Metabolic profiling of bacteria with the application of polypyrrole-MOF SPME fibers and plasmonic nanostructured LDI-MS substrates.

Here we present application of innovative lab-made analytical devices such as plasmonic silver nanostructured substrates and polypyrrole-MOF solid-phase microextraction fibers for metabolic profiling of bacteria. For the first time, comprehensive metabolic profiling of both volatile and non-volatile low-molecular weight compounds in eight bacterial strains was carried out with utilization of lab-made devices. Profiles of low molecular weight metabolites were analyzed for similarities and differences using principal component analysis, hierarchical cluster analysis and random forest algorithm. The results showed clear differentiation between Gram positive (G+) and Gram negative (G-) species which were identified as distinct clusters according to their volatile metabolites. In case of non-volatile metabolites, differentiation between G+ and G- species and clustering for all eight species were observed for the chloroform fraction of the Bligh & Dyer extract, while methanolic fraction failed to recover specific ions in the profile. Furthermore, the results showed correlation between volatile and non-volatile metabolites, which suggests that lab-made devices presented in the current study might be complementary and therefore, useful for species differentiation and gaining insights into bacterial metabolic pathways.

Electropolymerized polypyrrole-MOF composite as a coating material for SPME fiber for extraction VOCs liberated by bacteria.

The synthesis of efficient and low-cost coatings for solid-phase microextraction attracted much attention. Conductive polymers are excellent candidates for this purpose due to the possibility of electropolymerization, which results in the reproducible synthesis of films. A plethora of studies reported in the literature concluded that modification of conductive polymers with innovative materials could lead to an increase in sensitivity toward specific analytes. In this work, the metal-organic framework-polypyrrole composite was electrodeposited in one step directly onto a stainless-steel substrate. The effect of synthesis parameters on extraction efficiency was investigated. The obtained PPy@ZIF-8 coating was subjected to physical-chemical characterization using electron microscopy and Fourier-transform IR spectroscopy. The main finding of the study was that the values of the limit of detection and intra- and inter-day reproducibility for analytes with different chemical structures were found to be lower as compared to pure polypyrrole coating. Furthermore, the obtained polypyrrole-MOF coating was applied for the collection of profiles of volatile organic compounds liberated by bacteria. Hence, the polypyrrole@ZIF-8 coating synthesized using a low-cost and facile approach presented in this study can be useful for the profiling of VOCs liberated by bacteria.

Silver Nanostructured Substrates in LDI-MS of Low Molecular Weight Compounds.

Mass spectrometric techniques can provide data on the composition of a studied sample, utilizing both targeted and untargeted approaches to solve various research problems. Analysis of compounds in the low mass range has practical implications in many areas of research and industry. Laser desorption ionization techniques are utilized for the analysis of molecules in a low mass region using low sample volume, providing high sensitivity with low chemical background. The fabrication of substrates based on nanostructures to assist ionization with well-controlled morphology may improve LDI-MS efficiency for silver nanoparticles with plasmonic properties. In this work, we report an approach for the preparation of silver nanostructured substrates applied as laser desorption ionization (LDI) plates, using the chemical vapor deposition (CVD) technique. Depending on the mass of used CVD precursor, the approach allowed the synthesis of LDI plates with tunable sensitivity for various low molecular weight compounds in both ion-positive and ion-negative modes. Reduced chemical background and sensitivity to small biomolecules of various classes (fatty acids, amino acids and water-soluble metabolites) at nanomolar and picomolar detection levels for lipids such as triacylglycerols, phosphatidylethanolamines and lyso-phosphatidylcholines represent an emerging perspective for applications of LDI-MS plates for the collection of molecular profiles and targeted analysis of low molecular weight compounds for various purposes.

Nanostructured Layer of Silver for Detection of Small Biomolecules in Surface-Assisted Laser Desorption Ionization Mass Spectrometry.

A facile approach for the synthesis of a silver nanostructured layer for application in surface-assisted laser desorption/ionization mass spectrometry of low-molecular-weight biomolecules was developed using electrochemical deposition. The deposition was carried out using the following silver salts: trifluoroacetate, acetate and nitrate, varying the voltage and time. The plate based on trifluoroacetate at 10 V for 15 min showed intense SALDI-MS responses for standards of various classes of compounds: fatty acids, cyclitols, saccharides and lipids at a concentration of 1 nmol/spot, with values of the signal-to-noise ratio ≥50. The values of the limit of detection were 0.71 µM for adonitol, 2.08 µM for glucose and 0.39 µM for palmitic acid per spot. SEM analysis of the plate showed anisotropic flower-like microstructures with nanostructures on their surface. The reduced chemical background in the low-mass region can probably be explained by the absence of stabilizers and reducing agents during the synthesis. The plate synthesized with the developed approach showed potential for future use in the analysis of low-molecular-weight compounds of biological relevance. The absence of the need for the utilization of sophisticated equipment and the synthesis time (10 min) may benefit large-scale applications of the layer for the detection of various types of small biomolecules.

Micro-Chamber/Thermal Extractor (µ-CTE) as a new sampling system for VOCs emitted by feces.

VOCs (volatile organic compounds) are increasingly wished to be used in diagnosis of diseases. They present strategic advantages, when compared to classical methods used, such as simplicity and current availability of performant non-invasive sample collection methods/systems. However, standardized sampling methods are required in order to achieve reproducible results. In the current study we developed a method to be used for feces sampling using a Micro-Chamber/Thermal Extractor (µ-CTE). Design Expert software (with Box-Behnken design) was used to predict the solutions. Therefore, by using the simulation experimental plan that was further experimentally verified, extraction time of 19.6 min, at extraction temperature of 30.6 °C by using a flow rate of 48.7 mL/min provided the higher response. The developed method was validated by using correlation tests and Network analysis, which both proved the validity of the developed model.

Development of controlled film of polypyrrole for solid-phase microextraction fiber by electropolymerization.

The aim of the study was to develop an approach for synthesis of polypyrrole coating material and further fabrication of solid-phase microextraction fiber by direct electropolymerization on a stainless-steel wire. Surface morphology, porosity and thermal stability were evaluated by different physico-chemical methods. Synthesized polypyrrole fiber was successfully applied for extraction of VOCs such as benzene, toluene, ethylbenzene, p-xylene, phenol and dodecane. Utilization of polypyrrole fiber for extraction of VOCs was assessed by HS-SPME coupled to gas chromatography with flame ionization detection (GC-FID). Solid-phase microextraction parameters for their simultaneous analysis were optimized using Box-Behnken design and accounted for 49.7 min for extraction time, 30 °C for extraction temperature and 17.2 min for equilibration time. In addition, the coating showed good reproducibility (RSD < 12.22%), while the values of limit of detection were in the range of 0.59-283.33 ng/mL.

Evolution and Evaluation of GC Columns.

A chromatographic column is the fundamental element required for gas-chromatographic analysis. The separation of components coming from complex mixtures, prior to their detection was leading to a prominent revolution in different areas of science. Moreover, current advances in gas chromatographic (GC) columns technology and development have been providing almost unlimited possibilities for analysis employing diverse matrices. We aim through this review article to describe the evolution of chromatographic columns, by pointing the most important stages, as well as the new trends and future perspectives predicted for the new generation of GC columns. Furthermore, it was in our scope to present the main fundamentals regarding the theoretical relationships that describe the chromatographic separation, to introduce concepts related to columns selection in accordance with the required application as well as to discuss the available evaluation parameters for columns efficiency. Consequently, the early stages of first columns preparation up to the development of GC capillary columns used nowadays, together with examples of their applications are also reported and described in detail.

Temporal influence of different antibiotics onto the inhibition of Escherichia coli bacterium grown in different media.

Escherichia coli (E. coli) is a Gram-negative bacterium commonly found in the lower intestine of warm-blooded organisms, including humans. Although the majority of the strains are considerably harmless, some serotypes are pathogenic, frequently causing diarrhea and other illnesses outside the intestinal tract. The standard antidote against bacteria is the use of antibiotics. Depending on their type, the antibiotics have various mechanisms of action on bacteria. Moreover, in case of in-vitro cultivation of bacteria, the used growth media plays a crucial role, since it influences bacterial inhibition as well. In the present study, we emphasize the importance of cultivability in bacterial inhibition under the treatment with five different antibiotics belonging to different classes. Consequently, E. coli was cultivated in three different growth media: trypcase soy broth (TSB), Mueller Hinton (MH), and minimal salts (M9) enriched with glucose, respectively. MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) analyses, that were used for fast characterization of changes that occur in ribosomal protein profiles, revealed differentiation and similarities between investigated cases, while flow cytometry (FCM) tests better explained the given changes that occurred in the analyzed samples after 3, 24 and 48 h of experimental campaign.