Applied Spectroscopy最新文献

To better interpret the Raman spectra from mammalian cells, it is often desirable to reduce their complexity by decomposing them into the spectral contributions from individual macromolecules or types of macromolecules. Diverse methods exist for demixing complex spectra, each with different benefits and drawbacks. However, some methods require a library of component spectra that might not be available, while others are hampered by noise and peak congestion that includes many proximal overlapping peaks. Through rapid fitting of individual peaks in every spectrum of a Raman hyperspectral data set, we have obtained individual peak parameters from which we determined the trends for all the peak amplitudes. We then grouped similar trends with k-means clustering. Then we used the peak parameters of all the peaks in a given cluster to reconstruct a spectrum representative of that cluster. This method produced spectra that were less distorted by unrelated overlapping peaks or noise, were less congested than those in the hyperspectral set, and thereby improved peak identification and macromolecule recognition. We have demonstrated the application of the method with Raman spectra from a perchlorate-polystyrene model system and extended it to complex spectra from methanol-fixed mammalian cells. We were able to recover independent spectra of perchlorate and polystyrene in the model system and spectra pertaining to individual macromolecular types (proteins, nucleic acids, lipids) from the mammalian cell data. We discuss how imperfections in spectral preprocessing and peak fitting can adversely affect the results. In summary, we have provided a proof-of-concept for a novel mixture resolution method with different attributes than extant ones.

Surface immobilization of DNA for biosensing or separations applications requires covalent attachment chemistry that is efficient, reproducible, and stable. In this work, an approach to link thiol-functionalized DNA to thiol-modified silica surfaces using N,N'-1,4-phenylene-bismaleimide is optimized by developing an efficient, one-pot synthesis of the maleimide-conjugated DNA followed by its immediate reaction with thiolated porous silica particles. The methodology takes advantage of a Michael addition reaction that couples a phenyl-bismaleimide cross-linking reagent and thiol-modified DNA to form a monomeric DNA-maleimide conjugate. The 1:1 stoichiometry of this reaction must be carefully controlled to avoid excess thiol-DNA, which generates unreactive bismaleimide-linked DNA dimers, or excess bismaleimide, which competes with the DNA-maleimide conjugate for reaction with the thiolated silica surface. To achieve control over the reaction forming the DNA conjugate, we adapt a fluorescence assay for free-thiols using 7-diethylamino-3-(4-maleimidophenyl)-4-methyl-coumarin (CPM) to determine the concentration of thiol-modified DNA that emerges from its synthesis, disulfide labeling, reduction to a thiol, and purification. The fluorescence response of the CPM reagent was calibrated using reduced glutathione as a standard, which allowed determination of the concentrations of thiolated-DNA and control over the stoichiometry of its reaction with a bismaleimide linker. The maleimide-conjugated DNA product thus formed was then reacted with thiolated-silica in order to bind the DNA to the internal surfaces of porous silica, whose surface populations were determined in individual particles by confocal Raman microscopy. Self-modeling curve resolution of the Raman spectra of surface-bound molecules validated the efficiency of the bismaleimide:thiolated DNA reaction, which provided stoichiometric control over formation of the monomeric DNA-maleimide conjugate and its optimized reaction with thiolated-silica surfaces.

Physiological systems are not at equilibrium and undergo time-dependent fluctuations, making it challenging to relate in vitro studies to in vivo biomolecular behavior. To bridge this gap, enzyme dynamics can be studied in the presence of controlled perturbations that recapitulate the intracellular environment. Here, we report an approach to the study of reactive oxygen species (ROS) based on the in situ manipulation of hydrogen peroxide (H₂O₂) levels in functionalized nanopore-based electrochemical zero-mode waveguide (EZMW) arrays, with each nanopore presenting small numbers of immobilized horseradish peroxidase (HRP) enzyme molecules. H₂O₂ is generated or consumed within the attoliter volume of the EZMW nanopores by poising an embedded ring electrode to suitable potentials, and the resulting effect on apparent turnover of HRP under non-equilibrium conditions is monitored using the enzymatically accelerated conversion of the non-fluorescent probe Amplex Red to fluorescent resorufin. A Nafion membrane is placed on the top surface of the EZMW array, providing a cation permselective barrier to transport in, or out, of the EZMW nanopores, thereby improving the sensitivity of the experiment by sequestering enzymatically generated resorufin in the attoliter volume of the EZMW nanopores. By fabricating arrays presenting 441 individual reaction volumes in parallel, distinct changes in population dynamics in the presence of in situ H₂O₂ generation or consumption are characterized with respect to temporal evolution and magnitude of the H₂O₂ aliquot delivered. This approach presents a promising avenue for studying biomolecular reactions in spatiotemporally controlled chemical environments that can mimic the non-equilibrium conditions encountered in vivo.

The correlation filter (CF) technique is introduced as a versatile tool for data pretreatment to selectively attenuate interfering or overlapping signals of congested spectra. This technique leverages two-dimensional correlation spectroscopy (2D-COS) to create a filter multiplier that effectively addresses limitations inherent in traditional null-space projection (NSP) methods based on least-squares subtraction. We apply CF to the analysis of a model solution mixture system undergoing spontaneous evaporation, where volatile solvent concentrations change concurrently but at only slightly different rates. Despite the similarity of these parallel processes, CF successfully separates the overlapped dynamics of individual components by attenuating dominant signal contributions. CF also enables streamlined 2D codistribution spectroscopy (2D-CDS) analysis to determine the sequential order of component appearance. Multiple layers of CF can be applied to isolate individual component dynamics. Heterocomponent 2D correlation can then recover lost information by recombining CF-treated spectra. CF is applicable to two-trace two-dimensional (2T2D) correlation for comparative spectral analysis of a pair of spectra. CF treatment is expected to be a useful tool beyond 2D-COS applicable to many areas of spectral analyses, including the environmental and interfacial studies.

The ability to combine microscopy and spectroscopy is beneficial for directly monitoring physical and biological processes. Spectral imaging approaches, where a transmission diffraction grating is placed near an imaging sensor to collect both the spatial image and spectrum for each object in the field of view, provide a relatively simple method to simultaneously collect images and spectroscopic responses on the same sensor. Initially demonstrated with fluorescence spectroscopy, the use of spectral imaging in Raman spectroscopy and surface-enhanced Raman spectroscopy (SERS) can provide a vibrational spectrum containing molecularly specific information that can inform on chemical changes. However, a major complication to this approach is the spectral overlap that occurs when objects are spaced closely together horizontally. In this work, we add a dove prism to a spectral imaging instrument developed for SERS imaging, enabling rotation of the collected SERS image and dispersed spectrum onto the imaging complementary metal-oxide semiconductor (CMOS) sensor. We demonstrate that this effectively reduces spectral overlap for emitters with clear separation between them and emitters with slightly overlapping point spread functions thereby facilitating collection of unambiguous spectra from each emitter.

In this study, Raman spectra (3700-10 cm^-1) and attenuated total reflection infrared-far-infrared (ATR-IR/FIR) spectra (4000-50 cm^-1) including low-frequency region were measured for amorphous rocks, which were five types of obsidians whose formation ages and sources are different and pitchstone to clarify the differences in water content (free and bound water species), their Si-O bonds and possible linkage with a metal ion, and the mean atomic volume. In order to explore these points, we focused on infrared (IR) absorptions of hydroxyl (OH) groups that is observed in the 4000-3000 cm^-1 region, those of Si-O bond that is identified in the 1300-850 cm^-1 region and a Boson peak that appears in a low-frequency region of Raman spectra, respectively. IR absorption of Si-O stretching was detected for all samples and that of OH stretching and H-O-H bending was also detected in some rocks. Therefore, using IR spectroscopy was useful to discriminate each rock based on the water content and the environment of Si-O bonds. On the other hands, a Boson peak could be detected for the low-frequency region below 60 cm^-1 of Raman spectra, which appears in amorphous solids. This study is the first finding that the Raman shift of Boson peak was different among similar natural glassy rocks from multiple sources and it means that the mean atomic volume of samples was different. In addition, sharp bands of Raman scattering which came from inorganic substances such as feldspar helped to identify ingredients in samples. As a results, we made clear that using both IR and Raman including low-frequency regions is effective to identify the same types of natural amorphous rocks.

Hydroxyl-terminated polybutadiene (HTPB) is used in a variety of formulations, particularly for military and aerospace applications as a binder for energetic materials. This work investigates details of its curing process when formulated with isophorone diisocyanate (IPDI). Raman spectroscopy was used as a fast, sensitive, non-destructive technique to monitor the curing process of HTPB-IPDI. A significant feature at 777 cm^-1 was shown to grow over the course of the curing process. Ab initio calculations of the normal modes of the HTPB-IPDI dimer indicate that this feature is most likely connected to the urethane bond, which suggests that the feature at 777 cm^-1 is associated with formation of the urethane linkage as the formulation cures. Raman spectroscopy thus has potential to be used for quality assurance and other material state awareness measurements for HTPB-IPDI materials.

This paper reports on the application of infrared external reflection spectroscopy (IR-ERS) to the characterization of small surface area addresses prepared on smooth gold surfaces after modification for use as capture substrates in sandwich immunoassays based on surface-enhanced Raman scattering (SERS). Most of the past work with IR-ERS on analyzing coatings formed on highly reflective metals utilized relatively large area samples (e.g., 76 × 25  mm glass microscope slides and ∼51  mm diameter silicon wafers) to accommodate the large size of the elliptical IR beam reflected off the metal surface at grazing angles of incidence. Our interest in employing assay-sized (3  mm diameter) addresses for IR-ERS measurements arises from the need to minimize the consumption, and, thereby, the expense of rare biological reagents like the antibodies under development for immunoassays to detect tuberculosis. The obvious approach to achieving this goal would be to utilize the spatial resolution and sample scanning capabilities of Fourier transform infrared (FT-IR) microscopes. We, however, opted to re-examine the physical optics and geometric layout of the measurement through an analysis of the strength of the mean square electric field at the sample/substrate interface as a function of angle of incidence. These findings suggested that, given the high light throughput and low noise levels of today's FT-IR spectrometers, it may be possible to perform these measurements simply by collecting spectra at a lower angle of incidence when using the optical layout of a standard IR-ERS experiment. Herein, we report both the theoretical analysis and experimental results that demonstrate it is possible to obtain useful spectra from much smaller samples than those traditionally used, e.g., those employed in our SERS-based immunoassays, simply by decreasing the angle at which the IR beam is incident on the sample surface. We also demonstrate that these types of samples can be analyzed by constructing a small jig that allows for the careful positioning of the sample in the IR beam, rather than by extensively modifying the optics of the IR-ERS accessory.

Confocal Raman microscopy was applied to detect structural change within individual particles of low-density polyethylene (LDPE) following chemical and electrochemical processing steps that aimed to facilitate material decomposition. A high numerical aperture (NA) oil-immersion objective enabled depth-profiling through the near surface region (20 μm-40 μm) of irregularly shaped particles with an axial spatial resolution < 2 μm estimated from measurements of instrument detection efficiency profiles. Changes in vibrational bands sensitive to polyethylene crystallinity were evident following treatments and linked to the release of low molecular weight compounds present as additives and products of processing. Effects of processing were probed by monitoring the rise of Raman scattering intensity in vibrational modes associated with polyethylene chains in a zig-zag (trans) conformation near 1128 cm^-1, 1294 cm^-1, and 1418 cm^-1, signaling chain clustering and development of organized, crystalline-like assemblies. Pristine LDPE particles displayed a uniform structure across the near surface region, while particles treated initially with chemical extractant and then further processed displayed increasingly enhanced crystallinity up to the maximum depth probed (40 μm). As a step toward measurements on ensembles of particles, least squares modeling was adapted to derive pure component spectra reflecting crystallinity change within spectral datasets. The work demonstrates high spatial resolution Raman depth-profiling for the characterization of processed polymers using a high NA immersion objective to overcome the limitations of air-objectives often used for confocal Raman microscopy.