Fluorescence Anisotropy as a Temperature-Sensing Molecular Probe Using Fluorescein

Abstract

Fluorescence anisotropy, a technique to study the folding state of proteins or affinity of ligands, is used in this present work as a temperature sensor, to measure the microfluidic temperature field, by adding fluorophore in the liquid. Fluorescein was used as a temperature-sensing probe, while glycerol–aq. ammonia solution was used as a working fluid. Fluorescence anisotropy of fluorescein was measured by varying various parameters. Apart from this, a comparison of fluorescence anisotropy and fluorescence intensity is also performed to demonstrate the validity of anisotropy to be applied in a microfluidic field with non-uniform liquid thickness. Viscosity dependence and temperature dependence on the anisotropy are also clarified; the results indicate an appropriate selection of relation between molecule size and viscosity is important to obtain a large temperature coefficient in anisotropy. Furthermore, a practical calibration procedure of the apparatus constant is proposed. In addition, the potential of temperature imaging is confirmed by the measurement of temperature distribution under focused laser heating.

Influence of Viscosity and Molecular Size on Temperature Sensitivity of Fluorescence Anisotropy

“…The fluorescence lifetime is τ, Boltzmann's constant is k B , and the hydrodynamic volume of the fluorescent molecule is V. Equation ( 2) explicitly indicates that FA is a temperature-sensitive parameter. (11,18) It can also be confirmed that FA depends on the sample fluorescence lifetime, molecular size, and viscosity. In this study, we compared the temperature sensitivity of FA by changing the molecular size as a probe-dependent parameter and the viscosity as a solvent-dependent parameter.…”

“…Fluorescein derivatives were employed as a temperature-sensitive molecular probe in this study because fluorescein is a wellknown molecule with FA. (11,19) Here, fluorescein sodium salt (uranine, purchased from Fujifilm Wako) and fluorescein isothiocyanate (FITC) labeled with a polysaccharide dextran of different molecular weights, i.e., FITC-dextran conjugates (MW 10000, 20000, 40000, and 150000, purchased from Sigma-Aldrich), were used to investigate the effect of the molecular size on the temperature sensitivity of FA. The sample concentration was 0.01 mM under the diluted condition.…”

“…Our group has focused on the fluorescence anisotropy (FA) of fluorophores as an indicator of liquid temperature. (11)(12)(13) This technique analyzes the polarization characteristics of the fluorescence emission of a probe molecule. FA is independent of the solution pH (14) and is not affected by a nonuniform illumination intensity, molecular concentration, (15) or liquid film thickness, which may cause significant errors in LIF measurement.…”

“…In our previous study, we clarified that the size of the thermal probe influences the temperature coefficient. (11) In other studies, the temperature dependence of FA for various molecules was investigated on the basis of Perrin's law. (16,17) Recently, the important effect of the temperature-dependent viscosity on the rotational correlation time has been reported.…”

Temperature sensitivity of BODIPY dye (pyrromethene 597) over different linear organic solvents

“…[18][19][20][21] But recently, we have used fluorescence anisotropy to study the temperature of the fluid in a microchannel, by adding temperature sensitive fluorophore in the microfluid. 22,23) Then we found this parameter can be useful to explore the microscale water temperature. 23) In the present work, BODIPY C 22 H 33 BF 2 N 2 , is used as a potential thermal probe for organic solvents.…”