A Novel Quantum Dot-Based pH Probe for Long-Term Fluorescence Lifetime Imaging Microscopy Experiments in Living Cells

Area of Science:

• Nanotechnology
• Biomedical Engineering
• Cell Biology

Background:

• Accurate intracellular pH (pH(i)) measurement is crucial for understanding cellular processes.
• Existing probes often suffer from aggregation, toxicity, or limited sensitivity in complex biological environments.
• Fluorescence lifetime imaging microscopy (FLIM) offers a robust method for quantitative biological measurements.

Purpose of the Study:

• To investigate the utility of two distinct cadmium selenide/zinc sulfide (CdSe/ZnS) quantum dot (QD) nanoparticles for quantitative intracellular pH measurements using FLIM.
• To compare the performance of QDs functionalized with N-acetylcysteine (CdSe/ZnS-A) versus a peptide containing D-penicillamine and histidine (CdSe/ZnS-PH).
• To evaluate the potential of CdSe/ZnS-PH as a stable, non-toxic FLIM probe for live-cell pH monitoring.

Main Methods:

• Synthesis and functionalization of CdSe/ZnS quantum dots (QDs) with N-acetylcysteine (CdSe/ZnS-A) and a peptide (CdSe/ZnS-PH).
• Characterization of nanoparticle properties, including aggregation, pH sensitivity, stability, and cytotoxicity in C3H10T1/2 cells.
• Quantitative pH measurements in live C3H10T1/2 cells using fluorescence lifetime imaging microscopy (FLIM) with the developed nanoprobe.
• Assessment of probe stability and cellular response over a 24-hour period.

Main Results:

• CdSe/ZnS-A nanoparticles exhibited aggregation and nonlinear pH sensitivity in complex media.
• CdSe/ZnS-PH nanoparticles demonstrated excellent chemical stability, low toxicity, and efficient cellular uptake in C3H10T1/2 cells.
• CdSe/ZnS-PH outperformed a previously reported D-penicillamine-functionalized QD probe, showing longer fluorescence lifetimes and higher pH sensitivity.
• FLIM measurements using CdSe/ZnS-PH determined the intracellular pH of C3H10T1/2 cells to be 6.97 ± 0.14.
• The fluorescence lifetime signal from CdSe/ZnS-PH remained stable in cells treated for 24 hours.

Conclusions:

• The peptide-functionalized quantum dot, CdSe/ZnS-PH, is a highly promising nanoprobe for quantitative intracellular pH monitoring via FLIM.
• CdSe/ZnS-PH offers significant advantages in terms of stability, sensitivity, and low toxicity compared to other QD-based probes.
• This nanoprobe facilitates reliable assessment of intracellular pH and monitoring of cell state using FLIM techniques.