Signal limitations in tip-enhanced Raman scattering: the challenge to become a routine analytical technique

The fundamental parameters and limitations that determine the signal strength in tip-enhanced Raman scattering (TERS) are discussed. A semiquantitative analysis of the Raman signal expected in different experimental geometries and with different sample systems is presented, taking into account experimental parameters including Fresnel factor, numerical aperture of the illumination and collection optics, detection efficiency, and the Raman scattering cross section of the material. A top/side-on illumination geometry is essential for the study of nontransparent samples. It can yield the highest signal levels when strong tip–sample coupling using a metallic substrate provides large field enhancement. In contrast, axial/through-sample illumination is limited to transparent sample materials. Although conceptually simpler in experimental implementation and despite high numerical aperture signal collection efficiency, signals are generally weaker due to limited field enhancement. Crystalline solids with small Raman cross sections and dense molecular/biological systems with unavoidable far-field background provide the biggest challenge for TERS analysis yet at the same time hold the most exciting outstanding scientific questions TERS has the potential to answer.

Zundel-Type H-Bonding in Biomolecular Ions

Using quantum chemical calculations and infrared multiphoton dissociation (IRMPD) spectroscopy in the fingerprint and X-H stretching regions, we demonstrate here that the all-Ala b ₆ fragment ion features a macrocyclic structure with C₂ symmetry. For this structure, the ionizing proton is equally shared by the Ala(1) and Ala(4) amide oxygens in a Zundel-type symmetric (X…H⁺…X) H-bond. Figure

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Microfluidic channel with embedded SERS 2D platform for the aptamer detection of ochratoxin A

A selective aptameric sequence is adsorbed on a two-dimensional nanostructured metallic platform optimized for surface-enhanced Raman spectroscopy (SERS) measurements. Using nanofabrication methods, a metallic nanostructure was prepared by electron-beam lithography onto a glass coverslip surface and embedded within a microfluidic channel made of polydimethylsiloxane, allowing one to monitor in situ SERS fingerprint spectra from the adsorbed molecules on the metallic nanostructures. The gold structure was designed so that its localized surface plasmon resonance matches the excitation wavelength used for the Raman measurement. This optofluidic device is then used to detect the presence of a toxin, namely ochratoxin-A (OTA), in a confined environment, using very small amounts of chemicals, and short data acquisition times, by taking advantage of the optical properties of a SERS platform to magnify the Raman signals of the aptameric monolayer system and avoiding chemical labeling of the aptamer or the OTA target.

Chloroaluminum phthalocyanine thin films: chemical reaction and molecular orientation

The chemical transformation of the polar chloroaluminum phthalocyanine, AlClPc, to μ-(oxo)bis(phthalocyaninato)aluminum(III), (PcAl)₂O, in thin films on indium tin oxide is studied and its influence on the molecular orientation is discussed. The studies were conducted using complementary spectroscopic techniques: Raman spectroscopy, X-ray photoelectron spectroscopy, and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. In addition, density functional theory calculations were performed in order to identify specific vibrations and to monitor the product formation. The thin films of AlClPc were annealed in controlled environmental conditions to obtain (PcAl)₂O. It is shown that the chemical transformation in the thin films can proceed only in the presence of water. The influence of the reaction and the annealing on the molecular orientation was studied with Raman spectroscopy and NEXAFS spectroscopy in total electron yield and partial electron yield modes. The comparison of the results obtained from these techniques allows the determination of the molecular orientation of the film as a function of the probing depth.

Preparation of a nanocomposite film from poly(diallydimethyl ammonium chloride) and gold nanoparticles by in-situ electrochemical reduction, and its application to SERS spectroscopy and sensing of ascorbic acid

A nanocomposite film is described that is composed of alternating layers of poly(diallydimethyl ammonium chloride) and gold nanoparticles that interact through electrostatic forces. The films of varying thickness were prepared by the layer-by-layer technique, and Au-NPs were generated by electrochemical reduction of hexachloroauric acid. The composite films were characterized by UV?Cvis spectroscopy, X-ray photoelectron spectroscopy, and cyclic voltammetry. Most nanocomposite films exhibit linear, uniform, and regular layer-by-layer growth during the process of formation. The films exhibit unique performance in terms of surface enhanced Raman scattering and electrocatalytic activitiy towards the oxidation of ascorbic acid.

Higher order structure characterization of protein therapeutics by hydrogen/deuterium exchange mass spectrometry

Characterization of therapeutic drugs is a crucial step in drug development in the biopharmaceutical industry. Analysis of protein therapeutics is a challenging task because of the complexities associated with large molecular size and 3D structures. Recent advances in hydrogen/deuterium-exchange mass spectrometry (HDX-MS) have provided a means to assess higher-order structure of protein therapeutics in solution. In this review, the principles and procedures of HDX-MS for protein therapeutics characterization are presented, focusing on specific applications of epitope mapping for protein–protein interactions and higher-order structure comparison studies for conformational dynamics of protein therapeutics. Figure

HDX of protein backbone amide hydrogen     

New strategies for the chemical characterization of multi-walled carbon nanotubes and their derivatives

Industrially relevant characterization of multi-walled carbon nanotubes (MWCNT) is still a challenging task. The aim of this work is to show novel and fast concepts for the chemical characterization of carbon nanotubes (CNT) by a combination of analytical techniques. Information obtained by individual tools like Fourier transform infrared spectroscopy (FTIR), attenuated total reflection infrared spectroscopy or Raman spectroscopy is not providing a full picture of the functionalization of MWCNTs. However, a combination of tools such as FTIR or mass spectrometry with thermogravimetric methods proved to be very useful. Sample preparation for FTIR and Raman spectroscopy is another focus of this contribution because of its strong effect on the results obtained. We also are suggesting methods for sample preparation that lead to highly reproducibility results. Measurements have been carried out on typical CNT samples such as commercially available pristine, carboxylated and amino-functionalized MWCNTs, and on polystyrenegrafted MWCNTs. The results may serve as a guidance for the qualitative and quantitative characterization of CNT.

Recent progress in surface enhanced Raman spectroscopy for the detection of environmental pollutants

Surface enhanced Raman spectroscopy (SERS) has emerged as one of the most promising analytical tools in recent years. Due to advantageous features such as sensitivity, specificity, ease of operation and rapidity, SERS is particularly well suited for environmental analysis. We summarize here some considerations with respect to the detection of pollutants by SERS and provide an overview on recent achievements in the determination of organic pollutants, heavy metal ions, and pathogens. Following an introduction into the topic and considering aspects of sensitivity, selectivity, reproducibility and portability, we are summarizing applications of SERS in the detection of pollutants, with sections on organic pollutants (pesticides, PAHs and PCBs, explosives), on heavy metal ions, and on pathogens. In addition, we discuss current challenges and give an outlook on applications of SERS in environmental analysis. Contains 174 references.

Characterization and analysis of mycobacteria and Gram-negative bacteria and co-culture mixtures by Raman microspectroscopy, FTIR, and atomic force microscopy

The molecular composition of mycobacteria and Gram-negative bacteria cell walls is structurally different. In this work, Raman microspectroscopy was applied to discriminate mycobacteria and Gram-negative bacteria by assessing specific characteristic spectral features. Analysis of Raman spectra indicated that mycobacteria and Gram-negative bacteria exhibit different spectral patterns under our experimental conditions due to their different biochemical components. Fourier transform infrared (FTIR) spectroscopy, as a supplementary vibrational spectroscopy, was also applied to analyze the biochemical composition of the representative bacterial strains. As for co-cultured bacterial mixtures, the distribution of individual cell types was obtained by quantitative analysis of Raman and FTIR spectral images and the spectral contribution from each cell type was distinguished by direct classical least squares analysis. Coupled atomic force microscopy (AFM) and Raman microspectroscopy realized simultaneous measurements of topography and spectral images for the same sampled surface. This work demonstrated the feasibility of utilizing a combined Raman microspectroscopy, FTIR, and AFM techniques to effectively characterize spectroscopic fingerprints from bacterial Gram types and mixtures.

UV-polymerized butyl methacrylate monoliths with embedded carboxylic single-walled carbon nanotubes for CEC applications

The preparation of polymeric monoliths with embedded carboxy-modified single-walled carbon nanotubes (c-SWNTs) and their use for capillary electrochromatography (CEC) is described. Carbon nanotube composites were obtained by preparing a polymerization mixture in the presence of increasing c-SWNT concentrations, followed by UV initiation. The novel stationary phases were studied by optical microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Raman spectroscopy. Using short UV-polymerization times, the optimized porogenic solvent (a binary mixture of 1,4-butanediol and 2-propanol) gave rise to polymeric beds with homogenously dispersed embedded c-SWNTs. The CEC features of these monoliths were evaluated using polycyclic aromatic hydrocarbons (PAHs), non-steroidal anti-inflammatory drugs (NSAIDs) and chiral compounds. The monolith prepared in the presence of c-SWNTs showed enhanced resolution of the text mixtures, including a remarkable capability to separate enantiomers. Graphical Abstract

UV-polymerized polymeric monoliths with embedded c-SWNTs for CEC applications     

Spectroscopic detection and quantification of heme and heme degradation products

Heme and heme degradation products play critical roles in numerous biological phenomena which until now have only been partially understood. One reason for this is the very low concentrations at which free heme, its complexes and the partly unstable degradation products occur in living cells. Therefore, powerful and specific detection methods are needed. In this contribution, the potential of nondestructive Raman spectroscopy for the detection, quantification and discrimination of heme and heme degradation products is investigated. Resonance Raman spectroscopy using different excitation wavelengths (413, 476, 532, and 752?nm) is employed to estimate the limit of detection for hemin, myoglobin, biliverdin, and bilirubin. Concentrations in the low micromolar range (down to 3?μmol/L) could be reliably detected when utilizing the resonance enhancement effect. Furthermore, a systematic study on the surface-enhanced Raman spectroscopy (SERS) detection of hemin in the presence of other cellular components, such as the highly similar cytochrome c, DNA, and the important antioxidant glutathione, is presented. A microfluidic device was used to reproducibly create a segmented flow of aqueous droplets and oil compartments. Those aqueous droplets acted as model chambers where the analytes have to compete for the colloid. With the help of statistical analysis, it was possible to detect and differentiate the pure substances as well as the binary mixtures and gain insights into their interaction.

Cyanide–Arene Meisenheimer Complex Generated in Electrospray Ionization Mass Spectrometry Using Acetonitrile as a Solvent

The C – C bond formation activated under negative electrospray ionization of an acetonitrile solution of 1,3,5-trinitrobenzene is reported. The solvent function is to provide a source of cyanide ion, a highly problematic reagent, which is found to attack the electron-deficient aromatic ring to form a covalently bound anionic complex (Meisenheimer complex). The structure of the complex is elucidated by means of collision induced dissociation mass spectrometry and IR multiple photon dissociation spectroscopy in the ‘fingerprint’ region.

Complexity of fatty acid distribution inside human macrophages on single cell level using Raman micro-spectroscopy

Macrophages are phagocytic cells which are involved in the non-specific immune defense. Lipid uptake and storage behavior of macrophages also play a key role in the development of atherosclerotic lesions within walls of blood vessels. The allocation of exogenous lipids such as fatty acids in the blood stream dictates the accumulation and quantity of lipids within macrophages. In case of an overexposure, macrophages transform into foam cells because of the large amount of lipid droplets in the cytoplasm. Raman micro-spectroscopy is a powerful tool for studying single cells due to the combination of microscopic imaging with spectral information. With a spatial resolution restricted by the diffraction limit, it is possible to visualize lipid droplets within macrophages. With stable isotopic labeling of fatty acids with deuterium, the uptake and storage of exogenously provided fatty acids can be investigated. In this study, we present the results of time-dependent Raman spectroscopic imaging of single THP-1 macrophages incubated with deuterated arachidonic acid. The polyunsaturated fatty acid plays an important role in the cellular signaling pathway as being the precursor of icosanoids. We show that arachidonic acid is stored in lipid droplets but foam cell formation is less pronounced as with other fatty acids. The storage efficiency in lipid droplets is lower than in cells incubated with deuterated palmitic acid. We validate our results with gas chromatography and gain information on the relative content of arachidonic acid and its metabolites in treated macrophages. These analyses also provide evidence that significant amounts of the intracellular arachidonic acid is elongated to adrenic acid but is not metabolized any further. The co-supplementation of deuterated arachidonic acid and deuterated palmitic acid leads to a non-homogenous storage pattern in lipid droplets within single cells. Figure a

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Preparation of nitrogen-doped reduced graphene oxide and its use in a glassy carbon electrode for sensing 4-nitrophenol at nanomolar levels

We describe a novel procedure for the synthesis of nitrogen-doped reduced graphene oxide (N-rGO). It is based on the thermal reduction of GO (dispersed in water) with sodium diethyldithiocarbamate that acts as both the reducing agent and the source for nitrogen. The surface morphology of the N-rGO is characterized using high resolution transmission electron microscopy. X-ray photoelectron spectroscopy was carried out to study the composition of their surface, and Raman spectroscopy was performed to study the level of doping with nitrogen and the structural order. The N-rGO was deposited on a glassy carbon electrode (GCE), and the resulting electrode utilized as a sensing platform for 4-nitrophenol (4-NP). The modified GCE exhibits a well-defined oxidation peak current that is about ten times larger when compared to that of a bare GCE. The electron transfer number, proton transfer number and electron transfer rate constant (k_s 1.046 s^?1) were determined. At optimized conditions, the oxidation peak current is linearly related to the concentration of 4-NP in the 20–500 nM range, with a correlation coefficient of 0.9917. The detection limit (at an SNR of 3) is 7 nM. The method was successfully applied to the analysis of waters spiked with 4-NP. Recoveries range from 97.8 to 102.6 %, and no interferences are found for common inorganic cations and anions. Figure

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Hyperspectral unmixing of Raman micro-images for assessment of morphological and chemical parameters in non-dried brain tumor specimens

Hyperspectral unmixing is an unsupervised algorithm to calculate a bilinear model of spectral endmembers and abundances of components from Raman images. Thirty-nine Raman images were collected from six glioma brain tumor specimens. The tumor grades ranged from astrocytoma WHO II to glioblastoma multiforme WHO IV. The abundance plots of the cell nuclei were processed by an image segmentation procedure to determine the average nuclei size, the number of nuclei, and the fraction of nuclei area. The latter two morphological parameters correlated with the malignancy. A combination of spectral unmixing and non-negativity constrained linear least squares fitting is introduced to assess chemical parameters. First, endmembers of the most abundant and most dissimilar components were defined that represent all data sets. Second, the content of the obtained components’ proteins, nucleic acids, lipids, and lipid to protein ratios were determined in all Raman images. Except for the protein content, all chemical parameters correlated with the malignancy. We conclude that the morphological and chemical information offer new ways to develop Raman-based classification approaches that can complement diagnosis of brain tumors. The role of non-linear Raman modalities to speed-up image acquisition is discussed.

Direct Analysis of Large Living Organism by Megavolt Electrostatic Ionization Mass Spectrometry

A new ambient ionization method allowing the direct chemical analysis of living human body by mass spectrometry (MS) was developed. This MS method, namely Megavolt Electrostatic Ionization Mass Spectrometry, is based on electrostatic charging of a living individual to megavolt (MV) potential, illicit drugs, and explosives on skin/glove, flammable solvent on cloth/tissue paper, and volatile food substances in breath were readily ionized and detected by a mass spectrometer. Figure

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The use of Au@SiO2 shell-isolated nanoparticle-enhanced Raman spectroscopy for human breast cancer detection

This study uses the powerful fingerprint features of Raman spectroscopy to distinguish different types of breast tissues including normal breast tissues (NB), fibroadenoma (FD), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC). Thin frozen tissue sections of fresh breast tissues were measured by Raman spectroscopy. Due to the inherent low sensitivity of Raman spectra, Au@SiO₂ shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) technique was utilized to provide supplementary and more informative spectral features. A total of 619 Raman spectra were acquired and compared to 654 SHINERS spectra. The maximum enhancement effect of distinct and specific bands was characterized for different tissue types. When applying the new criteria, excellent separation of FD, DCIS, and IDC was obtained for all tissue types. Most importantly, we were able to distinguish ADH from DCIS. Although only a preliminary distinction was characterized between ADH and NB, the results provided a good foundation of criteria to further discriminate ADH from NB and shed more light toward a better understanding of the mechanism of ADH formation. This is the first report to detect the premalignant (ADH and DCIS) breast tissue frozen sections and also the first report exploiting SHINERS to detect and distinguish breast tissues. The results presented in this study show that SHINERS can be applied to accurately and efficiently identify breast lesions. Further, the spectra can be acquired in a minimally invasive procedure and analyzed rapidly facilitating early and accurate diagnosis in vivo/in situ. Figure

Human breast cancer detection with Au@SiO₂ SHINERS     

Detection of tobacco-related biomarkers in urine samples by surface-enhanced Raman spectroscopy coupled with thin-layer chromatography

The nicotine metabolites, cotinine and trans-3′-hydroxycotinine (3HC) are considered as superior biomarkers for identifying tobacco exposure. More importantly, the ratio of 3HC to cotinine is a good indicator to phenotype individuals for cytochrome P450 2A6 activity and to individualize pharmacotherapy for tobacco addiction. In this paper, a simple, robust and novel method based on surface-enhanced Raman spectroscopy coupled with thin-layer chromatography (TLC) was developed to directly quantify the biomarkers in human urine samples. This is the first time surface-enhanced Raman spectroscopy (SERS) was used to detect cotinine and 3HC in urine samples. The linear dynamic range for the detection of cotinine is from 40 nM to 8 μM while that of 3HC is from 1 μM to 15 μM. The detection limits are 10 nM and 0.2 μM for cotinine and 3HC, respectively. The proposed method was further validated by quantifying the concentration of both cotinine and 3HC in smokers’ urine samples. This TLC-SERS method allows the direct detection of cotinine in the urine samples of both active and passive smokers and the detection of 3HC in smokers.

Automated quantitative analysis of lipid accumulation and hydrolysis in living macrophages with label-free imaging

The accumulation of lipids in macrophages is a key factor that promotes the formation of atherosclerotic lesions. Several methods such as biochemical assays and neutral lipid staining have been used for the detection of lipids in cells. However, a method for real-time quantitative assessment of the lipid content in living macrophages has yet to be shown, particularly for its kinetic process with drugs, due to the lack of suitable tools for non-invasive chemical detection. Here we demonstrate label-free real-time monitoring of lipid droplets (LDs) in living macrophages by using coherent anti-Stokes Raman scattering (CARS) microscopy. In addition, we have established an automated image analysis method based on maximum entropy thresholding (MET) to quantify the cellular lipid content. The result of CARS image analysis shows a good correlation (R ²?>?0.9) with the measurement of biochemical assay. Using this method, we monitored the processes of lipid accumulation and hydrolysis in macrophages. We further characterized the effect of a lipid hydrolysis inhibitor (diethylumbelliferyl phosphate, DEUP) and determined the kinetic parameters such as the inhibition constant, K _i. Our work demonstrates that the automated quantitative analysis method is useful for the studies of cellular lipid metabolism and has potential for preclinical high-throughput screening of therapeutic agents related to atherosclerosis and lipid-associated disorders.

Analysis and characterization of heparin impurities

This review discusses recent developments in analytical methods available for the sensitive separation, detection and structural characterization of heparin contaminants. The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007?C2008 spawned a global crisis resulting in extensive revisions to the pharmacopeia monographs on heparin and prompting the FDA to recommend the development of additional physicochemical methods for the analysis of heparin purity. The analytical chemistry community quickly responded to this challenge, developing a wide variety of innovative approaches, several of which are reported in this special issue. This review provides an overview of methods of heparin isolation and digestion, discusses known heparin contaminants, including OSCS, and summarizes recent publications on heparin impurity analysis using sensors, near-IR, Raman, and NMR spectroscopy, as well as electrophoretic and chromatographic separations.