Dipstick based immunochemiluminescence biosensor for the analysis of vitamin B12 in energy drinks: A novel approach

In this article, we describe a dipstick based immunochemiluminescence (immuno-CL) biosensor for the detection of vitamin B12 in energy drinks. The method is a direct competitive type format involving the immobilization of vitamin B12 antibody on nitrocellulose membrane (NC) followed by treatment with vitamin B12 and vitamin B12–alkaline phosphatase conjugate to facilitate the competitive binding. The dipstick was further treated with substrate disodium 2-chloro-5-(4-methoxyspiro {1,2-dioxetane-3,2¢-(5¢-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)-1-phenyl phosphate (CDP-Star) to generate chemiluminescence (CL). The number of photons generated was inversely proportional to the vitamin B12 concentration. After systematic optimization, the limit of detection was 1 ng mL−1. The coefficient of variation was below 0.2% for both intra- and inter-assay precision. Vitamin B12 was extracted from energy drinks with recovery ranged from 90 to 99.4%. Two different energy drinks samples were analyzed, and a good correlation was observed when the data were compared with a reference enzyme linked immuno sorbent assay (ELISA) method. The developed method is suitable for an accurate, sensitive, and high-throughput screening of vitamin B12 in energy drinks samples. The dipstick technique based on immuno-CL is suitable for the detection of several analyte in food and environmental samples.

(a) Schematic representation of immunochemiluminescence based dipstick technique for detection of vitamin B12. (b) Enzymatic dephosphorylation of dioxetane by alkaline phosphatase.Figure optionsDownload as PowerPoint slideHighlights
► Dipstick based immunochemiluminescence biosensor proposed for vitamin B12 analysis.
► The limit of detection of vitamin B12 is 1 ng mL−1 and applied in energy drinks.
► Chemiluminescence generated was inversely proportional to vitamin B12 concentration.
► Chemiluminescence analytical procedure was compared with ELISA.
► Alkaline phosphatase was stable chemiluminescent enzyme than Horse Radish Peroxidase.