Time-resolved Fluorescence Intensity Research Articles

RNA-based liposomes for oral cancer: From biophysical characterization to biological evaluation

Oral cancer incidence and mortality are increasing over time. The most common therapies for oral cancers are surgery and radiotherapy, either used alone or combined, and immunotherapy can be also an option. Although there are several therapeutic options, none of them are completely effective, and in addition, there are numerous associated side effects. To overcome these limitations, researchers have been trying to reduce these drawbacks by using drug delivery systems that carry drugs for specific delivery to cancer cells. For that purpose, RNA-coated liposomes to selectively deliver the ligands C8 (acridine orange derivative) and dexamethasone to oral cancer cells were produced, characterized, and biologically evaluated.Firstly, the RNA structure and binding interaction with ligands (C8 and dexamethasone) were evaluated by circular dichroism (CD), thermal difference spectroscopy (TDS), nuclear magnetic resonance (NMR) and fluorescence titrations. The biophysical assays evidenced the formation of an RNA hairpin and duplex structure. Moreover, steady-state and time-resolved fluorescence intensity and anisotropy experiments show that C8 forms a complex with RNA and adopts an open conformation upon RNA binding.Then, RNA-coated liposomes were characterized by dynamic light scattering, and diameters near 160 nm were observed. Time-resolved anisotropy measurements of C8 loaded in RNA-functionalized liposomes indicate the co-existence of free C8 in solution (inside the liposome) and C8 bound to RNA at the external liposome surface. The RNA-functionalized liposomes loaded with C8 or dexamethasone mediated a significant reduction in the cell viability of malignant UPCI-SCC-154 cells while maintaining viable non-malignant NHDF cells. Additionally, the liposomes were able to internalize the cells, with higher uptake by the malignant cell line.Overall, the results obtained in this work can contribute to the development of new drug delivery systems based on RNA-coated liposomes.

A portable toolbox based on time-resolved fluoroimmunoassay and immunomagnetic separation for Cronobacter sakazakii on-site detection in dairy

A portable toolbox based on time-resolved fluoroimmunoassay and immunomagnetic separation was designed for on-site detection of Cronobacter sakazakii in dairy without advanced facilities. In this study, C. sakazakii was recognised and captured in milk by immunomagnetic beads, and europium chelated nanoparticles were used as signal probes in fluoroimmunoassays for quantitative analysis. Under optimal conditions, the capture efficiency of the immunomagnetic beads ranged from 77.76% to 98.26%. The time-resolved fluorescence intensity ratio was used to quantify C. sakazakii (y = 0.2615x – 0.1639, R2 = 0.9925) in the range of 102 to 107 cfu mL−1, and the limit of detection was as low as 73 cfu mL−1. The assay offered higher sensitivity for a total of 273 actual samples compared with the colloidal gold method. Our method can be completed within 2 h and may be a good model for on-site quantitation of food-borne pathogens with simplicity, rapidity, and cost-effectiveness.

Nedd4-2 binding to 14-3-3 modulates the accessibility of its catalytic site and WW domains

Neural precursor cells expressed developmentally downregulated protein 4-2 (Nedd4-2), a homologous to the E6-AP carboxyl terminus (HECT) ubiquitin ligase, triggers the endocytosis and degradation of its downstream target molecules by regulating signal transduction through interactions with other targets, including 14-3-3 proteins. In our previous study, we found that 14-3-3 binding induces a structural rearrangement of Nedd4-2 by inhibiting interactions between its structured domains. Here, we used time-resolved fluorescence intensity and anisotropy decay measurements, together with fluorescence quenching and mass spectrometry, to further characterize interactions between Nedd4-2 and 14-3-3 proteins. The results showed that 14-3-3 binding affects the emission properties of AEDANS-labeled WW3, WW4, and, to a lesser extent, WW2 domains, and reduces their mobility, but not those of the WW1 domain, which remains mobile. In contrast, 14-3-3 binding has the opposite effect on the active site of the HECT domain, which is more solvent exposed and mobile in the complexed form than in the apo form of Nedd4-2. Overall, our results suggest that steric hindrance of the WW3 and WW4 domains combined with conformational changes in the catalytic domain may account for the 14-3-3 binding-mediated regulation of Nedd4-2.

Development of an automated assay for accelerated in vitro detection of DNA adduct-inducing and crosslinking agents

Current techniques for the identification of DNA adduct-inducing and DNA interstrand crosslinking agents include electrophoretic crosslinking assays, electrophoretic gel shift assays, DNA and RNA stop assays, mass spectrometry-based methods and 32P-post-labelling. While these assays provide considerable insight into the site and stability of the interaction, they are relatively expensive, time-consuming and sometimes rely on the use of radioactively-labelled components, and thus are ill-suited to screening large numbers of compounds. A novel medium throughput assay was developed to overcome these limitations and was based on the attachment of a biotin-tagged double stranded (ds) oligonucleotide to Corning DNA-Bind plates. We aimed to detect anthracycline and anthracenedione DNA adducts which form by initial non-covalent intercalation with duplex DNA, and subsequent covalent adduct formation which is mediated by formaldehyde. Following drug treatment, DNA samples were subjected to a denaturation step, washing and then measurement by fluorescence to detect remaining drug-DNA species using streptavidin-europium. This dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) is a time–resolved fluorescence intensity assay where the fluorescence signal arises only from stabilised drug-DNA complexes. We applied this new methodology to the identification of anthracycline-like compounds with the ability to functionally crosslink double-strand oligonucleotides. The entire procedure can be performed by robotics, requiring low volumes of compounds and reagents, thereby reducing costs and enabling multiple compounds to be assessed on a single microtitre plate.

Branching ratios of electronic quenching of atomic sulfur S(3p34p 3PJ) by collisions with N2

Atomic sulfur S(3p43P) was generated in the photolysis of OCS at 248 nm and exited to the 3p34p3P state by ultraviolet two-photon absorption at 308 nm. Transfer from the 3p34p3P state to the 3p34s3S state by infrared radiation and/or collisions with ambient gases was followed by the vacuum ultraviolet (VUV) fluorescence via the 3p34s3S → 3p43P transition at 180 nm. The time-resolved VUV fluorescence intensity was recorded at varying pressures of N2. Kinetic analyses have given not only the overall rate coefficient for quenching of the 3p34p3P state by N2 but also the branching ratio of the quenching from the 3p34p3P state to the 3p34s3S state.

A "turnon" aptasensor for simultaneous and time-resolved fluorometric determination of zearalenone, trichothecenes A and aflatoxin B1 using WS2 as a quencher.

A "turn on" time-resolved fluorometric aptasensor is described for the simultaneous detection of zearalenone (ZEN), trichothecenes A (T-2), and aflatoxin B1 (AFB1). Multicolor-emissive nanoparticles doped with lanthanide ions (Dy3+, Tb3+, Eu3+) were functionalized with respective aptamers and applied as abioprobe, and tungsten disulfide (WS2) nanosheets are used as a quencher of time-resolved fluorescence. The assay exploits the quenching efficiency of WS2 and the interactions between WS2 and the respective DNA aptamers. The simultaneous recognition of the three mycotoxins can be performed in a single solution. In the absence of targets, WS2 is easily adsorbed by the mixed bioprobes via van der Waals forces between nucleobases and the WS2 basal plane. This brings the bioprobe and WS2 into close proximity and results in quenched fluorescence. In the presence of targets, thefluorescence of thebioprobes is restored because the analytes react with DNA probe and modify their molecular conformation to weaken the interaction between the DNAs and WS2. Under the optimum conditions and at an excitation wavelength of 273nm, the time-resolved fluorescence intensities (peakingat 488, 544 and 618nm andcorresponding to emissions of Dy3+, Tb3+ and Eu3+) were used to quantify ZEN, T-2 and AFB1, respectively, with detection limits of 0.51, 0.33 and 0.40pgmL-1 and a linear range from 0.001 to 100ngmL-1. The three mycotoxins can be detected simultaneously without mutual interference. The assay was applied to the quantification of ZEN, T-2 and AFB1 in (spiked) maize samples. This homogeneous aptamer based assay can be performed within 1h. Conceivably, it can become an alternative to other heterogeneous methods such as the respective enzyme-linked immunosorbent assays. Graphical abstract Schematic presentation of an aptasensor for simultaneous detection of zearalenone, trichothecenes A and aflatoxin B1 using aptamer modified time-resolved fluorescence nanoparticles as signalling probes and tungsten disulfide as thequencher. This assay shows lower detection limit and requires no washing steps.

3D-printed CuO nanoparticle-functionalized flow reactor enables online fluorometric monitoring of glucose.

An online flow reactor was fabricated by using a fused deposition modeling three-dimensional printing (3DP) technology along with thermoplastic poly(lactic acid) filaments incorporating copper oxide nanoparticles (CuO NPs). In the presence of glucose, the flow reactor displays multi-catalytic activities because accelerates the oxidation of 2',7'-dichlorodihydrofluorescein to form fluorescein which displays green fluorescence under 480nm excitation (emission wavelength: 530nm). The CuO NPs exert two functions to mediate electron transfer at a basic reaction condition, viz. direct oxidation of glucose to generate reactive oxygen species (ROS), and prompting the ROS to oxidize 2',7'-dichlorofluorescin diacetate. The flow reactor coupled to a microdialysis sampler and a fluorometer was applied for online fluorometric monitoring of brain extracellular glucose levels in living rats based on scanning of time-resolved fluorescence intensities. After optimization of (a) the manufacture of the flow reactor, (b) the reaction conditions (pH10; 50°C), and (c) the online analytical system, the detection limit of the method (when using 10-μL samples of microdialysate) is as low as 6.1μM (linear range: 0.05-5mM) with a sampling frequency of 7.5h-1. To illustrate the method's applicability, analyses of spiked off-line-collected rat brain microdialysates were conducted. In addition, rat brain extracellular glucose levels were monitored in-vivo and online upon neuronal depolarization triggered by perfusing a high-K+ medium. The results demonstrate that functionalizing raw 3DP materials with appropriate nanomaterials can simplify the manufacturing of analytical devices and related analytical procedures. This will extend the diversity and adaptability of current 3DP-enabling analytical strategies. Graphical abstract Schematic presentation of an online flow reactor fabricated using a fused deposition modeling 3D printer along with poly(lactic acid) (PLA) filaments incorporating CuO NPs. The manufactured flow reactor displays multi-catalytic activities and simplifies online fluorometric monitoring of living rat brain extracellular glucose.

Polarized Two-Photon Absorption and Heterogeneous Fluorescence Dynamics in NAD(P)H

Two-photon absorption (2PA) finds widespread application in biological systems, which frequently exhibit heterogeneous fluorescence decay dynamics corresponding to multiple species or environments. By combining polarized 2PA with time-resolved fluorescence intensity and anisotropy decay measurements, we show how the two-photon transition tensors for the components of a heterogeneous population can be separately determined, allowing structural differences between the two fluorescent states of the redox cofactor NAD(P)H to be identified. The results support the view that the two states correspond to alternate configurations of the nicotinamide ring, rather than folded and extended conformations of the entire molecule.

Simultaneous detection of fumonisin B1 and ochratoxin A using dual-color, time-resolved luminescent nanoparticles (NaYF4: Ce, Tb and NH2-Eu/DPA@SiO2) as labels

A rapid and highly sensitive time-resolved fluorescence (TRF)-based aptasensor for simultaneous recognition of mycotoxins ochratoxin A (OTA) and fumonisin B1 (FB1) was developed using multi-color, Ln3+-doped time-resolved fluorescence nanoparticles (TRF-NPs) (NaYF4: Ce, Tb and NH2-Eu/DPA@SiO2 NPs) coupled with complementary strand DNA (cDNA) as luminescence probe and aptamers-conjugated amine-functionalized Fe3O4 magnetic nanoparticles (MNPs) act as a capture probe. Under the optimized conditions, the time-resolved fluorescence intensities at 544 and 618nm corresponded with Tb3+ and Eu3+, respectively, were used to measure FB1 (Y = 19,177.1 + (- 12,054.4)x, R2 = 0.9917) and OTA (Y = 4138.8 + (- 11,182.6)x, R2 = 0.9924), respectively. The limits of detection (LODs) for FB1 and OTA were 0.019pgmL-1 and 0.015pgmL-1, respectively, which were much lower than previously described methods for simultaneous recognition of mycotoxins OTA and FB1 while detection range varied from 0.0001-0.5ngmL-1. This aptasensor was effectively applied to quantity FB1 and OTA in maize samples and results were compared with ELISA method. This is the first reported time-resolved fluorescence (TRF)-based aptasensor to detect two agriculturally important toxins in the maize. The developed aptasensor has potential to be used for detection of toxins in food safety fields. Graphical abstract.

A novel bioassay based on aptamer-functionalized magnetic nanoparticle for the detection of zearalenone using time resolved-fluorescence NaYF4: Ce/Tb nanoparticles as signal probe

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by fungi on stored grains. The earlier detection methods used for ZEN rely on expensive equipment, time-consuming sample preparation and temperature sensitive antibodies. The current work, proposed a novel strategy based on ZEN aptamer labeled with amine-functionalized magnetic nanoparticle (MNPs) as a capture probe and time-resolved fluorescence (TRFL) nanoparticles labeled with complementary DNA (cDNA) as a signal probe. Under the optimized conditions, TRFL intensity at 544 nm was used to measure ZEN (R2 = 0.9920) in the range of 0.001–10 ng mL−1 and limits of detection (LOD) for proposed method was 0.21 pg mL−1. The specificity of bioassay was also determined by using other mycotoxins (OTA, AFB2, DON and Patulin) and results showed that the aptamer are specific to recognize only ZEN. The analytical applications of the present bioassay in maize and wheat samples were also examined and results were compared with existing methods. Based on these findings, it is suggested to use current rapid and simple bioassay for the determination of ZEN in food and agricultural products.

Use of Interactive Graphical Tools to Demonstrate Changes in Time-Resolved Fluorescence Intensity Decays

Use of Interactive Graphical Tools to Demonstrate Changes in Time-Resolved Fluorescence Intensity Decays

Homogeneous time-resolved FRET assay for the detection of Salmonella typhimurium using aptamer-modified NaYF4:Ce/Tb nanoparticles and a fluorescent DNA label

The authors describe an aptasensor based on time-resolved fluorescence resonance energy transfer (TR-FRET) for the identification of Salmonella typhimurium (S. typhimurium). Aptamer-functionalized nanoparticles (NPs) is used as the energy donor, and a complementary oligonucleotide (cDNA) labeled with carboxyfluorescein (FAM) acts as the acceptor. The detection scheme is based on the hybridization between aptamer and cDNA, upon which photonic energy is transferred from NPs to FAM unless aptamer interacts with S. typhimurium. Due to the highly specific recognition ability of aptamer and the strong fluorescence intensity of NPs, the method is highly sensitive and selective for S. typhimurium. Under the optimal conditions, at excitation wavelength of 273 nm, a delay time of 100 μs and a gating time of 1 ms, the integrated time-resolved fluorescence intensity ratio (FAM520/Tb489) is linear in the 100 to 106 cfu·mL−1 range, and the limit of detection is as low as 25 cfu·mL−1. The assay was applied to the analysis of eggs and chicken meat for S. typhimurium, and the results were consistent with those of a plate-counting method.

Time-resolved and temperature tuneable measurements of fluorescent intensity using a smartphone fluorimeter.

A smartphone fluorimeter capable of time-based fluorescence intensity measurements at various temperatures is reported. Excitation is provided by an integrated UV LED (λex = 370 nm) and detection obtained using the in-built CMOS camera. A Peltier is integrated to allow measurements of the intensity over T = 10 to 40 °C. All components are controlled using a smartphone battery powered Arduino microcontroller and a customised Android application that allows sequential fluorescence imaging and quantification every δt = 4 seconds. The temperature dependence of fluorescence intensity for four emitters (rhodamine B, rhodamine 6G, 5,10,15,20-tetraphenylporphyrin and 6-(1,4,8,11-tetraazacyclotetradecane)2-ethyl-naphthalimide) are characterised. The normalised fluorescence intensity over time of the latter chemosensor dye complex in the presence of Zn2+ is observed to accelerate with an increasing rate constant, k = 1.94 min-1 at T = 15 °C and k = 3.64 min-1 at T = 30 °C, approaching a factor of ∼2 with only a change in temperature of ΔT = 15 °C. Thermally tuning these twist and bend associated rates to optimise sensor approaches and device applications is proposed.

Simultaneous detection of Staphylococcus aureus and Salmonella typhimurium using multicolor time-resolved fluorescence nanoparticles as labels

Foodborne illnesses caused by Staphylococcus aureus and Salmonella typhimurium are common public health issues worldwide, affecting both developing and developed countries. In this study, aptamers labeled with multicolor lanthanide-doped time-resolved fluorescence (TRFL) nanoparticles were used as signal probes, and immobilized by Fe3O4 magnetic nanoparticles were used as the capture probes. The signal probes were bonded onto the captured bacteria by the recognition of aptamer to form the sandwich-type complex. Under the optimal conditions, TRFL intensity at 544nm was used to quantify S. typhimurium (y=10,213×−12,208.92, R2=0.9922) and TRFL intensity at 615nm for S. aureus (y=4803.20×−1933.87, R2=0.9982) in the range of 102–105CFU/ml. Due to the magnetic separation and concentration of Fe3O4 nanoparticles, detection limits of the developed method were found to be 15, 20CFU/ml for S. typhimurium and S. aureus, respectively. The application of this bioassay in milk was also investigated, and results were consistent with those of plate-counting method. Therefore, this simple and rapid method owns a great potential in the application for the multiplex analysis in food safety.

Single-molecule enzymatic conformational dynamics: spilling out the product molecules.

Product releasing is an essential step of an enzymatic reaction, and a mechanistic understanding primarily depends on the active-site conformational changes and molecular interactions that are involved in this step of the enzymatic reaction. Here we report our work on the enzymatic product releasing dynamics and mechanism of an enzyme, horseradish peroxidase (HRP), using combined single-molecule time-resolved fluorescence intensity, anisotropy, and lifetime measurements. Our results have shown a wide distribution of the multiple conformational states involved in active-site interacting with the product molecules during the product releasing. We have identified that there is a significant pathway in which the product molecules are spilled out from the enzymatic active site, driven by a squeezing effect from a tight active-site conformational state, although the conventional pathway of releasing a product molecule from an open active-site conformational state is still a primary pathway. Our study provides new insight into the enzymatic reaction dynamics and mechanism, and the information is uniquely obtainable from our combined time-resolved single-molecule spectroscopic measurements and analyses.

Motor oil classification based on time-resolved fluorescence.

A time-resolved fluorescence (TRF) technique is presented for classifying motor oils. The system is constructed with a third harmonic Nd:YAG laser, a spectrometer, and an intensified charge coupled device (ICCD) camera. Steady-state and time-resolved fluorescence (TRF) measurements are reported for several motor oils. It is found that steady-state fluorescence is insufficient to distinguish the motor oil samples. Then contour diagrams of TRF intensities (CDTRFIs) are acquired to serve as unique fingerprints to identify motor oils by using the distinct TRF of motor oils. CDTRFIs are preferable to steady-state fluorescence spectra for classifying different motor oils, making CDTRFIs a particularly choice for the development of fluorescence-based methods for the discrimination and characterization of motor oils. The two-dimensional fluorescence contour diagrams contain more information, not only the changing shapes of the LIF spectra but also the relative intensity. The results indicate that motor oils can be differentiated based on the new proposed method, which provides reliable methods for analyzing and classifying motor oils.

Ion Intensity and Thermal Proton Transfer in Ultraviolet Matrix-Assisted Laser Desorption/Ionization

The ionization mechanism of ultraviolet matrix-assisted laser desorption/ionization (UV-MALDI) was investigated by measuring the total cation intensity (not including sodiated and potasiated ions) as a function of analyte concentration (arginine, histidine, and glycine) in a matrix of 2,4,6-trihydroxyacetophenone (THAP). The total ion intensity increased up to 55 times near the laser fluence threshold as the arginine concentration increased from 0% to 1%. The increases were small for histidine, and a minimal increase occurred for glycine. Time-resolved fluorescence intensity was employed to investigate how analytes affected the energy pooling of the matrix. No detectable energy pooling was observed for pure THAP and THAP/analyte mixtures. The results can be described by using a thermal proton transfer model, which suggested that thermally induced proton transfer is crucial in the primary ion generation in UV-MALDI.

Site-Specific Fluorescence Dynamics of α-Synuclein Fibrils Using Time-Resolved Fluorescence Studies: Effect of Familial Parkinson’s Disease-Associated Mutations

α-Synuclein (α-Syn) aggregation is directly implicated in both the initiation and spreading of Parkinson's Diseases (PD) pathogenesis. Although the familial PD-associated mutations (A53T, E46K, and A30P) are known to affect the aggregation kinetics of α-Syn in vitro, their structural differences in resultant fibrils are largely unknown. In this report we studied the site-specific dynamics of wild type (wt) α-Syn and its three PD mutant fibrils using time-resolved fluorescence intensity, anisotropy decay kinetics, and fluorescence quenching. Our data suggest that the N- and C-terminus are more flexible and exposed compared to the middle non-amyloid-β component (NAC) region of wt and PD mutant α-Syn fibrils. Yet the N-terminus showed great conformational heterogeneity compared to the C-terminus for all these proteins. 71 position of E46K showed more flexibility and solvent exposure compared to other α-Syns, whereas both E46K and A53T fibrils possess a more rigid C-terminus compared to wt and A30P. The present data suggest that wt and PD mutant fibrils possess large differences in flexibility and solvent exposure at different positions, which may contribute to their different pathogenicity in PD.

Quantifying exciton hopping in disordered media with quenching sites: Application to arrays of quantum dots

We present an analytical method for quantifying exciton hopping in an energetically disordered system with quenching sites. The method is subsequently used to provide a quantitative understanding of exciton hopping in a quantum dot (QD) array. Several statistical quantities that characterize the dynamics (survival probability, average number of distinct sites visited, average hopping distance, and average hopping rate in the initial stage) are obtained experimentally by measuring time-resolved fluorescence intensities at various temperatures. The time evolution of these quantities suggests in a quantitative way that at low temperature an exciton tends to be trapped at a local low-energy site, while at room temperature, exciton hopping occurs repeatedly, leading to a large hopping distance. This method will serve to facilitate highly efficient optoelectronic devices using QDs such as photovoltaic cells and light-emitting diodes, since exciton hopping is considered to strongly influence their operational parameters. The presence of a dark QD (quenching site) that exhibits fast decay is also quantified.

Octyl-β-D-Glucopyranoside Shows Composition Dependent Disordering Effects in Ternary Lipid Bilayers

Solubilization of lipid bilayers by the nonionic detergent OG has been well characterized in one and two component lipid systems, but has not been extensively studied in ternary membranes containing mixed fluid phases. In order to understand the effects of membrane order on solubilization by OG, LUVs of pure POPC were compared to those composed of POPC plus varying fractions of sphingomyelin (SM) and cholesterol. Detergent partitioning from the aqueous phase into the bilayer and membrane solubilization were monitored by ITC. The fluorescent membrane probe, DPH, was observed by time-resolved fluorescence intensity and polarization decay in the presence of increasing detergent concentrations to determine the effects on membrane order and dynamics. The results of partitioning experiments showed approximately a 50% decrease in the partition constant between pure POPC and the most ordered samples. ITC solubilization experiments showed clear boundaries for the micelle-bilayer mixed aggregate coexistence region in samples of low initial order, but the distinct thermodynamic signature associated with this coexistence region was not present in the cholesterol and SM rich samples. The average excited state lifetime of DPH, an indicator of water penetration, in vesicles rich in POPC showed a rapid increase at the onset of solubilization, whereas vesicles rich in SM and cholesterol showed a corresponding rapid decrease. Dynamic fluorescence depolarization data analyzed in terms of a Brownian rotational diffusion model revealed an increase in the occupancy of the bilayer midplane by DPH prior to solubilization in less ordered membranes coupled with a constant rotational correlation time. Membranes with higher initial order showed little increase in midplane occupancy and increasing rotational correlation time in the same detergent concentration range, suggesting that two modes of membrane disruption are possible and depend on the lipid makeup of the bilayer.