Towards on-site visual detection of proteases in food matrices.
Anal Chim Acta
; 1078: 182-188, 2019 Oct 31.
Article
em En
| MEDLINE
| ID: mdl-31358218
ABSTRACT
Early detection of toxic proteases in food matrices plays a major role in preventing the occurrence of diseases as well as outbreaks. However, on-site detection of proteases, for instance, botulinum, anthrax and cholera in food matrices remains challenging due to their extremely low lethal dose levels. Here, we report a lateral flow assay (LFA) in a dipstick format for on-site visual detection of proteases in food matrices. The light chain of BoNT serotype A (BoNT/A LC) is used as a model system for validation of the proposed assay using magnetic beads conjugated to a synthetic peptide that provide a specific cleavage site for BoNT/A LC. Magnetic beads serve as both reporters for visual detection and as facilitators for sample clean-up, owing to the efficient magnetic separation protocol adopted. Digestion of the peptide substrate by BoNT/A LC for 5â¯h followed by the dipstick assay yields a reduction in color intensity of the test line on the dipstick compared to the control line obtained using an un-cleaved peptide substrate. Concentration dependent responses for the assay in carrot juice were obtained with a limit of detection (LOD) of 1 nM/2.5â¯nM (with/without amplification), also supported by RGB (ΔE) analysis, indicating the potential of the proposed methodology for on-site assaying of proteases in food matrices. Unlike typical affinity-based assays that yield a collective response for the active and inactive forms of the proteases, the proposed functional LFA targets only the active form, thereby enabling a more precise analysis for preventing potential false-positives. The proposed approach could be extended for detection of BoNT serotypes and other proteases in food matrices, upon utilizing appropriate substrates with specific cleavage sites.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Bioensaio
/
Toxinas Botulínicas Tipo A
/
Sucos de Frutas e Vegetais
Tipo de estudo:
Diagnostic_studies
/
Screening_studies
Idioma:
En
Revista:
Anal Chim Acta
Ano de publicação:
2019
Tipo de documento:
Article