Dual Excitation Ratiometric Fluorescent pH Sensor for Noninvasive Bioprocess Monitoring: Development and Application

Abstract

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.

Design and performance of a 24‐station high throughput microbioreactor

“…In all cases, optical sensors were used to measure pH and DO as previously described (Kermis et al, 2002;Kostov et al, 2001). Cell mass was monitored by turbidity, and provision was also included to allow fluorescence intensity measurements of green fluorescent protein (GFP), which can be used to monitor protein expression and organism stress (Lu et al, 2003).…”

Cell Culture Processes for Biopharmaceutical Manufacturing

“…Once the initial high throughput screening step discards the low producers, clones are typically screened at higher culture volumes like, in shake flasks or TubeSpin® tubes. These culture platforms have an obvious limitation of not being able to control parameters like pH and dissolved oxygen (DO) concentration which are controlled in bioreactors, though single use sensors have been used in shake flasks to monitor these parameters 56 . These parameters are known to affect cellular metabolism and a lack of their control at the screening stage can increase uncertainty associated with the behaviour of the selected clone upon its scale-up to bioreactors.…”

Design of Fluorescent Materials for Chemical Sensing

“…These particles are easily transformed into sensing systems by simply staining them with dye molecule solutions. [71][72][73][74] Because sensing particles can act as an individual probe, they can easily be used for the fabrication of sensor arrays. 58 Their small size and their polymeric nature make them suitable candidates for the generation of micro or nanosensors for intracellular analysis.…”