Abstract
A robust method to immobilize a maltose biosensor is described using an engineered maltose periplasmic binding protein (PBP) covalently coupled to NBDamide, an environmentally sensitive fluorophore. A mesoporous silica sol-gel derived from diglycerylsilane (DGS) was constructed to embed the maltose biosensor, and the ligand reporting fluorescence properties were meas red. When sequestered in the DGS-derived silica matrix, the biosensor retained maltose-dependent fluorescence sensing capability with micromolar affinity, which is consistent with the protein free in solution. The MBP-NBD conjugate was further modified by covalent conjugation with poly(ethylene glycol)-5000 (PEG) to promote the retention of water molecules around the protein and to reduce possible steric effects between the silica matrix and protein. Bioconjugation with PEG molecules does not significantly affect the signaling response of the protein in solution. When immobilized in the DGS polymer, a consistent increase in fluorescence intensity was observed as compared to the protein not functionalized with PEG. To our knowledge, this report presents the first successful method to embed a PBP biosensor in a polymerized matrix and retain
signaling response using an environmentally sensitive probe. The immobilization method presented here should be easily adaptable to all conformation-dependent biosensors.