Nanoparticle Metrology of Silicates Using Time-Resolved Multiplexed Dye Fluorescence Anisotropy, Small Angle X-ray Scattering, and Molecular Dynamics Simulations.

We investigate the nanometrology of sub-nanometre particle sizes in industrially manufactured sodium silicate liquors at high pH using time-resolved fluorescence anisotropy. Rather than the previous approach of using a single dye label, we investigate and quantify the advantages and limitations of multiplexing two fluorescent dye labels. Rotational times of the non-binding rhodamine B and adsorbing rhodamine 6G dyes are used to independently determine the medium microviscosity and the silicate particle radius, respectively. The anisotropy measurements were performed on the range of samples prepared by diluting the stock solution of silicate to concentrations ranging between 0.2 M and 2 M of NaOH and on the stock solution at different temperatures. Additionally, it was shown that the particle size can also be measured using a single excitation wavelength when both dyes are present in the sample. The recovered average particle size has an upper limit of 7.0 ± 1.2 Å. The obtained results were further verified using small-angle X-ray scattering, with the recovered particle size equal to 6.50 ± 0.08 Å. To disclose the impact of the dye label on the measured complex size, we further investigated the adsorption state of rhodamine 6G on silica nanoparticles using molecular dynamics simulations, which showed that the size contribution is strongly impacted by the size of the nanoparticle of interest. In the case of the higher radius of curvature (less curved) of larger particles, the size contribution of the dye label is below 10%, while in the case of smaller and more curved particles, the contribution increases significantly, which also suggests that the particles of interest might not be perfectly spherical.

Keratin intrinsic fluorescence as a mechanism for non-invasive monitoring of its glycation.

We have studied the evolution of keratin intrinsic fluorescence as an indicator of its glycation. Steady-state and time-resolved fluorescence of free keratin and keratin-glucose samples were detected in PBS solutionsin vitro. The changes in the fluorescence response demonstrate that the effect of glucose is manifest in the accelerated formation of fluorescent cross-links with an emission peak at 460 nm and formation of new cross-links with emission peaks at 525 nm and 575 nm. The fluorescence kinetics of these structures is studied and their potential application for the detection of long-term complications of diabetes discussed.

Impact of the Flavonoid Quercetin on ß-Amyloid Aggregation Revealed by Intrinsic Fluorescence.

We report the effects of quercetin, a flavonoid present in the human diet, on early stage beta-amyloid (Aß) aggregation, a seminal event in Alzheimer's disease. Molecular level changes in Aß arrangements are monitored by time-resolved emission spectral (TRES) measurements of the fluorescence of Aß's single tyrosine intrinsic fluorophore (Tyr). The results suggest that quercetin binds ß-amyloid oligomers at early stages of their aggregation, which leads to the formation of modified oligomers and hinders the creation of ß-sheet structures, potentially preventing the onset of Alzheimer's disease.

Collagen Glycation Detected by Its Intrinsic Fluorescence.

Collagen's long half-life (in skin approximately 10 years) makes this protein highly susceptible to glycation and formation of the advanced glycation end products (AGEs). Accumulation of cross-linking AGEs in the skin collagen has several detrimental effects; thus, the opportunity for non-invasive monitoring of skin glycation is essential, especially for diabetic patients. In this paper, we report using the time-resolved intrinsic fluorescence of collagen as a biomarker of its glycation. Contrary to the traditional fluorescence intensity decay measurement at the arbitrarily selected excitation and detection wavelengths, we conducted systematic wavelength- and time-resolved measurements to achieve time-resolved emission spectra. Changes in the intrinsic fluorescence kinetics, caused by both collagen aggregation and glycation, have been detected.

Detecting beta-amyloid glycation by intrinsic fluorescence - Understanding the link between diabetes and Alzheimer's disease.

We monitor early stages of beta-amyloid (Aß1-40) aggregation, one of the key processes leading to Alzheimer's disease (AD), in the presence of high glucose concentrations by measuring Aß1- 40 intrinsic fluorescence. The multiple peaks and their shifts observed in the time-resolved emission spectra (TRES) reveal the impact of glycation on Aß1- 40 oligomerisation. The results show that formation of the advanced glycation end products (AGEs) alters the aggregation pathway. These changes are highly relevant to our understanding of the pathophysiology of AD and the implication of AGE and diabetes in these pathways.

Monitoring the Assembly and Aggregation of Polypeptide Materials by Time-Resolved Emission Spectra.

Polypeptide assembly and aggregation are the common forms of its physiological and pathological activity, and monitoring them on a molecular level is critical for resolving numerous medical (e.g., onset of neurodegenerative diseases) or biological problems. Sensitivity of the intrinsic fluorescence of protein to its assembly, aggregation, or complexation offers a noninvasive methodology for identifying and determining different stages of these processes. In this protocol, we present the approach based on the time-resolved emission spectra (TRES), which reveals the number of fluorescent residues, the presence of dielectric relaxation, and the changes in fluorescence kinetics during aggregation.

Cu2+ Effects on Beta-Amyloid Oligomerisation Monitored by the Fluorescence of Intrinsic Tyrosine.

A non-invasive intrinsic fluorescence sensing of the early stages of Alzheimer's beta amyloid peptide aggregation in the presence of copper ions is reported. By using time-resolved fluorescence techniques the formation of beta amyloid-copper complexes and the accelerated peptide aggregation are demonstrated. The shifts in the emission spectral peaks indicate that the peptides exhibit different aggregation pathways than in the absence of copper.

Tracking Insulin Glycation in Real Time by Time-Resolved Emission Spectroscopy.

The application of time-resolved fluorescence sensing to the study of heterogenic biomolecular systems remains challenging because of the complexity of the resulting photophysics. Measuring the time-resolved emission spectroscopy (TRES) spectra can provide a more informative alternative to the modeling of the fluorescence decay that is currently employed. Here, we demonstrate this approach by monitoring real-time changes in intrinsic insulin fluorescence by TRES as a straightforward probe to directly measure kinetics of insulin aggregation and glycation. Our findings hold promise for monitoring the storage of insulin and its application in the control of diabetes and may support the development of more effective therapeutics against amyloidosis.

Protein fibrillogenesis model tracked by its intrinsic time-resolved emission spectra.

The excited-state kinetics of the fluorescence of tyrosine in a de novo protein fibrillogenesis model was investigated as a potential tool for monitoring protein fibre formation and complexation with glucose (glycation). In stark contrast to insulin the time-resolved emission spectra (TRES) recorded over the period of 700 hours in buffered solutions of the model with and without glucose revealed no apparent changes in Tyr fluorescence responses. This indicates the stability of the model and provides a measurement-supported basis for its use as a reference material in fluorescence studies of protein aggregation.

Probing beta amyloid aggregation using fluorescence anisotropy: experiments and simulation.

The aggregation of beta amyloid (Ab) protein is associated with the development of Alzheimer's disease. In this work we monitor Ab aggregation using fluorescence anisotropy, a technique that provides information on the rotational diffusion of the fluorescing tyrosine (Tyr) side chains. We also perform Monte Carlo (MC) and fully atomistic Molecular Dynamics (MD) simulations to interpret the experiments. The experimental results show that there are two different rotational timescales contributing to the anisotropy. Our MC simulation captures this behaviour in a coarse-scale manner, and, more importantly, shows that the Tyr side chains must have their movements restricted in order to reproduce the anisotropy. The MD simulations provide a molecular scale view, and indeed show that aggregation restricts the Try side chains to yield anisotropy in line with the experimental results. This combination of experiment and simulation therefore provides a unique insight into the aggregation process, and we suggest how this approach might be used to gain further information on aggregating protein systems.

Tyrosine Rotamer States in Beta Amyloid: Signatures of Aggregation and Fibrillation.

During the early stages of ß amyloid (Ab) peptide aggregation, toxic oligomers form which have been recognized as a likely cause of Alzheimer's disease. In this work, we use fully atomistic molecular dynamics simulation to study the amorphous aggregation of the peptide as well as model ß-sheet protofibril structures. In particular, we study the rotamer states of the single fluorescent tyrosine (Tyr) residue present in each Ab. We find that the occupation of the four previously identified rotamers is different for monomeric and amorphous aggregates because of the differing environments of the Tyr side-chains. Surprisingly, we also identify two new rotamers that uniquely appear for the ß-sheet structures, so that together the rotamers provide distinct signatures for the different stages of aggregation and fibrillation. We propose that these rotamers could be identified in fluorescence spectroscopy, with each rotamer having a distinct fluorescence lifetime because of its different exposures to the solvent. The identification of the two new rotamers therefore provides a new means to probe amyloid formation kinetics and to monitor the effect of additives including prospective drugs.

Resolving environmental microheterogeneity and dielectric relaxation in fluorescence kinetics of protein.

The fluorescence intensity decay of protein is easily measurable and reports on the intrinsic fluorophore-local environment interactions on the sub-nm spatial and sub-ns temporal scales, which are consistent with protein activity in numerous biomedical and industrial processes. This makes time-resolved fluorescence a perfect tool for understanding, monitoring and controlling these processes at the molecular level, but the complexity of the decay, which has been traditionally fitted to multi-exponential functions, has hampered the development of this technique over the last few decades. Using the example of tryptophan in HSA we present the alternative to the conventional approach to modelling intrinsic florescence intensity decay in protein where the key factors determining fluorescence decay, i.e. the excited-state depopulation and the dielectric relaxation (Toptygin and Brand 2000 Chem. Phys. Lett. 322 496-502), are represented by the individual relaxation functions. This allows quantification of both effects separately by determining their parameters from the global analysis of a series of fluorescence intensity decays measured at different detection wavelengths. Moreover, certain pairs of the recovered parameters of tryptophan were found to be correlated, indicating the influence of the dielectric relaxation on the transient rate of the electronic transitions. In this context the potential for the dual excited state depopulation /dielectric relaxation fluorescence lifetime sensing is discussed.

Tyrosine Photophysics During the Early Stages of ß-Amyloid Aggregation Leading to Alzheimer's.

We have monitored the formation of toxic ß-amyloid oligomers leading to Alzheimer's disease by detecting changes in the fluorescence decay of intrinsic tyrosine. A new approach based on the non-Debye model of fluorescence kinetics resolves the complexity of the underlying photophysics. The gradual disappearance of nonmonotonic fluorescence decay rates, at the early stages of aggregation as larger, tighter-packed oligomers are formed, is interpreted in terms of tyrosine-peptide dielectric relaxation influencing the decay. The results demonstrate the potential for a new type of fluorescence lifetime sensing based on dual excited-state/dielectric relaxation, with application across a broad range of biological molecules. The results also reconcile previously conflicting models of protein intrinsic fluorescence decay based on rotamers or dielectric relaxation by illustrating conditions under which both are manifest.

Fluorescence kinetics of tryptophan in a heterogeneous environment.

The potentially highly informative, but complex fluorescence decay of amino acids in protein is not fully understood and presents a barrier to understanding. Here we have tested a new and general approach to describing experimentally measured the fluorescence decay in a heterogeneous macroscopic sample. The decay parameters carry information on the features of the kinetics induced by the environment's heterogeneity. Bayesian interference demonstrated that the model fits well to the fluorescence decay of tryptophan in human serum albumin (HSA). The approach has the potential to accelerate photophysical research of heterogeneous media and, specifically, to solve a critical outstanding problem in interpreting protein fluorescence, paving the way to further progress in biomedical research.

Initial stages of beta-amyloid Aß1-40 and Aß1-42 oligomerization observed using fluorescence decay and molecular dynamics analyses of tyrosine.

The development of Alzheimer's disease is associated with the aggregation of the beta-amyloid peptides Aß1-40 and Aß1-42. It is believed that the small oligomers formed during the early stages of the aggregation are neurotoxic and involved in the process of neurodegeneration. In this paper we use fluorescence decay measurements of beta-amyloid intrinsic fluorophore tyrosine (Tyr) and molecular dynamics (MD) simulations to study the early stages of oligomer formation for the Aß1-40 and Aß1-42 peptides in vitro. We demonstrate that the lifetime distributions of the amyloid fluorescence decay efficiently describe changes in the complex Tyr photophysics during the peptide aggregation and highlight the differences in aggregation performance of the two amyloids. Tyr fluorescence decay is found to be a more sensitive sensor of Aß1-40 aggregation than Aß1-42 aggregation. The MD simulation of the peptide aggregation is compared with the experimental data and supports a four-rotamer model of Tyr.

Beta-amyloid oligomerisation monitored by intrinsic tyrosine fluorescence.

Aggregation of the peptide beta-amyloid is known to be associated with Alzheimer's disease. According to recent findings the most neurotoxic aggregates are the oligomers formed in the initial stages of the aggregation process. Here we use beta-amyloid's (Aß's) intrinsic fluorophore tyrosine to probe the earliest peptide-to-peptide stages of aggregation, a region often merely labelled as a time lag, because negligible changes are observed by the commonly used probe ThT. Using spectrally resolved fluorescence decay time techniques and analysis we demonstrate how the distribution of 3 rotamer conformations of the single tyrosine in Aß tracks the aggregation across the time lag and beyond according to the initial peptide concentration. At low Aß concentrations (≤5 µM), negligible aggregation is observed and this is mirrored by little change in the fluorescence decay parameters, providing a useful baseline for comparison. At higher concentrations (≈50 µM), and contrary to what is generally accepted from ThT studies the rate of aggregation can be described by an exponential growth to a plateau in terms of the relative contributions of two of the three rotamers, with a characteristic aggregation time of ≈33 h.

Early detection of amyloid aggregation using intrinsic fluorescence.

Beta-amyloid (Abeta) aggregation, believed to be responsible for Alzheimer's disease, is monitored using its intrinsic fluorescence decay. Alterations in the fluorescence decay of tyrosine correlate with the Abeta aggregation at a much earlier stage than the traditionally used fluorescence intensity of Thioflavin T (ThT). Potentially the finding may underpin progress towards an earlier diagnosis of the onset of Alzheimer's disease and an improved approach to developing intervention therapies.

Fluorescence biosensing in nanopores.

Hydrated nanopores offer a unique environment for studying biological molecules under controlled conditions and fabricating sensors using fluorescence. Silica nanopores for example are non-toxic, biologically and optically compatible with protein, and can be easily synthesized to entrap protein and exclude potentially interfering macromolecules, while transmitting analytes of interest. A well known problem when polymerizing orthosilicates to fabricate silica sol-gel nanopores is the release of alcohol, which denatures proteins. We will describe how using the fluorescence of PRODAN (6-propionyl-2-(N,N-dimethylamino) naphthalene) to monitor methanol generated during polymerization has helped define a protocol with enhanced biocompatibility. The improved biocompatibility of sol-gel nanopores synthesized using tetramethyl orthosilicate (TMOS) has been demonstrated by preserving the unstable native trimer form of allophycocyanin (APC) for up to 500 Hrs without the need to covalently binding the subunits together. This has enabled the observation of native APC trimer by means of its fluorescence in a pore down to the single molecule level. In this paper we demonstrate how PRODAN and another polarity sensitive dye, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, Nile red (NR) report on pore polarity and successfully extend protein encapsulation to nano-channels of alumina (Al2O3). Improved biocompatibility of nanopores has potential impact in nanomedicine where the ability to study single biomolecules is a primary goal as it underpins our understanding of disease pathology and therapeutics at the most fundamental level. In sensing also the advantages of nanopore isolation of metabolite-specific protein for detecting non-fluorescent metabolites has been demonstrated. Similar approaches can in principle be developed for both single-molecules and lab-on-a-chip sensors.

Protein fluorescence decay: a gamma function description of thermally induced interconversion of amino acid rotamers.

We present a description of fluorescence decay kinetics in complex environments based on gamma functions rather than the conventional approach using exponentials. The gamma function description is tested in measurements on the temperature dependence of the protein human serum albumin (HSA), N-acetyl tryptophanamide (NATA), and 2, 5-dipenyl oxazole (PPO). The monitoring of macromolecular structure and dynamics is demonstrated by means of distinct tryptophan (Trp) rotamer populations and their interconversion in HSA.

Fluorescence lifetime spectroscopy and imaging of nano-engineered glucose sensor microcapsules based on glucose/galactose-binding protein.

We aimed to develop microsensors for eventual glucose monitoring in diabetes, based on fluorescence lifetime changes in glucose/galactose-binding protein (GBP) labelled with the environmentally sensitive fluorophore dye, badan. A mutant of GBP was labelled with badan near the binding site, the protein adsorbed to microparticles of CaCO(3) as templates and encapsulated in alternating nano-layers of poly-L-lysine and heparin. We used fluorescence lifetime imaging (FLIM) with two-photon excitation and time-correlated single-photon counting to visualize the lifetime changes in the capsules. Addition of glucose increased the mean lifetime of GBP-badan by a maximum of approximately 2 ns. Analysis of fluorescence decay curves was consistent with two GBP states, a short-lifetime component (approximately 0.8 ns), likely representing the open form of the protein with no bound glucose, and a long-lifetime component (approximately 3.1 ns) representing the closed form with bound glucose and where the lobes of GBP have closed round the dye creating a more hydrophobic environment. FLIM demonstrated that increasing glucose increased the fractional proportion of the long-lifetime component. We conclude that fluorescence lifetime-based glucose sensing using GBP encapsulated with nano-engineered layer-by-layer films is a glucose monitoring technology suitable for development in diabetes management.