Molecular biology and engineering of fluorescent proteins provide powerful tools to probe cellular physiology and the molecular mechanisms of disease. A variety of fluorescent genetically encodable biosensors have been developed in the recent years to detect calcium concentration (cameleons, camgaroos and pericams), chloride (clomeleon), cell redox state (rxyFP), kinase activity (AKA, ATOMIC, Erkus, Picchu, ...) and pH (phluorins and deGFP).

I've also developed a pH sensor in the past. Based on the design of CY11.5 tandem protein developed by Miyawaki's groups and CY11.5's pH sensitivity, I developed a family of FRET based biosensors for pH: pHlameleons.

In more recent times, I have focused on the development of FRET pairs tailored to multiplexed biochemical imaging by spectral FLIM or spectral FAIM techniques. More to come on this topic in the near future.



Milestones
Over the last few years, we have developped two families of FRET pairs:
  • FRET pairs for biochemical multiplexing (to be published soon...)
  • pHlameleonsfor pH measurements (~2008)


FRET pairs for biochemical multiplexing

SECTION TO BE COMPLETED - Follow me on Twitter for updates


pHlameleons: A Family of FRET-Based Protein Sensors for Quantitative pH Imaging

Click here if you wish to read the paper published in Biochemistry

This work was performed in the laboratory of Prof. Fred Wouters

Abstract | Intracellular pH is an important indicator for cellular metabolism and pathogenesis. pH sensing in living cells has been achieved using a number of synthetic organic dyes and genetically expressible sensor proteins, even allowing the specific targeting of intracellular organelles. Ideally, a class of genetically encodeable sensors need to cover relevant cellular pH ranges. We present a FRET-based pH sensor platform, based on the pH modulation of YFP acceptor fluorophores in a fusion construct with ECFP. The concurrent loss of the overlap integral upon acidification results in a proportionally reduced FRET coupling. The readout of FRET over the sensitized YFP fluorescence lifetime yields a highly sensitive and robust pH measurement that is self-calibrated. The principle is demonstrated in the existing high-efficiency FRET fusion Cy11.5, and tunability of the platform design is demonstrated by genetic alteration of the pH sensitivity of the acceptor moiety.

Not only I hope this sensing strategy will be useful to others, but this work also allowed me to receave more extensive training on molecular biology.