To ensure food safety and to prevent unnecessary foodborne complications this study reports fast, fully automated process for histamine determination. This method is based on magnetic separation of histamine with magnetic particles and quantification by the fluorescence intensity change of MSA modified CdSe Quantum dots. Formation of Fe2O3 particles was followed by adsorption of TiO2 on their surface. Magnetism of developed probe enabled rapid histamine isolation prior to its fluorescence detection. Quantum dots (QDs) of approx. 3 nm were prepared via facile UV irradiation. The fluorescence intensity of CdSe QDs was enhanced upon mixing with magnetically separated histamine, in concentration-dependent manner, with a detection limit of 1.6 μM. The linear calibration curve ranged between 0.07 and 4.5 mM histamine with a low LOD and LOQ of 1.6 μM and 6 μM. The detection efficiency of the method was confirmed by ion exchange chromatography. Moreover, the specificity of the sensor was evaluated and no cross-reactivity from nontarget analytes was observed. This method was successfully applied for the direct analysis of histamine in white wine providing detection limit much lower than the histamine maximum levels established by EU regulation in food samples. The recovery rate was excellent, ranging from 84 to 100% with an RSD of less than 4.0%. The main advantage of the proposed method is full automation of the analytical procedure that reduces the time and cost of the analysis, solvent consumption and sample manipulation, enabling routine analysis of large numbers of samples for histamine and highly accurate and precise results.

• MeSH
• fluorescence MeSH
• fluorescenční barviva chemie   MeSH
• fluorescenční spektrometrie metody   MeSH
• histamin analýza   MeSH
• kontaminace potravin analýza   MeSH
• kovové nanočástice chemie   MeSH
• kvantové tečky chemie   MeSH
• limita detekce MeSH
• magnetické jevy MeSH
• silany chemie   MeSH
• sloučeniny kadmia chemie   MeSH
• telur chemie   MeSH
• titan chemie   MeSH
• víno analýza   MeSH
• železité sloučeniny chemie   MeSH
• Publikační typ
• časopisecké články MeSH

Hydrophobic nonaggregating metal-free azaphthalocyanines (AzaPc) of the tetrapyrazinoporphyrazine type were synthesized, characterized, and used for oligonucleotide labeling. Both 3'-end and 5'-end labeling methods using solid phase synthesis suitable for automatic processes in the DNA/RNA synthesizer were developed. The hydrophobic character of AzaPc enabled the anchoring of the conjugates on reverse phase of the oligonucleotide purification cartridge, thus enabling their simple purification. AzaPc did not show any fluorescence and extremely low singlet oxygen quantum yields (Φ(Δ) = 0.015-0.018 in DMF) in a monomeric state due to ultrafast intramolecular charge transfer. That is why they were investigated as a new dark quencher structural type. They profit particularly from absorption in a wide range of wavelengths (300-740 nm) that covers all fluorophores used in hybridization assays nowadays. As an example, quenching efficiency was evaluated in a simple hybridization assay using monolabeled probes. AzaPc-based probes efficiently quenched both fluorescein and Cy5 fluorescence by both resonance energy transfer and contact quenching. The results were compared with three established dark quenchers, and the AzaPc exerted better (BHQ-1 and BHQ-2) or comparable (BBQ-650) quenching efficiencies for both fluorophores.

• MeSH
• absorpce MeSH
• fluorescenční barviva chemie   MeSH
• fluorescenční spektrometrie MeSH
• oligonukleotidové sondy chemická syntéza   chemie   genetika   MeSH
• oligonukleotidy chemická syntéza   chemie   genetika   MeSH
• pyraziny chemie   MeSH
• pyrroly chemie   MeSH
• sekvence nukleotidů MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Surface-enhanced Raman spectroscopy (SERS) is an extremely powerful analytical tool, which not only yields information about the molecular structure of the analyte in the form of characteristic vibrational spectrum but also gives sensitivities approaching those in fluorescence spectroscopy. The SERS measurement on the microfluidic platform provides possibility to manufacture the device with design perfectly fulfilling the needs of the application with minimal sample consumption. This review aims at describing basic strategies for SERS measurement in microfluidic devices published in the last decade and covers current trends in microfluidics with SERS detection in the field of bioanalysis and approaches toward on-line coupling of liquid-based separation techniques with SERS detection.

• MeSH
• DNA analýza   MeSH
• fyzikální jevy MeSH
• kovové nanočástice chemie   MeSH
• limita detekce MeSH
• mikrofluidní analytické techniky metody   MeSH
• povrchové vlastnosti MeSH
• proteiny analýza   MeSH
• Ramanova spektroskopie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

A combined approach to signal enhancement in fluorescence affinity biosensors and assays is reported. It is based on the compaction of specifically captured target molecules at the sensor surface followed by optical probing with a tightly confined surface plasmon (SP) field. This concept is utilized by using a thermoresponsive hydrogel (HG) binding matrix that is prepared from a terpolymer derived from poly(N-isopropylacrylamide) (pNIPAAm) and attached to a metallic sensor surface. Epi-illumination fluorescence and SP-enhanced total internal reflection fluorescence readouts of affinity binding events are performed to spatially interrogate the fluorescent signal in the direction parallel and perpendicular to the sensor surface. The pNIPAAm-based HG binding matrix is arranged in arrays of sensing spots and employed for the specific detection of human IgG antibodies against the Epstein-Barr virus (EBV). The detection is performed in diluted human plasma or with isolated human IgG by using a set of peptide ligands mapping the epitope of the EBV nuclear antigen. Alkyne-terminated peptides were covalently coupled to the pNIPAAm-based HG carrying azide moieties. Importantly, using such low-molecular-weight ligands allowed preserving the thermoresponsive properties of the pNIPAAm-based architecture, which was not possible for amine coupling of regular antibodies that have a higher molecular weight.

• MeSH
• akrylové pryskyřice chemie   MeSH
• biosenzitivní techniky metody   MeSH
• fluorescence MeSH
• hydrogely chemie   metabolismus   MeSH
• imunoglobulin G analýza   imunologie   MeSH
• infekce virem Epsteina-Barrové diagnóza   imunologie   metabolismus   virologie   MeSH
• lidé MeSH
• peptidové fragmenty imunologie   metabolismus   MeSH
• polymery chemie   MeSH
• virus Epsteinův-Barrové - jaderné antigeny imunologie   MeSH
• virus Epsteinův-Barrové imunologie   izolace a purifikace   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články 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.

• 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

Interaction between semiconductor nanocrystals, cadmium telluride quantum dots (CdTe QDs) capped with mercaptosuccinic acid (MSA) and metallothionein (MT) was investigated. MSA-capped CdTe QDs were synthesized in aqueous solution. Mixture of MT and MSA-capped CdTe QDs has been investigated by various analytical methods as follows: tris-tricine gel electrophoresis, fluorescence evaluation and electrochemical detection of catalysed hydrogen evolution. The obtained results demonstrate that MSA-capped CdTe QDs and MT do not create firmly bound stabile complex. However, weak electrostatic interactions contribute to the interaction of MT with MSA-capped CdTeQDs. It can be concluded that QDs size influences the QDs and MT interaction. The smallest QDs had the highest affinity to MT and vice versa.

• MeSH
• elektrochemické techniky MeSH
• fluorescenční spektrometrie MeSH
• králíci MeSH
• kvantové tečky * MeSH
• metalothionein chemie   MeSH
• molekulární struktura MeSH
• nanočástice * MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Antibodies to human sperm are useful diagnostic reagents for detection of changes in sperm protein expression and their relationship with sperm defects and male infertility. The specificity of Hs-16 monoclonal antibody (mAb) and the localization and frequency of the occurrence of Hs-16-recognized protein on human spermatozoa were investigated. METHODS: Samples from 30 fertile men with normal spermiograms and 30 men with pathological spermiograms were studied. The specificity of Hs-16 mAb was analysed by the western blotting technique and matrix-assisted laser desorption/ionization mass spectrometry. Indirect immunofluorescence with Hs-16 antibody was used to test sperm ejaculates. RESULTS: The Hs-16 antibody detected a human sperm and seminal plasma protein, which was determined to be secretory actin-binding protein (SABP). This specificity was also verified by co-localization of SABP and actin on spermatozoa with Hs-16 and anti-actin antibodies, and partial co-localization of these proteins was found. SABP was localized on the sperm tail, mainly in the midpiece of the tail. Other parts of spermatozoa were labelled with lower frequency. A significant difference was found in SABP labelling between men with normal spermiograms and donors with asthenozoospermia or oligoasthenoteratozoospermia (both P < 0.01), and asthenozoospermia versus oligoasthenoteratozoospermia (P < 0.05). Increased expression of SABP was observed in men with pathological spermiograms. CONCLUSIONS: Hs-16 antibody reacts specifically with SABP. SABP can serve as a marker of defective sperm and may be associated with fertility failure.

• MeSH
• aktiny analýza   MeSH
• dospělí MeSH
• financování organizované MeSH
• fluorescenční protilátková technika nepřímá MeSH
• lidé MeSH
• mikrofilamentové proteiny biosyntéza   sekrece   MeSH
• monoklonální protilátky MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• spermie imunologie   metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH

We emphasize the importance of dynamics and hydration for enzymatic catalysis and protein design by transplanting the active site from a haloalkane dehalogenase with high enantioselectivity to nonselective dehalogenase. Protein crystallography confirms that the active site geometry of the redesigned dehalogenase matches that of the target, but its enantioselectivity remains low. Time-dependent fluorescence shifts and computer simulations revealed that dynamics and hydration at the tunnel mouth differ substantially between the redesigned and target dehalogenase.

• MeSH
• bromované uhlovodíky chemie   MeSH
• fluorescenční spektrometrie MeSH
• hydrolasy chemie   genetika   MeSH
• katalytická doména MeSH
• katalýza MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• molekulární sekvence - údaje MeSH
• mutageneze cílená MeSH
• proteinové inženýrství * MeSH
• sekvence aminokyselin MeSH
• simulace molekulární dynamiky * MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• stereoizomerie MeSH
• voda chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Spheroids-three-dimensional aggregates of cells grown from a cancer cell line-represent a model of living tissue for chemotherapy investigation. Distribution of chemotherapeutics in spheroid sections was determined using the matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Proliferating or apoptotic cells were immunohistochemically labeled and visualized by laser scanning confocal fluorescence microscopy (LSCM). Drug efficacy was evaluated by comparing coregistered MALDI MSI and LSCM data of drug-treated spheroids with LSCM only data of untreated control spheroids. We developed a fiducial-based workflow for coregistration of low-resolution MALDI MS with high-resolution LSCM images. To allow comparison of drug and cell distribution between the drug-treated and untreated spheroids of different shapes or diameters, we introduced a common diffusion-related coordinate, the distance from the spheroid boundary. In a procedure referred to as "peeling", we correlated average drug distribution at a certain distance with the average reduction in the affected cells between the untreated and the treated spheroids. This novel approach makes it possible to differentiate between peripheral cells that died due to therapy and the innermost cells which died naturally. Two novel algorithms-for MALDI MS image denoising and for weighting of MALDI MSI and LSCM data by the presence of cell nuclei-are also presented.

• MeSH
• buněčné sféroidy účinky léků   MeSH
• konfokální mikroskopie metody   MeSH
• lidé MeSH
• nádory farmakoterapie   MeSH
• protinádorové látky farmakokinetika   farmakologie   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• teoretické modely MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The transient receptor potential channel TRPC6 is a non-selective cation channel which modulates the calcium level in eukaryotic cells (including sensory receptor cells) in response to external signals. Calmodulin (CaM) is a ubiquitously expressed Ca(2+) binding protein that is an important mediator of Ca(2+)-dependent regulation of the TRPC6 channel. One CaM binding site was identified within the C-tail of TRPC6. The aim of this study is to map in detail the CaM and inositol (1,4,5)-triphosphate receptor binding (CIRB) domain in the C-terminal region of mouse TRPC6 that is capable of interacting with CaM using in vitro binding assays. Besides the set of positively charged amino acid residues Arg852, Lys856, Arg864, Lys859/Arg860, a hydrophobic Ile857, at the position 1 in 1-5-10 motif, was located and the effect of replacing it with a neutral residue was tested using fluorescence anisotropy measurement. Participation of Ile857 could indicate a strong role of this conserved CaM binding motif.

• MeSH
• fluorescenční polarizace MeSH
• kalmodulin metabolismus   MeSH
• kationtové kanály TRPC chemie   genetika   metabolismus   MeSH
• klonování DNA MeSH
• molekulární modely MeSH
• mutageneze cílená MeSH
• myši MeSH
• retardační test MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH