Classification of spectroscopically encoded resins by Raman mapping and infrared hyperspectral imaging

Barcoded resins (BCRs) were recently introduced as a potential platform for pre-encoded multiplexed synthesis, screening, and biomedical diagnostics. A key step toward the development of this strategy is the ability to rapidly interrogate and classify the BCRs in a high-throughput, noninvasive manner. Here, we describe a one-step strategy based on Raman mapping and Fourier transform infrared imaging to classify and spatially resolve randomly distributed BCRs. To illustrate this methodology, mixtures of up to 25 different BCRs were imaged and classified with 100% confidence. This strategy can be readily extended to a larger pool of resins, provided each BCR features a unique vibrational fingerprint (spectroscopic barcode). We have also established that reliable single-bead Raman spectra can be recorded in 10 ms, thus confirming that Raman mapping, in particular, could be a very fast method to classify the BCRs.     

Preparation and infrared/raman classification of 630 spectroscopically encoded styrene copolymers

The barcoded resins (BCRs) were introduced recently as a platform for encoded combinatorial chemistry. One of the main challenges yet to be overcome is the demonstration that a large number of BCRs could be generated and classified with high confidence. Here, we describe the synthesis and classification of 630 polystyrene-based copolymers prepared from the combinatorial association of 15 spectroscopically active styrene monomers. Each of the 630 copolymers displayed a unique vibrational fingerprint (infrared and Raman), which was converted into a spectral vector. To each of the 630 copolymers, a vector of the known (reference) composition was assigned. Unknown (prediction) vectors were decoded using multivariate data analysis. From the inner product of the reference and prediction vectors, a correlation map comparing 396 900 copolymer pairs (630 x 630) was generated. In 100% of the cases, the highest correlation was obtained for polymer pairs in which the reference and prediction vectors correspond to copolymers prepared from identical styrene monomers, thus demonstrating the high reliability of this encoding strategy. We have also established that the spectroscopic barcodes generated from the Raman and infrared spectra are independent of the copolymers' morphology (beaded versus bulk polymers). Besides the demonstration of the generality of the polymer barcoding strategy, the analytical methods developed here could in principle be extended to the investigation of the composition and purity of any other synthetic polymer and biopolymer library, or even scaffold-based combinatorial libraries.     

Investigation of fungal deterioration of synthetic paint binders using vibrational spectroscopic techniques

The deterioration of synthetic polymers caused by biological process is usually evaluated by visual inspection and measuring physical effects. In contrast to this approach, we have applied vibrational spectroscopies to study the biodegradation of the synthetic resins. 29 synthetic resins used as paint binding media, including acrylic, alkyd and poly(vinyl acetate) polymers, were examined for potential susceptibility to fungal degradation using the standard method ASTM G21-96(2002). In addition, the degraded resins were analysed by Raman spectroscopy, FT-IR and FT-IR photoacoustic spectroscopy. Almost all the acrylic resins studied proved to be resistant to microbial attack, while all alkyd resins and some poly(vinyl acetates) turned out to be biodegradable. Within a few days of inoculation Aspergillus niger was the most copious fungus on the biodegraded resins. A comparison of the IR and Raman spectra of control and biodegraded resins did not show any differences, but photoacoustic spectroscopy revealed additional bands for the fungal-degraded resins, consistent with the presence of fungal-derived substances. The additional bands in the photoacoustic spectra were due to the presence of Aspergillus niger and melanin, a fungal pigment. Since IR photoacoustic spectroscopy can be also a suitable technique for the chemical characterisation of binding media, the same spectroscopic analysis can be employed to both characterise the material and obtain evidence for fungal colonization. Microbial growth on Sobral 1241ML (alkyd resin) after 28 d (growth rating 4) compared with the non-inoculated resin.     

Spectroscopically encoded microspheres for antigen biosensing

Here, we demonstrate the potential of barcoded resins (BCRs) as a reliable platform for immunoassays. Four BCRs were synthesized by dispersion polymerization of 4-methylstyrene, t-butylstyrene, 2,4-dimethylstyrene, and 2,5-dimethylstyrene. Methacrylic acid was included in the polymerization step to provide an anchor point for antibody conjugation. In addition to identifying the BCRs through their unique spectrum in an immunoassay experiment, Raman scattering spectroscopy confirmed the immunoreactivity of the bead-conjugated antibody by detecting 150 ng/mL ( approximately 150 pg/bead) of fluorescently labeled rabbit IgG antigen. The simplicity, versatility, and effectiveness of this platform demonstrate its potential for high-throughput multiplexed bioassays.     

Applications of Vibrational Spectroscopy for Analysis of Connective Tissues

Advances in vibrational spectroscopy have propelled new insights into the molecular composition and structure of biological tissues. In this review, we discuss common modalities and techniques of vibrational spectroscopy, and present key examples to illustrate how they have been applied to enrich the assessment of connective tissues. In particular, we focus on applications of Fourier transform infrared (FTIR), near infrared (NIR) and Raman spectroscopy to assess cartilage and bone properties. We present strengths and limitations of each approach and discuss how the combination of spectrometers with microscopes (hyperspectral imaging) and fiber optic probes have greatly advanced their biomedical applications. We show how these modalities may be used to evaluate virtually any type of sample (ex vivo, in situ or in vivo) and how “spectral fingerprints” can be interpreted to quantify outcomes related to tissue composition and quality. We highlight the unparalleled advantage of vibrational spectroscopy as a label-free and often nondestructive approach to assess properties of the extracellular matrix (ECM) associated with normal, developing, aging, pathological and treated tissues. We believe this review will assist readers not only in better understanding applications of FTIR, NIR and Raman spectroscopy, but also in implementing these approaches for their own research projects.     

Quantifiable correlation of ToF-SIMS and XPS data from polymer surfaces with controlled amino acid and peptide content

Peptide-coated surfaces are widely employed in biomaterial design, but quantifiable correlation between surface composition and biological response is challenging due to, for example, instrumental limitations, a lack of suitable model surfaces or limitations in quantitatively correlating data from different surface analytical techniques. Here, we first establish a reference material that allows control over amino acid content. Reversible addition-fragmentation chain-transfer (RAFT) polymerisation is used to prepare a copolymer containing alkyne and furan units with well-defined chain length and composition. Huisgen Cu(I)-catalysed azide-alkyne cycloaddition reaction is used to attach the model azido-polyethyleneglycol-amide-modified pentafluoro-l -phenylalanine to the polymer. Different compositional ratios of the polymer provide a surface with varying amino acid content that is analysed by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Nitrogen-related signals are compared with fluorine signals from both techniques. Fluorine and nitrogen signals from both techniques are found to be related to the copolymer compositions, but the homopolymer data deviate from this trend. The approach is then translated to a heparin-binding peptide that supports cell adhesion. Human embryonic stem cells cultured on copolymer surfaces presenting different amounts of heparin-binding peptide show strong cell growth while maintaining pluripotency after 72 h of culture. The early cell adhesion at 24 h can be correlated to the logarithm of the normalised CH₄N⁺ ion intensity from ToF-SIMS data, which is established as a suitable and generalisable marker ion for amino acids and peptides. This work contributes to the ability to use ToF-SIMS in a more quantitative manner for the analysis of amino acid and peptide surfaces.     

Vibrational relaxation pathways of amide I and amide II modes in N-methylacetamide

We studied the vibrational energy relaxation mechanisms of the amide I and amide II modes of N-methylacetamide (NMA) monomers dissolved in bromoform using polarization-resolved femtosecond two-color vibrational spectroscopy. The results show that the excited amide I vibration transfers its excitation energy to the amide II vibration with a time constant of 8.3 ± 1 ps. In addition to this energy exchange process, we observe that the excited amide I and amide II vibrations both relax to a final thermal state. For the amide I mode this latter process dominates the vibrational relaxation of this mode. We find that the vibrational relaxation of the amide I mode depends on frequency which can be well explained from the presence of two subbands with different vibrational lifetimes (~1.1 ps on the low frequency side and ~2.7 ps on the high frequency side) in the amide I absorption spectrum.     

Investigations of matrix-assisted laser desorption/ionization sample preparation by time-of-flight secondary ion mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) analyses are compared to gain insight into some of the details of sample preparation for MALDI analysis of synthetic polymers. ToF-SIMS imaging of MALDI samples shows segregation of the cationization agent from the matrix crystals. The amount of observed segregation can be controlled by the sample preparation technique. Electrospray sample deposition minimizes segregation. Comparing ToF-SIMS and MALDI mass spectra from the same samples confirms that ToF-SIMS is significantly more surface sensitive than MALDI. This comparison shows that segregation of the oligomers of a polymer sample can occur during MALDI sample preparation. Our data indicate that MALDI is not as sensitive to those species dominating the sample surface as to species better incorporated into the matrix crystals. Finally, we show that matrix-enhanced SIMS can be an effective tool to analyze synthetic polymers, although the sample preparation conditions may be different than those optimized for MALDI.     

Evidence for coupling between nitrile groups using DNA templates: a promising new method for monitoring structures with infrared spectroscopy

Infrared spectroscopy is a common method for monitoring biomolecular structures but suffers from spectral congestion. Non-natural vibrational probes provide a way to regain structural specificity because they provide a unique vibrational signature and can be incorporated into proteins or other biomolecules at specific locations. A popular probe is the nitrile group because its frequency is sensitive to the electrostatics of its environment. In this work, we show that pairs of nitrile groups can be used to directly probe distances and angles in dual labeled molecules. By labeling model DNA oligomers with pairs of nitrile tags, we demonstrate that the vibrational coupling between two nitrile groups is strong enough that Fourier transform infrared (FTIR) spectra can be used to probe relative nitrile distances >4.5 A. Our approach is similar in spirit to monitoring structures with fluorescence resonance energy transfer (FRET) using a pair of fluorescent labels or a pair of spin labels in electron spin resonance spectroscopy. The small sizes of nitrile groups make especially valuable probes of sterically confined regions like the inner cores of large biomolecules where other spectroscopic probes do not fit.     

Nanoscale imaging of hydrogen and sodium in alteration layers of corroded glass using ToF-SIMS: Is an auxiliary sputtering ion beam necessary?

The hydrogen (H)/sodium (Na) interface is of great interest in glass corrosion research. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is one of the few techniques that can provide nanoscale H and Na imaging simultaneously. However, the optimized condition for ToF-SIMS imaging of H in glass is still unclear. In H depth profiling using ToF-SIMS, H background control is a key, in which an analysis ion beam and a sputtering ion beam work together in an interlaced mode to minimize it. Therefore, it is of great interest to determine if an auxiliary sputtering ion beam is also necessary to control H background in ToF-SIMS imaging of H. In this study, H imaging with and without auxiliary sputtering beams (Cs⁺, O₂⁺, and Ar_n⁺) was compared on a corroded international simple glass (ISG). It was surprising that the H/Na interface could be directly imaged using positive ion imaging without any auxiliary sputtering ion beam under a vacuum of 2 to 3 × 10⁻⁸ mbar. The H⁺ background was about 5% atomic percent on the pristine ISG glass, which was significantly lower than the H concentration in the alteration layer (~15%). Moreover, positive ion imaging could show distributions of other interesting species simultaneously, providing more comprehensive information of the glass corrosion. If an auxiliary O₂⁺ sputtering ion beam was used, the H⁺ background could be reduced but still higher than that in the depth profiling. Besides, this condition could cause significant loss of signal intensities due to strong surface charging.     

A new colorimetric test for detection of hydroxyl groups in solid-phase synthesis

A new colorimetric test (methyl red-DIC test) for monitoring the presence of hydroxyl groups on resins for use in solid-phase synthesis was developed. The resin became orange-red when the carboxyl group of methyl red formed ester linkages with the resin’s hydroxyl groups. This new test can be used not only for qualitative analysis but also quantitative analysis by using pictures of the relationship between color depth and hydroxy loading rates.     

Thermal oxidation of 6 nm aerosolized silicon nanoparticles: size and surface chemistry changes

The earliest stages of thermal oxidation of 6 nm diameter silicon nanoparticles by molecular oxygen are examined using a tandem differential mobility analysis (TDMA) apparatus, Fourier-transform infrared (FTIR) spectroscopy, time-of-flight secondary ion mass spectroscopy (ToF-SIMS), and X-ray photoelectron spectroscopy (XPS). Particles are synthesized in and then extracted from a nonthermal RF plasma operating at approximately 20 Torr into the atmospheric pressure TDMA apparatus. The TDMA apparatus was used to measure oxidation-induced size changes over a broad range of temperature settings and N2-O2 carrier gas composition. Surface chemistry changes are evaluated in situ with an FTIR spectrometer and a hybrid flow-through cell, and ex situ with ToF-SIMS and XPS. Particle size measurements show that, at temperatures less than approximately 500 degrees C, particles shrink regardless of the carrier gas oxygen concentration, while FTIR and ToF-SIMS spectra demonstrate a loss of hydrogen from the particles and minimal oxide formation. At higher temperatures, FTIR and XPS spectra indicate that an oxide forms which tends toward, but does not fully reach, stoichiometric SiO2 with increasing temperature. Between 500 and 800 degrees C, size measurements show a small increase in particle diameter with increasing carrier gas oxygen content and temperature. Above 800 degrees C, particle growth rapidly reaches a plateau while FTIR and XPS spectra change little. ToF-SIMS signals associated with O-Si species also show an increase in intensity at 800 degrees C.     

Aromatic bisvinyl ethers: A new class of highly reactive thermosetting monomers

Aromatic bisvinyl ethers, prepared by the condensation of bisphenols with 2-chloroethyl vinyl ether in the presence of sodium hydroxide, are a new class of thermosetting monomers. These new materials can be polymerized cationically by using diaryliodonium salts as photo and thermal initiators to produce crosslinked resins whose physical and thermal characteristics resemble those of epoxy resins.     

Application of XPS and ToF-SIMS for surface chemical analysis of DNA microarrays and their substrates

The chemical composition of the functional surfaces of substrates used for microarrays is one of the important parameters that determine the quality of a microarray experiment. In addition to the commonly used contact angle measurements to determine the wettability of functionalized supports, X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) are more specific methods to elucidate details about the chemical surface constitution. XPS yields information about the atomic composition of the surface, whereas from ToF-SIMS, information on the molecular species on the surface can be concluded. Applied on printed DNA microarrays, both techniques provide impressive chemical images down to the micrometer scale and can be utilized for label-free spot detection and characterization. Detailed information about the chemical constitution of single spots of microarrays can be obtained by high-resolution XPS imaging. Figure Eye-catching image for the graphical online abstract     

Theory of time-resolved sum-frequency generation and its applications to vibrational dynamics of water

A molecular theory of time-resolved sum-frequency generation (SFG) has been developed. The theoretical framework is constructed using the coupled-oscillator model in the adiabatic approximation. This theory can treat not only the vibrational spectroscopy but also vibrational dynamics. An application of this theory is also provided for estimation of the time constants of the intermolecular vibrational energy transfer between water molecules. This approach can be used for molecular analysis of the experimental results of Shen at al. on the SFG studies of vibrational dynamics of water.     

Quantitative Analysis of Alpha‐D‐glucose in an Ionic Liquid by Using Infrared Spectroscopy

The applicability of infrared (IR) spectroscopy for quantitative concentration measurements in mixtures of carbohydrates and ionic liquids (ILs) is investigated. The compound 1‐ethyl‐3‐methylimidazolium acetate, [EMIM][OAc]—an “enzyme‐friendly” ionic liquid with great application potential in the dissolution of various biomasses—is used as solvent in combination with alpha‐D ‐glucose. Our study establishes a new way to monitor the concentration of sugars in ILs, thus providing a convenient method to follow the kinetics of, for example, enzymatic reactions in [EMIM][OAc]. As a first step, we present the IR spectrum of pure [EMIM][OAc] (this constitutes the first vibrational study of this particular IL). Although numerous lines overlap in the fingerprint region of the spectrum, characteristic features can be assigned to the corresponding vibrational modes of both ions. Secondly, we study different mixtures of the IL with alpha‐D ‐glucose (in the concentration range: 0–20 mass % glucose) and analyze them by means of IR spectroscopy, followed by computational methods, thus demonstrating the great potential of this spectroscopic technique in quantitative measurements.     

A new ab initio potential energy surface for studying vibrational relaxation in NO(v) + NO collisions

A new ab initio potential energy surface for the ground state of the NO-NO system has been calculated within a reduced dimensionality model. We find an unusually large vibrational dependence of the interaction potential which explains previous spectroscopic observations. The potential can be used to model vibrational energy transfer, and here we perform quantum scattering calculations of the vibrational relaxation of NO(v). We show that the vibrational relaxation for v = 1 is 4 orders of magnitude larger than that for the related O(2)(v) + O(2) system without having to invoke nonadiabatic mechanisms as had been suggested in the past. For highly vibrationally excited states, we predict a strong dependence of the rates on the vibrational quantum number as has been observed experimentally, although there remain important quantitative differences. The importance of a chemically bound isomer on the relaxation mechanism is analyzed, and we conclude it does not play a role for the values of v considered in the experiment. Finally, the intriguing negative temperature dependence of the vibrational relaxation rate constants observed in experiments was studied using an statistical model to include the presence of many asymptotically degenerate spin-orbit states.     

A new particle mounting method for surface analysis

The chemical analysis of microparticles is challenging due to the need to mount the particles on a substrate for analysis; double-sided adhesive tape is often used (sometimes conductive), however that is usually coated with poly (dimethyl siloxane) (PDMS) that is often used as a release agent. PDMS is a common surface contamination that can mask surface chemistries and hinder material performance where it is dependent on this contaminated interface. It is known that PDMS contains a very mobile oligomeric fraction that readily diffuses across surfaces resulting in the contamination of mounted particulate samples before and during surface chemistry analysis. This makes it impossible to determine whether the PDMS has arisen from the analysis procedure or from the sample itself. A new sample preparation method is proposed where polymer microparticles are mounted on a poly (hydroxyethyl methacrylate) (pHEMA) polymer solution, which we compare with particles that have been mounted on adhesive discs using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and 3D OrbiSIMS analysis. Particles mounted on the pHEMA substrate results in a reduction of PDMS signal by 99.8% compared with microparticles mounted on adhesive discs. This illustrates how a simple, quick and inexpensive polymer solution can be used to adhere particles for analysis by ToF-SIMS, or other surface chemical analysis techniques such as X-ray photoelectron spectroscopy (XPS), without introduction of large amounts of silicone contaminant.     

Vibrational spectroscopy: a 'vanishing' discipline?

The aim of this tutorial review is to convince a broad readership that vibrational spectroscopy, although according to some vibrational spectroscopists seemingly less in focus nowadays than in days past, is far from 'dead'. It may seem to some that infrared and Raman spectroscopy are less in focus than in times past, despite the unique analytical capabilities. Vibrational spectroscopy is particularly powerful for non-destructive characterisation of substances, including living material. But compared to the past, a shift in applications has taken place, bringing new opportunities. This is partly due to the introduction of new features, including imaging and 2D correlation spectroscopy. Another factor is the recognition that vibrational spectroscopy can play a role in new rather than only in the traditional fields of application, e.g. new applications in the life-science field (living cells, cancer research), the characterisation of soil. But also the traditional application in catalysis sees new development within the context of Operando spectroscopy.     

C-H stretching vibrations of methyl, methylene and methine groups at the vapor/alcohol (N = 1-8) interfaces

In IR and Raman spectral studies, the congestion of the vibrational modes in the C-H stretching region between 2800 and 3000 cm(-1) has complicated spectral assignment, conformational analysis, and structural and dynamics studies, even with quite a few of the simplest molecules. To resolve these issues, polarized spectra measurement on a well aligned sample is generally required. Because the liquid interface is generally ordered and molecularly thin, and sum frequency generation vibrational spectroscopy (SFG-VS) is an intrinsically coherent polarization spectroscopy, SFG-VS can be used for discerning details in vibrational spectra of the interfacial molecules. Here we show that, from systematic molecular symmetry and SFG-VS polarization analysis, a set of polarization selection rules could be developed for explicit assignment of the SFG vibrational spectra of the C-H stretching modes. These polarization selection rules helped assignment of the SFG-VS spectra of vapor/alcohol (n = 1-8) interfaces with unprecedented details. Previous approach on assignment of these spectra relied on IR and Raman spectral assignment, and they were not able to give such detailed assignment of the SFG vibrational spectra. Sometimes inappropriate assignment was made, and consequently misleading conclusions on interfacial structure, conformation and even dynamics were reached. With these polarization rules in addition to knowledge from IR and Raman studies, new structural information and understanding of the molecular interactions at these interfaces were obtained, and some new spectral features for the C-H stretching modes were also identified. Generally speaking, these new features can be applied to IR and Raman spectroscopic studies in the condensed phase. Therefore, the advancement on vibrational spectra assignment may find broad applications in the related fields using IR and Raman as vibrational spectroscopic tools.