Spatial pH jump measures chemical kinetics in a steady-state system

Area of Science:

• Chemical kinetics
• Photochemistry
• Fluorescence spectroscopy

Background:

• Studying fast chemical reactions requires high time resolution.
• Traditional methods often face limitations in achieving both high temporal resolution and sufficient signal averaging.
• Developing new techniques to probe transient chemical species is crucial.

Purpose of the Study:

• To develop and demonstrate a novel method for measuring chemical kinetics in a steady-state solution with a localized, transient reaction volume.
• To achieve high time resolution (<50 mus) for observing molecular changes within short residence times.
• To showcase the technique's versatility by measuring protonation-induced fluorescence changes in fluorescein.

Main Methods:

• Creation of a microscopic reaction volume with distinct conditions using multiphoton excitation of ortho-nitro benzaldehyde (o-NBA) to induce a pH jump.
• Utilizing fluorescence correlation spectroscopy (FCS) to determine molecular residence times (tau(D)) in the reaction volume.
• Employing time-correlated single-photon counting (TCSPC) for fluorescence lifetime measurements with long data averaging.

Main Results:

• Demonstrated a pH jump of >1 unit achievable via o-NBA multiphoton excitation.
• Measured a fluorescein residence time (tau(D)) of approximately 30 mus in the low-pH microvolume.
• Successfully captured fluorescence lifetime changes of the transient low-pH fluorescein species using TCSPC, proving the technique's ability to resolve short-lived states.
• Showcased tunable time resolution over three orders of magnitude by altering focal volume and solution viscosity.
• Confirmed the resolution of small, chemically induced fluorescence lifetime changes.

Conclusions:

• The developed method enables high time-resolution kinetic measurements in transient microenvironments.
• The technique is advantageous for studying fast, reversible reactions with minimal sample volume (<20 muL).
• This approach offers a powerful tool for investigating the dynamics of short-lived chemical species and subtle fluorescence changes.