Near-infrared fluorescence lifetime pH-sensitive probes

Area of Science:

• Biochemistry
• Molecular Probes
• Fluorescence Spectroscopy

Background:

• Developing pH-sensitive probes is critical for understanding biological processes.
• Existing probes often lack sensitivity or suitability for near-infrared biological imaging.
• Hexamethylindotricarbocyanine is a known fluorophore scaffold with potential for modification.

Purpose of the Study:

• To create the first near-infrared (NIR) pH-sensitive fluorescence lifetime molecular probe for biological applications.
• To investigate the effect of amine functionalization position on probe pK(a).
• To demonstrate the probe's utility in biological imaging using fluorescence-lifetime tomography.

Main Methods:

• Modification of hexamethylindotricarbocyanine with a tertiary amine functionality.
• Determination of pK(a) values using emission spectroscopy.
• Measurement of fluorescence lifetimes in protonated and deprotonated states.
• Demonstration of biological applicability using fluorescence-lifetime tomography.

Main Results:

• A novel NIR pH-sensitive probe was synthesized by electronically coupling a tertiary amine to the fluorophore.
• Probe pK(a) was dependent on amine substitution position, with values of ~4.9 and ~5.5 observed.
• The probe (LS482) exhibited distinct fluorescence lifetimes for protonated (~1.16 ns) and deprotonated (~1.4 ns) states.
• Successful demonstration of the probe's utility in biological imaging via fluorescence-lifetime tomography.

Conclusions:

• The developed NIR probe is suitable for biological applications and offers pH sensitivity in the physiological range.
• Fluorescence lifetime measurements provide a robust method for pH sensing with this probe.
• This probe has significant potential for interrogating cellular processes and in vivo biological systems.