The visual evaluation of data derived from screening and optimization experiments in the development of new analytical methods poses a considerable time investment and introduces the risk of subjectivity. This study presents a novel approach to processing such data, based on factor analysis of mixed data and hierarchical clustering - multivariate techniques implemented in the R programming language. The methodology is demonstrated in the early-stage screening and optimization of the chromatographic separation of 15 structurally diverse drugs that affect the central nervous system, using a custom R Language script. The presented explorative approach enabled the identification of key parameters affecting the separation and significantly reduced the time required to evaluate the comprehensive dataset from the screening experiments. Based on the data analysis results, the optimal combination of stationary phase and mobile phase composition was selected, considering retention, overall resolution, and peak shape of compounds. Additionally, compounds vulnerable to changes in selected chromatographic conditions were identified. As a complement to the presented R Language script, a web-based application ChromaFAMDeX has been developed to offer an intuitive interface that enhances the accessibility of the used statistical methods. Accompanying the publication, the R script and the link to the standalone application are provided, enabling replication and adaptation of the methodology.
This study investigates the efficacy of supramolecular solvent (SUPRAS) in extracting a diverse spectrum of organic contaminants from indoor dust. Initially, seven distinct SUPRAS were assessed across nine categories of contaminants to identify the most effective one. A SUPRAS comprising Milli-Q water, tetrahydrofuran, and hexanol in a 70:20:10 ratio, respectively, demonstrated the best extraction performance and was employed for testing a wider array of organic contaminants. Furthermore, we applied the selected SUPRAS for the extraction of organic compounds from the NIST Standard Reference Material (SRM) 2585. In parallel, we performed the extraction of NIST SRM 2585 with conventional extraction methods using hexane:acetone (1:1) for non-polar contaminants and methanol (100%) extraction for polar contaminants. Analysis from two independent laboratories (in Norway and the Czech Republic) demonstrated the viability of SUPRAS for the simultaneous extraction of twelve groups of organic contaminants with a broad range of physico-chemical properties including plastic additives, pesticides, and combustion by-products. However, caution is advised when employing SUPRAS for highly polar contaminants like current-use pesticides or volatile substances like naphthalene.
- Publikační typ
- časopisecké články MeSH
This work describes the intricacies of the determination of the trimethylselenonium ion (TMSe) in human urine via high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS). By definition, this technique requires that the separated TMSe can be online converted into a volatile compound. Literature data for the determination of TMSe via the hydride generation technique are contradictory; i.e., some authors claim that direct formation of volatile compounds is possible under reduction with NaBH4, whereas others reported that a digestion step is mandatory prior to conversion. We studied and optimized the conditions for online conversion by varying the mobile phase composition (pyridine, phosphate, and acetate), testing different reaction coils, and optimizing the hydride generation conditions, although technically no hydride (H2Se) is formed but a dimethylselenide (DMSe). The optimized conditions were used for the analysis of 64 urine samples of 16 (unexposed) volunteers and the determination of low amounts of TMSe (LOD = 0.2 ng mL-1). Total (specific gravity-corrected) selenium concentrations in the urine samples ranged from 7.9 ± 0.7 to 29.7 ± 5.0 ng mL-1 for individual volunteers. Four volunteers were characterized as TMSe producers (hINMT genotype GA) and 12 were non-producers (hINMT genotype GG). Urine of TMSe producers contained 2.5 ± 1.7 ng mL-1 of TMSe, compared to 0.2 ± 0.2 ng mL-1 for non-producers.
- MeSH
- fluorescenční spektrometrie MeSH
- lidé MeSH
- selen * moč MeSH
- sloučeniny selenu * MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Cancer is a genetic disease induced by mutations in DNA, in particular point mutations in important driver genes that lead to protein malfunctioning and ultimately to tumorigenesis. Screening for the most common DNA point mutations, especially in such genes as TP53, BRCA1 and BRCA2, EGFR, KRAS, or BRAF, is crucial to determine predisposition risk for cancer or to predict response to therapy. In this review, we briefly depict how these genes are involved in cancer, followed by a description of the most common techniques routinely applied for their analysis, including high-throughput next-generation sequencing technology and less expensive low-throughput options, such as real-time PCR, restriction fragment length polymorphism, or high resolution melting analysis. We then introduce benefits of electrochemical biosensors as interesting alternatives to the standard methods in terms of cost, speed, and simplicity. We describe most common strategies involved in electrochemical biosensing of point mutations, relying mostly on PCR or isothermal amplification techniques, and critically discuss major challenges and obstacles that, until now, prevented their more widespread application in clinical settings.
Direct infusion of lipid extracts into the ion source of a mass spectrometer is a well-established method for lipid analysis. In most cases, nanofluidic devices are used for sample introduction. However, flow injection analysis (FIA) based on sample infusion from a chromatographic pump can offer a simple alternative to shotgun-based approaches. Here, we describe important modification of a method based on FIA and tandem mass spectrometry (MS/MS). We focus on minimizing contamination of the FIA/MS both to render the lipidomic platform more robust and to increase its capacity and applicability for long-sequence measurements required in clinical applications. Robust validation of the developed method confirms its suitability for lipid quantitation in human plasma analysis. Measurements of standard human plasma reference material (NIST SRM 1950) and a set of plasma samples collected from kidney cancer patients and from healthy volunteers yielded highly similar results between FIA-MS/MS and ultra-high-performance supercritical fluid chromatography (UHPSFC)/MS, thereby demonstrating that all modifications have practically no effect on the statistical output. Newly modified FIA-MS/MS allows for the quantitation of 141 lipid species in plasma (11 major lipid classes) within 5.7 min. Finally, we tested the method in a clinical laboratory of the General University Hospital in Prague. In the clinical setting, the method capacity reached 257 samples/day. We also show similar performance of the classification models trained based on the results obtained in clinical settings and the analytical laboratory at the University of Pardubice. Together, these findings demonstrate the high potential of the modified FIA-MS/MS for application in clinical laboratories to measure plasma and serum lipid profiles.
- MeSH
- krevní plazma chemie MeSH
- lidé MeSH
- lipidomika * metody MeSH
- lipidy analýza MeSH
- průtoková injekční analýza MeSH
- tandemová hmotnostní spektrometrie * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Micro/nanomotors are nanoscale devices that have been explored in various fields, such as drug delivery, environmental remediation, or biosensing and diagnosis. The use of micro/nanomotors has grown considerably over the past few years, partially because of the advantages that they offer in the development of new conceptual avenues in biosensing. This is due to their propulsion and intermixing in solution compared with their respective static forms, which enables motion-based detection methods and/or decreases bioassay time. This review focuses on the impacts of micro/nanomotors on biosensing research in the last 2 years. An overview of designs for bioreceptor attachment to micro/nanomotors is given. Recent developments have focused on chemically propelled micromotors using external fuels, commonly hydrogen peroxide. However, the associated fuel toxicity and inconvenience of use in relevant biological samples such as blood have prompted researchers to explore new micro/nanomotor biosensing approaches based on biocompatible propulsion sources such as magnetic or ultrasound fields. The main advances in biocompatible propulsion sources for micro/nanomotors as novel biosensing platforms are discussed and grouped by their propulsion-driven forces. The relevant analytical applications are discussed and representatively illustrated. Moreover, envisioning future biosensing applications, the principal advantages of micro/nanomotor synthesis using biocompatible and biodegradable materials are given. The review concludes with a realistic drawing on the present and future perspectives.
In this study, a mercury meniscus-modified silver solid amalgam electrode was used for the first time for the detection of UV-induced DNA damage. The integrity of the double-stranded DNA (dsDNA) layer was detected indirectly using the evaluation of the methylene blue reduction within its accumulation into dsDNA after the UV irradiation of the biosensor surface with two different wavelengths (254 nm and 365 nm), monitored by differential pulse voltammetry. Moreover, a simple electrochemical characterization of the biosensor surface was performed using cyclic voltammetry of the redox indicator hexaammineruthenium chloride (RuHex) present in the solution. Electrochemical impedance spectroscopy (EIS) was used in both cases for the verification of results. Individual electrochemical signals depend on the time of biosensor exposure to UV irradiation as well as on the selected wavelengths and are different for both used types of dsDNA (salmon sperm and calf thymus). The highest degradation degree up to 60% was observed using sensitive EIS of methylene blue after 10 min irradiation of the biosensor at 254 nm. The use of RuHex seems to be less sensitive for the detection of dsDNA structural changes, when the degradation degree up to 40% was observed, using EIS at the same conditions.
- MeSH
- biosenzitivní techniky * metody MeSH
- DNA chemie MeSH
- elektrochemické techniky metody MeSH
- elektrody MeSH
- lidé MeSH
- methylenová modř chemie MeSH
- poškození DNA MeSH
- sperma MeSH
- stříbro * MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
This article will debate the usefulness of POCT measurements and the contribution microdialysis can make to generating valuable information. A particular theme will be the rarely considered difference between ex vivo sampling, which typically generates only a static measure of concentration, and in vivo measurements that are subject to dynamic changes due to mass transfer. Those dynamic changes provide information about the patients' physiological state.
The protein heterogeneity at the single-cell level has been recognized to be vital for an understanding of various life processes during animal development. In addition, the knowledge of accurate quantity of relevant proteins at cellular level is essential for appropriate interpretation of diagnostic and therapeutic results. Some low-copy-number proteins are known to play a crucial role during cell proliferation, differentiation, and also in apoptosis. The fate decision is often based on the concentration of these proteins in the individual cells. This is likely to apply also for caspases, cysteine proteases traditionally associated with cell death via apoptosis but recently being discovered also as important factors in cell proliferation and differentiation. The hypothesis was tested in bone-related cells, where modulation of fate from apoptosis to proliferation/differentiation and vice versa is particularly challenging, e.g., towards anti-osteoporotic treatments and anti-cancer strategies. An ultrasensitive and highly selective method based on bioluminescence photon counting was used to quantify activated caspase-3/7 in order to demonstrate protein-level heterogeneity in individual cells within one population and to associate quantitative measurements with different cell fates (proliferation, differentiation, apoptosis). The results indicate a gradual increase of caspase-3/7 activation from the proliferative status to differentiation (more than three times) and towards apoptosis (more than six times). The findings clearly support one of the putative key mechanisms of non-apoptotic functions of pro-apoptotic caspases based on fine-tuning of their activation levels.
- MeSH
- aktivace enzymů MeSH
- apoptóza MeSH
- buněčná diferenciace MeSH
- buněčné linie MeSH
- kaspasa 3 chemie genetika metabolismus MeSH
- kaspasa 7 chemie genetika metabolismus MeSH
- myši MeSH
- osteoblasty cytologie fyziologie MeSH
- proliferace buněk MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
We adapted a radioligand receptor binding assay for measuring insulin levels in unknown samples. The assay enables rapid and accurate determination of insulin concentrations in experimental samples, such as from insulin-secreting cells. The principle of the method is based on the binding competition of insulin in a measured sample with a radiolabeled insulin for insulin receptor (IR) in IM-9 cells. Both key components, radiolabeled insulin and IM-9 cells, are commercially available. The IR binding assay was used to determine unknown amounts of insulin secreted by MIN6 β cell line after stimulation with glucose, arginine, ornithine, dopamine, and serotonin. The experimental data obtained by the IR binding assay were compared to the results determined by RIA kits and both methods showed a very good agreement of results. We observed the stimulation of glucose-induced insulin secretion from MIN6 cells by arginine, weaker stimulation by ornithine, but inhibitory effects of dopamine. Serotonin effects were either stimulatory or inhibitory, depending on the concentration of serotonin used. The results will require further investigation. The study also clearly revealed advantages of the IR binding assay that allows the measuring of a higher throughput of measured samples, with a broader range of concentrations than in the case of RIA kits. The IR binding assay can provide an alternative to standard RIA and ELISA assays for the determination of insulin levels in experimental samples and can be especially useful in scientific laboratories studying insulin production and secretion by β cells and searching for new modulators of insulin secretion.
- MeSH
- arginin metabolismus MeSH
- beta-buňky metabolismus MeSH
- buněčné linie MeSH
- dopamin metabolismus MeSH
- glukosa metabolismus MeSH
- inzulin analýza metabolismus MeSH
- krysa rodu rattus MeSH
- Langerhansovy ostrůvky metabolismus MeSH
- lidé MeSH
- myši MeSH
- ornithin metabolismus MeSH
- potkani Wistar MeSH
- radioimunoanalýza metody MeSH
- radioligandová zkouška metody MeSH
- sekrece inzulinu * MeSH
- serotonin metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
