Microfluidic Pneumatic Cages: A Novel Approach for In-chip Crystal Trapping, Manipulation and Controlled Chemical Treatment

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. ...

WITec GmbH

Tip-enhanced Raman spectroscopy of single RNA strands: towards a novel direct-sequencing method.

Tip-enhanced Raman scattering (TERS) is a technique that provides molecular information on the nanometre scale. Using a nanometre-sized metal particle results in a strong signal enhancement and a lateral resolution similar to the dimensions of the particle. As TERS is in a way the ultimate SERS experiment, the theoretical background will be briefly discussed with respect to the unique features and the specific effects that occur when only a single nanoparticle is used as a probe. All the major parts of the instrument will be revealed and the specific advantages of the different instrumental set ups will be investigated with respect to the particular requirements of the sample. Selected examples ranging from material science to cell biological applications demonstrate the capabilities and the potential of TERS in this tutorial review.

Tip-enhanced Raman scattering.

Nano-objects and cellular components are of great interest in biological sciences. Tip-enhanced Raman scattering (TERS) is a tool that allows addressing of such features for structural investigations without the need of further labelling. After brief introduction to the basic aspects of the technique an overview of present application of TERS is given. For this contribution we picked TERS experiments with emphasis on its application in life sciences. Based on these experiments, the future perspective of this analytical method and its limitations is discussed.

Perspectives for spatially resolved molecular spectroscopy--Raman on the nanometer scale.

Surface-enhanced Raman scattering was used as a spectroscopic tool to investigate the changes brought upon cytochrome P450BSss after fatty acid binding. Differences in the spectra of substrate-free and substrate-bound enzyme were observed indicating the potential for this method to be used in the screening of P450 substrates. In particular, the binding characteristics of myristic acid, an inherent substrate, and hydroxylauric acid, a product of fatty acid oxidation, towards P450BSss in the presence of H(2)O(2) were investigated. Specific spectral changes could be assigned to changes in the heme environment only for myristic acid, indicating an occurrence of oxidation process characteristic for the enzymatic substrate.

Surface-enhanced Raman scattering as a tool to probe cytochrome P450-catalysed substrate oxidation.

Tip-enhanced Raman scattering (TERS) spectra of a short immobilised peptide on a single transparent gold nanoplate are presented.

Tip-enhanced Raman scattering (TERS) of oxidised glutathione on an ultraflat gold nanoplate.

The aim of this survey was to evaluate the current practice regarding pain assessment and pain management strategies adopted in commonly performed minor painful procedures in Northern Italian Neonatal Intensive Care Units (NICUs).

Analgesic techniques in minor painful procedures in neonatal units: a survey in northern Italy.

Tip-enhanced Raman scattering (TERS) is a powerful technique to obtain molecular information on a nanometer scale, however, the technique has been limited to cell surfaces, viruses, and isolated molecules. Here we show that TERS can be used to probe hemozoin crystals at less than 20 nm spatial resolution in the digestive vacuole of a sectioned malaria parasite-infected cell. The TERS spectra clearly show characteristic bands of hemozoin that can be correlated to a precise position on the crystal by comparison with the corresponding atomic force microscopy (AFM) image. These are the first recorded AFM images of hemozoin crystals inside malaria-infected cells and clearly show the hemozoin crystals protruding from the embedding medium. TERS spectra recorded of these crystals show spectral features consistent with a five-coordinate high-spin ferric heme complex, which include the electron density marker band ν(4) at 1373 cm(-1) and other porphyrin skeletal and ring breathing modes at approximately 1636, 1557, 1412, 1314, 1123, and 1066 cm(-1). These results demonstrate the potential of the AFM/TERS technique to obtain nanoscale molecular information within a sectioned single cell. We foresee this approach paving the way to a new independent drug screening modality for detection of drugs binding to the hemozoin surface within the digestive vacuole of the malaria trophozoite.

Tip-enhanced Raman scattering (TERS) from hemozoin crystals within a sectioned erythrocyte.

The development of novel DNA sequencing methods is one of the ongoing challenges in various fields of research seeking to address the demand for sequence information. However, many of these techniques rely on some kind of labeling or amplification steps. Here we investigate the intrinsic properties of tip-enhanced Raman scattering (TERS) towards the development of a novel, label-free, direct sequencing method. It is known that TERS allows the acquisition of spectral information with high lateral resolution and single-molecule sensitivity. In the presented experiments, single stranded adenine and uracil homopolymers were immobilized on different kinds of substrates (mica and gold nanoplates) and TERS experiments were conducted, which demonstrated the reproducibility of the technique. To elucidate the signal contributions from the specific nucleobases, TERS spectra were collected on single stranded calf thymus DNA with arbitrary sequence. The results show that, while the Raman signals with respect to the four nucleobases differ remarkably, specific markers can be determined for each respective base. The combination of sensitivity and reproducibility shows that the crucial demands for a sequencing procedure are met.

Distinction of nucleobases - a tip-enhanced Raman approach.

Hemoglobin nanocrystals were analyzed with tip-enhanced Raman scattering (TERS), surface-enhanced resonance Raman scattering (SERRS) and conventional resonance Raman scattering (RRS) using 532 nm excitation. The extremely high spatial resolution of TERS enables selective enhancement of heme, protein, and amino acid bands from the crystal surface not observed in the SERRS or RRS spectra. Two bands appearing at 1378 and 1355 cm(-1) assigned to the ferric and ferrous oxidation state marker bands, respectively, were observed in both TERS and SERRS spectra but not in the RRS spectrum of the bulk sample. The results indicate that nanoscale oxidation changes are occurring at the hemoglobin crystal surface. These changes could be explained by oxygen exchange at the crystal surface and demonstrate the potential of the TERS technique to obtain structural information not possible with conventional Raman microscopy.

Detection of nano-oxidation sites on the surface of hemoglobin crystals using tip-enhanced Raman scattering.

The applications of spectroscopic methods in cancer detection open new possibilities in early stage diagnostics. Raman spectroscopy and Raman imaging represent novel and rapidly developing tools in cancer diagnosis. In the study described in this paper Raman spectroscopy has been employed to examine noncancerous and cancerous human breast tissues of the same patient. The most significant differences between noncancerous and cancerous tissues were found in regions characteristic for the vibrations of carotenoids, lipids and proteins. Particular attention was paid to the role played by unsaturated fatty acids in the differentiation between the noncancerous and the cancerous tissues. Comparison of Raman spectra of the noncancerous and the cancerous tissues with the spectra of oleic, linoleic, α-linolenic, γ-linolenic, docosahexaenoic and eicosapentaenoic acids has been presented. The role of sample preparation in the determination of cancer markers is also discussed in this study.

Raman spectroscopy and imaging: applications in human breast cancer diagnosis.

Tip-enhanced Raman Scattering (TERS) has recently emerged as a powerful spectroscopic technique capable of providing subdiffraction morphological and chemical information on samples. In this work, we apply TERS spectroscopy for surface analysis of the Bacillus subtilis spore, a very attractive biosystem for a wide range of applications regulated by the spore surface properties. The observed spectra reflect the complex and heterogeneous environment explored by the plasmonic tip, therefore exhibiting significant point-to-point variations at the nanoscale. Herein, we demonstrate that TERS data processing via principal component analysis allows handling such spectral changes, thus enabling an unbiased correlative imaging based on TERS. Our experimental outcomes suggest a denser arrangement of both proteins and carbohydrates on specific spore surface regions simultaneously revealed by AFM phase imaging. Successful TERS analysis of spores' surface is useful for bacterial surface-display systems and drug delivery applications.

Elena Bailo

WITec GmbH

Microfluidic Pneumatic Cages: A Novel Approach for In-chip Crystal Trapping, Manipulation and Controlled Chemical Treatment