Conjugation of 5(6)-carboxyfluorescein and 5(6)-carboxynaphthofluorescein with bovine serum albumin and their immobilization for optical pH sensing

An approach for the immobilization of a pH indicator in optical pH sensors and biosensors was developed. Fluorescent dyes 5(6)-carboxyfluorescein and 5(6)-carboxynaphthofluorescein were conjugated with bovine serum albumin as a non-enzymatic scaffold protein and the conjugation procedure was optimized. Fluorescent properties, sensitivity to temperature and photostability of conjugates have been studied and characterized into details. The conjugates were immobilized on glass support by: (i) glutaraldehyde cross-linking or (ii) entrapment in sol–gel matrix ORMOCER with subsequent glutaraldehyde cross-linking. The response to pH and leaching potential were evaluated. The sensor layer, based on immobilized conjugates of 5(6)-carboxyfluorescein and bovine serum albumin, displayed rapid response over the pH range 4.0–9.0, making it compatible with a range of applications such as bioprocessing, clinical diagnostics or environmental monitoring. Immobilized conjugates of 5(6)-carboxynaphthofluorescein and bovine serum albumin showed an enhanced sensitivity in alkaline pH levels, response for glutaraldehyde cross-linking ranged from pH 9.1 to 11.0 and from 8.3 to 10.0 for the ORMOCER entrapment, however, control of the indicator leaching was essential.