Camelid-derived nanobody (Nb) is attractive for monitoring environmental compounds in complicated matrix due to its small size, thermal stability, high binding affinity and resilience to organic solvents. Superparamagnetic bacteria magnetic particles (BMPs) with membrane-enclosed magnetite nanocrystals synthesized by magnetotactic bacteria was of great interest as magnetic carrier for immunomagnetic assays. In this study, alpaca-derived nanobodies specific to tetrabromobisphenol A (TBBPA), a brominated flame retardant, in monovalent (Nb1), bivalent (Nb2) and trivalent (Nb3) formats were genetically biotinylated to construct the complex Nb1-biotin, Nb2-biotin and Nb3-biotin, respectively. The biotinylated Nbs were then immobilized on the BMPs through streptavidin (SA) which was previously conjugated with the NH2 group of the membrane using glutaraldehyde. The formed immunoparticles (BMP-SA-biotin-Nbs) combining with a tracer of hapten-horseradish peroxidase were used to develop ELISAs for the detection of TBBPA from landfill leachate, sewage and sludge. Under the optimal assay condition with PBS (pH 10) containing 10% methanol and 0.137 moL L-1 NaCl, the assay with the immunoparticle BMP-SA-biotin-Nb3, showing a half maximal inhibition concentration (IC50) values of 0.37 ng mL-1, had a higher sensitivity than those with BMP-SA-biotin-Nb1 (IC50 = 1.02 ng mL-1) and BMP-SA-biotin-Nb2 (IC50 = 0.64 ng mL-1). It indicates that the binding affinity of Nb was improved after the multivalent and exhibited in the following order: Nb3＞Nb2＞Nb1. BMP-SA-biotin-Nb3 is quite stable as this immunoparticle-based assay could even be performed in 100% methanol or in solution with high pH (12) and ionic strength (2.18 moL L-1 NaCl) without the significant loss of sensitivity, having IC50 values in a range of 0.44–0.95 ng mL-1. Recoveries of TBBPA from spiked landfill leachate, sewage and sludge samples at 0.1–2 ng mL-1 (or ng g-1 (dw)) were in a range of 72–88%, 85–114% and 109–127%, respectively. TBBPA residues in real landfill leachate, sewage and sludge samples collected from Beijing area by this assay were detectable, with the highest concentration of 1.17 ng mL-1, 0.68 ng mL-1 and 3.65 ng g-1 (dw), respectively. The BMP-SA-biotin-Nb3-based assay, with a total time of 30 min, shows promising for the rapid monitoring of environmental chemicals.

Immobilization of multivalent nanobodies onto bacteria magnetic particles for the rapid detection of tetrabromobisphenol A in the environment

2019 Fall National Meeting

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