Fluorescence Correlation Spectroscopy

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 "evanescent 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 observation area, or diffuse along the surface through it, the measured fluorescence fluctuates. The average rate of decay of the fluctuations, measured by minicomputer autocorrelation of the photmultiplier output, depends on the rates of association, dissociation, and surface diffusion. FCS without internal reflection has been proposed for or applied to the measurement of diffusion coefficients and chemical reaction rates in solution (Elson and Magde, 1974; Magde et al., 1974; Phillies, 1975; Koppel, et al., 1976, Rigler et al., 1979; Borejdo, 1979), molecular weights (Weissman et al., 1976), rotational diffusion coefficients (Aragon and Pecora, 1976; Ehrenberg and Rigler, 1976; Yardley and Specht, 1976), laminar flow rates (Magde et al., 1978), diffusion in lipid bilayers (Fahey et al., 1976; Fahey and Webb, 1978; Dragsten and Webb, 1978), specific immunoglobulin concentration (Nicoli et al., 1980), and the radius of focused laser beams (Sorscher and Klein, 1980). FCS has also found applications in cell biology, to study motions of muscle cross-bridges during contraction (Borejdo et al., 1979), binding of ethidium bromide to DNA in the cell nucleus (Sorscher et al., 1980), and microaggregation of surface receptors (Peterson et al., 1981). Expressions for the statistical accuracy of FCS autocorrelation functions have been derived (Koppel, 1974). FCS has been combined with internal reflection to detect viruses in biological fluids (Hirschfeld et al., 1977). Fluorescence excitation by total internal reflection (TIRF) has been used to study solutions of fluorescein (Hirschfeld, 1965), serum protein adsorption to solid surfaces (Watkins and Robertson, 1977; Harrick and Loeb, 1973; Lok et al., 1983 a; Lok et al., 1983 b), antibody binding to immobilized antigen (Kronick and Little, 1975; Kronick, 1974), and cell-substrate contact regions (Axelrod, 1981; Axelrod et al., 1983; Weis et al., 1982). Recently, TIRF has been combined with the fluorescence photobleaching recovery technique (Axelrod et al., 1976) to measure the adsorption kinetics and surface diffusion of serum albumin at glass (TIR/FPR; Thompson et al., 1981; Burghardt and Axelrod, 1981). TIRF has also been combined with singlet-singlet energy transfer methods to detect conformational changes of serum albumin upon adsorption to glass (Burghardt and Axelrod, 1983), and with anisotropy measurements to study the rotational diffusion of adsorbed fluorophores (Burghardt, 1983). We apply TIR/FCS to the nonspecific reversible adsorption of rhodamine-labeled immunoglobulin molecules (R-IgG) to fused silica coated with irreversibly adsorbed bovine serum albumin (BSA-glass). This system was chosen as a first test of TIR/FCS both because of its ease of preparation and because of the importance of nonspecific binding of serum proteins in the interaction of molecular and cellular blood components with solid surfaces. As a test of the TIR/FCS technique on a different system, autocorrelation functions are also obtained from rhodamine-labeled insulin reversibly adsorbing to BSAglass. These measurements are preliminary to the measurement of the binding kinetics of fluorescent-labeled hormones to biological or model cell surfaces and to surface-immobilized receptors. A potential application of TIR/FCS is to the measure of specific binding kinetics between two chemical species, one of which is immobilized on a surface. We have attempted to measure binding rates of rhodamine-labeled mouse myeloma protein MOPC 315 (with known specificity for dinitrophenyl) or of its univalent hapten-binding fragments at a fused-silica surface coated with dinitrophenylconjugated BSA (DNP-BSA-glass). We find that nonspecific binding dominates specific binding at equilibrium unless the characteristic desorption time of specific binding is very long.