Whole cell biosensing via recA::mCherry and LED-based flow-through fluorometry.
A miniature flow-through optical cell has been developed with the potential for integration into a stand-alone, potentially disposable whole-cell biosensor platform. The compact and inexpensive optical system is comprised of closely coupled light-emitting diodes (LEDs), light-to-frequency (LTF) photodiodes, and celluloid filters. The system has been optimized to measure fluorescent reporters produced by cultures of biosensor cells in liquid suspension. As demonstration subjects, Escherichia coli cells carrying medium-copy plasmids with fluorescent reporter fusions to the rec promoter were exposed to the DNA-damaging agent mitomycin C (MMC). As reporter proteins, green fluorescent protein (GFP) and red fluorescent protein (RFP) were compared for suitability in the compact instrument. The RFP mCherry outperformed GFP (GFPmut3.1) as a reporter protein in the developed system on two counts. First, measurement distortions due to high optical density suspensions are minimal using RFP compared to GFP. Second, the limit of detection for MMC is estimated at 0.25nM for recA::mCherry and 2.0nM for recA::gfpmut3.1. Finally, a measurement method is presented whereby multiple channels of optical data are calibrated in an integrated fashion to allow simultaneous measurement of fluorescence and biomass concentration. The method substantially eliminates optical distortions due to dense samples and thus obviates the conventional need for sample dilution prior to measurement.
Harrington Department of Bioengineering, Arizona State University, Tempe, AZ 85287-9709, USA. Rhett.Martineau@gmail.com
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D001681:Biological Assay / Q000295:instrumentation*; D015374:Biosensing Techniques / Q000295:instrumentation; D004867:Equipment Design; D019544:Equipment Failure Analysis; D004926:Escherichia coli / Q000302:isolation & purification* / Q000378:metabolism*; D005434:Flow Cytometry / Q000295:instrumentation*; D008029:Lighting / Q000295:instrumentation; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D010783:Photometry / Q000295:instrumentation; D011938:Rec A Recombinases / Q000032:analysis*; D012666:Semiconductors; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*
D011938:Rec A Recombinases