Whole cell biosensing via recA::mCherry and LED-based flow-through fluorometry

R. L. Martineau, V. Stout, B. C. Towe

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)759-766
Number of pages8
JournalBiosensors and Bioelectronics
Volume25
Issue number4
DOIs
StatePublished - Dec 15 2009

Fingerprint

Fluorometry
Green Fluorescent Proteins
Light emitting diodes
Mitomycin
Biosensing Techniques
Proteins
Light
Suspensions
Biosensors
Optical Devices
Biomass
Density (optical)
Limit of Detection
Plasmids
Cell Culture Techniques
Photodiodes
Fluorescence
Optical systems
Escherichia coli
Dilution

Keywords

  • Escherichia coli
  • Green fluorescent protein
  • Light-emitting diode
  • mCherry
  • Red fluorescent protein
  • Whole-cell biosensor

ASJC Scopus subject areas

  • Biophysics
  • Biomedical Engineering
  • Biotechnology
  • Electrochemistry

Cite this

Whole cell biosensing via recA::mCherry and LED-based flow-through fluorometry. / Martineau, R. L.; Stout, V.; Towe, B. C.

In: Biosensors and Bioelectronics, Vol. 25, No. 4, 15.12.2009, p. 759-766.

Research output: Contribution to journalArticle

Martineau, R. L. ; Stout, V. ; Towe, B. C. / Whole cell biosensing via recA::mCherry and LED-based flow-through fluorometry. In: Biosensors and Bioelectronics. 2009 ; Vol. 25, No. 4. pp. 759-766.
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