• Development of sensor systems for application in cryopreservation

      Jahangir, Jahanbeen (University of BedfordshireUniversity of Bedfordshire, 2014-12)
      This work describes the development, validation and application of sensor systems to monitor phase transition events of cryoprotectant mixtures in samples and cryopreservation profiles and post-thaw recovery of Lactobacillus delbrueckii subsp. bulgaricus CFL1. Ice nucleation and glass transition (Tg) temperatures influence cell viability during cryopreservation. Knowledge of these phase changes for cryoprotectant mixtures is an essential step in optimising cryopreservation protocols for cell survival. Differential scanning calorimetry (DSC) is used to determine Tg, but the expensive nature of such instrumentation limits its widespread use. Cost-effective sensor systems have been designed to monitor ice-initiation and Tg events in small volume samples of cryoprotectants solutions. Tg values were measured for glycerol, sucrose and Me2SO (with and without NaCl supplement and ice-nucleators) in cryotubes and cryostraws, using temperature and screen-printed impedance sensors. The effect of changes to ice-initiation temperature on Tg was also investigated at different cooling and warming rates by using a Grant Asymptote (EF600) controlled rate freezer. The resulting Tg values obtained by single-channel transition monitoring system (TMS 1) were not significantly different from the values obtained by DSC reported in the literature. However multiple channelled transition monitoring system (TMS 2) requires further circuit modification and multiple screen-printed temperature probes to study the phase-change temperatures and to determine transition events in more than one sample at a time. The lactic acid bacterium (LAB) Lactobacillus delbrueckii was investigated as a model system to monitor the effect of different cryopreservation protocols on post-thaw cell metabolic activity. An important parameter for monitoring the post-thaw quality of LAB for starter culture preparation is the change in pH of the culture medium during incubation at 40 oC. Glass pH combination electrodes are the most common and widely used sensors. However, they are fragile, must be conditioned before use and are not disposable. An alternative to conventional glass electrodes are screen-printed carbon-metal electrodes. Different percentage mixtures of ruthenium and antimony pastes were tested and 54.5% carbon-antimony electrodes gave the best sensitivity and consistency in potentials at fixed pH with a screen-printed salt-bridged Ag/AgCl reference. LAB cultures were cryo-preserved at very rapid, moderate and very slow cooling rates and their post-thaw metabolic activity after overnight incubation in MRS broth was determined using screen-printed pH electrodes. Back to back testing with conventional glass pH sensors was performed to compare responses. Results indicated that early ice-initiation (by means of nucleators) prevents the cells from extensive dehydration (during cooling) and enables maximum post-thaw recovery after incubation (due to equilibrium ice formation and ice melting). In future, screen-printed pH sensors require development with integrated salt-bridged Ag/AgCl reference to make it robust in signalling response. The availability of low cost, disposable, non-fragile sensors and sensor systems to monitor transition events allows the determination of Tg of cryopreservation media during both cooling and warming cycles. A combined screen-printed (impedance + temperature) sensor is proposed for this purpose. A combined screen-printed (pH + reference) sensor would allow the monitoring of metabolic activity in post-thaw and fresh starter cultures of LAB. At present the salt-bridged pH reference is manually attached to the screen-printed pH working electrode but it requires further modifications to the method of attachment. The two sensor systems would enable optimisation of cryopreservation protocols for LAB and could enable such measurements to become routine at commercial scale.