Abstract
The use of two nanoparticles for quantitative pH measurements in live cells by means of fluorescence lifetime imaging microscopy (FLIM) is investigated here. These nanoparticles are based on CdSe/ZnS quantum dots (QDs), functionalized with N-acetylcysteine (CdSe/ZnS-A) and with a small peptide containing D-penicillamine and histidine (CdSe/ZnS-PH). CdSe/ZnS-A has tendency to aggregate and nonlinear pH sensitivity in a complex medium containing salts and macromolecules. On the contrary, CdSe/ZnS-PH shows chemical stability, low toxicity, efficient uptake in C3H10T1/2 cells, and good performance as an FLIM probe. CdSe/ZnS-PH also has key advantages over a recently reported probe based on a CdSe/ZnS QD functionalized with D-penicillamine (longer lifetimes and higher pH-sensitivity). A pH(±2σ) of 6.97 ± 0.14 was determined for C3H10T1/2 cells by FLIM employing this nanoprobe. In addition, the fluorescence lifetime signal remains nearly constant for C3H10T1/2 cells treated with CdSe/ZnS-PH for 24 h. These results show the promising applications of this nanoprobe to monitor the intracellular pH and cell state employing the FLIM technique.
Highlights
The use of two nanoparticles for quantitative pH measurements in live cells by means of fluorescence lifetime imaging microscopy (FLIM) is investigated here
Few examples of quantitative intracellular pH measurements by FLIM in live cells, employing fluorescent nanoparticle probes, have been reported.[9,13−15] Different functionalized CdSe/ZnS core−shell quantum dots (QDs) and carbon dots (CDs), as well as a perylene bisimide derivative encapsulated in a nanopolymer, were used as pH probes in those studies.[9,13,14]
It is well known that the linkage between CdSe/ZnS and thiolated compounds is produced by Zn-S covalent bonds.[9,21−23] The fluorescence emission of nanoparticles increased as a function of the ligand/QD ratio because ligands improve the water solubility and reduce the quenching
Summary
Obtained at different times maintain a Gaussian profile (see Figure S15). All the intracellular pH values calculated in the 24-hour experiment are within the 6.9−7.1 pH range as shown in Figure 5b (assuming that the linear dependence of τav vs pH is maintained at different times). These results illustrate the promising applications of the probe CdSe/ZnS-PH in fluorescence lifetime bioimaging. The effect of diverse biomolecules, drugs, and toxic substances on the cell state can be monitored by pH measurements with FLIM and a suitable probe
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