fluorescence correlation spectroscopy
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Laser-induced breakdown spectroscopy (LIBS) is a promising technique for the readout of immunochemical assays utilizing indirect detection of labels (Tag-LIBS), typically based on nanoparticles. We have previously demonstrated that Tag-LIBS immunoassay employing yttrium-based photon-upconversion nanoparticles (UCNPs) can reach sensitivity similar to commonly used enzyme and fluorescence immunoassays. In this study, we report on further increasing the sensitivity of UCNP-based Tag-LIBS immunoassay by employing magnetic microbeads (MBs) as the solid phase in the determination of cancer biomarker prostate-specific antigen. Due to the possibility of analyte preconcentration, MBs enabled achieving a limit of detection (LOD) of 4.0 pg·mL-1, representing two orders of magnitude improvement compared with equivalent microtiter plate-based assay (LOD of 460 pg·mL-1). In addition, utilizing MBs opens up the possibility of an internal standardization of the LIBS readout by employing iron spectral lines, which improves the assay robustness by compensating for LIBS signal fluctuations and bead-bound immunocomplexes lost throughout the washing steps. Finally, the practical applicability of the technique was confirmed by the successful analysis of clinical samples, showing a strong correlation with the standard electrochemiluminescence immunoassay. Overall, MB-based Tag-LIBS was confirmed as a promising immunoassay approach, combining fast readout, multiplexing possibilities, and high sensitivity approaching upconversion luminescence scanning while avoiding the requirement of luminescence properties of labels.
- MeSH
- imunoanalýza metody MeSH
- lasery * MeSH
- lidé MeSH
- limita detekce * MeSH
- mikrosféry MeSH
- prostatický specifický antigen * analýza imunologie krev MeSH
- spektrální analýza metody MeSH
- ytrium chemie účinky záření MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In response to the growing need for development of modern biomaterials for applications in regenerative medicine strategies, the research presented here investigated the biological potential of two types of polymer nanocomposites. Graphene oxide (GO) and partially reduced graphene oxide (rGO) were incorporated into a poly(ε-caprolactone) (PCL) matrix, creating PCL/GO and PCL/rGO nanocomposites in the form of membranes. Proliferation of osteoblast-like cells (human U-2 OS cell line) on the surface of the studied materials confirmed their biological activity. Fluorescence microscopy was able to distinguish the different patterns of interaction between cells (depending on the type of material) after 15 days of the test run. Raman micro-spectroscopy and two-dimensional correlation spectroscopy (2D-COS) applied to Raman spectra distinguished the nature of cell-material interactions after only 8 days. Combination of these two techniques (Raman micro-spectroscopy and 2D-COS analysis) facilitated identification of a much more complex cellular response (especially from proteins) on the surface of PCL/GO. The presented approach can be regarded as a method for early study of the bioactivity of membrane materials.
- MeSH
- grafit * farmakologie chemie MeSH
- lidé MeSH
- osteoblasty MeSH
- polyestery chemie MeSH
- polymery MeSH
- Ramanova spektroskopie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The aim of the study is to investigate the differences in the interaction of three structurally diverse anthocyanidins, namely peonidin, petunidin, and delphinidin, as well as their glucosides with model biological membranes, human albumin, and plasmid DNA in order to look into their structure-activity relationships. Fluorimetric studies, as well as ATR-FTIR analyses, were jointly used in order to determine the changes observed in both the hydrophilic and hydrophobic layers of cell-mimic membranes (MM) which reflected the membrane lipid composition of tumour cells and red blood cell membranes (RBCM). Our results showed that anthocyanins and anthocyanidins can cause an increase in the packing order of the polar heads of lipids, as well as interact with their deeper layers by reducing the fluidity of lipid chains. The results presented here indicate that all compounds tested here possessed the ability to bind to human serum albumin (HSA) and the presence of a glucose molecule within the structures formed by anthocyanidin reduces their ability to bind to proteins. Using fluorescence correlation spectroscopy, it was demonstrated that the compounds tested here were capable of forming stable complexes with plasmid DNA and, particularly, strong DNA conformational changes were observed in the presence of petunidin and corresponding glucoside, as well as delphinidin. The results we obtained can be useful in comprehending the anthocyanins therapeutic action as molecular antioxidants and provide a valuable insight into their mechanism of action.
Antenna protein aggregation is one of the principal mechanisms considered effective in protecting phototrophs against high light damage. Commonly, it is induced, in vitro, by decreasing detergent concentration and pH of a solution of purified antennas; the resulting reduction in fluorescence emission is considered to be representative of non-photochemical quenching in vivo. However, little is known about the actual size and organization of antenna particles formed by this means, and hence the physiological relevance of this experimental approach is questionable. Here, a quasi-single molecule method, fluorescence correlation spectroscopy (FCS), was applied during in vitro quenching of LHCII trimers from higher plants for a parallel estimation of particle size, fluorescence, and antenna cluster homogeneity in a single measurement. FCS revealed that, below detergent critical micelle concentration, low pH promoted the formation of large protein oligomers of sizes up to micrometers, and therefore is apparently incompatible with thylakoid membranes. In contrast, LHCII clusters formed at high pH were smaller and homogenous, and yet still capable of efficient quenching. The results altogether set the physiological validity limits of in vitro quenching experiments. Our data also support the idea that the small, moderately quenching LHCII oligomers found at high pH could be relevant with respect to non-photochemical quenching in vivo.
- MeSH
- chlorofyl chemie genetika účinky záření MeSH
- fluorescence MeSH
- fluorescenční spektrometrie MeSH
- fotosyntéza genetika MeSH
- fotosystém II (proteinový komplex) genetika účinky záření MeSH
- fototrofní procesy genetika MeSH
- homeodoménový protein Antennapedia chemie genetika MeSH
- koncentrace vodíkových iontů MeSH
- proteinové agregáty genetika MeSH
- shluková analýza MeSH
- světlo škodlivé účinky MeSH
- světlosběrné proteinové komplexy chemie genetika MeSH
- tylakoidy chemie genetika účinky záření MeSH
- zeaxanthiny genetika MeSH
- Publikační typ
- časopisecké články MeSH
The importance of macromolecules paves the way towards a detailed molecular level investigation as all most all cellular processes occurring at the interior of cells in the form of proteins, enzymes, and other biological molecules are significantly affected because of their crowding. Thus, exploring the role of crowding environment on the denaturation and renaturation kinetics of protein molecules is of great importance. Here, CRABP I (cellular retinoic acid binding protein I) is employed as a model protein along with different molecular weights of Polyethylene glycol (PEG) as molecular crowders. The experimental evaluations are done by accessing the protein secondary structure analysis using circular dichroism (CD) spectroscopy and unfolding kinetics using intrinsic fluorescence of CRABP I at 37 °C to mimic the in vivo crowding environment. The unfolding kinetics results indicated that both PEG 2000 and PEG 4000 act as stabilizers by retarding the unfolding kinetic rates. Both kinetic and stability outcomes presented the importance of crowding environment on stability and kinetics of CRABP I. The molecular dynamics (MD) studies revealed that thirteen PEG 2000 molecules assembled during the 500 ns simulation, which increases the stability and percentage of β-sheet. The experimental findings are well supported by the molecular dynamics simulation results.
Adenine nucleotide translocase (ANT) is a well-known mitochondrial exchanger of ATP against ADP. In contrast, few studies have shown that ANT also mediates proton transport across the inner mitochondrial membrane. The results of these studies are controversial and lead to different hypotheses about molecular transport mechanisms. We hypothesized that the H+-transport mediated by ANT and uncoupling proteins (UCP) has a similar regulation pattern and can be explained by the fatty acid cycling concept. The reconstitution of purified recombinant ANT1 in the planar lipid bilayers allowed us to measure the membrane current after the direct application of transmembrane potential ΔΨ, which would correspond to the mitochondrial states III and IV. Experimental results reveal that ANT1 does not contribute to a basal proton leak. Instead, it mediates H+ transport only in the presence of long-chain fatty acids (FA), as already known for UCPs. It depends on FA chain length and saturation, implying that FA's transport is confined to the lipid-protein interface. Purine nucleotides with the preference for ATP and ADP inhibited H+ transport. Specific inhibitors of ATP/ADP transport, carboxyatractyloside or bongkrekic acid, also decreased proton transport. The H+ turnover number was calculated based on ANT1 concentration determined by fluorescence correlation spectroscopy and is equal to 14.6 ± 2.5 s-1. Molecular dynamic simulations revealed a large positively charged area at the protein/lipid interface that might facilitate FA anion's transport across the membrane. ANT's dual function-ADP/ATP and H+ transport in the presence of FA-may be important for the regulation of mitochondrial membrane potential and thus for potential-dependent processes in mitochondria. Moreover, the expansion of proton-transport modulating drug targets to ANT1 may improve the therapy of obesity, cancer, steatosis, cardiovascular and neurodegenerative diseases.
- MeSH
- iontový transport MeSH
- konformace proteinů MeSH
- mastné kyseliny metabolismus MeSH
- membránový potenciál mitochondrií MeSH
- mitochondrie metabolismus MeSH
- myši MeSH
- protony * MeSH
- translokátor adeninových nukleotidů 1 chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
We have prepared a candidate biocompatible construct for skin wound healing based on electrospun polycaprolactone (PCL) nanofibrous membranes. The membrane material was loaded either with L-arginine or with alaptide, or with a mixture of both bioactive components. Alaptide is a spirocyclic synthetic dipeptide, an analogue of melanocyte-stimulating hormone release-inhibiting factor. L-arginine is an amino acid with a basic guanidine side chain. It is a direct precursor of nitric oxide, which plays a pivotal role in skin repair. The presence and the distribution of the additives were proved with high-performance liquid chromatography, Fourier-transform infrared spectroscopy and Raman spectroscopy. The influence of L-arginine and alaptide on the morphology of the membrane was characterized using scanning electron microscopy. No statistically significant correlation between fiber diameter and drug concentration was observed. The membranes were then tested in vitro for their cytotoxicity, using primary human dermal fibroblasts, in order to obtain the optimal concentrations of the additives for in vivo tests in a rat model. The membranes with the highest concentration of L-arginine (10 wt. %) proved to be cytotoxic. The membranes with alaptide in concentrations from 0.1 to 2.5 wt.%, and with the other L-arginine concentrations (1 and 5 wt.%), did not show high toxicity. In addition, there was no observed improvement in cell proliferation on the membranes. The in vivo experiments revealed that membranes with 1.5 wt.% of alaptide or with 1.5 wt.% of alaptide in combination with 5 wt.% of L-arginine markedly accelerated the healing of skin incisions, and particularly the healing of skin burns, i.e. wounds of relatively large extent. These results indicate that our newly-developed nanofibrous membranes are promising for treating wounds with large damaged areas, where a supporting material is needed.
- MeSH
- arginin chemie MeSH
- biokompatibilní materiály chemie MeSH
- cyklické peptidy chemie MeSH
- elektrochemie MeSH
- elektrody MeSH
- fibroblasty účinky léků MeSH
- fluorescenční mikroskopie MeSH
- hojení ran účinky léků MeSH
- krysa rodu rattus MeSH
- kůže patologie MeSH
- lékové transportní systémy MeSH
- lidé MeSH
- nanovlákna chemie MeSH
- neuropeptidy chemie MeSH
- peptidy chemie MeSH
- potkani Wistar MeSH
- proliferace buněk MeSH
- Ramanova spektroskopie MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- techniky in vitro MeSH
- testování materiálů MeSH
- tkáňové inženýrství metody MeSH
- tkáňové podpůrné struktury chemie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lipid transfer from lipoprotein particles to cells is essential for lipid homeostasis. High-density lipoprotein (HDL) particles are mainly captured by cell membrane-associated scavenger receptor class B type 1 (SR-B1) from the bloodstream, while low-density and very-low-density lipoprotein (LDL and VLDL, respectively) particles are mostly taken up by receptor-mediated endocytosis. However, the role of the target lipid membrane itself in the transfer process has been largely neglected so far. Here, we study how lipoprotein particles (HDL, LDL, and VLDL) interact with synthetic lipid bilayers and cell-derived membranes and transfer their cargo subsequently. Employing cryo-electron microscopy, spectral imaging, and fluorescence (cross) correlation spectroscopy allowed us to observe integration of all major types of lipoprotein particles into the membrane and delivery of their cargo in a receptor-independent manner. Importantly, the biophysical properties of the target cell membranes change upon delivery of cargo. The concept of receptor-independent interaction of lipoprotein particles with membranes helps us to better understand lipoprotein particle biology and can be exploited for novel treatments of dyslipidemia diseases.
Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. More than half of BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, the BBSome, which mediates trafficking of ciliary cargoes. In this study, we elucidated the mechanisms of the BBSome assembly in living cells and how this process is spatially regulated. We generated a large library of human cell lines deficient in a particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We analyzed these cell lines utilizing biochemical assays, conventional and expansion microscopy, and quantitative fluorescence microscopy techniques: fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. Our data revealed that the BBSome formation is a sequential process. We show that the pre-BBSome is nucleated by BBS4 and assembled at pericentriolar satellites, followed by the translocation of the BBSome into the ciliary base mediated by BBS1. Our results provide a framework for elucidating how BBS-causative mutations interfere with the biogenesis of the BBSome.
- MeSH
- Bardetův-Biedlův syndrom genetika metabolismus patologie MeSH
- buněčné linie MeSH
- cilie metabolismus MeSH
- CRISPR-Cas systémy genetika MeSH
- cytoplazma metabolismus MeSH
- editace genu MeSH
- fluorescenční mikroskopie MeSH
- FRAP MeSH
- lidé MeSH
- mutace MeSH
- podjednotky proteinů genetika metabolismus MeSH
- proteiny asociované s mikrotubuly nedostatek genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
DNA nanodevices have been developed as platforms for the manipulation of gene expression, delivery of molecular payloads, and detection of various molecular targets within cells and in other complex biological settings. Despite efforts to translate DNA nanodevices from the test tube (in vitro) to living cells, their intracellular trafficking and functionality remain poorly understood. Herein, quantitative and super-resolution microscopy approaches were employed to track and visualise, with nanometric resolution, the molecular interactions between a synthetic DNA nanosensor and transcription factors in intracellular compartments. Specifically, fluorescence resonance energy transfer microscopy, fluorescence correlation spectroscopy, fluorescence lifetime imaging microscopy and multicolour single-molecule localisation microscopy were employed to probe the specific binding of the DNA nanosensor to the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). We monitored the mobility, subcellular localisation and degradation of the DNA nanosensor inside living prostate cancer PC3 cells. Super-resolution imaging enabled the direct visualisation of the molecular interactions between the synthetic DNA nanosensors and the NF-κB molecules in cells. This study represents a significant advance in the effective detection as well as understanding of the intracellular dynamics of DNA nanosensors in a complex biological milieu.
- MeSH
- DNA vazebné proteiny MeSH
- DNA MeSH
- lidé MeSH
- NF-kappa B * genetika metabolismus MeSH
- rezonanční přenos fluorescenční energie MeSH
- signální transdukce * MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
