Design and validation of a new ratiometric intracellular pH imaging probe using lanthanide-doped upconverting nanoparticles

Abstract

pH homeostasis is strictly controlled at a subcellular level. A deregulation of the intra/extra/subcellular pH environment is associated with a number of diseases and as such, the monitoring of the pH state of cells and tissues is a valuable diagnostic tool. To date, only a few tools have been developed to measure the pH in living cells with the spatial resolution needed for intracellular imaging. Among the techniques available, only optical imaging offers enough resolution and biocompatibility to be proposed for subcellular pH monitoring. We present herein a ratiometric probe based on upconversion nanoparticles modified with a pH sensitive moiety for the quantitative imaging of pH at the subcellular level in living cells. This system provides the properties required for live cell quantitative imaging i.e. positive cellular uptake, biocompatibility, long wavelength excitation, sensitive response to pH within a biologically relevant range, and self-referenced signal.

Upconversion nanoparticles as intracellular pH messengers

“…As shown by us and others previously, the multiple emissions of the UCNPs at different wavelengths allow ratiometric sensing to be achieved. Depending on the chemistry of the indicator and distance from the UCNP linked via surface modification, it is possible to use one band of the UCNP (eg green emission) to excite a pH-sensitive moiety through the inner filter effect or energy transfer, while the other UCNP band (eg red emission) acts as a reference for quantitative ratiometric measurements [18,23,24,31]. This could also "turn-on" a separate analyte-dependent peak relative to a reference UCNP emission band.…”

“…In the earlier publication, Li et al were able to obtain a change in a ratio of 3.63 unit per unit of pH between pH 3.0-7.0 in buffers but failed to show quantitative pH-weighted images in the cells, only images of how the nanosensors colocalized with a lysosome dye in QBC939 cells [25]. In the later report by Du et al, the live cell quantitative imaging was only achievable down to pH 5.0 [24], which is above the pH attained by late endosomes and lysosomes for degrading internalized material [26,27].…”

“…Measurement of intracellular pH via UC nanoparticle (UCNP) has been described in the literature [18,[23][24][25], where the pH-sensing ability is bestowed by the presence of a pH dye, which modulates the ratio of the UCNP emission peaks depending on the pH of the surrounding environment. However, currently reported UC-based sensors fall short on mapping pH quantitatively at the subcellular level or their range does not cover the lower end of pH found in lysosomes.…”

Upconversion Luminescence Sensitized pH-Nanoprobes

“…The NIR light falls within the biological transparency window (700–1300 nm) and its deep-penetration and low auto-fluorescence give low photostability in comparison to UV and visible light. Most of the optical pH-sensors are investigated to perform within this window [ 30 , 31 , 32 , 33 , 34 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 ]. A list of the UC materials speculating pH-sensing is given in Table 2 .…”

“…However, engineering the structure of UCNPs to achieve high UC emission and FRET efficiency is one of the most required criteria for figuring out efficient pH-nanoprobe. An effort to the light manipulation at the nanoscale is evident in the recent report by Du et al [ 87 ]. The system is composed of NaGdF 4 :Tm 3+ /Yb 3+ @NaGdF 4 :Nd 3+ /Yb 3+ @NaYF 4 , coated with PEI (polyethyleneimine) and combined with fluorescein.…”