TY - JOUR
T1 - Whole cell biosensing via recA::mCherry and LED-based flow-through fluorometry
AU - Martineau, R. L.
AU - Stout, V.
AU - Towe, B. C.
N1 - Funding Information:
This work was supported by NASA under grant NAG9#1359 . This grant provided funding but did not provide other support in terms of experimental design, analysis, or reporting.
PY - 2009/12/15
Y1 - 2009/12/15
N2 - 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 0.25 nM for recA::mCherry and 2.0 nM 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.
AB - 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 0.25 nM for recA::mCherry and 2.0 nM 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.
KW - Escherichia coli
KW - Green fluorescent protein
KW - Light-emitting diode
KW - Red fluorescent protein
KW - Whole-cell biosensor
KW - mCherry
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U2 - 10.1016/j.bios.2009.08.042
DO - 10.1016/j.bios.2009.08.042
M3 - Article
C2 - 19800215
AN - SCOPUS:70350411158
SN - 0956-5663
VL - 25
SP - 759
EP - 766
JO - Biosensors and Bioelectronics
JF - Biosensors and Bioelectronics
IS - 4
ER -