Development of Aequorin-Based Mast Cell Nanosensor for Rapid Identification of Botulinum Neurotoxin Type B

Abstract

An aequorin-based mast cell sensor was developed for rapid identification and detection of protein toxins. To monitor mast cell activation and to improve the sensitivity of detection, aequorin was stably expressed in the cells and used as an indicator of calcium flux and the stable cell line was created. The procedures for preparing cells were optimized to improve the quantum yield and sensitivity of detection. The cells sensitized with anti-DNP (dinitrophenyl) IgE were capable of detecting as little as 0.1 ng/mL DNP-BSA in less than 2 min. To demonstrate the utility of this mast cell sensor in detecting protein toxins, an IgE chimeric protein (p21-Fc was created. This was achieved by fusing the Fc region of IgE antibody to a 21-amino acid peptide (p21) derived from residues 40-60 of synaptotagmin II (Syt II), the protein receptor of botulinum neurotoxin type B (BoNT/B). In addition, magnetic beads linked with anti-BoNT/B polyclonal antibody were prepared to capture BoNT/B molecules in solution to make targets multivalent and concentrated. The p21-Fc binds to Fc RI receptors on RBL (Rat basophilic leukemia) cells and can be cross-linked by BoNT/B captured by the magnetic beads. This led to cell activation and Ca 2+ flux indicated by an increase of quantum yield. This assay can detect as little as 100 pM (15 ng/mL) BoNT/B in less than 1 hr, which included the initial sample preparation time. This study lays a foundation for detecting other protein toxins using mast cell sensors.

Emerging biosensors in detection of natural products

“…They used immobilized nano-magnetic beads coated with anti-BoNT/B polyclonal antibody for concentrated absorption of targets in sample solutions. The sensor detects 100 pM BoNT/B in less than an hour [ 132 ]. Lin et al developed a field-effect enzyme biosensor for quantification of urea, glucose, hydrogen ions, and specific proteins in solutions.…”

Fluorescent magnetic bead-based mast cell biosensor for electrochemical detection of allergens in foodstuffs

“…This triggers a sequence of intracellular events, including cellular degranulation and the release of chemical mediators such as histamine, serotonin, and β-hexosaminidase (Brockow, 2013;Corry and Kheradmand, 1999). Hence,RBL-2H3 mast cells show excellent potential for biosensor applications (Xin et al, 2014), due to their dramatic exocytotic response to antigen within minutes.They can detect target antigens and convert biological recognition into a signal that can be recorded and quantified quite easily (Kawakami and Galli,2002) .…”