Dosage of chemotherapeutic drugs is a tradeoff between efficacy and side-effects. Liposomes are nanocarriers that increase therapy efficacy and minimize side-effects by delivering otherwise difficult to administer therapeutics with improved efficiency and selectivity. Still, variabilities in liposome preparation require assessing drug encapsulation efficiency at the single liposome level, an information that, for non-fluorescent therapeutic cargos, is inaccessible due to the minute drug load per liposome. Photothermal induced resonance (PTIR) provides nanoscale compositional specificity, up to now, by leveraging an atomic force microscope (AFM) tip contacting the sample to transduce the sample’s photothermal expansion. However, on soft samples (e.g. liposomes) PTIR effectiveness is reduced due to the likelihood of tip-induced sample damage and inefficient AFM transduction. Here, individual liposomes loaded with the chemotherapeutic drug cytarabine are deposited intact from suspension via nES-GEMMA (nano-electrospray gas-phase electrophoretic mobility molecular analysis) collection and characterized at the nanoscale with the chemically-sensitive PTIR method. A new tapping-mode PTIR imaging paradigm based on heterodyne detection is shown to be better adapted to measure soft samples, yielding cytarabine distribution in individual liposomes and enabling classification of empty and drug-loaded liposomes. The measurements highlight PTIR capability to detect ≈ 103 cytarabine molecules (≈ 1.7 zmol) label-free and non-destructively.

This work was sparked by the reported identification of man-made cellulosic fibers (rayon/viscose) in the marine environment as a major fraction of plastic litter by Fourier transform infrared (FT-IR) transmission spectroscopy and library search. To assess the plausibility of such findings, both natural and man-made fibers were examined using FT-IR spectroscopy. Spectra acquired by transmission microscopy, attenuated total reflection (ATR) microscopy, and ATR spectroscopy were compared. Library search was employed and results show significant differences in the identification rate depending on the acquisition method of the spectra. Careful selection of search parameters and the choice of spectra acquisition method were found to be essential for optimization of the library search results. When using transmission spectra of fibers and ATR libraries it was not possible to differentiate between man-made and natural fibers. Successful differentiation of natural and man-made cellulosic fibers has been achieved for FT-IR spectra acquired by ATR microscopy and ATR spectroscopy, and application of ATR libraries. As an alternative, chemometric methods such as unsupervised hierarchical cluster analysis, principal component analysis, and partial least squares-discriminant analysis were employed to facilitate identification based on intrinsic relationships of sample spectra and successful discrimination of the fiber type could be achieved. Differences in the ATR spectra depending on the internal reflection element (Ge versus diamond) were observed as expected; however, these did not impair correct classification by chemometric analysis. Moreover, the effects of different levels of humidity on the IR spectra of natural and man-made fibers were investigated, too. It has been found that drying and re-humidification leads to intensity changes of absorption bands of the carbohydrate backbone, but does not impair the identification of the fiber type by library search or cluster analysis.

Against the outdated belief that inclusion bodies (IBs) in Escherichia coli are only inactive aggregates of misfolded protein, and thus should be avoided during recombinant protein production, numerous biopharmaceutically important proteins are currently produced as IBs. To obtain correctly folded, soluble product, IBs have to be processed, namely, harvested, solubilized, and refolded. Several years ago, it was discovered that, depending on cultivation conditions and protein properties, IBs contain partially correctly folded protein structures, which makes IB processing more efficient. Here, we present a method of tailored induction of recombinant protein production in E. coli by a mixed feed system using glucose and lactose and its impact on IB formation. Our method allows tuning of IB amount, IB size, size distribution, and purity, which does not only facilitate IB processing, but is also crucial for potential direct applications of IBs as nanomaterials and biomaterials in regenerative medicine.Electronic supplementary materialThe online version of this article (10.1007/s00253-017-8641-6) contains supplementary material, which is available to authorized users.

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.

Worldwide, multiresistant bacterial strains are emerging at unprecedented rates. This development seriously threatens the ability of humanity to treat even common infections, resulting in disability and death. Furthermore, this development endangers all medical achievements including cancer therapy or organ transplantations. Therefore, the World Health Organization has endorsed antimicrobial resistance as a great threat to humanity. To still allow effective treatment of patients, rapid, automated, and reliable antibiotic susceptibility testing (AST) of bacterial pathogens is essential. Thereby, speed and sensitivity of the AST results are crucial for improving patient care. Here, Raman spectroscopy as a nondestructive technique providing chemical-specific information is employed to monitor the deuterium uptake of metabolically active bacteria during antibiotic treatment, enabling fast and reliable AST. For this purpose, a bulk sample-preparation method was developed, allowing a high-throughput analysis of a significant number of cells. A protocol was developed for Gram-positive (Enterococcus faecalis) and Gram-negative (Escherichia coli) reference strains and was tested on 51 clinical isolates with wellcharacterized resistance phenotypes against ampicillin, ciprofloxacin, meropenem, and vancomycin. Borderline resistant and heteroresistant phenotypes were observed and further investigated. This is of critical importance as the sensitive detection of lowfrequency heteroresistance in bacterial populations is a huge challenge. Such isolates seem susceptible but are resistant to treatment in vivo. Automatable analysis detects strong phenotypes within 3 h. On the basis of experimental and modeled data, heteroresistance is estimated to be detectable down to frequencies of 10 −6 and investigated on clinical isolates as a proof-of-concept study, but requiring longer incubation time.

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 + .

We developed a top-irradiated, liquid-phase attenuated total reflectance Fourier transform infrared (ATR-FTIR) setup that allows time-resolved investigations of both Pt particle growth during in situ photodeposition via monitoring of the Pt 0 −CO ads band on TiO 2 thin films as well as the photooxidation of methanol in aqueous environments. Obtained ATR-FTIR data sets were analyzed via multivariate curve resolution-alternating least squares (MCR-ALS), which enabled us to clearly differentiate various reaction pathways for different Pt loadings at otherwise fixed reaction conditions (i.e., methanol concentration, UV intensity). At the highest Pt loading (nominal concentration of 2.7 wt %), photooxidation of methanol occurs via direct oxidation through a formaldehyde intermediate to CO 2 , whereas the lower Pt loadings of 0.7 and 1.4 wt % favor a side reaction that includes methyl formate as an intermediate. These findings were correlated with the formation of different CO binding sites on Pt during photodeposition, and we presume that changes in the reaction pathway depend on the number rather than the nature of active available Pt sites. Complementary ex situ characterizations of the thin films by transmission electron microscopy (TEM), Raman spectroscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and inductively coupled plasma mass spectrometry (ICP-MS) were performed, delivering information on the generated Pt nanoparticles and structural changes of TiO 2 . The presented optical setup paves the way for fundamental studies of heterogeneous catalytic reactions as close as possible to their actual use in aqueous systems.

BackgroundThe well-known inflammatory and fibrogenic changes of the lung upon crystalline silica are accompanied by early changes of the phospholipid composition (PLC) as detected in broncho-alveolar lavage fluid (BALF). Amorphous silica nanoparticles (NPs) evoke transient lung inflammation, but their effect on PLC is unknown. Here, we compared effects of unmodified and phosphonated amorphous silica NP and describe, for the first time, local changes of the PLC with innovative bioimaging tools.MethodsUnmodified (SiO2-n), 3-(trihydroxysilyl) propyl methylphosphonate coated SiO2-n (SiO2-p) as well as a fluorescent surrogate of SiO2-n (SiO2-FITC) nanoparticles were used in this study. In vitro toxicity was tested with NR8383 alveolar macrophages. Rats were intratracheally instilled with SiO2-n, SiO2-p, or SiO2-FITC, and effects on lungs were analyzed after 3 days. BALF from the right lung was analyzed for inflammatory markers. Cryo-sections of the left lung were subjected to fluorescence microscopy and PLC analyses by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MS), Fourier transform infrared microspectroscopy (FT-IR), and tandem mass spectrometry (MS/MS) experiments.ResultsCompared to SiO2-p, SiO2-n NPs were more cytotoxic to macrophages in vitro and more inflammatory in the rat lung, as reflected by increased concentration of neutrophils and protein in BALF. Fluorescence microscopy revealed a typical patchy distribution of SiO2-FITC located within the lung parenchyma and alveolar macrophages. Superimposable to this particle distribution, SiO2-FITC elicited local increases of phosphatidylglycerol (PG) and phosphatidylinositol (PI), whereas phoshatidylserine (PS) and signals from triacylgyceride (TAG) were decreased in the same areas. No such changes were found in lungs treated with SiO2-p or particle-free instillation fluid.ConclusionsPhosphonate coating mitigates effects of silica NP in the lung and abolishes their locally induced changes in PLC pattern. Bioimaging methods based on MALDI-MS may become a useful tool to investigate the mode of action of NPs in tissues.Electronic supplementary materialThe online version of this article (10.1186/s12989-018-0267-z) contains supplementary material, which is available to authorized users.