A standard 96-well based high throughput microfluidic perfusion biofilm reactor for in situ optical analysis

Biofilm infections represent a major public health threat due to their high tolerance to antimicrobials and the lack of specific anti-biofilm drugs. To develop such drugs, it is crucial to have high-throughput biofilm growth systems that can emulate in vivo conditions without the cost and complexity of animal models. However, no current biofilm reactor can provide in vivo -like conditions in a high throughput standard microtiter format. This paper demonstrates a novel high-throughput (HT) microfluidic perfusion biofilm reactor (HT-μPBR) compatible with a standard 96-well microtiter plate for in situ optical analysis. A snap-on liquid-tight cover for standard microtiter plates was designed and fabricated with fluidic channels to provide closed-loop recirculating perfusion. Our system takes steps toward providing in vivo -like conditions with controlled shear stress and nutrient delivery. We describe the system fabrication and usage in optical analysis of biomass and viability of Escherichia coli ( E. coli ) biofilms. The HT-μPBR was set to perfuse at 1 mL/min corresponding to an average shear rate of approximately 5.7s^-1 on the bottom surface of a single well. Biofilms were detected on well plate bottoms and measured using a fluorescence microscope and plate reader to determine biomass and viability. Samples cultured in the HT-μPBR showed increased biomass while maintaining viability after 24 h. The HT-μPBR can further be combined with HT antibiotic susceptibility testing and additional optical techniques such as time-lapse imaging to improve understanding of the drug reaction mechanism as well as the optimization of drug combinations and delivery profiles.