Tanja Deckert-Gaudig

Catalytic processes monitored at the nanoscale with tip-enhanced Raman spectroscopy

Heterogeneous catalysts play a pivotal role in the chemical industry, but acquiring molecular insights into functioning catalysts remains a significant challenge. Recent advances in micro-spectroscopic approaches have allowed spatiotemporal information to be obtained on the dynamics of single active sites and the diffusion of single molecules. However, these methods lack nanometre-scale spatial resolution and/or require the use of fluorescent labels. Here, we show that time-resolved tip-enhanced Raman spectroscopy can monitor photocatalytic reactions at the nanoscale. We use a silver-coated atomic force microscope tip to both enhance the Raman signal and to act as the catalyst. The tip is placed in contact with a self-assembled monolayer of p-nitrothiophenol molecules adsorbed on gold nanoplates. A photocatalytic reduction process is induced at the apex of the tip with green laser light, while red laser light is used to monitor the transformation process during the reaction. This dual-wavelength approach can also be used to observe other molecular effects such as monolayer diffusion.

Tip-Enhanced Raman Scattering (TERS) from Hemozoin Crystals within a Sectioned Erythrocyte

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.

Structure and Composition of Insulin Fibril Surfaces Probed by TERS

Amyloid fibrils associated with many neurodegenerative diseases are the most intriguing targets of modern structural biology. Significant knowledge has been accumulated about the morphology and fibril-core structure recently. However, no conventional methods could probe the fibril surface despite its significant role in the biological activity. Tip-enhanced Raman spectroscopy (TERS) offers a unique opportunity to characterize the surface structure of an individual fibril due to a high depth and lateral spatial resolution of the method in the nanometer range. Herein, TERS is utilized for characterizing the secondary structure and amino acid residue composition of the surface of insulin fibrils. It was found that the surface is strongly heterogeneous and consists of clusters with various protein conformations. More than 30% of the fibril surface is dominated by β-sheet secondary structure, further developing Dobsons model of amyloid fibrils (Jimenez et al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9196-9201). The propensity of various amino acids to be on the fibril surface and specific surface secondary structure elements were evaluated. β-sheet areas are rich in cysteine and aromatic amino acids, such as phenylalanine and tyrosine, whereas proline was found only in α-helical and unordered protein clusters. In addition, we showed that carboxyl, amino, and imino groups are nearly equally distributed over β-sheet and α-helix/unordered regions. Overall, this study provides valuable new information about the structure and composition of the insulin fibril surface and demonstrates the power of TERS for fibril characterization.

Ultraflat Transparent Gold Nanoplates—Ideal Substrates for Tip‐Enhanced Raman Scattering Experiments

Ultraflat gold nanoplates were found to be very attractive substrates for tip-enhanced Raman scattering (TERS) measurements. The transparent flat triangular or hexagonal nanoplates were synthesized by citrate reduction of HAuCl4 in aqueous solution. The obtained nanoparticles with a height of 15–20 nm had a smooth homogeneous surface with a roughness of about 100–200 pm. After spreading the gold plates on glass slides, cystine was successfully immobilized on them and the first TERS spectra of an amino acid were recorded. The spectra revealed a local variation of the attachment of cystine on the gold surface in two conformers. The production of well-defined gold particles is of general interest due to their geometry-dependent physical properties. Controlling and tuning the properties of these particles down to the nanometer regime extends their present applications, such as fluorescence quenching of organic molecules, surface-enhanced Raman scattering (SERS), and, as will be presented here, TERS. In SERS, molecules are adsorbed on rough metal substrates like colloids, electrodes, or evaporated films, resulting in a Raman signal-enhancement of several orders of magnitude. The irregular surface structure of those metallic surfaces, however, restricts the application of the SERS technique. With SERS, chemical information on low sample concentrations down to single molecules is accessible but the spatial information is lost. This argument is also valid for defined and highly reproducible SERS structures like electronbeam lithographic masks (see for instance Reference [5]) and for such structures the field enhancement invariably varies across the surfaces. Due to this lack of spatial resolution, a quantitative molecular analysis is hardly possible. If such information is required, TERS is the method of choice. In TERS a scanning tunneling microscope (STM) or an atomic force microscope (AFM) is coupled with a Raman spectrometer. Here the field-enhancing feature is confined to the very end of the probe. Usually a sharp metal tip or a single small metal particle acts as the sole source of enhancement.

Distinction of nucleobases – a tip-enhanced Raman approach

Summary 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.

Amide I vibrational mode suppression in surface (SERS) and tip (TERS) enhanced Raman spectra of protein specimens

Surface- and tip-enhanced Raman spectroscopy (SERS and TERS) are modern spectroscopic techniques, which are becoming widely used and show a great potential for the structural characterisation of biological systems. Strong enhancement of the Raman signal through localised surface plasmon resonance enables chemical detection at the single-molecule scale. Enhanced Raman spectra collected from biological specimens, such as peptides, proteins or microorganisms, were often observed to lack the amide I band, which is commonly used as a marker for the interpretation of the secondary protein structure. The cause of this phenomenon was unclear for many decades. In this work, we investigated this phenomenon for native insulin and insulin fibrils using both TERS and SERS and compared these spectra to the spectra of well-defined homo peptides. The results indicate that the appearance of the amide I Raman band does not correlate with the protein aggregation state, but is instead determined by the size of the amino acid side chain. For short model peptides, the absence of the amide I band in TERS and SERS spectra correlates with the presence of a bulky side chain. Homo-glycine and -alanine, which are peptides with small side chain groups (H and CH(3), respectively), exhibited an intense amide I band in almost 100% of the acquired spectra. Peptides with bulky side chains, such as tyrosine and tryptophan, exhibited the amide I band in 70% and 31% of the acquired spectra, respectively.

Tracking of nanoscale structural variations on a single amyloid fibril with tip‐enhanced Raman scattering

Amyloid fibrils are known to be responsible for diseases such as Alzheimers disease. A detailed insight into the structure of amyloid fibrils is fundamental since it is not yet understood what triggers the misfolding of proteins to the fiber like structures. The molecular structure of fibril surfaces on a single amino acid level has not been revealed so far but would present a valuable contribution to this question. Here we demonstrate the direct molecular distinction of selected amino acids on insulin fibril surfaces with a lateral resolution better than 2 nm by applying tip-enhanced Raman spectroscopy (TERS). This approach provides simultaneously a way to directly reveal conformational changes in the secondary structure, namely α-helix, β-sheet, on the fibril surface with nanometer resolution.

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 Characterization of Insulin Protofilaments and Fibril Polymorphs Using Tip-Enhanced Raman Spectroscopy (TERS)

Amyloid fibrils are β-sheet-rich protein aggregates that are strongly associated with a variety of neurodegenerative maladies, such as Alzheimers and Parkinsons diseases. Even if the secondary structure of such fibrils is well characterized, a thorough understanding of their surface organization still remains elusive. Tip-enhanced Raman spectroscopy (TERS) is one of a few techniques that allow the direct characterization of the amino acid composition and the protein secondary structure of the amyloid fibril surface. Herein, we investigated the surfaces of two insulin fibril polymorphs with flat (flat) and left-twisted (twisted) morphology. It was found that the two differ substantially in both amino acid composition and protein secondary structure. For example, the amounts of Tyr, Pro, and His differ, as does the number of carboxyl groups on the respective surfaces, whereas the amounts of Phe and of positively charged amino and imino groups remain similar. In addition, the surface of protofilaments, the precursors of the mature flat and twisted fibrils, was investigated using TERS. The results show substantial differences with respect to the mature fibrils. A correlation of amino acid frequencies and protein secondary structures on the surface of protofilaments and on flat and twisted fibrils allowed us to propose a hypothetical mechanism for the propagation to specific fibril polymorphs. This knowledge can shed a light on the toxicity of amyloids and define the key factors responsible for fibril polymorphism. Finally, this work demonstrates the potential of TERS for the surface characterization of amyloid fibril polymorphs.

Nanoscale structural analysis using tip-enhanced Raman spectroscopy.

Tip-enhanced Raman scattering (TERS) enables the label-free investigation of biochemical interfaces with nanometer lateral resolution by combining the benefits of the intrinsic molecular specificity of Raman spectroscopy, the sensitivity because of signal enhancing capabilities of plasmonic nanoparticles, and the precision of scanning probe microscopy. The structural differentiation of constituents based on inherent molecular information is possible even down to a few nanometer spatial resolution and consequently, nucleobases, proteins, lipids, and carbohydrates can be identified and localized in a single measurement. This has been shown in the last few years for different biological samples ranging from single DNA strand investigations to cell membrane studies.