TIME RESOLVED QD PH NANOSENSORS WITH EXTENDED RANGE

MENENDEZ, G., JOVIN, T., JARES-ERIJMAN, E.,

Linz, Austria.

Workshop; XI Winter Workshop. Advances in single-molecule research for biology and nanoscience; 2009

Semiconductor nanocrystals (quantum dots, QDs) have emerged as robust and stable probes with high potential for use in a variety of technological applications, ranging from electronic devices to biotechnology. QDs display high fluorescence quantum yields, are highly lumines-cent, stable over a broad range of biological conditions, and compatible with simple conjuga-tion techniques. Due to these properties, an increasing interest in their application in living cells and organism has been developed1,2. Recently, a variety of FRET-based QDot biosensors have been reported in which the nanopar-ticles act as efficient FRET donors. Quantum dots can also serve as energy acceptors both in fluorescence as in bioluminescence resonance energy transfer. FRET-based sensors comprise a central donor NC attached to chromophores displaying ab-sorption spectra sensitive to the microenvironment (i.e. pH). Changes in the spectral region due to microenviroment results in changes in the emission properties of the central QD. One strategy to conjugate the dye molecules to the NC is covalent binding3, which offers the ad-vantage of placing sensors close to the central donor. The disadvantadge of this approach is that obtaining sensors with different contents in dye is laborious and relies on a new synthesis.In this work, we introduce nanosensors based on streptavidin-biotin technology, a non cova-lent method with several advantages, such as a ratio dye/NC variable, perfectly controllable and reproducible. It also allows conjugation of the QD with different biotinilated molecules, such as specific ligand receptors in order to internalize the nanosensors in live cells4 (EGF, NGF, etc), or with other sensors for simultaneous sensing of various parameters (pH, polarity, ion concentration, etc). In addition, the new nanosensors were designed to perform in the physiological range. We performed the calibration curves and determination of pH by emission intensity and/or fluorescence lifetime of the NC. The latter permits the evaluation of wide field and confocal microscope images in which the local concentration of NCs in a given pixel is unknown. Time resolved measurements show that the nanosensors can perform in a pH range from 5.0 to 9.0, extending the usual dye-based sensors range of 2 units of pH. A direct and back mechanism of Energy Transfer QD-dye is proposed to account for the observed results.