Enzymes Immobilized on Glass

Lartigue, Donald J.; Yaverbaum, Sidney

doi:10.1016/b978-0-12-571810-3.50010-7

Cited by 2 publications

(1 citation statement)

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“…Biochemical reactions involving association of a molecule dissolved in solution with a target confined to a two-dimensional surface are of considerable industrial, medical, and biological importance. For example, biochemical products may be manufactured from substrate reactions with surface-immobilized enzymes which can be easily separated from the reaction and reused (1,2). Surface immobilized antigens can be used to assay for the presence of specific antibodies in blood serum (3,4).…”

Section: Introductionmentioning

confidence: 99%

Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy

Thompson¹,

Burghardt²,

Axelrod³

1981

Biophysical Journal

307

289

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The theoretical basis of a new technique for measuring equilibrium adsorption/desorption kinetics and surface diffusion of fluorescent-labeled solute molecules at solid surfaces has been developed. The technique combines total internal reflection fluorescence (TIR) with either fluorescence photobleaching recovery (FPR) or fluorescence correlation spectroscopy (FCS). A laser beam totally internally reflects at a solid/liquid interface; the shallow evanescent field in the liquid excites the fluorescence of surface adsorbed molecules. In TIR/FPR, adsorbed molecules are bleaching by a flash of the focused laser beam; subsequent fluorescence recovery is monitored as bleached molecules exchange with unbleached ones from the solution or surrounding nonilluminated regions of the surface. In TIR/FCS, spontaneous fluorescence fluctuations due to individual molecules entering and leaving a well-defined portion of the evanescent field are autocorrelated. Under appropriate experimental conditions, the rate constants and surface diffusion coefficient can be readily obtained from the TIR/FPR and TIR/FCS curves. In general, the shape of the theoretical TIR/FPR and TIR/FCS curves depends in a complex manner upon the bulk and surface diffusion coefficients, the size of the iluminated or observed region, and the adsorption/desorption/kinetic rate constants. The theory can be applied both to specific binding between immobilized receptors and soluble ligands, and to nonspecific adsorption processes. A discussion of experimental considerations and the application of this technique to the adsorption of serum proteins on quartz may be found in the accompanying paper (Burghardt and Axelrod. 1981. Biophys. J. 33:455).

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Section: Introductionmentioning

confidence: 99%