Artificial clickase-triggered fluorescence "turn on" based on a click bio-conjugation strategy for the immunoassay of food allergenic protein. (1st January 2023) - Zhang, Xianlong
Graphical abstract: Highlights: An artificial clickase was prepared for the click bio-conjugation. The possible mechanism of click bio-conjugation of oligonucleotides was proposed. A new FCLISA based on the click bio-conjugation was established. The FCLISA was well applied in the detection of allergenic protein in food samples. Abstract: Herein, based on an artificial clickase-catalyzed bio-conjugation strategy, we established a sensitive fluorescent clickase-linked immunosorbent assay (FCLISA) platform using an oligonucleotide-molecular beacon (Oligo-MB) hairpin structure as a fluorescence switch for detection of food allergenic protein. Firstly, a highly stable Cu(I)-containing nanocube was prepared for usage as an artificial clickase, which could catalyze the bio-conjugation of two short oligonucleotides (i.e., Oligo-A and Oligo-B labeled by a 5′-alkyne and a 3′-azide group, respectively) through clickase-catalyzed azide/alkyne cycloaddition reaction. Subsequently, the formed long-chain oligonucleotide (Oligo-A-B) could hybridize with Oligo-MB hairpin to open hairpin structure, leading to its fluorescence turn on. By using clickase as an alternative enzymatic label in conventional ELISAs, the established FCLISA showed high sensitivity and accuracy in detection of casein, achieving a limit of detection as low as 1.5 × 10 −8 g/mL. Additionally, FCLISA has been challenged by detecting the casein in real samples, indicating a great potential in food safety assay.