The development and application of a fluorescent excitation-ratiometric, noninvasive pH sensor for continuous on-line fermentation monitoring is presented. The ratiometric approach is robust and insensitive to factors such as source intensity, photobleaching, or orientation of the patch, and since measurements can be made with external instrumentation and without direct contact with the patch, detection is completely noninvasive. The fluorescent dye 8-hydroxy-1,3,6-pyrene trisulfonic acid was immobilized onto Dowex strongly basic anion-exchange resin, which was subsequently entrapped into a proton-permeable hydrogel layer. The sensor layer was polymerized directly onto a white microfiltration membrane backing that provided an optical barrier to the fluorescence and scatter of the fermentation medium. The ratio of emission intensity at 515 nm excited at 468 nm to that excited at 408 nm correlated well with the pH of clear buffers, over the pH range of 6-9. The sensor responded rapidly (<9 min) and reversibly to changes in the solution pH with high precision. The sterilizable HPTS sensor was used for on-line pH monitoring of an E. coli fermentation. The output from the indwelling sensor patch was always in good agreement with the pH recorded off-line with an ISFET probe, with a maximum discrepancy of 0.05 pH units. The sensor is easily adaptable to closed-loop feedback control systems.
A simple and rapid method for the preparation of a fluorescence-based optical pH sensor is described. The sensor is based on excitation ratiometric detection of a methacryloyl-modified analog of the well-known fluorescent pH indicator dye, 8-hydroxy-1,3,6-pyrene trisulfonic acid (HPTS). The modified dye, 6-methacryloyl-8-hydroxy-1,3-pyrene disulfonic acid (MA-HPDS), is similar in structure and function to HPTS. However, unlike HPTS, the presence of the methacrylate moiety allows MA-HPDS to participate as a comonomer in a free radical polymerization reaction. Covalent immobilization by this method is simple, as the preparation of the modified dye is accomplished in a single reaction step and its subsequent reaction with a comonomer results in simultaneous immobilization and purification. Sensors were prepared by copolymerization of the MA-HPDS with poly(ethylene glycol) diacrylate. Minimal leaching of the immobilized dye was observed from the HPDS-PEG matrix. The copolymer of HPDS and PEG-DA is fully autoclavable. The sensor is useful over the pH range of 6-9, with excellent reproducibility. Ionic strength effects on the apparent pKa of the immobilized dye are small and predictable.
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