title: Specific capture of the hydrolysate on magnetic beads for sensitive detecting plant vacuolar processing enzyme activity.
abstract: Conventional plant protease detection always suffers from high background interference caused by the complex coloring metabolites in plant cells. In this study, a bio-modified magnetic beads-based strategy was developed for sensitive and quantitative detection of plant vacuolar processing enzyme (VPE) activity. Cleavage of the peptide substrate (ESENCRK-FITC) after asparagine residue by VPE resulted in the 2-cyano-6-amino-benzothiazole (CABT)-functionalized magnetic beads capture of the severed substrate CRK-FITC via a condensation reaction between CABT and cysteine (Cys). The catalytic activity was subsequently obtained by the confocal microscopy imaging and flow cytometry quantitative analysis. The sensor system integrated advantages of (i) the high efficient enrichment and separation capabilities of magnetic beads and (ii) the catalyst-free properties of the CABT-Cys condensation reaction. It exhibited a linear relationship between the fluorescence signal and the concentration of severed substrate in the range of 10-600 pM. The practical results showed that, compared with normal growth conditions, VPE activity was increased by 2.7-fold (307.2 ± 25.3 μM min(-1)g(-1)) upon cadmium toxicity stress. This platform effectively overcame the coloring metabolites-caused background interference, showing fine applicability for the detection of VPE activity in real samples. The strategy offers great sensitivity and may be further extended to other protease activity detection.
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhou
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Cheng
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, China.
authors.forename: Lizhang
authors.initials: L
authors.lastname: Zeng
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, China.
authors.forename: Weipeng
authors.initials: W
authors.lastname: Liu
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Zhang
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, China. Electronic address: xingda@scnu.edu.cn.
authors.forename: Da
authors.initials: D
authors.lastname: Xing
mesh_terms: D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology*; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D006868:Hydrolysis; D008280:Magnetics / Q000379:methods; D059346:Magnets / Q000737:chemistry; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D010455:Peptides; D003546:Cysteine Endopeptidases; C089278:vacuolar processing enzyme
keywords: Condensation reaction; Confocal microscopy; Flow cytometry; Magnetic beads; Protease activity; Vacuolar processing enzyme