Advances in TERS (tip-enhanced Raman scattering) for biochemical applications.

TERS (tip-enhanced Raman scattering) provides exceptional spatial resolution without any need for labelling and has become a versatile tool for biochemical analysis. Two examples will be highlighted here. On the one hand, TERS measurements on a single mitochondrion are discussed, monitoring the oxidation state of the central iron ion of cytochrome c, leading towards a single protein characterization scheme in a natural environment. On the other hand, a novel approach of single molecule analysis is discussed, again based on TERS experiments on DNA and RNA, further highlighting the resolution capabilities of this method.

Surface-enhanced Raman spectroscopy (SERS): progress and trends.

Surface-enhanced Raman spectroscopy (SERS) combines molecular fingerprint specificity with potential single-molecule sensitivity. Therefore, the SERS technique is an attractive tool for sensing molecules in trace amounts within the field of chemical and biochemical analytics. Since SERS is an ongoing topic, which can be illustrated by the increased annual number of publications within the last few years, this review reflects the progress and trends in SERS research in approximately the last three years. The main reason why the SERS technique has not been established as a routine analytic technique, despite its high specificity and sensitivity, is due to the low reproducibility of the SERS signal. Thus, this review is dominated by the discussion of the various concepts for generating powerful, reproducible, SERS-active surfaces. Furthermore, the limit of sensitivity in SERS is introduced in the context of single-molecule spectroscopy and the calculation of the 'real' enhancement factor. In order to shed more light onto the underlying molecular processes of SERS, the theoretical description of SERS spectra is also a growing research field and will be summarized here. In addition, the recording of SERS spectra is affected by a number of parameters, such as laser power, integration time, and analyte concentration. To benefit from synergies, SERS is combined with other methods, such as scanning probe microscopy and microfluidics, which illustrates the broad applications of this powerful technique.

Nanoscale distinction of membrane patches--a TERS study of Halobacterium salinarum.

The structural organization of cellular membranes has an essential influence on their functionality. The membrane surfaces currently are considered to consist of various distinct patches, which play an important role in many processes, however, not all parameters such as size and distribution are fully determined. In this study, purple membrane (PM) patches isolated from Halobacterium salinarum were investigated in a first step using TERS (tip-enhanced Raman spectroscopy). The characteristic Raman modes of the resonantly enhanced component of the purple membrane lattice, the retinal moiety of bacteriorhodopsin, were found to be suitable as PM markers. In a subsequent experiment a single Halobacterium salinarum was investigated with TERS. By means of the PM marker bands it was feasible to identify and localize PM patches on the bacterial surface. The size of these areas was determined to be a few hundred nanometers.

Characterizing cytochrome c states--TERS studies of whole mitochondria.

Protein structures (cytochrome c) were visualized by TERS measurements on whole mitochondria referring to specific spectral features describing the electronic state of the heme moiety.

Towards multiple readout application of plasmonic arrays.

In order to combine the advantages of fluorescence and surface-enhanced Raman spectroscopy (SERS) on the same chip platform, a nanostructured gold surface with a unique design, allowing both the sensitive detection of fluorescence light together with the specific Raman fingerprint of the fluorescent molecules, was established. This task requires the fabrication of plasmonic arrays that permit the binding of molecules of interest at different distances from the metallic surface. The most efficient SERS enhancement is achieved for molecules directly adsorbed on the metallic surface due to the strong field enhancement, but where, however, the fluorescence is quenched most efficiently. Furthermore, the fluorescence can be enhanced efficiently by careful adjustment of the optical behavior of the plasmonic arrays. In this article, the simultaneous application of SERS and fluorescence, through the use of various gold nanostructured arrays, is demonstrated by the realization of a DNA detection scheme. The results shown open the way to more flexible use of plasmonic arrays in bioanalytics.

Biochemical imaging below the diffraction limit--probing cellular membrane related structures by tip-enhanced Raman spectroscopy (TERS).

A first vibrational mapping on the nanometer scale was performed on a protein (streptavidin) labelled supported phospholipid film by means of tip-enhanced Raman spectroscopy (TERS). For this purpose a TERS spectral map was measured on the biomembrane model, using a step size far below the diffraction limit. Considering the model composition, spectra were classified as either typical for lipids, proteins or both simultaneously, in a qualitative manner. Subsequently, the spectroscopic information was assigned with respect to the topographic features. Since a spatial differentiation between different compositional domains is difficult to achieve by topographic features only, the combination of morphology and spectral data enables a much more detailed characterization of biomembranes.