Efficient separation and sensitive identification of pathogenic bacterial strains is essential for a prosperous modern society, with direct applications in medical diagnostics, drug discovery, biodefense, and food safety. We developed a fast and reliable method for antibody-based selective immobilization of bacteria from suspension onto a gold-plated glass surface, followed by detection using strain-specific antibodies linked to gold nanoparticles decorated with a reporter molecule. The reporter molecules are subsequently detected by surface-enhanced Raman spectroscopy (SERS). Such a multi-functionalized nanoparticle is called a SERS-tag. The presented procedure uses widely accessible and cheap materials for manufacturing and functionalization of the nanoparticles and the immobilization surfaces. Here, we exemplify the use of the produced SERS-tags for sensitive single-cell detection of opportunistic pathogen Escherichia coli, and we demonstrate the selectivity of our method using two other bacterial strains, Staphylococcus aureus and Serratia marcescens, as negative controls. We believe that the described approach has a potential to inspire the development of novel medical diagnostic tools for rapid identification of bacterial pathogens.

• Klíčová slova
• Escherichia coli, SERS-tag, sandwich immunoassay, single-cell detection,
• MeSH
• Escherichia coli MeSH
• kovové nanočástice * chemie   MeSH
• protilátky chemie   MeSH
• Ramanova spektroskopie * metody   MeSH
• Staphylococcus aureus MeSH
• zlato chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protilátky MeSH
• zlato MeSH

Highly complex nanoparticles combining multimodal imaging with the sensing of physical properties in biological systems can considerably enhance biomedical research, but reports demonstrating the performance of a single nanosized probe in several imaging modalities and its sensing potential at the same time are rather scarce. Gold nanoshells with magnetic cores and complex organic functionalization may offer an efficient multimodal platform for magnetic resonance imaging (MRI), photoacoustic imaging (PAI), and fluorescence techniques combined with pH sensing by means of surface-enhanced Raman spectroscopy (SERS). In the present study, the synthesis of gold nanoshells with Mn-Zn ferrite cores is described, and their structure, composition, and fundamental properties are analyzed by powder X-ray diffraction, X-ray fluorescence spectroscopy, transmission electron microscopy, magnetic measurements, and UV-Vis spectroscopy. The gold surface is functionalized with four different model molecules, namely thioglycerol, meso-2,3-dimercaptosuccinate, 11-mercaptoundecanoate, and (11-mercaptoundecyl)-N,N,N-trimethylammonium bromide, to analyze the effect of varying charge and surface chemistry on cells in vitro. After characterization by dynamic and electrophoretic light scattering measurements, it is found that the particles do not exhibit significant cytotoxic effects, irrespective of the surface functionalization. Finally, the gold nanoshells are functionalized with a combination of 4-mercaptobenzoic acid and 7-mercapto-4-methylcoumarin, which introduces a SERS active pH sensor and a covalently attached fluorescent tag at the same time. 1H NMR relaxometry, fluorescence spectroscopy, and PAI demonstrate the multimodal potential of the suggested probe, including extraordinarily high transverse relaxivity, while the SERS study evidences a pH-dependent spectral response.

• Klíčová slova
• cell viability, gold nanoshells, magnetic nanoparticles, photoacoustic imaging, surface-enhanced Raman spectroscopy, transverse relaxivity,
• Publikační typ
• časopisecké články MeSH

Detection of trace amounts of poorly water-soluble pharmaceuticals or related (bio)solutions represents a key challenge in environment protection and clinical diagnostics. However, this task is complicated by low concentrations of pharmaceuticals, complex sample matrices, and sophisticated sample preparative routes. In this work, we present an alternative approach on the basis of an on-line flow extraction procedure and SERS measurements performed in a microfluidic regime. The advantages of our approach were demonstrated using ibuprofen (Ibu), which is considered as a common pharmaceutical contaminant in wastewater and should be monitored in various bioliquids. The extraction of Ibu from water to the dichloromethane phase was performed with an optimized microfluidic mixer architecture. As SERS tags, lipophilic functionalized gold multibranched nanoparticles (AuMs) were added to the organic phase. After microfluidic extraction, Ibu was captured by the functionalized AuM surface and recognized by on-line SERS measurements with up to 10-8 M detection limit. The main advantages of the proposed approach can be regarded as its simplicity, lack of need for preliminary sample preparation, high reliability, the absence of sample pretreatment, and low detection limits.

• MeSH
• léčivé přípravky * MeSH
• mikrofluidika * MeSH
• Ramanova spektroskopie MeSH
• reprodukovatelnost výsledků MeSH
• voda MeSH
• zlato MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• léčivé přípravky * MeSH
• voda MeSH
• zlato MeSH

Endodontic treatment of immature permanent teeth with necrotic pulp poses several clinical challenges and is one of the most demanding interventions in endodontics. Recently, with new discoveries in the field of tissue engineering, novel treatment protocols have been established. The most promising treatment modality is revascularization, whose integral part is the exposure of collagen matrix and embedded growth factors. However, optimization of the treatment protocol requires a development of analytical procedures able to analyze growth factors directly on the sample surface. In this work, method based on surface-enhanced Raman spectroscopy (SERS) was developed to investigate the influence of the time of the medical treatment using EDTA on exposure and accessibility of the growth factors, namely TGF-ß1, BMP-2, and bFGF on the dentine surface. The nanotags, which consist of magnetic Fe3O4@Ag nanocomposite covalently functionalized by tagged antibodies (anti-TGF-ß1-Cy3, anti-BMP-2-Cy5, and anti-bFGF-Cy7), were employed as a SERS substrate. Each antibody was coupled with a unique label allowing us to perform a parallel analysis of all three growth factors within one analytical run. Developed methodology presents an interesting alternative to a fluorescence microscopy and in contrary allows evaluating a chemical composition and thus minimizing possible false-positive results. Graphical abstract.

• Klíčová slova
• Growth factors, Imaging, Nanocomposites, SERS,
• MeSH
• dentin chemie   MeSH
• fibroblastový růstový faktor 2 analýza   MeSH
• kavita zubní dřeně chemie   MeSH
• kostní morfogenetický protein 2 analýza   MeSH
• lidé MeSH
• nanokompozity chemie   MeSH
• oxid železnato-železitý chemie   MeSH
• Ramanova spektroskopie metody   MeSH
• stříbro chemie   MeSH
• transformující růstový faktor beta analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• BMP2 protein, human MeSH Prohlížeč
• fibroblastový růstový faktor 2 MeSH
• kostní morfogenetický protein 2 MeSH
• oxid železnato-železitý MeSH
• stříbro MeSH
• transformující růstový faktor beta MeSH

Protein phosphorylation is the most abundant and best studied protein posttranslational modification, dedicated to the regulation of protein function and subcellular localization as well as to protein-protein interactions. Identification and quantitation of the dynamic, conditional protein phosphorylation can be achieved by either metabolic labeling of the protein of interest with (32)P-labeled ATP followed by autoradiographic analysis, the use of specific monoclonal or polyclonal antibodies against the phosphorylated protein species and finally by phosphoproteome delineation using mass spectrometry.Hereby we present a fourth alternative which relies on the enforced-affinity-based-electrophoretic separation of phosphorylated from non-phosphorylated protein species by standard SDS-PAGE systems co-polymerized with Phos-Tag™ and Mn(2+) or Zn(2+) cations. Phosphate groups of phosphorylated Ser, Thr, and Tyr residues form complexes with Mn(2+) and Zn(2+) cations with polyacrylamide immobilized Phos-Tag™. Following appropriate treatment of the gels, separated proteins can be quantitatively transferred to PVDF or nitrocellulose membranes and probed with common-not phosphorylation state specific-antibodies and delineate the occurrence of a certain phosphoprotein species against its non-phosphorylated counterpart.

• MeSH
• akrylamid chemie   MeSH
• Arabidopsis enzymologie   růst a vývoj   MeSH
• bakteriofág lambda enzymologie   MeSH
• elektroforéza v polyakrylamidovém gelu metody   MeSH
• fenol chemie   MeSH
• fosfatasy metabolismus   MeSH
• fosfoproteiny izolace a purifikace   metabolismus   MeSH
• fosforylace MeSH
• kultivační techniky MeSH
• membrány umělé MeSH
• mitogenem aktivované proteinkinasy izolace a purifikace   metabolismus   MeSH
• polyvinyly chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• akrylamid MeSH
• fenol MeSH
• fosfatasy MeSH
• fosfoproteiny MeSH
• membrány umělé MeSH
• mitogenem aktivované proteinkinasy MeSH
• polyvinylidene fluoride MeSH Prohlížeč
• polyvinyly MeSH

WD-repeat proteins are found in all eukaryotes and are implicated in a variety of regulatory functions as a result of protein-protein interactions. PkwA from Thermomonospora curvata CCM3352 is a first potential example of a WD-repeat protein in a prokaryotic actinomycete. A mAb (3G2) was generated against the carboxy terminus of PkwA and was used to analyse the expression of PkwA in T. curvata. PkwA was detected in exponential growth phase following inoculation with spores, but could not be found at any stage of growth following inoculation with vegetative mycelium. PkwA and its WD domain were expressed in Escherichia coli as His-tag derivatives and purified on a Talon metal affinity matrix. The WD domain was phosphorylated by Pkg2, a membrane-spanning protein Ser/Thr kinase from 'Streptomyces granaticolor'. A membrane fraction from an exponential, spore-derived culture of T. curvata was found to phosphorylate the WD domain specifically in the presence of Mn(2+). These data confirm that PkwA is expressed in spore-derived exponential growth phase of T. curvata and could play a role as a molecular switch in a signalling pathway.

• MeSH
• Actinomycetales genetika   růst a vývoj   metabolismus   MeSH
• bakteriální proteiny * MeSH
• buněčná membrána metabolismus   MeSH
• fosforylace MeSH
• histidin chemie   MeSH
• monoklonální protilátky imunologie   MeSH
• northern blotting MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• protein-serin-threoninkinasy chemie   genetika   imunologie   metabolismus   MeSH
• signální transdukce MeSH
• spory bakteriální genetika   metabolismus   MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny * MeSH
• histidin MeSH
• monoklonální protilátky MeSH
• PkwA protein, Thermomonospora curvata MeSH Prohlížeč
• protein-serin-threoninkinasy MeSH

Diazirine-tagged d- and l-adrenaline derivatives formed abundant noncovalent gas-phase ion complexes with peptides N-Ac-SSIVSFY-NH2 (peptide S) and N-Ac-VYILLNWIGY-NH2 (peptide V) upon electrospray ionization. These peptide sequences represent the binding motifs in the β2-adrenoreceptor. The structures of the gas-phase complexes were investigated by selective laser photodissociation of the diazirine chromophore at 354 nm, which resulted in a loss of N2 and formation of a transient carbene intermediate in the adrenaline ligand without causing its expulsion. The photolyzed complexes were analyzed by collision-induced dissociation (CID-MS3 and CID-MS4) in an attempt to detect cross-links and establish the binding sites. However, no cross-linking was detected in the complexes regardless of the peptide and d- or l-configuration in adrenaline. Cyclic ion mobility measurements were used to obtain collision cross sections (CCS) in N2 for the peptide S complexes. These showed identical values, 334 ± 0.9 Å2, for complexes of the l- and d-adrenaline derivatives, respectively. Identical CCS were also obtained for peptide S complexes with natural l- and d-adrenaline, 317 ± 1.2 Å2, respectively. Born-Oppenheimer molecular dynamics (BOMD) in combination with full geometry optimization by density functional theory calculations provided structures for the complexes that were used to calculate theoretical CCS with the ion trajectory method. A close match (337 Å2) was found for a single low Gibbs energy structure that displayed a binding pocket with Ser 2 and Ser 5 residues forming hydrogen bonds to the adrenaline catechol hydroxyls. Analysis of the BOMD trajectories revealed a small number of contacts between the incipient carbene carbon atom in the ligand and X-H bonds in the peptide, which was consistent with the lack of cross-linking. Temperature dependence of the internal dynamics of peptide S-adrenaline complexes as well as the specifics of the adrenaline carbene reactions are discussed. In particular, peptide amide hydrogen transfer to the carbene carbon atom was calculated to require crossing a potential energy barrier, which may hamper cross-linking in competition with carbene internal rearrangements.

• MeSH
• adrenalin metabolismus   MeSH
• aminokyselinové motivy MeSH
• beta-2-adrenergní receptory metabolismus   MeSH
• fotochemie MeSH
• iontová mobilní spektrometrie metody   MeSH
• lidé MeSH
• methan analogy a deriváty   MeSH
• molekulární struktura MeSH
• peptidové fragmenty metabolismus   účinky záření   MeSH
• plyny MeSH
• reagencia zkříženě vázaná MeSH
• stereoizomerie MeSH
• teorie funkcionálu hustoty MeSH
• teplota MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• adrenalin MeSH
• beta-2-adrenergní receptory MeSH
• carbene MeSH Prohlížeč
• methan MeSH
• peptidové fragmenty MeSH
• plyny MeSH
• reagencia zkříženě vázaná MeSH