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.

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 solutions. 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.

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

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

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

We monitor early stages of beta-amyloid (Aβ) aggregation, one of the key processes leading to Alzheimer's disease (AD), in the presence of high glucose concentrations by measuring Aβ intrinsic fluorescence. The multiple peaks and their shifts observed in the time-resolved emission spectra (TRES) reveal the impact of glycation on Aβ oligomerisation. The results show that formation of the advanced glycation end products (AGEs) alters the aggregation pathway.

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.

Cu 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.

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

During the early stages of β 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 β-sheet protofibril structures. In particular, we study the rotamer states of the single fluorescent tyrosine (Tyr) residue present in each Ab.

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.

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.

Detecting beta-amyloid aggregation from time-resolved emission spectra.

The aggregation of beta-amyloids is one of the key processes responsible for the development of Alzheimer's disease. Early molecular-level detection of beta-amyloid oligomers may help in early diagnosis and in the development of new intervention therapies. Our previous studies on the changes in beta-amyloid's single tyrosine intrinsic fluorescence response during aggregation demonstrated a four-exponential fluorescence intensity decay, and the ratio of the pre-exponential factors indicated the extent of the aggregation in the early stages of the process before the beta-sheets were formed.

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.

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

We have monitored the formation of toxic β-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.

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.

Initial stages of beta-amyloid Aβ and Aβ 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 Aβ and Aβ. 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 Aβ and Aβ peptides in vitro.

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 (Aβ'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.

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.

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.

Optical spectroscopic methods for probing the conformational stability of immobilised enzymes.

We report the development of biophysical techniques based on circular dichroism (CD), diffuse reflectance infrared Fourier transform (DRIFT) and tryptophan (Trp) fluorescence to investigate in situ the structure of enzymes immobilised on solid particles. Their applicability is demonstrated using subtilisin Carlsberg (SC) immobilised on silica gel and Candida antartica lipase B immobilised on Lewatit VP.OC 1600 (Novozyme 435).

Nonextensive kinetics of fluorescence resonance energy transfer.

Some fluorescence dyes in complex media, such as those found in biology, demonstrate nonextensive kinetics, which implies representing their fluorescence decays in terms of lifetime distributions rather than simple exponentials. Complex kinetics usually discourage application to lifetime sensors, as it is believed, that additional molecular mechanisms employed for detection of an analyte will make the resulting kinetics ambiguous and the sensor response inconclusive. In this paper we investigate theoretically the applicability of complex dye kinetics as a fluorescence resonance energy transfer based lifetime sensor and demonstrate that the nonextensive nature of its kinetics does not decrease the sensing performance, and indeed even provides richer structural information than a simple exponential behavior.

Human serum albumin-flavonoid interactions monitored by means of tryptophan kinetics.

A nonextensive model of decay kinetics has been used to describe fluorescence behavior of tryptophan in human serum albumin on binding two flavonoids, quercetin and morin. We demonstrate that this approach, alternative to multiexponential representation of usually complex decays of tryptophan, is more adequate and can be beneficial in noninvasive lifetime sensing based on intrinsic fluorescence.

Toward single-metal-ion sensing by Förster resonance energy transfer.

Here we describe progress toward our objective of detecting single nonfluorescent hydrated metal ions. Single-ion detection represents detection and spectroscopy at the ultimate sensitivity level of approximately 1.6 x 10(-24) M.