Fluorescence-lifetime-based pH sensing using resorufin

Abstract

Accurate, non-contact pH sensing is of particular importance in the biological and clinical sciences. Fluorescence lifetime based pH sensing is potentially more useful than intensity based methods because of the reduced sensitivity to excitation source intensity variations, scattering effects, and photobleaching. In this work, we investigate the variation of fluorescence lifetime with pH for resorufin. The intensity averaged lifetime ( τ ) of resorufin sodium salt in 0.1M phosphate buffer shows an increase of > 3 ns over the 2 -10 pH range, with 90% of the signal change occurring between pH 4 and 8. The fluorescence is not quenched by chloride or oxygen and was unaffected by the ionic strength of the buffer. Resorufin is relatively insoluble in non-alkaline phosphate buffered solutions, but τ was estimated to increase by ~2 ns between pH 6 and 8. Resorufin and its sodium salt were both incorporated into sol-gels by either acid or base hydrolysis of tetramethoxysilane (TMOS). Various surfactants were also added to the sol-gels in an attempt to optimise the fluorescence properties and pH sensitivity of the dyes, and to prevent cracking. The sols were then cast from petri-dishes or dip-coated onto acrylic and glass slides. The dyes retained their pH sensitivity, with τ showing an increase of approximately 2 ns over the pH range 6 -8. However, leaching of the dye is observed at higher pH and attempt to minimise dye leaching and solgel cracking, poly(vinyl alcohol) (PVA) was cross-linked to the silica gel to form a more flexible matrix.

Single-Molecule Enzymatic Conformational Dynamics: Spilling Out the Product Molecules

“…At the enzymatic active site and at the particular time of point 3 the product most likely exists as the resorufin anion (R – ) state since it is dramatically different in lifetime compared to that of the protonated form of resorufin (RH). 54 , 64 …”

“…The assignment of loose state and tight state of HRP from the rotation correlation time of nascent resorufin molecules is also consistent with the quenching effect of the heme group to the florescence lifetime of resorufin. 54 , 69 , 70 As in the loose states, the average distance from the nascent resorufin to the heme group is larger than that in tight states, which results in the decrease of the quenching effect from the heme and thus makes the lifetime longer than that in tight states. Furthermore, the local dielectric constant variation also influences the fluorescence lifetime of the enzymatic product that is embedded in the active site of the HRP enzyme.…”

Model-based experimental analysis of enzyme kinetics in aqueous–organic biphasic systems

“…In each of those samples, the decay of resorufin fluorescence intensity after the excitation pulses was recorded tenfold using all wavelengths larger than 500 nm. The acidic and basic forms of resorufin have different pH independent lifetimes (Ryder et al, 2003). At a defined pH value, the decay curve is composed of lifetime components of both forms.…”

A long-wavelength fluorescent substrate for continuous fluorometric determination of α-mannosidase activity: Resorufin α-d-mannopyranoside

“…However, previous assays with Res-CB [20] suggest that the kinetic parameters obtained are essentially unaffected relative to an assay performed using a stop buffer. Furthermore, since resorufin is a relatively pH-insensitive fluorophore, fluorescence emission and excitation wavelengths remain fairly uniform over a large range of pH values from 3-9 [26] which allow the same filter sets and instrumental setup methods to be used for assays run with different samples having a variety of pH values. It is noted that fluorescence measurements of free resorufin standards should be measured over the range of concentrations and at the same pH as those used for test samples, in order to obtain quantitative turnover rates at specific pH values.…”