Isotopomeric Peak Assignment for N2O in Cross-Labeling Experiments by Fiber-Enhanced Raman Multigas Spectroscopy.

Human intervention in nature, especially fertilization, greatly increased the amount of N2O emission. While nitrogen fertilizer is used to improve nitrogen availability and thus plant growth, one negative side effect is the increased emission of N2O. Successful regulation and optimization strategies require detailed knowledge of the processes producing N2O in soil. Nitrification and denitrification, the main processes responsible for N2O emissions, can be differentiated using isotopic analysis of N2O. The interplay between these processes is complex, and studies to unravel the different contributions require isotopic cross-labeling and analytical techniques that enable tracking of the labeled compounds. Fiber-enhanced Raman spectroscopy (FERS) was exploited for sensitive quantification of N2O isotopomers alongside N2, O2, and CO2 in multigas compositions and in cross-labeling experiments. FERS enabled the selective and sensitive detection of specific molecular vibrations that could be assigned to various isotopomer peaks. The isotopomers 14N15N16O (2177 cm-1) and 15N14N16O (2202 cm-1) could be clearly distinguished, allowing site-specific measurements. Also, isotopomers containing different oxygen isotopes, such as 14N14N17O, 14N14N18O, 15N15N16O, and 15N14N18O could be identified. A cross-labeling showed the capability of FERS to disentangle the contributions of nitrification and denitrification to the total N2O fluxes while quantifying the total sample headspace composition. Overall, the presented results indicate the potential of FERS for isotopic studies of N2O, which could provide a deeper understanding of the different pathways of the nitrogen cycle.

Spectrally Resolved and Highly Parallelized Raman Difference Spectroscopy for the Analysis of Drug-Target Interactions between the Antimalarial Drug Chloroquine and Hematin.

Malaria is a severe disease caused by cytozoic parasites of the genus Plasmodium, which infiltrate and infect red blood cells. Several drugs have been developed to combat the devastating effects of malaria. Antimalarials based on quinolines inhibit the crystallization of hematin into hemozoin within the parasite, ultimately leading to its demise. Despite the frequent use of these agents, there are unanswered questions about their mechanisms of action. In the present study, the quinoline chloroquine and its interaction with the target structure hematin was investigated using an advanced, highly parallelized Raman difference spectroscopy (RDS) setup. Simultaneous recording of the spectra of hematin and chloroquine mixtures with varying compositions enabled the observation of changes in peak heights and positions based on the altered molecular structure resulting from their interaction. A shift of (-1.12 ± 0.05) cm-1 was observed in the core-size marker band ν(CαCm)asym peak position of the 1:1 chloroquine-hematin mixture compared to pure hematin. The oxidation-state marker band ν(pyrrole half-ring)sym exhibited a shift by (+0.93 ± 0.13) cm-1. These results were supported by density functional theory (DFT) calculations, indicating a hydrogen bond between the quinolinyl moiety of chloroquine and the oxygen atom of ferric protoporphyrin IX hydroxide (Fe(III)PPIX-OH). The consequence is a reduced electron density within the porphyrin moiety and an increase in its core size. This hypothesis provided further insights into the mechanism of hemozoin inhibition, suggesting chloroquine binding to the monomeric form of hematin, thereby preventing its further crystallization to hemozoin.

Ultrasensitive Raman Gas Spectroscopy for Dinitrogen Sensing at the Parts-per-Billion Level.

Sensing small changes in the concentration of dinitrogen (N2) is a difficult analytical task. As N2-sensing is crucial for nitrogen cycle research in general and studies of denitrification in particular, researchers went to great lengths to develop techniques like the gas-flow-soil-core method, which achieves a precision of 200 ppb at 20 ppm of N2. Here, we present a Raman gas spectroscopic technique based on high pressure, high laser power, and high-NA signal collection, which achieves a limit of detection (LoD) of 59 ppb N2 and a precision of 27 ppb at 10 ppm of N2. This improves the lowest LoD for N2 reported for Raman gas spectroscopy by 2 orders of magnitude. Furthermore, this constitutes an improvement in precision by 1 order of magnitude compared to the GC-MS-based gas-flow-soil-core method currently established in denitrification research. We show that the presented setup is both stable and tight enough to ensure highly sensitive, precise, and repeatable measurements of N2. As Raman gas spectroscopy is a versatile and comprehensive method, the described technique could be easily expanded to other relevant gases like nitrous oxide or to simultaneous multigas sensing. In summary, our method offers possibilities for N2-sensing and could eventually enable denitrification studies with increased sensitivity and a larger scope.

Comprehensive multi-gas study by means of fiber-enhanced Raman spectroscopy for the investigation of nitrogen cycle processes.

The extensive use of synthetic fertilizers has led to a considerable increase in reactive nitrogen input into agricultural and natural systems, resulting in negative effects in multiple ecosystems, the so-called nitrogen cascade. Since the global population relies on fertilization for food production, synthetic fertilizer use needs to be optimized by balancing crop yield and reactive nitrogen losses. Fiber-enhanced Raman spectroscopy (FERS) is introduced as a unique method for the simultaneous quantification of multiple gases to the study processes related to the nitrogen cycle. By monitoring changes in the headspace gas concentrations, processes such as denitrification, nitrification, respiration, and nitrogen fixation, as well as fertilizer addition were studied. The differences in concentration between the ambient and prepared process samples were evident in the Raman spectra, allowing for differentiation of process-specific spectra. Gas mixture concentrations were quantified within a range of low ppm to 100% for the gases N2, O2, CO2, N2O, and NH3. Compositional changes were attributed to processes of the nitrogen cycle. With help of multivariate curve resolution, it was possible to quantify N2O and CO2 simultaneously. The impact of fertilizers on N-cycle processes in soil was simulated and analyzed for identifying active processes. Thus, FERS was proven to be a suitable technique to optimize fertilizer composition and to quantify N2O and NH3 emissions, all with a single device and without further sample preparation.

Molecular Interactions Identified by Two-Dimensional Analysis-Detailed Insight into the Molecular Interactions of the Antimalarial Artesunate with the Target Structure ß-Hematin by Means of 2D Raman Correlation Spectroscopy.

A thorough understanding of the interaction of endoperoxide antimalarial agents with their biological target structures is of utmost importance for the tailored design of future efficient antimalarials. Detailed insights into molecular interactions between artesunate and ß-hematin were derived with a combination of resonance Raman spectroscopy, two-dimensional correlation analysis, and density functional theory calculations. Resonance Raman spectroscopy with three distinct laser wavelengths enabled the specific excitation of different chromophore parts of ß-hematin. The resonance Raman spectra of the artesunate-ß-hematin complexes were thoroughly analyzed with the help of high-resolution and highly sensitive two-dimensional correlation spectroscopy. Spectral changes in the peak properties were found with increasing artesunate concentration. Changes in the low-frequency, morphology-sensitive Raman bands indicated a loss in crystallinity of the drug-target complexes. Differences in the high-wavenumber region were assigned to increased distortions of the planarity of the structure of the target molecule due to the appearance of various coexisting alkylation species. Evidence for the appearance of high-valent ferryl-oxo species could be observed with the help of differences in the peak properties of oxidation-state sensitive Raman modes. To support those findings, the relaxed ground-state structures of ten possible covalent mono- and di-meso(Cm)-alkylated hematin-dihydroartemisinyl complexes were calculated using density functional theory. A very good agreement with the experimental peak properties was achieved, and the out-of-plane displacements along the lowest-frequency normal coordinates were investigated by normal coordinate structural decomposition analysis. The strongest changes in all data were observed in vibrations with a high participation of Cm-parts of ß-hematin.

Investigations on the Novel Antimalarial Ferroquine in Biomimetic Solutions Using Deep UV Resonance Raman Spectroscopy and Density Functional Theory.

Deep ultraviolet (DUV) resonance Raman experiments are performed, investigating the novel, promising antimalarial ferroquine (FQ). Two buffered aqueous solutions with pH values of 5.13 and 7.00 are used, simulating the acidic and neutral conditions inside a parasite's digestive vacuole and cytosol, respectively. To imitate the different polarities of the membranes and interior, the buffer's 1,4-dioxane content was increased. These experimental conditions should mimic the transport of the drug inside malaria-infected erythrocytes through parasitophorous membranes. Supporting density functional theory (DFT) calculations on the drug's micro-speciation were performed, which could be nicely assigned to shifts in the peak positions of resonantly enhanced high-wavenumber Raman signals at λexc = 257 nm. FQ is fully protonated in polar mixtures like the host interior and the parasite's cytoplasm or digestive vacuole (DV) and is only present as a free base in nonpolar ones, such as the host's and parasitophorous membranes. Additionally, the limit of detection (LoD) of FQ at vacuolic pH values was determined using DUV excitation wavelengths at 244 and 257 nm. By applying the resonant laser line at λexc = 257 nm, a minimal FQ concentration of 3.1 µM was detected, whereas the pre-resonant excitation wavelength 244 nm provides an LoD of 6.9 µM. These values were all up to one order of magnitude lower than the concentration found for the food vacuole of a parasitized erythrocyte.

Towards therapeutic drug monitoring of antibiotic levels - analyzing the pharmacokinetics of levofloxacin using DUV-resonance Raman spectroscopy.

Therapeutic drug monitoring (TDM) plays an important role in clinical practice. Here, pharmacokinetics has a decisive influence on the effective antibiotic concentration during treatment. Moreover, different kinetics exist for different administration forms. Accordingly, adjusting the correct concentration depends, in addition to gender, age, weight, clinical picture, etc., on the dosage form of the antibiotic. This study investigates the capability of deep UV resonance Raman spectroscopy (DUV-RRS) to simulate the pharmacokinetics of fluoroquinolone levofloxacin after two different administration forms (intravenous and oral). Three different pre-processing methods were applied, and the best agreement with the simulation was achieved using the extended multiplicative scatter correction. The resulting spectra were used for partial least squares (PLS) regression and ordinary least squares (OLS) regression. The kinetic parameters were compared with the simulated data, with PLS showing the best performance for intravenous administration and a comparable result to OLS for oral administration, while the errors are smaller. The acquired results show the potential of DUV-RRS in combination with PLS regression as a promising supportive method for TDM.

Parallelized Raman Difference Spectroscopy for the Investigation of Chemical Interactions.

Raman spectroscopy provides an extremely high chemical selectivity. Raman difference spectroscopy is a technique to reveal even the smallest differences that occur due to weak interactions between substances and changes in the molecular structure. To enable parallelized and highly sensitive Raman difference spectroscopy in a microtiter-array, a diffractive optical element, a lens array, and a fiber bundle were integrated into a Raman spectroscopy setup in a unique fashion. The setup was evaluated with a microtiter-array containing pyridine-water complexes, and subwavenumber changes below the spectrometer's resolution could be resolved. The spectral changes were emphasized with two-dimensional correlation analysis. Density functional theory calculation and "atoms in molecule" analysis were performed to simulate the intermolecular long-range interactions between water and pyridine molecules and to get insight into the involved noncovalent interactions, respectively. It was found that by the addition of pyridine, the energy portion of hydrogen bonds to the total complexation energy between pyridine and water reduces. These results demonstrate the unique abilities of the new setup to investigate subtle changes due to biochemically important molecular interactions and opens new avenues to perform drug binding assays and to monitor highly parallelized chemical reactions.

Activity and electron donor preference of two denitrifying bacterial strains identified by Raman gas spectroscopy.

Human activities have greatly increased the input of reactive nitrogen species into the environment and disturbed the balance of the global N cycle. This imbalance may be offset by bacterial denitrification, an important process in maintaining the ecological balance of nitrogen. However, our understanding of the activity of mixotrophic denitrifying bacteria is not complete, as most research has focused on heterotrophic denitrification. The aim of this study was to investigate substrate preferences for two mixotrophic denitrifying bacterial strains, Acidovorax delafieldii and Hydrogenophaga taeniospiralis, under heterotrophic, autotrophic or mixotrophic conditions. This complex analysis was achieved by simultaneous identification and quantification of H2, O2, CO2, 14N2, 15N2 and 15N2O in course of the denitrification process with help of cavity-enhanced Raman spectroscopic (CERS) multi-gas analysis. To disentangle electron donor preferences for both bacterial strains, microcosm-based incubation experiments under varying substrate conditions were conducted. We found that Acidovorax delafieldii preferentially performed heterotrophic denitrification in the mixotrophic sub-experiments, while Hydrogenophaga taeniospiralis preferred autotrophic denitrification in the mixotrophic incubation. These observations were supported by stoichiometric calculations. The results demonstrate the prowess of advanced Raman multi-gas analysis to study substrate use and electron donor preferences in denitrification, based on the comprehensive quantification of complex microbial gas exchange processes.

Hydrogen and C2-C6 Alkane Sensing in Complex Fuel Gas Mixtures with Fiber-Enhanced Raman Spectroscopy.

Power-to-gas is a heavily discussed option to store surplus electricity from renewable sources. Part of the generated hydrogen could be fed into the gas grid and lead to fluctuations in the composition of the fuel gas. Consequently, both operators of transmission networks and end users would need to frequently monitor the gas to ensure safety as well as optimal and stable operation. Currently, gas chromatography-based analysis methods are the state of the art. However, these methods have several downsides for time-resolved and distributed application and Raman gas spectroscopy is favorable for future point-of-use monitoring. Here, we demonstrate that fiber-enhanced Raman gas spectroscopy (FERS) enables the simultaneous detection of all relevant gases, from major (methane, CH4; hydrogen, H2) to minor (C2-C6 alkanes) fuel gas components. The characteristic peaks of H2 (585 cm-1), CH4 (2917 cm-1), isopentane (765 cm-1), i-butane (798 cm-1), n-butane (830 cm-1), n-pentane (840 cm-1), propane (869 cm-1), ethane (993 cm-1), and n-hexane (1038 cm-1) are well resolved in the broadband spectra acquired with a compact spectrometer. The fiber enhancement achieved in a hollow-core antiresonant fiber enables highly sensitive measurements with limits of detection between 90 and 180 ppm for different hydrocarbons. Both methane and hydrogen were quantified with high accuracy with average relative errors of 1.1% for CH4 and 1.5% for H2 over a wide concentration range. These results show that FERS is ideally suited for comprehensive fuel gas analysis in a future, where regenerative sources lead to fluctuations in the composition of gas.