News
ANALYTICAL CURRENTS served. But when cholesterol molecules were present, the recorded emission intenViewing multilayered thin films sity trajectories revealed stochastic blinkMuch of our understanding of selfmultilayer assemblies. The zrr sheets ing behavior. assembled, multilayered, thin-film ma- formed a tiled structure, which covered The researchers attribute the blinking terials is based on bulk analysis techmore than 95% of the surface. The to the toggling of the flavin between the niques, which provide results averheight of the assembly measured from a oxidized state (FAD) and the reduced state aged over macroscopic distances. Paul shear-force image, agreed with the theo(FADH2), with each on-off cycle correF. Barbara of the University of Minneretical estimate of 48 A. sponding to an enzymatic turnover. The sota, Thomas E. Mallouk of PennsylvaThe NSOM and AFM images demonturnover rate was independent of excitation nia State University, and their costrate that the single sheets of ZrP pack intensity, which suggests that it is not a workers used closely and lay flat photoinduced phenomenon. Statistical fluorescence on die substrate, analyses of single-molecule trajectories near-field scanthat the polymer revealed a slow fluctuation in the rate of ning optical adheres primarily cholesterol oxidation by FAD. microscopy to the ZrP sheets The single-molecule approach also (NSOM) atomic rather than the allowed the researchers to examine the force microscracks between the validity of chemical kinetics at the singlecopy (AFM) sheets, and that molecule level and to distinguish static and and bulk fluoresthe structure of the dynamic disorders—a level of detail that is cence microsfilm ii maintained very difficult with ensemble-averaged excopy to investiin the multilayer periments. The studies revealed a memory gate the morassemblies. Within effect: a short on-time tends to be followed pholosv of the optical spatial by another short on-time, whereas a long zirconium phosresolution of on-time tends to be followed by another phate (ZrP) mul50 nm no preferlong on-time. After a separation of 10 turntilater filmsm ential ordering of overs, the distribution becomes indepenA shear-force topographic image of a which were prethe dyes occurred multilayered film structure of the form dent. The researchers concluded that this pared by dipping glass/anchor/ZrP/Rhodamine B/ZrP/ within the polveffect arises from a slowly varying rate (k2 the substrate in electrolyte layers Texas Red. and is not photoinduced. Instead, they atsolutions of epFluorescence tribute thefluctuationof k to conformasositely sharged polyelectrolytes. efficiency was used tional changes of the protein. The simplest estimate the distance between the doThe microscopic techniques were explanation they say is that the enzyme nor ('Rhodamine K) and the accentor used to study ZrP multilayers, which has two conformational states which have f T W a c R o r ) l a v p r c T h p hi ah enprcnr were tonnected to one another by laydifferent rates for the activation step But ers of dye-labeled poly(allylamine hyfr„n f p r p ffi ie rvof0Q^ translates to they note that additional conformational ersofloride). Spatially resolved fluoresan average separation of 20 A between states could exist cench energy-transfey efficiencies were the chromophores. {]. Phys. them, B Evidence from ensemble-averaged ex1 Q Q S 7/)9 Q/151 Rf\\ used tenetermine the integrity of the periments had already suggested that this kind of molecular memory phenomenon, which deviates from Michaelis-Menten mechanisms, could be at work in other moThe active site of cholesterol oxidase Single-molecule nomelic enzyme systems. And a sigmoidal (COx) includes a flavin adenine dinucleenzymatic dynamics otide (FAD), which is naturally fluorescent dependence of the enzymatic reaction velocity on substrate concentration—a conseAdvances influorescencemicroscopy have in its oxidized form but not in its reduced quence of the memory effect—had been form. Individual COx molecules were conallowed studies of single molecules, includfined in an agarose gel of 99% water, allow- observed for COx. But the single-molecule ing enzymatic turnovers of a few motor approach permitted a more definitive ing them to rotate freely. Fluorescence improtein systems. Now, X. Sunney Xie and study, and the researchers expect detailed ages of FAD emission were taken with an colleagues at Pacific Northwest National microscopic pictures of conformational inverted fluorescence microscope by Laboratory and Washington State Univerdynamics and their influence on enzymatic raster-scanning with a fixed He-Cd laser s i t y lldVC oUSCI VCLl Cll^ylllilLlv. (.Ulllv/Vcis Ui single molecules of the navoenzyme cho- focus. In the absence of substrate, no mod- reactions to follow. (Science1998,282, 1877-82) ulation of emission intensity could be oblesterol oxidase. Analytical Chemistry News & Features, February 1, 1999 8 1 A
