Electroabsorption and Stark Fluorescence Spectroscopies of Thioflavin T

Abstract

Thioflavin T (ThT) is a typical fluorescent marker for detecting the formation of amyloid fibrils, because its fluorescence intensity increases by more than 2 orders of magnitude upon complexation with the fibrils. Strong electrostatic fields on protein surfaces are known to be a significant factor in chemical reactions and biological functions. Therefore, ThT bound to amyloid fibrils must experience strong electric fields. This study employed electroabsorption and Stark fluorescence spectroscopies to clarify the effects of external electric fields on the photophysics of ThT. The absorption spectrum shows two bands ascribed to locally excited (LE) and charge transfer (CT) states. Coupling between the LE and CT states is enhanced in the presence of an external electric field, resulting in fluorescence quenching. The electric field strength of the amyloid fibril surface was inferred from the fluorescence quenching efficiency of ThT.

Charge-transfer state and state mixing in tetracyanoquinodimethane probed using electroabsorption spectroscopy

“…This indicates the overlapping of multiple transitions, which have different molecular parameters Dm and D a. 23,25 The excited states of TCNQ were studied using second-order approximate coupled-cluster singles and doubles (CC2) calculations. 35 In this work, the two excited states were predicted within the measured energy range from 2.0 Â 10 4 to 3.2 Â 10 4 cm À1 .…”

“…A similar pairing of the zeroth derivative components of the two absorption bands was reported for the EA spectrum of thioflavin T, which exhibited the mixing of two different excited states. 23 The zeroth-derivative coefficient A w includes the contributions from the transition polarizability (A) and transition hyperpolarizability (B) (eqn ( 2) and ( 7)). According to eqn (2), the contribution from A is expected to be positive, whereas that from B can be either positive or negative, depending on the magnitude and sign of its components.…”

“…This indicates the overlapping of multiple transitions, which have different molecular parameters Δ μ and Δ. 23,25…”

“…Electroabsorption (EA) and Stark spectroscopies directly provide information about the nature of excited states and their mixing. [20][21][22][23][24] EA spectroscopy, based on the principle of the Stark effect, quantifies the perturbation of the absorption spectra owing to an external electric field by measuring the peak shift and lineshape modifications. 25 It is an optical absorption technique that relies on third-order nonlinear susceptibility and measures only instantaneous changes, thereby mitigating the impact of excited-state interactions with polar environments, such as relaxation after excitation.…”

“…A large dipole moment is associated with the ICT state, which leads to a large Stark effect. Therefore, EA spectroscopy is a powerful technique for probing the ICT state 21,23,26,27 and provides valuable insights into the interactions in excited states.…”