Applied Spectroscopy

The capabilities of using gold nanoparticle based near-infrared surface-enhanced Raman scattering (SERS) to obtain biochemical information with high spatial resolution from human nasopharyngeal tissue were presented in this paper. The gold nanoparticles used have a mean diameter of 43 nm with a standard deviation of 6 nm. The SERS bands of nasopharyngeal tissue were assigned to known molecular vibrations of nucleic acids, amino acids, proteins, and metabolites. We also observed the blinking phenomenon at the tissue level when measuring the nasopharyngeal tissue SERS spectra, most frequently in signal intensity but also occasionally in peak positions. This phenomenon is excitation light intensity dependent. This work demonstrated great potential for using SERS imaging for distinguishing cancerous and normal nasopharyngeal tissues on frozen sections without using any dye labeling or other chemical species as functionalized binding sites.

Surface-enhanced Raman scattering (SERS) is a powerful technique for characterization of biological samples. SERS spectra from healthy brain tissue and tumors are obtained by sudden freezing of tissue in liquid nitrogen and crashing and mixing it with a concentrated silver colloidal suspension. The acquired spectra from tissues show significant spectral differences that can be used to identify whether it is from a healthy region or tumor. The most significant change on SERS spectra from the healthy/peripheral brain tissue to tumor is the increase of the ratio of the peaks at around 723 to 655 cm⁻¹. In addition, the spectral changes indicate that the protein content in tumors increases compared to the peripheral/healthy tissue as observed with tumor invasion. The preliminary results show that SERS spectra can be used for a quick diagnosis due to the simplicity of the sample preparation and the speed of the spectral acquisition.

A novel method for batch fabrication of substrates for surface-enhanced Raman scattering (SERS) has been developed. A modified platen that fits in a commercial electron beam evaporator enables the simultaneous deposition of Ag nanorod arrays onto six microscope slides by glancing angle deposition. Following removal of substrates from the evaporator, patterned wells are formed by contact printing of a polymer onto the surface. Well dimensions are defined by penetration of the polymer into the nanorod array and subsequent photochemical curing. Inherent advantages of this method include: (1) simultaneous production of several nanorod array substrates with high structural uniformity, (2) physical isolation of nanorod arrays from one another to minimize cross contamination during sample loading, (3) dimensional compatibility of the patterned array with existing SERS microscope, (4) large SERS enhancement afforded by the nanorod array format, (5) small fluid volumes, and (6) ease of use for manual delivery of fluids to each element in the patterned array. In this article, the well-to-well, slide-to-slide, and batch-to-batch variability in physical characteristics and SERS response of substrates prepared via this method is critically examined.

We have developed a rapid, sensitive, and quantitative method for identification of microRNA (miRNA) sequences in multicomponent mixtures using surface-enhanced Raman spectroscopy (SERS). The method uses Ag nanorod array substrates prepared by oblique angle vapor deposition as the SERS platform. We show that Ag nanorod-based SERS spectra are uniquely characteristic for each miRNA sequence studied, and that the spectral reproducibility is sufficient for quantitative analysis of miRNA profiles in multicomponent mixtures using partial least squares (PLS) regression analysis. This method was applied to individual sample mixtures consisting of two, three, and five miRNAs. Separate PLS models were generated for the two-, three-, and five-component mixtures from >150 calibration spectra covering a concentration range of 6 to 150 μM for each miRNA. The PLS models were externally validated with independent test samples resulting in root mean square errors of prediction (RMSEP) of 7.4, <7.4, and <10 μM for the two-, three-, and five-component models, respectively. These results demonstrate the applicability of SERS for quantitative detection and profiling of miRNAs and suggest that SERS may prove to be a novel, label-free method for identification of disease biomarkers.

The light sheet direct Raman (LSDR) imaging technique is used to obtain wide scope, simultaneous images of samples emitting Raman scattered light, without mapping their point-to-point Raman scattering intensities. A prototype system consisting of a background-free electronically tuned Ti:sapphire laser (BF-ETL), band-pass (BP) filters, and a charge-coupled device (CCD) detector is developed in the present study. The LS excitation method enables us to obtain a wide field of Raman view. The BF-ETL allows us to obtain direct Raman images with multiple Raman bands without the need for rearranging the optical settings. The system is used to observe the mixing of pure solvents: carbon tetrachloride (CCl₄) and chloroform (CHCl₃), and ethylene glycol (EG) and polyethylene glycol (PEG). LSDR images are successfully obtained within an exposure time of 0.5 s. EG and PEG, whose Raman spectra appear similar, can be distinguished clearly in the images, suggesting that the system has high spectral resolution.

Low-field nuclear magnetic resonance (NMR) spectroscopy is applied to study the hydrogenation of toluene in a lab-scale reactor. A conventional benchtop NMR system was modified to achieve chemical shift resolution. After an off-line validity check of the approach, the reaction product is analyzed on-line during the process, applying chemometric data processing. The conversion of toluene to methylcyclohexane is compared with off-line gas chromatographic analysis. Both classic analytical and chemometric data processing was applied. As the results, which are obtained within a few tens of seconds, are equivalent within the experimental accuracy of both methods, low-field NMR spectroscopy was shown to provide an analytical tool for reaction characterization and immediate feedback.

This paper develops the theoretical basis behind the transmittance ratio test method for determining the relative stray radiant energy level in a double-beam dispersive spectrophotometer so as to allow for the non-transparency of a test solution towards the stray radiant energy for all sample beam-to-reference beam cuvette path length ratios. Non-transparency is defined as the transmittance of the reference beam solution, whose monochromatic absorbance is unity, towards stray radiant energy. The proposed method has the same concentration absorbing sample placed in the beams of the scanning spectrophotometer, the sample-beam cuvette being a known factor longer than the reference-beam cuvette. While scanning towards shorter wavelengths, an apparent differential absorbance Mielenz peak is recorded. An exact formula is derived in this paper relating the relative stray radiant energy level to the Mielenz peak absorbance, to the known cuvette path length ratio, to the observed monochromatic absorbance of the test sample at the Mielenz peak wavelength, and to the sample transmittance towards the stray radiant energy. Sample transmittance towards stray radiant energy cannot be determined experimentally. However, the derived formula only allows the other experimental quantities to tie in together for a single numerically calculated value for the sample-transmittance towards stray radiant energy. The formulae are tedious to derive and cumbersome to handle, but their application is facilitated greatly by a Microsoft Office Excel 2007 spreadsheet. The test method was applied to an ultraviolet–visible (UV/VIS) scanning spectrophotometer at nine wavelengths in the range 713 < γ (nm) >; 649 for a sample beam-to-reference beam cuvette path length ratio of 10 mm/5 mm and using blue food dye (E123) as the test material. Sample transparency to stray radiant energy fluctuated in wavelength between 0.819 and 0.948, while the relative stray radiant energy level fluctuated between 1.283 × 10⁻³ and 2.516 × 10⁻³. The investigation was repeated at 665.6 nm for all fifteen sample beam-to-reference beam cuvette path length ratios, which it was possible to establish using combinations of quartz-glass cuvettes with path lengths of 100, 50, 20, 10, 5, 2, and 1 mm. The sample transparency to stray radiant energy at 665.6 nm was 0.95 ± 0.5, while the relative stray radiant energy was (1.5 ± 0.33) × 10⁻³.

Minerals play an important role in the structure and stability of casein micelles: minerals and caseins in milk are in dynamic equilibrium. Front-face synchronous fluorescence and mid-infrared spectra in combination with multivariate statistical analysis have been used to investigate, at a molecular level, the effects of added minerals (calcium, phosphate, or citrate) on mineral equilibria and casein micelle structure. Synchronous fluorescence spectra were recorded in the 250–500 nm excitation wavelength range using an offset of 80 nm between the excitation and emission monochromators for skim-milk samples fortified with 0, 3, 6, and 9 mM of calcium, phosphate, or citrate at 30 °C and 4 °C. Regarding mid-infrared spectroscopy, the region located between 1700–1500 cm⁻¹, corresponding to the amide I and II bands, and the 1500–900 cm⁻¹ region, called the fingerprint region, were considered for the characterization of the fortified skim-milk samples at the two considered temperatures. Principal component analysis (PCA) was applied to the collections of fluorescence and infrared spectral data of the two systems to optimize their description. The results show that the phenomena induced by the addition of phosphate were different from the ones observed following the addition of calcium or citrate, a calcium-chelating agent. Finally, common components and specific weights analysis was applied to infrared spectra and fluorescence data collected on fortified skim-milk samples. This analysis enabled the relationship between the different data tables to be established.

Frequency displacement, or spectral shift, is commonly observed in industrial spectral measurements. It can be caused by many factors such as sensor de-calibration or by external influences, which include changes in temperature. The presence of frequency displacement in spectral measurements can cause difficulties when statistical techniques, such as independent component analysis (ICA), are used to analyze it. Using simulated spectral measurements, this paper initially highlights the effect that frequency displacement has on ICA. A post-processing technique, employing particle swarm optimization (PSO), is then proposed that enables ICA to become robust to frequency displacement in spectral measurements. The capabilities of the proposed approach are illustrated using several simulated examples and using tablet data from a pharmaceutical application.

A simple method for obtaining phase transition temperatures is proposed. It is based on absolute variations of a baseline in a temperature-dependent mid-infrared transmittance spectral data set recorded for a sample KBr pellet. The method is rapid, inexpensive, and completely free of any personal bias. The method efficiency was corroborated by measuring the phase transitions of mesomorphic (trans-4-heptyl cyclohexanecarboxylic acid) and polymorphic compounds (polycyclic aromatic hydrocarbons and phenylacetylenes). In addition, the exothermic decomposition of phenylacetylenes was also monitored, thereby opening the possibility for simple and rapid monitoring of chemical reactions in solid and liquid phase. A great prospect of the method as an economical way of establishing critical temperature regions for analytical and quality control purposes is foreseen.

In situ surface-enhanced infrared absorption spectroscopy (SEIRAS) in attenuated total reflection (ATR) configuration has been extended to a Co electrode fabricated by potentiostatic deposition of a 50-nm-thick Co overlayer onto a Au underlayer chemically preformed on the reflecting plane of an ATR Si hemi-cylindrical prism. The as-prepared Co-on-Au film was characterized with atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). The AFM images of the films before and after Co coating revealed island structures facilitating the SEIRA effect with Co nanoparticles much smaller than the underlying Au ones. The XPS spectrum did not contain any characteristic peaks related to Au, suggestive of a virtually pinhole-free nature of the Co overlayer. The voltammetric response of the as-prepared films in phosphate buffer solution (PBS, pH 6.9) was characteristic of a polycrystalline bulk Co electrode. Normally directed unipolar bands were found for surface probe CO molecules on Co surfaces in the PBS with their major band (CO_l) intensity being one order of magnitude higher than that obtained with conventional IR reflection–absorption spectroscopy (IRRAS). By taking advantage of the higher detection sensitivity, the bands for linearly bonded CO (CO_l) at 1965–2005 cm⁻¹ and the multi-bonded (CO_m) band at 1845–1875 cm⁻¹ were clearly detected with their Stark tuning rates being 59 and 63 cm^−1•V⁻¹, respectively, which would be otherwise unobtainable with the conventional IRRAS in the neutral solution.

Nitrification and mineralization of organic nitrogen (N) are important N transformation processes in soil, and mass spectrometry is a suitable technique for tracing changes of ¹⁵N isotopic species of mineral N and estimating the rates of these processes. However, mass spectrometric methods for tracing N dynamics are costly, time consuming, and require long and laborious preparation procedures. This study investigates mid-infrared attenuated total reflection (ATR) spectroscopy as an alternative method for detecting changes in ¹⁴NO₃–N and ¹⁵NO₃–N concentrations. There is a significant shift of the v₃ absorption band of nitrate according to N species, namely from the 1275 to 1460 cm⁻¹ region for ¹⁴NO₃⁻ to the 1240–1425 cm⁻¹ region for ¹⁵NO₃⁻. This shift makes it possible to quantify the N isotopes using multivariate calibration methods. Partial least squares regression (PLSR) models with five factors yielded a determination error of 6.7–9.2 mg N L⁻¹ for aqueous solutions and 5.9–7.8 mg N kg⁻¹ (dry soil) for pastes of a Terra rossa soil. These PLSR models were used to monitor the changes of ¹⁵NO₃–N and ¹⁴NO₃–N content in the same Terra rossa soil during an incubation experiment in which [¹⁵NH₄]₂SO₄ was applied to the soil, allowing the estimation of the contributions of applied N and mineralized N to the net nitrification rate, the potential losses of the applied ¹⁵NH₄–N, and the net mineralization of soil organic N.

The temperature-dependent dissociation behavior of 2-pyrrolidinone in the pure liquid state was investigated via perturbation-correlation moving-window two-dimensional (PCMW2D) correlation spectroscopy and two-dimensional near-infrared (2D-NIR) correlation spectroscopy. Absorption bands in the region of 6900–6000 cm⁻¹ assigned to the first overtones of stretching modes of NH groups provided detailed information about the dissociation process from 25 °C to 95 °C. On the basis of PCMW2D analysis, the sequence of dissociation events occurring during the temperature variation were elucidated in two temperature ranges (25–60 °C and 65–95 °C). Specific dissociation order of the hydrogen bond under temperature perturbation was disclosed by 2D-NIR correlation spectroscopy. As temperature increased, the dissociation of larger oligomers occurred firstly with the generation of smaller oligomers, which were then consumed later by the decomposition of hydrogen bonds. The dissociation of stable cyclic dimers occurred mainly at higher temperature through gradually weakened hydrogen-bonding of NH groups.

We have developed a liquid/flow cell/chamber allowing infrared measurements of living biological specimens with high spatial resolution under a controlled aqueous environment. This flow chamber features sub-micrometer thick diamond windows exhibiting low spherical and chromatic aberrations. Diamond has excellent transmission properties and minimal dispersion over the entire mid-infrared and visible spectral ranges. In contrast to current commercially available infrared liquid chambers, the flow chamber has a slim profile, which accommodates high resolution/magnification microscope objectives with small working distances, down to 0.6 mm above the chamber and 6 mm below the flow chamber. We have coupled a pump to the flow chamber to provide medium exchange. As an example, we present microspectroscopic infrared maps and spectra of the freshwater green alga Micrasterias sp. in the new flow chamber and compare them to maps obtained with a conventional liquid chamber. Pulse-amplitude-modulated fluorescence measurements on Micrasterias sp. cells inside the new flow chamber have been evaluated to demonstrate the viability of the algal cells.