Application of Electromodulated Fluorescence to the Study of the Dynamics of Alexa 488 Fluorophore Immobilized on a Gold Electrode

Published

May 16, 2000

Author(s)

L. Li, Curtis W. Meuse, Vitalii I. Silin, Adolfas Gaigalas, Y. Z. Zhang

Abstract

Alexa 488 fluorophores was immobilized on a gold electrode. The formation of the layer of fluorophore was followed in real time using surface plasmon resonance (SPR). Both SPR and spectroscopic ellipsometry (SE) gave a thickness of about} 0.7 nm for the fluorochrome layer. SE showed a strong optical absorption peak at 512 nm which is red-shifted relative to the absorption peak at 490 nm for fluorophores in solution. The electromodulated fluorescence(EmF) intensity was measured and found to be predominantly out-of-phase with the modulating potential. The amplitude of the EmF depended inversely on the modulating frequency, and was large only for frequencies less than 5 Hz. The EmF amplitude had a large dependence on solution ionic strength. Measurements of the electromodulated reflectance (EmR) indicated minimal changes in absorbency so that the EmF is most likely due to quantum yield modulation. We assumed that the underlying cause of the fluorescence modulation was local surface reorganization, which led to changes in the separation between the surface and the fluorochrome. The EmF response was described by a model which assumed that the fluorochrome had two locations relative to the surface, and a potential dependent rate constant, described the transition between the two locations. The rate constant was found to be less than 0.002s^-1. orientations relative to the surface, and a potential dependent rate constant described the transition between the two orientations. The rate constant was found to be less than 0.002 s^-1. We also considered a second model which assumed fluorochrome electrophoretic transport. The transport model did not reproduce the measured EmF response. Electron transfer from the excited state was also considered.

Citation

Langmuir

Volume

16

Issue

No. 10

Pub Type

Journals

Keywords

Alexa 488, electromodulated fluorescence, electroreflectance, ellipsometry, frequency response, SPR

Citation

Li, L. , Meuse, C. , Silin, V. , Gaigalas, A. and Zhang, Y. (2000), Application of Electromodulated Fluorescence to the Study of the Dynamics of Alexa 488 Fluorophore Immobilized on a Gold Electrode, Langmuir (Accessed December 12, 2024)