The intracellular pH (pHi
) plays critical roles in the function of the cell, and its regulation is essential for most cellular processes, including cell volume regulation, vesicle trafficking, cellular metabolism, cell membrane polarity, cellular signaling, cell activation, growth, and proliferation.1, 2
Cellular dysfunction is often associated with abnormal pH values in organelles, and low intracompartmental pH values can denature proteins or activate enzymes.3
can also affect human physiology such as the nervous system, and pathophysiology including cancer4
and Alzheimer's disease.5
Monitoring pH changes inside living cells, therefore, is important for studying cellular functions and gaining a better understanding of physiological and pathological processes.
Intracellular pH can be measured with a variety of techniques, including the use of H+
permeable microelectrodes, nuclear magnetic resonance (NMR), absorbance spectroscopy, and fluorescence imaging and spectroscopy.2, 6, 7
Fluorescence spectroscopy using pH-sensitive indicators provides a powerful tool to assess the pHi
of intact cells and sub-cellular regions, which has several technical and practical advantages over other methods, including high sensitivity and excellent spatial and temporal resolution.3
In particular, ratiometric measurements, i.e.
, ratios obtained from simultaneous (or near simultaneous) fluorescence measurements at two (or more) excitation or emission wavelengths of the pH-sensitive probe, can eliminate the influence of variations in the local probe concentration, temperature, and optical path length.8
High spatial resolution of pHi
indicators is critically important, since pHi
may vary significantly between subcellular compartments, including the cytosol, mitochondria, endoplasmic reticulum, endosome, lysosome, and nucleus.
While fluorescent indicators based on small organic dyes have been used to study the intracellular environment for some time, severe limitations based of the rapid photobleaching of these dyes disallow the tracking of cellular processes, and how they relate to pH, over time. Fluorescent indicators with higher sensitivity, improved signal-to-noise ratios, and better photostability could enable studies into subtle changes in the cytosolic pH with changes in the environment, cell health, or cell type. In addition, the ability to track pH temporally and spatially in a living cell could be utilized for visualizing the endosomal release of nanoparticle drug carriers, thus providing new insights into nanoparticle-based targeted drug delivery approaches9, 10
. This information is crucial since endosomal release of drug carriers is necessary to enhance the efficacy of the drug being administered.
Our nanoparticle-based ratiometric pH sensor comprises a bright and photostable semiconductor quantum dot (QD) and pH-sensitive fluorescent proteins (FPs). The QD donor and pH-sensitive FP acceptors constitute a unique FRET pair wherein the environmental sensitivity of the acceptor fluorophore modulates the emission intensity of the donor. QDs are particularly useful FRET donors due to their exceptional brightness, high quantum yields and photostability, the capacity to bind multiple acceptor molecules, and their broad excitation spectra and narrow, tunable emission spectra.11, 12
FPs are versatile FRET acceptors as the polypeptide sequence can be genetically modified to include structural and functional elements necessary for protein purification, signal transduction, and probe assembly, as well as intracellular delivery and localization. FRET pairs comprising GFP-like FPs and QDs exhibit high energy transfer efficiencies and enable ratiometric measurements, resulting in heightened sensitivity by eliciting opposing changes in fluorescence emission at two wavelengths, while maintaining an internal control at an isosbestic point.13-15