Fluorescence Correlation Spectroscopy

Abstract

An experimental application of total internal reflection with fluorescence correlation spectroscopy (TIR/FCS) is presented. TIR/FCS is a new technique for measuring the binding and unbinding rates and surface diffusion coefficient of fluorescent-labeled solute molecules in equilibrium at a surface. A laser beam totally internally reflects at the solid-liquid interface, selectively exciting surface-adsorbed molecules. Fluorescence collected by a microscope from a small, well-defined surface area -5 ,gm2 spontaneously fluctuates as solute molecules randomly bind to, unbind from, and/or diffuse along the surface in chemical equilibrium. The fluorescence is detected by a photomultiplier and autocorrelated on-line by a minicomputer. The shape of the autocorrelation function depends on the bulk and surface diffusion coefficients, the binding rate constants, and the shape of the illuminated and observed region. The normalized amplitude of the autocorrelation function depends on the average number of molecules bound within the observed area. TIR/FCS requires no spectroscopic or thermodynamic change between dissociated and complexed states and no extrinsic perturbation from equilibrium. Using TIR/FCS, we determine that rhodamine-labeled immunoglobulin and insulin each nonspecifically adsorb to serum albumin-coated fused silica with both reversible and irreversible components. The characteristic time of the most rapidly reversible component measured is -5 ms and is limited by the rate of bulk diffusion. Rhodamine-labeled bivalent antibodies to dinitrophenyl (DNP) bind to DNP-coated fused silica virtually irreversibly. Univalent Fab fragments of these same antibodies appear to specifically bind to DNP-coated fused silica, accompanied by a large amount of nonspecific binding. TIR/FCS is shown to be a feasible technique for measuring absorption/desorption kinetic rates at equilibrium. In suitable systems where nonspecific binding is low, TIR/FCS should prove useful for measuring specific solute-surface kinetic rates. INTRODUCTION Association/dissociation reactions between biological molecules in solution and sites on a surface are involved in many important processes. When blood encounters a foreign surface, serum proteins adsorb, affecting subsequent interactions of biological molecules (Vroman and Adams, 1971; Brash and Lyman, 1971; Macritchie, 1978) and cells (Horbett and Weathersby, 1981; Grinnell and Feld, 1982; Neumann et al., 1979; Lahav et al., 1982) with the surface. For medical applications, substrate-immobilized antigens can be used to detect specific antibodies in a patient's serum (Giaver, 1978; Weetal, 1972). In industry, the manufacture and purification of biochemical products for medicine and biological research can use immobilized enzymes acting on substrates in solution (Mosbach, 1976; Lartigue and Yaverbaum, 1976). In cellular biology, hormones and neurotransmitters stimulate response by binding to specific receptors embedded in the plasma membrane of their target cells (Cuatrecasas, 1975; Whittaker, N. Thompson's present address is the Department of Chemistry, Stanford University, Stanford, CA 94305. 1975; Kahn, 1976). Similarly, an immune response is triggered after viruses or antigens attach to immunological cells (Eisen, 1974; Clark, 1980). Aside from its prominence in biology, medicine, and industry, the association/dissociation process itself holds theoretical interest. Nonspecific adsorption of solute molecules followed by surface diffusion can, under certain conditions, enhance reaction rates with specific sites on a surface (Adam and Delbriick, 1968; Berg and Purcell, 1977). Data gathered on model biochemical systems (Roberts and Hess, 1977; Wong et al., 1978) indirectly suggest that such rate enhancement indeed occurs in living systems. We describe the application of a technique for directly measuring rate parameters critical to the association/ dissociation process at a planar target, called total internal reflection with fluorescence correlation spectroscopy (TIR/FCS; Thompson, 1982 a). The theoretical and conceptual basis of TIR/FCS has previously been described (Thompson et al., 1981; Thompson, 1982 b). In TIR/FCS, fluorescent-labeled molecules are in chemical and thermodynamic equilibrium between a solution and a surface to 103 BIOPHYS. J. © Biophysical Society * 0007-3495/83/07/103/12 $1.00 Volume43 July 1983 103-114 which they reversibly adsorb. A laser beam totally internally reflects at the surface-solution interface, forming an electromagnetic field called the wave in a very thin layer of solution immediately adjacent to the surface. Those fluorescent solute molecules that are adsorbed to the surface are selectively excited by the evanescent wave. A microscope collects the fluorescence arising from a small surface area, defined by an image plane aperture, and a photomultiplier measures the fluorescence intensity in time. As individual molecules bind and unbind within the observatio