Multiplex assays for detection of biomarkers, provide advantageous analyses of different factors related to diagnoses of diseases. The Alzheimer’s disease (AD) is one of the most common disease in old people in societies which is increasing, significantly. A group of microRNAs (miRNAs) play an important role in developing the disease which can be considered as early stage biomarkers. Since, selective, sensitive, simple and rapid method for detection of these miRNAs in a single test is critical for early diagnosis and efficient therapy of the disease, herein, we report a sensitive fluorescence assay based on enzyme-free and isothermal hybridization chain reaction with SYBR Green and graphene oxide (GOX) for early detection of miR-137 and miR-142, as two Alzheimer’s biomarkers. Fluorescence spectrophotometry based on SYBR Green signal and GOX as the fluorescence quencher was used for detection and quantification of targets’ miRNAs and change in fluorescence intensity due to absence and presence of the targets was measured. The limit of detection in the newly designed nanobiosensor was achieved as 82 pM with a sensitive detection of the miRNAs from 0.05 to 5 nM, that is critical for detecting the biomarkers. Given the real range of concentrations of miRNAs in blood (from nanomolar to femtomolar values), the method holds great promise in dual and multiple targets detection due to its sensitivity, rapidness, inexpensive and specificity which provides a convenient detection method of Alzheimer’s in early stage.

Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of labelling with relatively minimal perturbation to biological samples compared with many competing biophysical techniques. Improvements in detector and dye technologies coupled to advances in image analysis methods have fuelled recent development towards single-molecule fluorescence microscopy, which can utilize light microscopy tools to enable the faithful detection and analysis of single fluorescent molecules used as reporter tags in biological samples. For example, the discovery of GFP, initiating the so-called ‘green revolution’, has pushed experimental tools in the biosciences to a completely new level of functional imaging of living samples, culminating in single fluorescent protein molecule detection. Today, fluorescence microscopy is an indispensable tool in single-molecule investigations, providing a high signal-to-noise ratio for visualization while still retaining the key features in the physiological context of native biological systems. In this review, we discuss some of the recent discoveries in the life sciences which have been enabled using single-molecule fluorescence microscopy, paying particular attention to the so-called ‘super-resolution’ fluorescence microscopy techniques in live cells, which are at the cutting-edge of these methods. In particular, how these tools can reveal new insights into long-standing puzzles in biology: old problems, which have been impossible to tackle using other more traditional tools until the emergence of new single-molecule fluorescence microscopy techniques.

Drug–drug interactions (DDIs) and associated toxicity from cardiovascular drugs represents a major problem for effective co-administration of cardiovascular therapeutics. A significant amount of drug toxicity from DDIs occurs because of drug interactions and multiple cardiovascular drug binding to the efflux transporter P-glycoprotein (Pgp), which is particularly problematic for cardiovascular drugs because of their relatively low therapeutic indexes. The calcium channel antagonist, verapamil and the cardiac glycoside, digoxin, exhibit DDIs with Pgp through non-competitive inhibition of digoxin transport, which leads to elevated digoxin plasma concentrations and digoxin toxicity. In the present study, verapamil-induced ATPase activation kinetics were biphasic implying at least two verapamil-binding sites on Pgp, whereas monophasic digoxin activation of Pgp-coupled ATPase kinetics suggested a single digoxin-binding site. Using intrinsic protein fluorescence and the saturation transfer double difference (STDD) NMR techniques to probe drug–Pgp interactions, verapamil was found to have little effect on digoxin–Pgp interactions at low concentrations of verapamil, which is consistent with simultaneous binding of the drugs and non-competitive inhibition. Higher concentrations of verapamil caused significant disruption of digoxin–Pgp interactions that suggested overlapping and competing drug-binding sites. These interactions correlated to drug-induced conformational changes deduced from acrylamide quenching of Pgp tryptophan fluorescence. Also, Pgp-coupled ATPase activity kinetics measured with a range of verapamil and digoxin concentrations fit well to a DDI model encompassing non-competitive and competitive inhibition of digoxin by verapamil. The results and previous transport studies were combined into a comprehensive model of verapamil–digoxin DDIs encompassing drug binding, ATP hydrolysis, transport and conformational changes.

Scorpion probes, specific DNA probe sequences maintained in a hairpin–loop, can be modified to carry the components of an exciplex for use as a novel fluorescence-based method for specific detection of DNA. The exciplex partners (5′-pyrenyl and 3′-naphthalenyl) were attached to oligonucleotides via phosphoramidate links to terminal phosphate groups. Hybridization of the probe to a complementary target in a buffer containing trifluoroethanol produced an obvious fluorescence change from blue (pyrene locally excited state emission) to green (exciplex emission).

The entry of enveloped viruses into host cells is accomplished by fusion ofthe viral envelope and target plasma membrane and is mediated by fusionproteins. Recently, several functional domains within fusion proteins fromdifferent viral families were identified. Some are directly involved inconformational changes after receptor binding, as suggested by the recentrelease of crystallographically determined structures of a highly stablecore structure of the fusion proteins in the absence of membranes. However, in the presence of membranes, this core binds strongly to the membrane's surface and dissociates therein. Other regions, besides the N-terminal fusionpeptide, which include the core region and an internal fusion peptide inparamyxoviruses, are directly involved in the actual membrane fusion event, suggesting an “umbrella” like model for the membrane inducedconformational change of fusion proteins. Peptides resembling these regionshave been shown to have specific antiviral activity, presumably because theyinterfere with the corresponding domains within the viruses. Overall, thesestudies shed light into the molecular mechanism of membrane fusion induced byenvelope glycoproteins and suggest that fusion proteins from different viralfamilies share common structural and functional motifs.

Receptor aggregation is believed to be an important step in the attachmentof membrane enveloped virus' to target cell membranes. A likely receptorfor Sendai virus is the ganglioside GD1a. In this work we have studied themembrane diffusion of the fluorescent ganglioside NBD-GD1a on the surfaceof CV-1 cells with standard photobleaching techniques. Using confocallaser scanning microscopy (CLSM) and Image Correlation Spectroscopy(ICS) NBD-GD1a is shown to exist in at least two populations: dispersedand aggregated. By quantifying the distribution of NBD-GD1a pre- andpost-incubation with Sendai virus it is shown that the virus inducesa dose-dependent clustering of NBD-GD1a. Image cross-correlationspectroscopy (ICCS) is used to further quantitatively characterizethis clustering by demonstrating that it occurs due to binding ofvirus to the dispersed population of NBD-GD1a.

The effect of ligands (glucose, ATP and Mg2+) and zwitterionic micellesof lysophosphatidylcholine (LPC) or N-hexadecyl-N,N-dimethyl-3-ammoniumpropanesulfonate (HPS) in the yeast hexokinase (HK) stability was studied at35°C. The thermal inactivation kinetics followed one-exponentialdecay. The effect of ligands on protecting the enzyme against inactivationfollowed the order: glucose>glucose/Mg2+>ATP/Mg2+≌Mg2+≌bufferonly. Both LPC and HPS micelles increased the enzyme stability only whenthe incubation medium contained glucose or glucose/Mg2+,suggesting that the protein conformation is a key prerequisite for theenzyme-micelle interaction to take place. This enzyme-micelle interactionresulted in an increased catalytic efficiency (with a decrease in Km forATP and increase in Vmax as well as in changes on the tertiary (intrinsicfluorescence) structure of the yeast hexokinase.

Applications of intrinsic fluorescence measurements in the study of Ca 2+ -transport ATPases are reviewed. Since the initial reports showing that the fluorescence emission was sensitive to Ca 2+ binding, a substantial amount of work has focused on the use of both steady-state and time-resolved fluorescence spectroscopy to investigate structure-function relationships in sarcoplasmic reticulum and plasma membrane Ca 2+ -ATPases. These studies have revealed ligand-induced conformational changes, as well as provided information on protein-protein, protein-solvent and/or protein-lipid interactions in different functional states of these proteins. The main results of these studies, as well as possible future prospects are discussed.

Changes in the fluoresence of N -acetyl- N ′-(5-sulfo-1-naphthyl)ethylenediamine (EDANS), being attached to Cys-674 of sarcoplasmic reticulum Ca 2+ -ATPase without affecting the catalytic activity, as well as changes in the intrinsic tryptophan fluorescence were followed throughout the catalytic cycle by the steady-state measurements and the stopped-flow spectrofluorometry. EDANS-fluorescence changes reflect conformational changes near the ATP binding site in the cytoplasmic domain, while tryptophan-fluorescence changes most probably reflect conformational changes in or near the transmembrane domain in which the Ca 2+ binding sites are located. Formation of the phosphoenzyme intermediates ( E P) was also followed by the continuous flow-rapid quenching method. The kinetic analysis of EDANS-fluorescence changes and E P formation revealed that, when ATP is added to the calcium-activated enzyme, conformational changes in the ATP binding site occur in three successive reaction steps; conformational change in the calcium enzyme substrate complex, formation of ADP-sensitive E P, and transition of ADP-sensitive E P to ADP-insensitive E P. In contrast, the ATP-induced tryptophan-fluorescence changes occur only in the latter two steps. Thus, we conclude that conformational changes in the ATP binding site in the cytoplasmic domain are transmitted to the Ca 2+ -binding sites in the transmembrane domain in these latter two steps.

The kinetics of the fusion process between erythrocyte ghosts, as induced by Sendal virus, were readily revealed by a simple fluorescence procedure previously employed to characterize the fusion of viruses with biological membranes. The method relies on the relief of fluorescence selfquenching of the membrane-inserted probe octadecyl Rhodamine B chloride (R 18 ) as occurs when labeled membranes fuse with unlabeled counterparts. The kinetics of R 18 insertion into ghost membranes, the non-exchangeable properties of the fluorophore and the kinetics, and some characteristics of Sendai virus-induced fusion of ghosts, are described. We propose that the experimental approach may be particularly advantageous to obtain insight into the efficiency and mechanism of a wide range of fusogens, capable of inducing fusion of erythrocyte membranes.

The combining region of Artocarpus integrifolia lectin has been studied by using the ligand-induced changes in the fluorescence of the lectin. The saccharide binding properties of the lectin show that C-l, C-2, C-4, and C-6 hydroxyl groups of D-galactose are important loci for sugar binding. The α-anorner of galactose binds more strongly than its β-counterpart. Inversion in the configuration at C-4 as in glucose results in a loss of binding to the lectin. The C-6 hydroxyl group is also presumably involved in binding as D-fucose does not bind to the lectin. The lectin binds to the Thomsen-Friedenreich antigen (Galβ(1→3)GalNAc) more strongly than the other disaccharides studied, viz. Gal/β (1→4) Gal and Galβ (1→3) GlcNAc, which are topographically similar to T-antigen. This observation suggests that the combining region of Artocarpus lectin is complementary to that of T-antigen. Solvent accessibility of the protein fluorophores have been probed by the quenching of protein fluorescence by Iodide ion in the absence and presence of sugar. In the presence of sugar a slight inaccessibility of the fluorophores to the solvent has been observed.