Surface-enhanced fluorescence (SEF) requires the absorption/emission band of the fluorophore, the localized surface plasmon resonance (LSPR) of the nanostructure and the excitation wavelength to fall in the same (or very close) spectral range. In this paper, we monitor the SEF intensity and lifetime dependence of riboflavin (vitamin B2) adsorbed on a spacer-modified Ag substrate with respect to the thickness of the spacer. The substrates were formed by silver nanoislands deposited onto magnetron-sputtered polytetrafluoroethylene (ms-PTFE). The spacer was formed by the ms-PTFE layer with the thickness ranging from ~5 to 25 nm. The riboflavin dissolved in dimethylsulfoxide (DMSO) at a 10 µM concentration forms, at the ms-PTFE surface, a homogeneous layer of adsorbed molecules corresponding to a monomolecular layer. The microspectroscopic measurements of the adsorbed layer were performed through a sessile droplet; our study has shown the advantages and limitations of this approach. Time-resolved fluorescence enabled us to determine the enhanced fluorescence quantum yield due to the shortening of the radiative decay in the vicinity of the plasmonic surface. For the 5 nm ms-PTFE layer possessing the largest (estimated 4×) fluorescence enhancement, the quantum yield was increased 2.3×.

• Klíčová slova
• enhancement factor, lifetime, riboflavin, surface-enhanced fluorescence (SEF), time-resolved,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• Microfluidics, Nanoparticles, Separation, Surface-enhanced Raman spectroscopy,
• 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
• Názvy látek
• DNA MeSH
• proteiny MeSH

Here a novel digital bioassay readout concept is reported that does not rely on enzymatic amplification nor compartmenting of an analyzed liquid sample. Rather, it is based on counting individual affinity-captured target biomolecules via the use of a tethered catalytic hairpin assembly (tCHA) deployed on a solid sensor surface with spatial confinement utilized by a flexible polymer linker (FPL). Wide-field plasmon-enhanced fluorescence (PEF) imaging is employed for optical real-time probing of the reaction kinetics, where affinity-captured target molecules are manifested as spatially distinct bright fluorescent spots. The effect of the length of the FPLs is investigated, and the analytical performance of the dual amplification tCHA-PEF concept is tested by using a model short single-stranded DNA analyte. When applied in a sandwich immunoassay, the detection of target proteins at sub-femtomolar concentrations is demonstrated. The reported experiments are supported by diffusion-limited mass transfer models and document the potential of tCHA-PEF as a new class of generic enzyme-free bioanalytical tools enabling the ultrasensitive analysis of trace amounts of protein and nucleic acid analytes, making it attractive for future molecular diagnostics and research applications.

• Klíčová slova
• catalytic hairpin assembly, flexible DNA linker, plasmon‐enhanced fluorescence, sandwich immunoassay, single molecule detection,
• MeSH
• biosenzitivní techniky * metody   MeSH
• fluorescence MeSH
• imunoanalýza metody   MeSH
• jednovláknová DNA chemie   analýza   MeSH
• katalýza MeSH
• povrchová plasmonová rezonance * metody   MeSH
• zobrazení jednotlivé molekuly * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• jednovláknová DNA MeSH

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.

• Klíčová slova
• Food safety, Histamine, Ion exchange chromatography, Maghemite, Quantum dots,
• 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
• Názvy látek
• cadmium telluride MeSH Prohlížeč
• ferric oxide MeSH Prohlížeč
• fluorescenční barviva MeSH
• histamin MeSH
• silany MeSH
• sloučeniny kadmia MeSH
• telur MeSH
• tetraethoxysilane MeSH Prohlížeč
• titan MeSH
• titanium dioxide MeSH Prohlížeč
• železité sloučeniny 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
• Názvy látek
• fluorescenční barviva MeSH
• oligonukleotidové sondy MeSH
• oligonukleotidy MeSH
• pyraziny MeSH
• pyrroly MeSH

This study focused on the antifouling effect of copper oxide (Cu2O)- and zineb-based coatings against Cyanothece sp. ATCC 51142 by analysing photosynthetic activity using chlorophyll fluorescence. The photoautotrophically grown cyanobacterium was exposed to toxic coatings over a short-term period of 32 h. The study showed that Cyanothece cultures are particularly sensitive to biocides (i) released from antifouling paints and (ii) exhibited by contact with the coated surfaces. Changes in the maximum quantum yield of photosystem II (FV/FM) were observed within the first 12 h of exposure to the coatings. Partial recovery of FV/FM in Cyanothece was revealed 24 h post exposure to a copper- and zineb-free coating. In this research, we proposed an analysis of the evaluation of fluorescence data to study the initial response of cyanobacterial cells to copper- and non-copper-based antifouling coatings formulated with zineb. We evaluated the dynamics of coating toxicity by determining the characteristic time constants of changes in the FV/FM. Within the most toxic paints studied, those formulated with the highest concentration of Cu2O and zineb, the estimated time constants were 3.9 times lower compared to the copper- and zineb-free paint. The use of zineb in copper-based antifouling coatings enhanced the toxic effect of paints and contributed to a faster decline in photosystem II activity in Cyanothece cells. The analysis we proposed, along with the fluorescence screening results, may be useful in evaluating the initial antifouling dynamic action against photosynthetic aquacultures.

• Klíčová slova
• Cyanothece, antifouling coatings, chlorophyll fluorescence, cyanobacteria, photosystem II (PSII) efficiency, toxicity,
• MeSH
• bioznečištění * prevence a kontrola   MeSH
• dezinficiencia * analýza   MeSH
• fluorescence MeSH
• fotosystém II (proteinový komplex) MeSH
• lodě MeSH
• nátěrové hmoty MeSH
• sinice * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dezinficiencia * MeSH
• fotosystém II (proteinový komplex) 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

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.

• Klíčová slova
• biomarkers, click chemistry, microarrays, pNIPAAm, peptide, plasmon-enhanced fluorescence, serotesting, thermoresponsive hydrogel,
• 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
• Názvy látek
• akrylové pryskyřice MeSH
• hydrogely MeSH
• imunoglobulin G MeSH
• peptidové fragmenty MeSH
• poly-N-isopropylacrylamide MeSH Prohlížeč
• polymery MeSH
• virus Epsteinův-Barrové - jaderné antigeny MeSH

When exposed to the intracellular environment fluorescent probes sensitive to pH exhibit changes of photophysical characteristics as a result of an interaction of the dye molecule with cell constituents such as proteins, lipids or nucleic acids. This effect is reflected in calibration curves different from those found with the same dye in pure buffer solutions. To study an interaction of the probe 5'(and 6')-carboxy-10-dimethylamino-3-hydroxy- spiro[7H-benzo[c]xanthene-7,1'(3H)-isobenzofuran]-3'-one (carboxy SNARF-1) with membrane lipids, we measured its fluorescence in model systems of large unilamellar vesicles (LUV) prepared by extrusion. When the dye was removed from the bulk solution by gel filtration the relative fluorescence intensity of the lipid-bound dye form was enhanced, showing a strong interaction of the dye molecule with LUV membrane lipids. Surprisingly, the dye molecules seem to be bound predominantly to the outer surface of the lipid bilayer. The same situation was found with small unilamellar vesicles prepared by sonication. This effect makes it difficult to use carboxy SNARF-1 for measurements of the internal pH in suspensions of liposomes.

• MeSH
• benzopyrany MeSH
• fluorescenční barviva chemie   MeSH
• fluorescenční spektrometrie MeSH
• fotochemie MeSH
• koncentrace vodíkových iontů MeSH
• liposomy MeSH
• naftoly chemie   MeSH
• rhodaminy chemie   MeSH
• techniky in vitro MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• benzopyrany MeSH
• carboxy-seminaphthorhodaminefluoride MeSH Prohlížeč
• fluorescenční barviva MeSH
• liposomy MeSH
• naftoly MeSH
• rhodaminy MeSH

Gliomas present one of the most prevalent malignant tumors related to the central nervous system. Surgical extraction is still a preferred route for glioma treatment. Nonetheless, neurosurgeons still have a considerable challenge to detect actual margins of the targeted glioma intraoperatively and correctly because of its great natural infiltration. Here we evaluated the possibility of using surface-enhanced Raman spectroscopy to analyze freshly resected brain tissues. The developed method is based on the application of Au@ZrO2 nanosensor. The plasmonic properties of the sensor were first tested on the analysis of Rhodamine 6G, where concentrations down to 10-7 mol/L can be successfully detected. We also compared the performance of the nanosensor with silver plasmonic nanoparticles, where similar results were obtained regarding the reduction of the fluorescence background and enhancement of the intensity of the measured analytical signal. However, application of silver nanospheres led to increased variations in spectral data due to its probable aggregation. Applied ZrO2@Au nanosensor thus dramatically lowers the fluorescence present in the Raman data, and considerably improves the quality of the measured signal. The developed method allows for rapid discrimination between the glioma's periphery and central parts, which could serve as a steppingstone toward highly precise neurosurgery.

• Klíčová slova
• Au nanospheres, Gliomas, Nanomaterials, Raman spectroscopy, SERS, ZrO(2),
• MeSH
• gliom * MeSH
• kovové nanočástice * chemie   MeSH
• lidé MeSH
• nanokuličky * chemie   MeSH
• Ramanova spektroskopie metody   MeSH
• stříbro chemie   MeSH
• zlato chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• stříbro MeSH
• zlato MeSH