Comparison of formats for the development of fiber-optic biosensors utilizing sol-gel derived materials entrapping fluorescently-labelled protein

The development of fiber-optic biosensors requires that a biorecognition el ement and a fluorescent reporter group be immobilized at or near the surfac e of an optical element such as a planar waveguide or optical fiber. In thi s study, we examined a model biorecognition element-reporter group couple c onsisting of human serum albumin that was site-selectively labelled at Cys 34 with iodoacetoxy-nitrobenzoxadiazole (HSA-NBD). The labelled protein was encapsulated into sol-gel derived materials that were prepared either as m onoliths, as beads that were formed at the distal tip of a fused silica opt ical fiber, or as thin films that were dipcast along the length of a glass slide or optical fiber. For fiber-based studies, the entrapped protein was excited using a helium-cadmium laser that was launched into a single optica l fiber, and emission was separated from the incident radiation using a per forated mirror beam-splitter, and detected using a monochromator-photomulti plier tube assembly. Changes in fluorescence intensity were generated by de naturant-induced conformational changes in the protein or by iodide quenchi ng. The analytical parameters of merit for the different encapsulation form ats, including minimum protein loading level, response time and limit-of-de tection, were examined, as were factors such as protein accessibility, leac hing and photobleaching. Overall, the results indicated that both beads and films were suitable for biosensor development. In both formats, a substant ial fraction of the entrapped protein remained accessible, and the entrappe d protein retained a large degree of conformational flexibility. Thin films showed the most rapid response times, and provided good detection limits f or a model analyte. However, the entrapment of proteins into beads at the d istal tip of fibers provided better signal-to-noise and signal-to-backgroun d ratios, and required less protein for preparation. Hence, beads appear to be the most viable method for interfacing of proteins to optical fibers.