Spectral Reproducibility Research Articles

Basic radiological characteristics of a non-scattering gel dosimeter for 3D dosimetry

We used a spectrophotometer to compare the dosimetric properties of two non-scattering (radiochromic) gel dosimeters: a non-scattering gel dosimeter developed in-house and a PRESAGE™ gel dosimeter. We evaluated the dosimetric characteristics, including spectral absorption, dose linearity, reproducibility, and dose rate dependency of the two gel dosimeters. The non-scattering gel and the PRESAGE™ gel dosimeters showed peak sensitivity at wavelengths of 600 nm and 630 nm, respectively. Over a range of doses the best dose linearities of the non-scattering and the PRESAGE™ gel dosimeters resulted in R2 values of 0.99 at wavelengths of 600 nm and 630 nm, respectively. The reproducibility and dose-rate dependence of each of the two gel dosimeters were within the range of ±3 %. Our results revealed that the peak sensitivities of the two radiochromic gel dosimeters were significantly different; the in-house non-scattering gel dosimeter demonstrated peak sensitivity at a wavelength of 600 nm while the PRESAGE™ gel dosimeter had peak sensitivity at a wavelength of 630 nm. We confirmed that for 3D gel dosimetry, the in-house non-scattering gel dosimeter had a more stable dose response compared with a commercial non-scattering gel dosimeter.

Electrochemical SERS observation of molecular adsorbates on Ru/Pt-modified Au(111) surfaces using sphere-plane type gap-mode plasmon excitation

In-situ electrochemical SERS observation was conducted at Ru/Pt alloyed overlayers formed on Au substrates with and without surface roughness. When the overlayer was formed on an electrochemically roughened Au substrate having SERS activity, spectral features of molecular adsorbates were significantly influenced by structural and compositional inhomogeneity of the surface, resulting in low spectral reproducibility under electrochemical conditions. On the other hand, when the overlayer was formed on an atomically defined Au(111), highly reproducible SERS spectra were obtained with the assistance of SERS-active sphere-plane type nano-gap structures. Application of electrochemical potentials clearly induced spectral variations such as electrochemical Stark shift of the internal molecular vibrations or appearance of molecule-to-metal charge transfer-induced Raman bands. The use of well-defined interfaces in SERS observation enabled us to obtain information on electron-vibration coupling at metal-molecule interfaces as well as vibrational information on molecular adsorption geometry.

Shotgun Lipidomic Profiling of the NCI60 Cell Line Panel Using Rapid Evaporative Ionization Mass Spectrometry.

Rapid evaporative ionization mass spectrometry (REIMS) was used for the rapid mass spectrometric profiling of cancer cell lines. Spectral reproducibility was assessed for three different cell lines, and the extent of interclass differences and intraclass variance was found to allow the identification of these cell lines based on the REIMS data. Subsequently, the NCI60 cell line panel was subjected to REIMS analysis, and the resulting data set was investigated for its distinction of individual cell lines and different tissue types of origin. Information content of REIMS spectral profiles of cell lines were found to be similar to those obtained from mammalian tissues although pronounced differences in relative lipid intensity were observed. Ultimately, REIMS was shown to detect changes in lipid content of cell lines due to mycoplasma infection. The data show that REIMS is an attractive means to study cell lines involving minimal sample preparation and analysis times in the range of seconds.

Towards quantitative multi‐color nanodiagnostics: spectral multiplexing with six silica‐encapsulated SERS labels

Quantitative multiplexing experiments using mixtures of up to six spectrally distinct surface‐enhanced Raman scattering (SERS) nanoparticle labels are demonstrated. The SERS labels comprise gold nanoparticles, Raman reporter molecules chemisorbed onto the metal surface, and a silica shell providing chemical and mechanical stability as well as spectral reproducibility. Spectral unmixing is performed with a simple least‐square algorithm because the overall SERS signal of the mixtures can be represented as a linear combination of the known spectral signatures from the individual silica‐protected labels. In contrast, simple spectral unmixing using unprotected SERS particles, i.e. without a silica shell, was not successful because of band shifts and alternating peak heights in the mixtures, which is indicative of perturbing interactions between the SERS labels. Copyright © 2016 John Wiley & Sons, Ltd.

Application of distance correction to ChemCam laser-induced breakdown spectroscopy measurements

Laser-induced breakdown spectroscopy (LIBS) provides chemical information from atomic, ionic, and molecular emissions from which geochemical composition can be deciphered. Analysis of LIBS spectra in cases where targets are observed at different distances, as is the case for the ChemCam instrument on the Mars rover Curiosity, which performs analyses at distances between 2 and 7.4m is not a simple task. In our previous work we showed that spectral distance correction based on a proxy spectroscopic standard created from first-shot dust observations on Mars targets ameliorates the distance bias in multivariate-based elemental-composition predictions of laboratory data. In this work, we correct an expanded set of neutral and ionic spectral emissions for distance bias in the ChemCam data set. By using and testing different selection criteria to generate multiple proxy standards, we find a correction that minimizes the difference in spectral intensity measured at two different distances and increases spectral reproducibility. When the quantitative performance of distance correction is assessed, there is improvement for SiO2, Al2O3, CaO, FeOT, Na2O, K2O, that is, for most of the major rock forming elements, and for the total major-element weight percent predicted. However, for MgO the method does not provide improvements while for TiO2, it yields inconsistent results. In addition, we have observed that many emission lines do not behave consistently with distance, evidenced from laboratory analogue measurements and ChemCam data. This limits the effectiveness of the method.

Introducing Discrete Frequency Infrared Technology for High-Throughput Biofluid Screening.

Accurate early diagnosis is critical to patient survival, management and quality of life. Biofluids are key to early diagnosis due to their ease of collection and intimate involvement in human function. Large-scale mid-IR imaging of dried fluid deposits offers a high-throughput molecular analysis paradigm for the biomedical laboratory. The exciting advent of tuneable quantum cascade lasers allows for the collection of discrete frequency infrared data enabling clinically relevant timescales. By scanning targeted frequencies spectral quality, reproducibility and diagnostic potential can be maintained while significantly reducing acquisition time and processing requirements, sampling 16 serum spots with 0.6, 5.1 and 15% relative standard deviation (RSD) for 199, 14 and 9 discrete frequencies respectively. We use this reproducible methodology to show proof of concept rapid diagnostics; 40 unique dried liquid biopsies from brain, breast, lung and skin cancer patients were classified in 2.4 cumulative seconds against 10 non-cancer controls with accuracies of up to 90%.

Bacterial Suspensions Deposited on Microbiological Filter Material for Rapid Laser-Induced Breakdown Spectroscopy Identification.

Four species of bacteria, E. coli, S. epidermidis, M. smegmatis, and P. aeruginosa, were harvested from agar nutrient medium growth plates and suspended in water to create liquid specimens for the testing of a new mounting protocol. Aliquots of 30 µL were deposited on standard nitrocellulose filter paper with a mean 0.45 µm pore size to create highly flat and uniform bacterial pads. The introduction of a laser-based lens-to-sample distance measuring device and a pair of matched off-axis parabolic reflectors for light collection improved both spectral reproducibility and the signal-to-noise ratio of optical emission spectra acquired from the bacterial pads by laser-induced breakdown spectroscopy. A discriminant function analysis and a partial least squares-discriminant analysis both showed improved sensitivity and specificity compared to previous mounting techniques. The behavior of the spectra as a function of suspension concentration and filter coverage was investigated, as was the effect on chemometric cell classification of sterilization via autoclaving.

Matrix assisted laser desorption ionization mass spectrometry linear time-of-flight method for white wine fingerprinting and classification

A fast and simple method for white wine differentiation based on fingerprinting by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI) combined with linear time-of-flight (TOF) mass spectrometry (MS) and statistical interpretation of the obtained data using principal component as well as cluster analysis is presented. A systematic development was performed to allow a direct comparison and classification of 33 Croatian white wines based on MALDI linear TOF mass spectra (“assuming protein/peptide fingerprints”) in the positive ion mode. α-Cyano-4-hydroxycinnamic acid as a MALDI matrix in solution mixed with the unmodified (without any dilution or preconcentration or purification/isolation) white wine sample in a volume ratio 1:1 was applied at ambient conditions and this volume sample preparation technique provided the highest mass spectral information content and optimal reproducibility. Several MALDI matrices, solvents and preparation techniques were evaluated with the selection of the above mentioned as the optimum, prior to large scale investigations. Data obtained by statistical analysis of the aligned mass spectrometric data are shown to provide an efficient way to differentiate white wines of Croatian origin.

Interpretation of infrared spectra for OM characterization of soil structural surfaces of Bt‐horizons

AbstractThe surfaces of macropores or aggregates can act as hot spots for biogeochemical processes and solute transport during preferential flow. For the characterization of organic matter (OM) at macropore surfaces non‐destructive methods have been applied such as diffuse reflectance infrared Fourier transform spectroscopy (DRIFT). However, effects of organic components on DRIFT signal intensities are often difficult to distinguish from those of mineral components. Here, DRIFT spectra from intact earthworm burrow walls and coated cracks were re‐evaluated to improve the interpretation of C–H and C=O bands. We compared DRIFT and transmission Fourier transform infrared (FTIR) spectra of entire samples that were from the same pedogenetic soil horizon (Bt) but different in mineral composition and texture (i.e., glacial till vs. loess). Spectra of incinerated samples were subtracted from the original spectra. Transmission FTIR and DRIFT spectra were almost identical for entire soil samples. However, the DRIFT spectra were affected by the bulk mode bands (i.e., wavenumbers 2000 to 1700 cm−1). These bands affected spectral resolution and reproducibility. The ratios between C–H and C=O band intensities as indicator for OM quality obtained with DRIFT were smaller than those obtained from transmission FTIR. The results demonstrated that DRIFT and transmission FTIR data required separate interpretations. DRIFT spectroscopy as a non‐destructive method for analyzing OM composition at intact surfaces in structured soils could be calibrated with information obtained with the more detailed transmission FTIR and complementary methods. Spectral subtraction procedure was found useful to reduce effects of mineral absorption bands. The improved DRIFT data may be related to other soil properties (e.g., cation exchange capacity) of hot spots in structured soils.

Humidity Effects on Fragmentation in Plasma-Based Ambient Ionization Sources.

Post-plasma ambient desorption/ionization (ADI) sources are fundamentally dependent on surrounding water vapor to produce protonated analyte ions. There are two reports of humidity effects on ADI spectra. However, it is unclear whether humidity will affect all ADI sources and analytes, and by what mechanism humidity affects spectra. Flowing atmospheric pressure afterglow (FAPA) ionization and direct analysis in real time (DART) mass spectra of various surface-deposited and gas-phase analytes were acquired at ambient temperature and pressure across a range of observed humidity values. A controlled humidity enclosure around the ion source and mass spectrometer inlet was used to create programmed humidity and temperatures. The relative abundance and fragmentation of molecular adduct ions for several compounds consistently varied with changing ambient humidity and also were controlled with the humidity enclosure. For several compounds, increasing humidity decreased protonated molecule and other molecular adduct ion fragmentation in both FAPA and DART spectra. For others, humidity increased fragment ion ratios. The effects of humidity on molecular adduct ion fragmentation were caused by changes in the relative abundances of different reagent protonated water clusters and, thus, a change in the average difference in proton affinity between an analyte and the population of water clusters. Control of humidity in ambient post-plasma ion sources is needed to create spectral stability and reproducibility.

Investigations of Some Liquid Matrixes for Analyte Quantification by MALDI.

Sample inhomogeneity is one of the obstacles preventing the generation of reproducible mass spectra by MALDI and to their use for the purpose of analyte quantification. As a potential solution to this problem, we investigated MALDI with some liquid matrixes prepared by nonstoichiometric mixing of acids and bases. Out of 27 combinations of acids and bases, liquid matrixes could be produced from seven. When the overall spectral features were considered, two liquid matrixes using α-cyano-4-hydroxycinnamic acid as the acid and 3-aminoquinoline and N,N-diethylaniline as bases were the best choices. In our previous study of MALDI with solid matrixes, we found that three requirements had to be met for the generation of reproducible spectra and for analyte quantification: (1) controlling the temperature by fixing the total ion count, (2) plotting the analyte-to-matrix ion ratio versus the analyte concentration as the calibration curve, and (3) keeping the matrix suppression below a critical value. We found that the same requirements had to be met in MALDI with liquid matrixes as well. In particular, although the liquid matrixes tested here were homogeneous, they failed to display spot-to-spot spectral reproducibility unless the first requirement above was met. We also found that analyte-derived ions could not be produced efficiently by MALDI with the above liquid matrixes unless the analyte was sufficiently basic. In this sense, MALDI processes with solid and liquid matrixes should be regarded as complementary techniques rather than as competing ones.

Detection analysis of particle size effects in overtone and combination region of NIR based on multi-types of Chinese material medica

Near-infrared (NIR) diffuse reflectance spectroscopy was used to analyze the impact of multi-class particle size of Chinese material medica (CMM) based on the spectral characteristics in overtone and combination region. Several types of CMM (60, 80, 100,120 mesh) were subjected to NIR spectra analysis. Spectral reproducibility was examined after sample repackage. The result showed that the effects of particle size on the NIR spectra were different according to different bands, in the combination region and first combination-overtone region. Spectroscopy intensity was proportional to the particle size and influence of particle size was greater as the wavelength increased. While in the second combination-overtone region, it was inversely proportional to particle size. To the sampling loading error, the result indicated that when the mesh number was larger than 60 mesh, the error was small. The appropriate particle size was clarified to guarantee the accuracy and reliability of NIR diffuse reflectance spectroscopy in CMM.

Ag Nanorod Arrays for SERS: Aspects of Spectral Reproducibility, Surface Contamination, and Spectral Sensitivity

Ag nanorod arrays prepared by oblique angle vapor deposition (OAD) represent regular, large area substrates for surface‐enhanced Raman scattering (SERS) spectroscopy. We studied uniformity and spectral reproducibility of silver OAD‐fabricated substrates (AgOADs) by spectral mapping of methylene blue. The results demonstrate good reproducibility apart from occasional “hot‐spot” sites where the intensity is higher. The number of “hot‐spots” represents 2%–6% of SERS‐active sites of mapping substrate area. We were able to obtain good SERS spectra of testing amino acid tryptophan at 1 × 10−5 M concentration and three different free‐base porphyrins down to ∼10−7 M concentration. We found out that keeping the AgOADs in a vacuum chamber overnight prevents the surface from binding any contaminants from the ambient atmosphere, without significant reduction in the SERS enhancement. Such substrates provide stable SERS enhancement even when stored for 1 year after preparation.

Design and characterization of protein films for modeling near-infrared spectra of human tissue.

Near-infrared (near-IR) spectroscopy has been investigated for use in direct measurements of human tissue for the purpose of quantifying clinically relevant analytes such as glucose. Spectra collected by transmitting near-IR light through human tissue reveal the presence of both aqueous components and solid structures within the optical path of the measurement. Developing technology for use in making these measurements requires either the availability of human subjects or an in vitro experimental platform that can effectively simulate the spectroscopic properties of tissue. This paper describes the preparation and testing of films of collagen and keratin, the two proteins that comprise the bulk of the solid material in the human epidermis and dermis. By placing these films in the optical path of a near-IR spectrometer together with an aqueous sample cell, a phantom can be constructed that allows experiments to be performed that simulate noninvasive measurements of tissue. In this work, both constant and variable thickness films are prepared and evaluated by use of a regression fit to spectra of human tissue. The stability and spectral reproducibility of the prepared films are also assessed. The regression fits to the human subject spectra yield values of R(2) ranging from 0.97 to 0.99 and the films are found to yield spectra that vary by less than a 2% relative standard deviation under three different reproducibility tests.

Large-scale Ag nanoislands stabilized by a magnetron-sputtered polytetrafluoroethylene film as substrates for highly sensitive and reproducible surface-enhanced Raman scattering (SERS)

Cheap and repeatable fabrication of large-scale Ag nanostructures with high stability, sensitivity and spectral reproducibility for SERS.

Temperature effect on laser-induced breakdown spectroscopy spectra of molten and solid salts

Laser-induced breakdown spectroscopy (LIBS) has been investigated as a potential analytical tool to improve operations and safeguards for electrorefiners, such as those used in processing spent nuclear fuel. This study set out to better understand the effect of sample temperature and physical state on LIBS spectra of molten and solid salts by building calibration curves of cerium and assessing self-absorption, plasma temperature, electron density, and local thermal equilibrium (LTE). Samples were composed of a LiCl–KCl eutectic salt, an internal standard of MnCl2, and varying concentrations of CeCl3 (0.1, 0.3, 0.5, 0.8, and 1.0wt.% Ce) under different temperatures (773, 723, 673, 623, and 573K). Analysis of salts in their molten form is preferred as plasma plumes from molten samples experienced less self-absorption, less variability in plasma temperature, and higher clearance of the minimum electron density required for local thermal equilibrium. These differences are attributed to plasma dynamics as a result of phase changes. Spectral reproducibility was also better in the molten state due to sample homogeneity.

Sensitivity gains, linearity, and spectral reproducibility in nonuniformly sampled multidimensional MAS NMR spectra of high dynamic range.

Recently, we have demonstrated that considerable inherent sensitivity gains are attained in MAS NMR spectra acquired by nonuniform sampling (NUS) and introduced maximum entropy interpolation (MINT) processing that assures the linearity of transformation between the time and frequency domains. In this report, we examine the utility of the NUS/MINT approach in multidimensional datasets possessing high dynamic range, such as homonuclear (13)C-(13)C correlation spectra. We demonstrate on model compounds and on 1-73-(U-(13)C,(15)N)/74-108-(U-(15)N) E. coli thioredoxin reassembly, that with appropriately constructed 50% NUS schedules inherent sensitivity gains of 1.7-2.1-fold are readily reached in such datasets. We show that both linearity and line width are retained under these experimental conditions throughout the entire dynamic range of the signals. Furthermore, we demonstrate that the reproducibility of the peak intensities is excellent in the NUS/MINT approach when experiments are repeated multiple times and identical experimental and processing conditions are employed. Finally, we discuss the principles for design and implementation of random exponentially biased NUS sampling schedules for homonuclear (13)C-(13)C MAS correlation experiments that yield high-quality artifact-free datasets.

Profiling Convoluted Single-Dimension Proton NMR Spectra: A Plackett–Burman Approach for Assessing Quantification Error of Metabolites in Complex Mixtures with Application to Cell Culture

Single-dimension hydrogen, or proton, nuclear magnetic resonance spectroscopy (1D-(1)H NMR) has become an attractive option for characterizing the full range of components in complex mixtures of small molecular weight compounds due to its relative simplicity, speed, spectral reproducibility, and noninvasive sample preparation protocols compared to alternative methods. One challenge associated with this method is the overlap of NMR resonances leading to "convoluted" spectra. While this can be mitigated through "targeted profiling", there is still the possibility of increased quantification error. This work presents the application of a Plackett-Burman experimental design for the robust estimation of precision and accuracy of 1D-(1)H NMR compound quantification in synthetic mixtures, with application to mammalian cell culture supernatant. A single, 20 sample experiment was able to provide a sufficient estimate of bias and variability at different metabolite concentrations. Two major sources of bias were identified: incorrect interpretation of singlet resonances and the quantification of resonances from protons in close proximity to labile protons. Furthermore, decreases in measurement accuracy and precision could be observed with decreasing concentration for a small fraction of the components as a result of their particular convolution patterns. Finally, the importance of a priori concentration estimates is demonstrated through the example of interpreting acetate metabolite trends from a bioreactor cultivation of Chinese hamster ovary cells expressing a recombinant antibody.

Techniques for the Analysis of Cysteine Sulfhydryls and Oxidative Protein Folding

Modification of cysteine thiols dramatically affects protein function and stability. Hence, the abilities to quantify specific protein sulfhydryl groups within complex biological samples and map disulfide bond structures are crucial to gaining greater insights into how proteins operate in human health and disease. Many different molecular probes are now commercially available to label and track cysteine residues at great sensitivity. Coupled with mass spectrometry, stable isotope-labeled sulfhydryl-specific reagents can provide previously unprecedented molecular insights into the dynamics of cysteine modification. Likewise, the combined application of modern mass spectrometers with improved sample preparation techniques and novel data mining algorithms is beginning to routinize the analysis of complex protein disulfide structures. Proper application of these modern tools and techniques, however, still requires fundamental understanding of sulfhydryl chemistry as well as the assumptions that accompany sample preparation and underlie effective data interpretation. The continued development of tools, technical approaches, and corresponding data processing algorithms will, undoubtedly, facilitate site-specific protein sulfhydryl quantification and disulfide structure analysis from within complex biological mixtures with ever-improving accuracy and sensitivity. Fully routinizing disulfide structure analysis will require an equal but balanced focus on sample preparation and corresponding mass spectral dataset reproducibility.

Surface enhanced Raman scattering of molecules related to highly ordered gold cavities

We presented a controlled particles‐in‐cavity (PIC) pattern for surface‐enhanced Raman scattering (SERS) detection. The periodic gold cavity array was fabricated by electrodeposition using highly ordered polystyrene spheres as a template. The as‐prepared gold cavities can be used as a SERS active substrate with significant spectral enhancement and reproducibility, which was evaluated by SERS signals using 4‐mercaptobenzoic acid (4‐MBA) as probe molecules. The surface of these gold cavities was further functionalized with cetyltrimethylammonium bromide molecules, which may immobilize the 4‐MBA‐modified silver nanoparticles in the gold cavity to form a PIC structure via the electrostatic interaction. We have demonstrated that there exists a pH window for the immobilization of the nanoparticles inside cavities. Therefore, the silver nanoparticles can be selectively immobilized into the functionalized gold cavities under the optimized pH value of the media. Further enhancement of the Raman scattering of the labeled molecules can be achieved due to the interconnection between the silver nanoparticles and gold cavity. Copyright © 2013 John Wiley & Sons, Ltd.