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Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry
Published on: September 28, 2016
Whole cell biosensing via recA::mCherry and LED-based flow-through fluorometry.
R L Martineau1, V Stout, B C Towe
1Harrington Department of Bioengineering, Arizona State University, Tempe, AZ 85287-9709, USA. Rhett.Martineau@gmail.com
Biosensors & Bioelectronics
|October 6, 2009
Summary
A new optical cell enables whole-cell biosensors using red fluorescent protein (RFP) for sensitive DNA damage detection. This compact system minimizes distortions in dense samples, improving accuracy for environmental monitoring.
Area of Science:
- Biotechnology
- Biosensor Technology
- Optical Engineering
Background:
- Whole-cell biosensors require sensitive detection methods.
- Existing optical systems can suffer from distortions in dense samples.
Purpose of the Study:
- Develop a miniature, cost-effective optical cell for biosensor integration.
- Compare red fluorescent protein (RFP) and green fluorescent protein (GFP) as reporters.
- Establish a method for simultaneous fluorescence and biomass measurement.
Main Methods:
- Designed a compact optical system with LEDs, photodiodes, and filters.
- Utilized Escherichia coli with recA promoter-fusions to GFP and RFP.
- Exposed cultures to the DNA-damaging agent mitomycin C (MMC).
- Developed a multi-channel calibration method for optical data.
Main Results:
- RFP (mCherry) showed fewer measurement distortions than GFP (GFPmut3.1) in dense cultures.
- Lower limit of detection for MMC: 0.25nM (recA::mCherry) vs. 2.0nM (recA::gfpmut3.1).
- The integrated calibration method effectively eliminated optical distortions, negating sample dilution.
Conclusions:
- The developed optical cell is suitable for disposable biosensor platforms.
- RFP is a superior reporter protein for this compact biosensor system.
- The novel calibration method enhances accuracy and simplifies sample handling.
