Intracellular pH (pH i ) plays a critical role in the physiological and pathophysiological processes of cells, and fluorescence imaging using pH-sensitive indicators provides a powerful tool to assess the pH i of intact cells and subcellular compartments. Here we describe a nanoparticlebased ratiometric pH sensor, comprising a bright and photostable semiconductor quantum dot (QD) and pH-sensitive fluorescent proteins (FPs), exhibiting dramatically improved sensitivity and photostability compared to BCECF, the most widely used fluorescent dye for pH imaging.We found that Förster resonance energy transfer between the QD and multiple FPs modulates the FP/QD emission ratio, exhibiting a >12-fold change between pH 6 and 8. The modularity of the probe enables customization to specific biological applications through genetic engineering of the FPs, as illustrated by the altered pH range of the probe through mutagenesis of the fluorescent protein. The QD-FP probes facilitate visualization of the acidification of endosomes in living cells following polyarginine-mediated uptake. These probes have the potential to enjoy a wide range of intracellular pH imaging applications that may not be feasible with fluorescent proteins or organic fluorophores alone.

The conjugation of biomolecules such as proteins and peptides to semiconductor quantum dots (QD) is a critical step in the development of QD-based imaging probes and nanocarriers. Such protein-QD assemblies can have a wide range of biological applications including in vitro protein assays and live-cell fluorescence imaging. One conjugation scheme that has a number of advantages is the self-assembly of biomolecules on a QD surface via polyhistidine coordination. This approach has been demonstrated using QDs that have different coating types, resulting in different interactions between the biomolecule and QD surface. Here we report the use of a fluorescence resonance energy transfer (FRET) assay to evaluate the self-assembly of fluorescent proteins on the surface of QDs with eight distinct coatings, including two used in commercial preparations. The results of this systematic comparison can provide a basis for rational design of self-assembled biomolecule-QD complexes for biomedical applications.