A dual beam total internal reflection fluorescence spectrometer for dynamic depth resolved measurements of biochemical liquid-solid interface binding reactions in opaque solvents

Abstract

The initiation of many physiologically important biochemical reactions is dependent on the binding of a molecule or molecules from solution to an appropriate solid surface such as a cell membrane. The quantitative study of dynamic interactions of molecules in solution with immobilized surfaces under physiologically relevant conditions presents a significant physical problem. Traditionally these measurements have been performed by discontinuous, nonequilibrium assays that cannot accurately model in vivo reaction mechanisms. Total internal reflection fluorescence spectroscopy (TIRFS) is an analytical technique that is based on the excitation of fluorescence by means of an exponentially decaying surface energy wave called the evanescent wave. The limited ‘‘penetration depth’’ of the evanescent wave leads to excitation of fluorescent molecules at or near the surface. This phenomena makes TIRFS uniquely qualified for the dynamic fluorescence study of liquid-solid interface binding reactions. The instrument described here permits the powerful technique of TIRFS to be used by biomedical researchers in their investigation of biochemical interface reactions. The fully automated, menu driven instrument allows the real time dynamic measurement of binding of fluorescent-labeled molecules in solution at physiologically relevant concentrations to appropriate immobilized surfaces. The novel design of the total internal reflection element enables the study of binding from essentially opaque and highly scattering solutions such as whole blood. Measurements may be performed under static, stirred, or flow conditions. The presently described TIRFS instrument is capable of tracking a binding reaction with evanescent fields established by two identical reflecting light beams with different reflection angles. This instrumental feature allows the dynamic measurement not only of the primary binding event but also of fluctuations in the distance between the optical interface and the point of signal generation. Data from experiments involving fluorescently labeled molecules binding to cell monolayers as well as binding experiments performed in whole blood are shown to verify the instrument performance. Due to its extraordinary capabilities and its user friendly design, the developed TIRFS instrument opens up a large number of relevant but previously unmeasurable experimental systems for investigation.