news
ANALYTICAL CURRENTS
AFM imaging and recognition Stuart Lindsay and colleagues at Arizona State University, the University of Linz (Austria), and Molecular Imaging Corporation have developed a technique to generate single-molecule maps of specific molecules in complex biological samples while simultaneously obtaining high-resolution topographic images using atomic force microscopy (AFM). The ability to separate composition signals from topography signals opens up the door for new AFM applications just as fluorescent labels have led to new applications in optical microscopy. The new technique relies on the use of an antibody attached to an atomic force microscope tip. A highly specific recognition reaction between the antibody on the tip and an antigen in the sample is used to identify a specific molecule. When the antibody reacts with the antigen, a slight reduction in the oscillation amplitude of the
Measuring distances with evanescent waves Movements of a single fluorescent molecule in the x–y plane can be resolved to 1.5 nm by optical techniques. However, the resolution of movements in the z direction is typically limited to ~10 nm. Julio 4-quadrant Fernandez and colleagues at Columbia Uniphotodetector versity and the Mayo Clinic College of Med690-nm laser icine now present a technique called “evanescent nanometry”, which can measure any Piezo fluorescent particle’s vertical displacement in real time with subnanometer resolution. Fernandez and colleagues generated an evanescent wave by total internal reflection fluorescence. They found that the intensity of the wave decayed as a function of vertical distance, which allowed them to use the wave as 488-nm a “ruler” to translate fluorescent intensity into TIRF laser length. The investigators calibrated the decay of the wave’s intensity with an atomic force microscope, in which a fluorescently labeled CCD camera cantilever was moved vertically by a piezoactuator. The piezoactuator could accurately track An atomic force microscope was the z displacement of the cantilever. combined with total internal reflecThe investigators demonstrated the ability tion fluorescence (TIRF) to generof the calibrated evanescent wave to measure ate calibrated evanescent waves. changes in z displacement of the protein ubiq(Adapted with permission. Copyright 2004 National Academy of Sciences.) uitin as it was unfolded by a cantilever. Fernandez and colleagues point out that atomic force microscopy is not the only way to calibrate the evanescent wave; techniques like magnetic tweezers can do the same. They also say that errors in z displacement measurements may occur due to photobleaching, but this problem can be overcome by using relatively large fluorescent reporters. (Proc. Natl. Acad. Sci. U.S.A. 2004, doi 10.1073/pnas0403534101)
tip occurs. This change in amplitude is restored, but it results in a small shift in the absolute dc level of the cantileverdeflection signal. The ability to monitor specific components in complex mixtures at the singlemolecule level greatly extends the capability of AFM. The technique can now be used to monitor compositional changes over time or to determine the distribution of specific components in biological samples. (Proc. Natl. Acad. Sci. U.S.A. 2004, doi 10.1073/pnas.0403538101)
384 A
Gold nanoshell molecular sensors By replacing gold nanospheres with gold nanoshells, Gunnar Raschke and colleagues at Ludwig-Maximilians-Universität München and Roche Diagnostics (both in Germany) have improved the performance of single-nanoparticle molecular sensors. Such sensors rely on scattered light from a single nanoparticle to signal molecular binding and greatly reduce the number of binding events needed for detection. The use of gold nanoshells instead of nanospheres in single-nanoparticle molecular sensors has three advantages. First, the particle plasmon resonance of the nanoshells occurs at lower energies than that of nanospheres of the same diameter. As a result, the particle plasmon resonance is closer to the biological spectral window (700–1100 nm) of high optical transmission in blood and tissue. The second advantage is that nanoshells show a larger plasmon shift for a given change in refractive index of the surrounding environment. Finally, the scattering spectra of nanoshells show sharper resonances. This third advantage stems from the smaller full width at half-maximum of the scattering spectrum. (Nano Lett. 2004, doi 10.1021/nl049038q)
A N A LY T I C A L C H E M I S T R Y / N O V E M B E R 1 , 2 0 0 4
