News
ANALYTICAL CURRENTS particle, respectively. However, most of the R6G molecules are adsorbed on inactive particles and are not detected. The authors Raman spectroscopy reveals far more struc- ruled out fluorescence as a source of the observed signals because they were able to tural information about molecules than obtain vibrational spectra of pyridine comdoes fluorescence spectroscopy, but the pounds and nonfluorescent biomolecules, inefficiency of Raman scattering usually hinders the analysis of single molecules. In such as tryptophan derivatives, that had fact, the normal efficiency of Raman scatter- also been adsorbed on single silver particles. Additional evidence for singleing must be enhanced by 1014 or more bemolecule detection was derived from Rafore single-molecule detection is possible. Shuming Nie and Steven R. Emory of Indi- man polarization measurements. Comparisons of relative fluorescence and SERS ana University report that by exploiting signal intensities obtained under identical surface and resonance enhancement efexperimental conditions revealed that (affects, they can collect Raman spectra of ter background subtraction) the Raman single rhodamine 6G (R6G) molecules adsignals were ~ 4-5 times higher than the sorbed on colloidal silver nanoparticles fluorescence The authors attributed the Nie and Emory discovered that a handful of the nanoparticles, termed "hot parti- large enhancements to the removal of pooulation-averaging effects cles", exhibited unusually high enhancement efficiencies and emitted bright, The authors commented that optimizaStokes-shifted light. Atomic force microstion of the colloid preparation and activacopy measurements revealed that the hot tion procedures should allow SERS singleparticles have an average particle diamemolecule studies of a wider range of moleter of 110-120 nm, approximately three cules, such as hemoproteins, nucleotides, times that of the overall average particle and pyridyl compounds. They also sugdiameter of 35 nm. gest that the resonance enhancement effect may be unnecessary because the surThe silver colloid is prepared with R6G concentrations of 2 x 10"10 and 2 x 10~n M, face enhancement effect appears to be an average of 1 and 0.1 molecule per nano- dominant. (Science e997,275,1102-6)
Single-molecule SERS
Continuous singlemolecule monitoring In a new twist on single-molecule fluorescence detection, Edward S. Yeung and Xiao-Hong Xu of Ames Laboratory at Iowa State University have reported the realtime continuous monitoring with video microscopy of individual rhodamine 6G and rhodamine-labeled 30-base singlestranded DNA (DNA-R6G) in aqueous solution. Measuring single-molecule photodecomposition lifetimes (t) requires careful selection of the laser power and exposure time. The power must be low enough to allow the fluorophore to emit over a longer period of time, yet high enough to overcome the dark counts of the intensified CCD. A small volume is defined by using a high-resolution microscope objective to image a thin layer of solution that has been excited with an
evanescent wave at the immediate quartzliquid layer. The spread of fluorescence spots reflects molecular diffusion, but individual molecules must be followed continuously
Continuously recorded fluorescence images of 0.5 nM R6G (a) and 0.5 nM R6G-DNA (b). The width of each zone measures one-dimensional diffusion during the static exposure time and the length measures photobleaching times clocked by the shift rate of the camera. (Adapted with permission from the American Association for the Advancement of Science.)
A comparison of laser-induced fluorescence (A) and SERS (B) of single R6G molecules. The fluorescence measurements are of free molecules in solution, whereas the SERS spectrum is of an adsorbed R6G molecule. (Adapted with permission from the American Association for the Advancement of Science.)
because there is not necessarily a one-toone correspondence between successive images in terms of trajectories and because photobleaching might be confused with molecules leaving the evanescentwave region. The camera shutter is left open during readout, smearing the image in the column direction and providing a means for measuring the diffusion coefficient and x independently. The value of t can be calculated from the digitization rate, the shift and exposure times, and the tail length in pixels. Direct measurement of molecular diffusion coefficients and photodecomposition rates makes it possible to determine the characteristics of individual molecules, not just the population average. The authors suggest that a similar method can be used to follow biomolecular reactions to compare individual values with macroscopic measurements. (Science e997, 275,1106-9)
Analytical Chemistry News & Features, April 1, 1997 223 A
