Starch-based C-dots from natural Gadong tuber as pH fluorescence label for optical biosensing of arginine

摘要

A fluorescence bi-enzyme optical arginine biosensor based on novel fluorophore carbon nanodots (C-dots) pH indicator derived from natural Gadong (Dioscorea hispida) tuber was developed. The brightly luminescent pH sensitive carbon-based quantum dots (QDs) exploited from exotic wild yam polysaccharides via aqueous-based synthesis method were immobilized on the acrylic-N-acryloxysuccinimide copolymer microspheres. Recombinant Glaciozyma antarctica arginase (GaArg) cloned in E. coli expression system and commercial urease were grafted on the acrylic microspheres (AMS) via peptide covalent link. During fluorescence arginine detection, the immobilized arginase catalyzed hydrolysis conversion of arginine to ornithine and urea, and subsequent enzymatic hydrolysis of urea by immobilized urease rendering deprotonation of starch-based C-dots, resulted in the heightening of C-dots' photoluminescence intensity due to elevated localized pH. Under 380 nm excitation, the C-dots fluorescence intensity at 420 nm increased linearly with arginine over the concentration range of 1-10 mM, with the limit of detection (LOD) estimated at 0.9 mM. The Gadong starch-based C-dots, which prepared from different natural tuber samples showed high reproducible physico-chemical properties, and gave reproducible fluorescence intensity at different pH values with relative standard deviation (RSD) values obtained at < 1.0%, which suggests high reproducibility of the synthesis of starch-based luminecent C-dots. The optical enzymatic biosensor doped with C-dots pH label demonstrated high selectivity towards fluorescence biorecognition of arginine compared to some other amino acids normally present in packaged fruit juices. No significant difference was found between C-dots-labeled enzymatic fluorescence microsensor and HPLC standard method for arginine content detection in fruit juice samples of grapes, mangoes and oranges.