu Guozhen Liu Study on Factors Affecting the Performance of a CRISPR/Cas-Assisted New Immunoassay: Detection of Salivary Insulin as an Example
description	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.
format	article
author	Xiaoting Lin Gonglei Wang Long Ma Guozhen Liu Guozhen Liu
author_facet	Xiaoting Lin Gonglei Wang Long Ma Guozhen Liu Guozhen Liu
author_sort	Xiaoting Lin
title	Study on Factors Affecting the Performance of a CRISPR/Cas-Assisted New Immunoassay: Detection of Salivary Insulin as an Example
title_short	Study on Factors Affecting the Performance of a CRISPR/Cas-Assisted New Immunoassay: Detection of Salivary Insulin as an Example
title_full	Study on Factors Affecting the Performance of a CRISPR/Cas-Assisted New Immunoassay: Detection of Salivary Insulin as an Example
title_fullStr	Study on Factors Affecting the Performance of a CRISPR/Cas-Assisted New Immunoassay: Detection of Salivary Insulin as an Example
title_full_unstemmed	Study on Factors Affecting the Performance of a CRISPR/Cas-Assisted New Immunoassay: Detection of Salivary Insulin as an Example
title_sort	study on factors affecting the performance of a crispr/cas-assisted new immunoassay: detection of salivary insulin as an example
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/2a62b3911a59413ab049641ab75ba058
work_keys_str_mv	AT xiaotinglin studyonfactorsaffectingtheperformanceofacrisprcasassistednewimmunoassaydetectionofsalivaryinsulinasanexample AT gongleiwang studyonfactorsaffectingtheperformanceofacrisprcasassistednewimmunoassaydetectionofsalivaryinsulinasanexample AT longma studyonfactorsaffectingtheperformanceofacrisprcasassistednewimmunoassaydetectionofsalivaryinsulinasanexample AT guozhenliu studyonfactorsaffectingtheperformanceofacrisprcasassistednewimmunoassaydetectionofsalivaryinsulinasanexample AT guozhenliu studyonfactorsaffectingtheperformanceofacrisprcasassistednewimmunoassaydetectionofsalivaryinsulinasanexample
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Study on Factors Affecting the Performance of a CRISPR/Cas-Assisted New Immunoassay: Detection of Salivary Insulin as an Example

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