Abstract
The function of proteins strongly depends on their conformations, which alters with the temperatures. Misfolding and aggregation might lead to numerous diseases. Particular attention thus has been given to protein folding and unfolding kinetics in numerous researches. Among miscellaneous relaxation approaches of protein kinetics, temperature jump is frequently used to initiate the protein conformational change. In this work, a confocal fluorescent thermometer was developed to detect the temperature evolution in a small excitation volume of ca. 10–3 mm3 and provide a spatial resolution of 100 μm. Photoexcitation of SiO2-coated gold nanorods (AuNR@SiO2) with a 1,064 nm pulse of ca. 100 μs duration leads to a temperature jump of 5 °C, as determined by the evolution of the fluorescence intensity of tryptophan. In addition, a continuous-wave laser at 1,550 nm was employed to increase the initial temperature to 44 °C by heating H2O, providing alternative initial temperatures for temperature jump experiments to reveal the unfolding kinetics of bovine serum albumin (BSA). The evolution of fluorescence of BSA upon temperature jump when the stationary temperature was raised to 44 °C differed from that at room temperature, suggesting a dynamical unfolding kinetics, and a rate coefficient of 75 ± 15 s–1 was derived. In this work, we successfully demonstrated the applicability of AuNR@SiO2 as the photothermal material for the temperature jump and employment of a confocal fluorescent thermometer to precisely monitor the minuscule heating volume. It would be advantageous to utilize this apparatus as an alternative tool for studying the kinetics of protein unfolding.