Assessing Lanthanide-Dependent Methanol Dehydrogenase Activity: The Assay Matters.

Artificial dye-coupled assays have been widely adopted as a rapid and convenient method to assess the activity of methanol dehydrogenases (MDH). Lanthanide(Ln)-dependent XoxF-MDHs are able to incorporate different lanthanides (Lns) in their active site. Dye-coupled assays showed that the earlier Lns exhibit a higher enzyme activity than the late Lns. Despite widespread use, there are limitations: oftentimes a pH of 9 and activators are required for the assay. Moreover, Ln-MDH variants are not obtained by isolation from the cells grown with the respective Ln, but by incubation of an apo-MDH with the Ln. Herein, we report the cultivation of Ln-dependent methanotroph Methylacidiphilum fumariolicum SolV with nine different Lns, the isolation of the respective MDHs and the assessment of the enzyme activity using the dye-coupled assay. We compare these results with a protein-coupled assay using its physiological electron acceptor cytochrome cGJ (cyt cGJ ). Depending on the assay, two distinct trends are observed among the Ln series. The specific enzyme activity of La-, Ce- and Pr-MDH, as measured by the protein-coupled assay, exceeds that measured by the dye-coupled assay. This suggests that early Lns also have a positive effect on the interaction between XoxF-MDH and its cyt cGJ thereby increasing functional efficiency.

Rapid desorption and analysis for illicit drugs and chemical profiling of fingerprints by SICRIT ion source.

Forensic analysis can encompass a wide variety of analytes from biological samples including DNA, blood, serum, and fingerprints to synthetic samples like drugs and explosives. In order to analyze this variety, there are various sample preparation techniques, which can be time-consuming and require multiple analytical instruments. With recent advancements in ambient ionization mass spectrometry (MS), plasma-based dielectric barrier discharge ionization (DBDI) sources have demonstrated to cover a wide range of these analytes. The flow-through design of this source also allows for easy connection to a thermal desorption type of sample introduction. We present an in-house built thermal desorption device where the sample is introduced via a glass slide, which gets heated and transferred to the DBDI-MS with nitrogen for identification and semi-quantification. Using a glass slide as an inexpensive sampling device, detection limits as low as 20 pg for fentanyl are demonstrated. Additionally, a very precise (>96% accuracy) identification of persons based on the chemical profile of their fingerprints is possible, establishing a direct analytical link of the drug trace to the individual in one measurement. We compared the DAG, TAG, sterol, and (semi-)volatile region of the averaged fingerprint spectra over multiple days, showing the best model accuracy for identification based on the DAG region. The combination of thermal desorption and DBDI-MS minimized sample preparation, leading to an ultrasensitive and rapid analysis of illicit drug traces and the identification of underlying personas based on fingerprints.

Effect of Dopants and Gas-Phase Composition on Ionization Behavior and Efficiency in Dielectric Barrier Discharge Ionization.

Cold plasma-based ionization techniques allow for soft ionization of a wide variety of chemical compounds. In this chemical ionization mechanism, the atmosphere plays a crucial role in ionization. Knowing its influence is critical for the optimization of analysis conditions and interpretation of resulting spectra. This study uses soft ionization by chemical reaction in transfer (SICRIT), a variant of dielectric barrier discharge ionization (DBDI), that allows for a controlled atmosphere to investigate atmosphere and dopant effects. The influence of eight makeup gas compositions (dry nitrogen, room air, and nitrogen-enriched with either water, HCl, MeOH, hexane, NH3, and fluorobenzene) on the ionization with SICRIT was investigated. Fifteen compound classes, comprising alkanes, polyaromatic hydrocarbons (PAHs), terpenes, oxygen-containing terpenes, alkylphenols, chlorophenols, nitrophenols, trialkylamines, triazines, phthalates with or without ether groups, aldehydes, ketones, fatty acid methyl esters (FAMEs), and polyoxy-methylene ethers (OMEs) were measured via gas chromatography SICRIT high-resolution mass spectrometry (GC-SICRIT-HRMS). The different atmospheres were compared in terms of generated ions, ion intensities and fragmentation during ionization. Measurements of reactant ions were performed for a better understanding of the underlying mechanisms. All 15 compound classes were mostly softly ionized. For most compound classes and atmospheres, protonation is the dominant ionization mode. The highest number of compounds ionized via protonation was observed in dry nitrogen, followed by room air and humid nitrogen. The study should work as a guideline for the choice of atmosphere for specific compound classes and the interpretation of spectra generated under a specific atmosphere.

A Multi-Analytical Approach for Studying the Effect of New LED Lighting Systems on Modern Paints: Chemical Stability Investigations.

This study aims to investigate the chemical stability of some modern paint samples exposed to a new Light Emitting Diode (LED)-lighting system and a halogen lamp by using micro-attenuated total reflectance of Fourier transform infrared spectroscopy (µ-ATR-FTIR), µ-Raman, pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), and thermally assisted hydrolysis and methylation of GC/MS (THM-GC/MS). Those investigations were performed before and after the exposure of the samples to lightings for 1250, 2400, 3300, and 5000 h. The results obtained with µ-Raman spectroscopy show the high stability of the selected inorganic pigments after the exposure to the lighting systems; while similar to the UV/Vis/NIR results reported in a previous study, µ-ATR-FTIR and THM-GC/MS results evidence greater chemical changes occurring principally on the linseed oil binder-based mock-ups among the acrylic and alkyd-based samples. Moreover, principal component analyses (PCA) and hierarchical cluster analyses (HCA) of THM-GC/MS results highlight that those changes were mostly dependent on the exposure time and on the type of pigment, while being independent of the lighting system used. Finally, semi-quantitative µ-ATR-FTIR results show slight pigment enrichment at the paint surface due to the auto and photo-oxidative degradation of the linseed oil binder.

Plug-and-play laser ablation-mass spectrometry for molecular imaging by means of dielectric barrier discharge ionization.

The plug-and-play hyphenation of UV-laser ablation (LA) and mass spectrometry is presented, using dielectric barrier discharge ionization (DBDI). The DBDI source employed here is characterized by its unique geometry, being directly mounted onto the inlet capillary of a mass spectrometer. In the literature, this particular kind of DBDI source is also referred to as active capillary plasma ionization. It has been commercialized as soft ionization by chemical reaction in transfer (SICRIT) and will be addressed as DBDI in this study. LA-DBDI-MS was used for the direct, molecule-specific and spatially resolved analysis of various solid samples, such as coffee beans and pain killer tablets without extensive sample preparation. The combination of fast washout UV-laser ablation and the principle of the DBDI source used here allowed for highly efficient soft ionization as well as high spatial resolution down to 10 µm for molecular imaging.

Ultrasensitive and Simultaneous SERS Detection of Multiplex MicroRNA Using Fractal Gold Nanotags for Early Diagnosis and Prognosis of Hepatocellular Carcinoma.

Sensitive and simultaneous detection of multiple cancer-related biomarkers in serum is essential for diagnosis, therapy, prognosis, and staging of cancer. Herein, we proposed a magnetically assisted sandwich-type surface-enhanced Raman scattering (SERS)-based biosensor for ultrasensitive and multiplex detection of three hepatocellular carcinoma-related microRNA (miRNA) biomarkers. The biosensor consists of an SERS tag (probe DNA-conjugated DNA-engineered fractal gold nanoparticles, F-AuNPs) and a magnetic capture substrate (capture DNA-conjugated Ag-coated magnetic nanoparticles, AgMNPs). The proposed strategy achieved simultaneous and sensitive detection of three miRNAs (miRNA-122, miRNA-223, and miRNA-21), and the limits of detection of the three miRNAs in human serum are 349 aM for miRNA-122, 374 aM for miRNA-223, and 311 aM for miRNA-21. High selectivity and accuracy of the SERS biosensor were proved by practical analysis in human serum. Moreover, the biosensor exhibited good practicability in multiplex detection of three miRNAs in 92 clinical sera from AFP-negative patients, patients before and after hepatectomy, recurred and relapse-free patients after hepatectomy, and hepatocellular carcinoma patients at distinct Barcelona clinic liver cancer stages. The experiment results demonstrate that our SERS-based assay is a promising candidate in clinical application and exhibited potential for the prediction, diagnosis, monitoring, and staging of cancers.

Gas Chromatography-Atmospheric Pressure Inlet-Mass Spectrometer Utilizing Plasma-Based Soft Ionization for the Analysis of Saturated, Aliphatic Hydrocarbons.

Soft ionization by a chemical reaction in transfer (SICRIT) is applied to couple gas chromatography (GC) to a high-resolution atmospheric pressure inlet mass spectrometer. These instruments are generally used in combination with liquid chromatography systems (LC-MS). Ionization of alkanes is not possible here with conventional electrospray ionization. Alternatively, separate GC-electron ionization (EI)-MS is employed for the analysis of nonpolar substances like alkanes, however, with the inherent challenge of strong fragmentation. In the case of alkanes, the determination of molecular masses becomes nearly impossible in complex hydrocarbon mixtures because of the wealth of similar fragment ions and the absence of the molecular ion signal. SICRIT, a soft ionization technique based on dielectric barrier discharge (DBDI), produces characteristic oxidized cations from alkanes that can be directly correlated to their molecular mass. Isotope labeling experiments reveal an ionization mechanism via hydride abstraction and reaction with water. Soft ionization can be achieved for iso- and n-alkanes, with very little fragmentation, enabling the determination of their molecular mass. Calibrations for n-alkanes from C10 to C30 were performed exhibiting high linearity, reproducibility, and sensitivity with an average LOD of 69 pg (on column). Measurements of diesel fuel samples are compared to traditional GC-EI-MS. The presented method combines sensitivity and easy handling of a GC-EI-MS with the determination of molecular mass commonly only achieved with field ionization (FI)-MS, while using existing and highly optimized mass spectrometers commonly coupled with LC. Additionally, many other analytes such as (alkylated-) PAHs could be detected simultaneously in the diesel sample.

Non-invasive Raman spectroscopy for time-resolved in-line lipidomics.

Oil-producing yeast cells are a valuable alternative source for palm oil production and, hence, may be one important piece of the puzzle for a more sustainable future. To achieve a high-quality product, the lipid composition inside oil-producing yeast cells is a crucial parameter for effective process control. Typically, the lipid composition is determined by off-line gas chromatography. A faster, less cumbersome approach is proposed here, by using non-invasive in-line Raman spectroscopy. A fed-batch fermentation of C. oleaginosus - a well-known oleaginous yeast - is used as model experiment to highlight the potential of Raman spectroscopy for in-line lipidomics. The temporal progression of biomass formation, lipid production and glucose consumption are determined based on PLS-regression models allowing process-relevant information on time to be accessed. Additionally, Gaussian curve fitting was applied to extract increasing and decreasing trends of saturated and unsaturated fatty acids produced by C. oleaginosus throughout the fermentation process.

Finding the Needle in the Haystack: High-Resolution Techniques for Characterization of Mixed Protein Particles Containing Shed Silicone Rubber Particles Generated During Pumping.

During the manufacturing process of biopharmaceuticals, peristaltic pumps are employed at different stages for transferring and dosing of the final product. Commonly used silicone tubings are known for particle shedding from the inner tubing surface due to friction in the pump head. These nanometer sized silicone rubber particles could interfere with proteins. Until now, only mixed protein particles containing micrometer-sized contaminations such as silicone oil have been characterized, detected, and quantified. To overcome the detection limits in particle sizes of contaminants, this study aimed for the definite identification of protein particles containing nanometer sized silicone particles in qualitative and quantitative manner. The mixed particles consisted of silicone rubber particles either coated with a protein monolayer or embedded into protein aggregates. Confocal Raman microscopy allows label free chemical identification of components and 3D particle imaging. Labeling the tubing enables high-resolution imaging via confocal laser scanning microscopy and counting of mixed particles via Imaging Flow Cytometry. Overall, these methods allow the detection and identification of particles of unknown origin and composition and could be a forensic tool for solving problems with contaminations during processing of biopharmaceuticals.

Bacteria Detection: From Powerful SERS to Its Advanced Compatible Techniques.

The rapid, highly sensitive, and accurate detection of bacteria is the focus of various fields, especially food safety and public health. Surface-enhanced Raman spectroscopy (SERS), with the advantages of being fast, sensitive, and nondestructive, can be used to directly obtain molecular fingerprint information, as well as for the on-line qualitative analysis of multicomponent samples. It has therefore become an effective technique for bacterial detection. Within this progress report, advances in the detection of bacteria using SERS and other compatible techniques are discussed in order to summarize its development in recent years. First, the enhancement principle and mechanism of SERS technology are briefly overviewed. The second part is devoted to a label-free strategy for the detection of bacterial cells and bacterial metabolites. In this section, important considerations that must be made to improve bacterial SERS signals are discussed. Then, the label-based SERS strategy involves the design strategy of SERS tags, the immunomagnetic separation of SERS tags, and the capture of bacteria from solution and dye-labeled SERS primers. In the third part, several novel SERS compatible technologies and applications in clinical and food safety are introduced. In the final part, the results achieved are summarized and future perspectives are proposed.

The Earlier the Better: Structural Analysis and Separation of Lanthanides with Pyrroloquinoline Quinone.

Lanthanides (Ln) are critical raw materials, however, their mining and purification have a considerable negative environmental impact and sustainable recycling and separation strategies for these elements are needed. In this study, the precipitation and solubility behavior of Ln complexes with pyrroloquinoline quinone (PQQ), the cofactor of recently discovered lanthanide (Ln) dependent methanol dehydrogenase (MDH) enzymes, is presented. In this context, the molecular structure of a biorelevant europium PQQ complex was for the first time elucidated outside a protein environment. The complex crystallizes as an inversion symmetric dimer, Eu2 PQQ2 , with binding of Eu in the biologically relevant pocket of PQQ. LnPQQ and Ln1Ln2PQQ complexes were characterized by using inductively coupled plasma mass spectrometry (ICP-MS), infrared (IR) spectroscopy, 151 Eu-Mössbauer spectroscopy, X-ray total scattering, and extended X-ray absorption fine structure (EXAFS). It is shown that a natural enzymatic cofactor is capable to achieve separation by precipitation of the notoriously similar, and thus difficult to separate, lanthanides to some extent.

Kinetic and Mechanistic Investigation of the Photocatalyzed Surface Reduction of 4-Nitrothiophenol Observed on a Silver Plasmonic Film via Surface-Enhanced Raman Scattering.

Hot electrons generated by photoinduced plasmon decay from a plasmonic metal surface can reduce 4-nitrothiophenol (4-NTP) to 4-aminothiophenol (4-ATP). Compared to the reduction with a reducing agent such as sodium borohydride, surface-enhanced Raman scattering (SERS) measurements were performed here to elucidate the complex molecular mechanism of the reduction in the presence of halide ions and hydrogen ions. The SERS measurements were performed using a simply prepared silver plasmonic film (AgPF), which enables monitoring of the reaction under different conditions at a solid-liquid surface and eliminates the need for the use of a reducing agent. As the concentration of H+ and Cl- could be controlled, the observation of the reaction under a systematic set of conditions was possible. Based on the kinetic traces of the intermediates, a reaction mechanism for the 4-NTP to 4-ATP reduction is suggested. Rate constants for the individual reactions are presented that fit the measured kinetic traces, and the role of hydrogen in each reaction step is characterized. This work provides clarification on the molecular transformation directly using protons as the hydrogen source and demonstrates an effective method of applying a simple and low-cost silver surface catalyst for SERS studies. Moreover, the monitoring of Cl--concentration-dependent spectra provides insight into the hot-electron conversion process during the photoreduction and strongly supports the formation of AgCl for the activation of H+.

Metal-Organic Framework Nanoparticles Induce Pyroptosis in Cells Controlled by the Extracellular pH.

Ion homeostasis is essential for cellular survival, and elevated concentrations of specific ions are used to start distinct forms of programmed cell death. However, investigating the influence of certain ions on cells in a controlled way has been hampered due to the tight regulation of ion import by cells. Here, it is shown that lipid-coated iron-based metal-organic framework nanoparticles are able to deliver and release high amounts of iron ions into cells. While high concentrations of iron often trigger ferroptosis, here, the released iron induces pyroptosis, a form of cell death involving the immune system. The iron release occurs only in slightly acidic extracellular environments restricting cell death to cells in acidic microenvironments and allowing for external control. The release mechanism is based on endocytosis facilitated by the lipid-coating followed by degradation of the nanoparticle in the lysosome via cysteine-mediated reduction, which is enhanced in slightly acidic extracellular environment. Thus, a new functionality of hybrid nanoparticles is demonstrated, which uses their nanoarchitecture to facilitate controlled ion delivery into cells. Based on the selectivity for acidic microenvironments, the described nanoparticles may also be used for immunotherapy: the nanoparticles may directly affect the primary tumor and the induced pyroptosis activates the immune system.

Quenching of nonlinear photoacoustic signal generation in gold nanoparticles through coating.

The photoacoustic signal generated from specific gold nanoparticles increases nonlinearly with respect to fluence. We demonstrate experimentally that this nonlinear behavior can be quenched with a particle coating, and present a theoretical analysis to explain this behavior. This effect has the potential to be developed into a photoacoustic-based biochemical sensor.

Comprehensive Multi-Analytical Investigations on the Vietnamese lacquered Wall-Panel "The Return of the Hunters" by Jean Dunand.

This work presents a comprehensive, multi-analytical scientific approach for determining the type of lacquer and artistic materials used by Jean Dunand on his work "The Return of the Hunters" (1935). For this purpose, thermally assisted hydrolysis and methylation - gas chromatography/mass spectrometry (THM-GC/MS), optical microscopy (OM) in visible (Vis) and ultraviolet light (UV), and scanning electron microscopy - energy-dispersive X-ray spectroscopy (SEM-EDX) were selected. Furthermore, a novel application of micro attenuated total reflection Fourier transform infrared (µATR-FTIR) spectroscopic mapping by univariate and multivariate analysis was applied for studying the complex lacquer paint stratigraphy. The results show that Vietnamese lacquer was used as a binder, mixed together with linseed oil and pine resins as additives in combination with inorganic pigments, and that shellac was included on the top of the paint; they document an important step in the story of the transfer of Vietnamese lacquer painting techniques to Europe.

Yeast cell wall - Silver nanoparticles interaction: A synergistic approach between surface-enhanced Raman scattering and computational spectroscopy tools.

Candida species are becoming one of the pathogens developing antifungal resistance due to inappropriate treatment and overuse of antimycotic drugs in building construction and agriculture. Further, fungal infections are often difficult to detect, also due to slow in vitro growth of the organisms from clinical specimens. Thus, fast detection and discrimination of yeast cells in direct patient materials is essential for an adequate treatment and success rate. In this work, we investigated Candida species isolated from patients, by using surface-enhanced Raman scattering (SERS) combined with computational spectroscopy tools, aiming to detect and discriminate between the three considered species, Candida albicans, Candida glabrata, and Candida parapsilosis. Density functional theory (DFT) was used to calculate Raman spectra of yeasts' main cell wall components for elucidating the origin of the observed bands. Accurate assignments of normal modes helped for a better understanding of the interaction between silver nanoparticles with yeasts' cell wall. Further, SERS spectra were used as samples in a database on which we performed multivariate analyses. By Principal component analysis (PCA), we obtained a maximum variation of 79% between the three samples. Linear discriminant analysis (LDA) was successfully used to discriminate between the three species.

Correction: Antimicrobial peptide based magnetic recognition elements and Au@Ag-GO SERS tags with stable internal standards: a three in one biosensor for isolation, discrimination and killing of multiple bacteria in whole blood.

[This corrects the article DOI: 10.1039/C8SC04637A.].

Preliminary survey of matrix effects in the Microwave-sustained, Inductively Coupled Atmospheric-pressure Plasma (MICAP).

Matrix effects caused by Na and Al in the nitrogen Microwave-sustained, Inductively Coupled, Atmospheric-pressure Plasma (MICAP) were investigated. Easily ionizable elements, such as Na, can suppress or enhance the analyte signal; Al is shown here to produce a similar effect. The influence of these matrices was examined for 18 emission lines of 8 analyte atoms and ions having a wide range of excitation and ionization energies. The plasma operating conditions were fixed during all experiments at a total nitrogen flow of 19.4Lmin-1 and a microwave power of 1.5kW. An Fe solution was used to determine the excitation temperature of the plasma by the Boltzmann plot method at selected matrix concentrations. In addition, vertical emission profiles of the plasma were measured. The matrix effect becomes worse at higher concentrations of an easily ionizable element. The effect is caused not only by a shift in ionization equilibrium but also by a possible change in plasma ionization temperature. Correction methods to reduce the matrix effects were tested and are discussed.

Characterization of Clinically Relevant Fungi via SERS Fingerprinting Assisted by Novel Chemometric Models.

Nonculture-based tests are gaining popularity and upsurge in the diagnosis of invasive fungal infections (IFI) fostered by their main asset, the reduced analysis time, which enables a more rapid diagnosis. In this project, three different clinical isolates of relevant filamentous fungal species were discriminated by using a rapid (less than 5 min) and sensitive surface-enhanced Raman scattering (SERS)-based detection method, assisted by chemometrics. The holistic evaluation of the SERS spectra was performed by employing appropriate chemometric tools-classical and fuzzy principal component analysis (FPCA) in combination with linear discriminant analysis (LDA) applied to the first relevant principal components. The efficiency of the proposed robust algorithm is illustrated on the data set including three fungal isolates (Aspergillus fumigatus sensu stricto, cryptic A. fumigatus complex species, and Rhizomucor pusillus) that were isolated from patient materials. The accurate and reliable discrimination between species of common fungal pathogen strains suggest that the developed method has the potential as an alternative, spectroscopic-based routine analysis tool in IFI diagnosis.

Antimicrobial peptide based magnetic recognition elements and Au@Ag-GO SERS tags with stable internal standards: a three in one biosensor for isolation, discrimination and killing of multiple bacteria in whole blood.

In this study, a new biosensor based on a sandwich structure has been developed for the isolation and detection of multiple bacterial pathogens via magnetic separation and SERS tags. This novel assay relies on antimicrobial peptide (AMP) functionalized magnetic nanoparticles as "capturing" probes for bacteria isolation and gold coated silver decorated graphene oxide (Au@Ag-GO) nanocomposites modified with 4-mercaptophenylboronic acid (4-MPBA) as SERS tags. When different kinds of bacterial pathogens are combined with the SERS tags, the "fingerprints" of 4-MPBA show corresponding changes due to the recognition interaction between 4-MPBA and different kinds of bacterial cell wall. Compared with the label-free SERS detection of bacteria, 4-MPBA here can be used as an internal standard (IS) to correct the SERS intensities with high reproducibility, as well as a Raman signal reporter to enhance the sensitivity and amplify the differences among the bacterial "fingerprints". Thus, three bacterial pathogens (Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa) were successfully isolated and detected, with the lowest concentration for each of the strains detected at just 101 colony forming units per mL (CFU mL-1). According to the changes in the "fingerprints" of 4-MPBA, three bacterial strains were successfully discriminated using discriminant analysis (DA). In addition, the AMP modified Fe3O4NPs feature high antibacterial activities, and can act as antibacterial agents with low cellular toxicology in the long-term storage of blood for future safe blood transfusion applications. More importantly, this novel method can be applied in the detection of bacteria from clinical patients who are infected with bacteria. In the validation analysis, 97.3% of the real blood samples (39 patients) could be classified effectively (only one patient infected with E. coli was misclassified). The multifunctional biosensor presented here allows for the simultaneous isolation, discrimination and killing of bacteria, suggesting its high potential for clinical diagnosis and safe blood transfusions.