Participant: PROMISE AGEP Research Symposium
Department: Chemical, Biochemical and Environmental Engineering
Institution: University of Maryland, Baltimore County (UMBC)
Fast Microdialysis as a Minimally-Invasive approach to Continuous Glucose Monitoring in Cell Culture
Our research group studies continuous bioprocess development in minibioreactors (50mL) and develops novel sensors and techniques for continuous monitoring of essential analytes. We focus on continuously monitoring essential analytes in up-stream processes with the objective of improving process control. Tighter parameter control can improve product quality, product quantity and ultimately decrease the cost of manufacturing with higher throughput and smaller culture volumes. This body of work aims to explore the efficacy of fast microdialysis as a minimally invasive sampling technique in cell culture. We hypothesize that by using fast microdialysis techniques in a loop microdialysis probe, we can detect glucose within the sensitivity range of our sensor. The sensor used in this study was a fluorescently tagged, monocysteine mutant, glucose binding protein (GBP, L255C) with micromolar sensitivity (LOD .04uM). Results showed that increasing the flow rate of the perfusion buffer, phosphate-buffered saline (PBS), decreased the mass transfer efficiency of the loop probe. At a flow rate of 100µL/min the dialysate did not require an additional dilution step. However, we also found that at high flow rates, this single-use loop probe fails/leaks. To verify the findings from our dynamic experiment, we ran a static experiment using dialysis cassettes. Our findings matched that of the dynamic experiment. Further development is needed to design a probe with improvements to the material choice, mechanical properties and dimensions. This work can be used to develop a minimally invasive, continuous glucose monitoring system for bioprocess monitoring and development.
Validating the Efficacy of Microdialysis in an Automated Transdermal Glucose Monitoring System
Conventional glucose monitoring techniques incorporate sensors with millimolar sensitivity. In recent years, interest has sparked in sensors with micromolar sensitivity for applications in minimally invasive sampling techniques such as fast microdialysis. Our lab is developing an automated transdermal glucose monitoring sensor. The sensor itself is an immobilized, fluorescent labeled, HIS-tagged glucose binding protein (GBP). GBP is a soluble protein found in the periplasmic space of Gram-negative bacteria. Glucose binding to GBP induces a conformational change that is the basis for attaching the fluorescent probe to an allosterically responsive site on the protein. GBP has a binding constant for glucose in the micromolar range. Previous results have also shown the reversibility of this interaction within this context, resulting in a reusable biosensor. Progression towards a fully automated device continues with the analysis of microdialysis in this setup. Microdialysis is a potentially useful method we have proposed for this system such that millimolar transdermal glucose samples will undergo microdialysis and fall within a detectable micromolar range. In this presentation we establish the feasibility of incorporating microdialysis to monitor glucose in the GBP biosensor setup. The effect of perfusion rate was determined.
Valencia Watson is a second-year graduate student in the department of Chemical and Biochemical Engineering. Valencia is a native of Denver, CO with a B.S. in Biomedical Engineering from the Georgia Institute of Technology. Valencia is proud to be a part of the UMBC community as a graduate student researcher and mentor. In her second year she is a continuing member of the Bridge to Doctorate Fellows program as well as the Meyerhoff Graduate Fellows program. She is an advocate for young women/men in STEM and has served on several teaching cohorts geared towards increasing the number of disadvantaged youths to attend college. She volunteers in the community and is currently teaching sign language to young children. She is motivated by the ideology that all persons deserve a shot at living a healthy and happy lifestyle, regardless of socioeconomic status. Valencia hopes to pursue a career in academia with entrepreneurial opportunities.
GENERAL SUMMARY OF GRADUATE RESEARCH
Under the advisory of Dr. Govind Rao and Dr. Leah Tolosa, our project focuses on developing an automated, continuous glucose monitoring device for bioprocesses. During the bioprocess it is important that the cells have access to essential nutrients, such as glucose, to maintain cell viability. Cell viability can correlate with the glucose concentration in the culture medium. The current gold standard method for glucose monitoring in (fed-)batch cell cultures requires direct sampling from the batch, after which the samples are quantified using an amperometric sensor device/machine. Our research group studies bioprocesses in minibioreactors (50mL) and develops novel sensors and techniques for continuous monitoring of essential analytes. We focus on continuously monitoring essential analytes in up-stream processes with the objective of improving process control.
SELECTED LIST OF PRESENTATIONS AND PUBLICATIONS
- Wilder CL, Walton C, Watson V, et al. Differential cathepsin responses to inhibitor-induced feedback: E-64 and cystatin C elevate active cathepsin S and suppress active cathepsin L in breast cancer cells. Int J Biochem Cell Biol. 2016; 79:199-208.
- Holzberg T, Watson V, Brown S et al. Sensors for biomanufacturing process development: facilitating the shift from batch to continuous manufacturing. Curr Opin in Chem Engin. 2018; 22: 115-127
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