Towards on-site visual detection of proteases in food matrices
Klisara, Nevena ; Yu, You Mei ; Palaniappan, Alagappan ; Liedberg, Bo - \ 2019
Analytica Chimica Acta 1078 (2019). - ISSN 0003-2670 - p. 182 - 188.
Food safety - Magnetic beads - Peptide - Proteases - Visual detection
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 5 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/2.5 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.
Woltering, E.J. - \ 2016
In: Encyclopedia of Applied Plant Sciences Elsevier Inc. Academic Press - ISBN 9780123948076 - p. 292 - 299.
Abscission - ACC - Ethylene - Ethylene sensitivity - Flower petals - Interorgan signaling - Ion leakage - Membrane integrity - Pollination - Programmed cell death - Proteases - Senescence - Vase life
Current knowledge indicates that flower petal senescence is a form of programmed cell death called vacuolar cell death. In this process the cell first degrades most of the cytoplasm and organelles using an array of degradative enzymes present in the vacuole for reuse of the nutrients. The final step in this process is disruption of the vacuolar membrane by which the hydrolytic enzymes are released to 'finish' what is left of the cell. In ethylene-sensitive flowers, ethylene triggers the cell death process, and flower life can be greatly extended by blocking production or perception of ethylene. This has led to development of very effective chemical treatments with broad application and molecular genetic strategies of potential commercial value. In ethylene-insensitive flowers, the senescence program may be similar to that of ethylene-induced senescence. However, the hormonal or developmental events that trigger the cell death processes in ethylene-insensitive species have not been identified. Many genes and transcriptional regulators with putative roles in senescence have been identified using transcriptomic approaches. Testing their functions in transgenic plants is necessary to design new concepts and treatments for prolonging the life of flowers.