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Abstract Thioflavin-T (THT) is a common and indispensable tool for the study of amyloid pathologies and protein aggregation, both in vitro and for histological samples. In this study we expand the use of THT beyond its canonical usage for staining amyloid plaques and demonstrate its novel use as an easy and rapid stain comparable to fluorescent Nissl staining, allowing for clear discernment of neuronal cell bodies and also nucleoli in fixed tissue and live cells. We believe that this is of value for any lab that studies central nervous system (CNS) tissues. Furthermore, we show that THT could potentially be used as a an alternative to the use of fluorescent reporters or other more costly RNA binding compounds in the study of nucleolar dynamics owing to its ability to clearly stain nucleoli in live cells. We also discovered the previously unreported effect of blue light exposure on the photo enhancement of THT excited by a 488 nm laser in stained tissue sample and how to avoid complications arising from this effect. Finally, we provide a simple protocol that can be easily adjusted either for using THT as a neuronal cell body and nucleoli stain, compatible with antibody based staining methods tested up to 4 fluorophores, or alternatively by using an additional washing step the protocol may be used for amyloid plaque detection in fixed brain tissue.

Coordination polymer nanoparticles (CPNs) are a unique class of nanoparticles designed via the self-aggregation of a metal ion and a polydentate ligand. Among them, lanthanide-based CPNs are considered to be the most promising due to their intrinsic luminescent properties, stability in different oxidation states, and high coordination flexibility. In the present study, cerium-based coordination polymer nanoparticles (Ce-CPNs) have been synthesized using adenosine triphosphate (ATP) as a cross-linking ligand. A weakly emissive molecular-rotor-type dye, Thioflavin-T (ThT), was incorporated into the porous polymeric network of CPNs, which led to a drastic enhancement in the emission intensity of ThT. The emission intensity of ThT-Ce-(III)-ATP-Tris CPNs is severely quenched in the presence of Cu-(II) ions, which has been utilized for the detection of Cu-(II). The sensing mechanism was established by using photophysical techniques such as absorbance, emission, excited-state lifetime, and particle size measurements. The selectivity of this methodology for Cu-(II) ions was checked by studying the interference of several metal ions, anions and biomolecules. The limit of detection (LOD) obtained for Cu-(II) in the present methodology was about 660 nM (42 ppb), which is far below the U.S. EPA prescribed limit (20 μM) in water. The present methodology was successfully demonstrated to determine the Cu-(II) concentration in e-waste leachate and also in spiked human serum samples. The LOD of Cu-(II) in diluted e-waste leachate was found to be marginally higher (730 nM) compared to that in the case of water, whereas the LOD in 10% (v/v) serum was to be 5 μM (320 ppb). [ABSTRACT FROM AUTHOR]

ABSTRACT The infection cycle of phage λ terminates in lysis mediated by three types of lysis proteins, each disrupting a layer in the bacterial envelope: the S105 holin, the R endolysin, and the Rz/Rz1 spanin complex targeting the inner membrane, cell wall or peptidoglycan, and the outer membrane, respectively. Video microscopy has shown that in most infections, lysis occurs as a sudden, explosive event at a cell pole, such that the initial product is a less refractile ghost that retains rod-shaped morphology. Here, we investigate the molecular basis of polar lysis using time-lapse fluorescence microscopy. The results indicate that the holin determines the morphology of lysis by suddenly forming two-dimensional rafts at the poles about 100 s prior to lysis. Given the physiological and biochemical similarities between the lambda holin and other class I holins, dynamic redistribution and sudden concentration may be common features of holins, probably reflecting the fitness advantage of all-or-nothing lysis regulation.IMPORTANCEIn this study, we use fluorescent video microscopy to track -green fluorescent protein (GFP)-labeled holin in the minutes prior to phage lysis. Our work contextualizes prior genetic and biochemical data, showing when hole formation starts and where holin oligomers form in relation to the site of lytic rupture. Furthermore, prior work showed that the morphology of lambda-infected cells is characterized by an explosive event starting at the cell pole; however, the basis for this was not clear. This study shows that holin most often oligomerizes at cell poles and that the site of the oligomerization is spatially correlated with the site of lytic blowout. Therefore, the holin is the key contributor to polar lysis morphology for phage lambda.

Thioflavin-T (THT) is a common and indispensable tool for the study of amyloid pathologies and protein aggregation, both in vitro and for histological samples. In this study we expand the use of THT beyond its canonical usage for staining amyloid plaques and demonstrate its novel use as an easy and rapid stain comparable to fluorescent Nissl staining, allowing for clear discernment of neuronal cell bodies and also nucleoli in fixed tissue and live cells. We believe that this is of value for any lab that studies central nervous system (CNS) tissues. Furthermore, we show that THT could potentially be used as a an alternative to the use of fluorescent reporters or other more costly RNA binding compounds in the study of nucleolar dynamics owing to its ability to clearly stain nucleoli in live cells. We also discovered the previously unreported effect of blue light exposure on the photo enhancement of THT excited by a 488 nm laser in stained tissue sample and how to avoid complications arising from this effect. Finally, we provide a simple protocol that can be easily adjusted either for using THT as a neuronal cell body and nucleoli stain, compatible with antibody based staining methods tested up to 4 fluorophores, or alternatively by using an additional washing step the protocol may be used for amyloid plaque detection in fixed brain tissue., (© 2024. The Author(s).)

Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of a glutamine repeat in the protein ataxin-3, which is deposited as intracellular aggregates in affected brain regions. Despite the controversial role of ataxin-3 amyloid structures in SCA3 pathology, the identification of molecules with the capacity to prevent aberrant self-assembly and stabilize functional conformation(s) of ataxin-3 is a key to the development of therapeutic solutions. Amyloid-specific kinetic assays are routinely used to measure rates of protein self-assembly in vitro and are employed during screening for fibrillation inhibitors. The high tendency of ataxin-3 to assemble into oligomeric structures implies that minor changes in experimental conditions can modify ataxin-3 amyloid assembly kinetics. Here, we determine the self-association rates of ataxin-3 and present a detailed study of the aggregation of normal and pathogenic ataxin-3, highlighting the experimental conditions that should be considered when implementing and validating ataxin-3 amyloid progress curves in different settings and in the presence of ataxin-3 interactors. This assay provides a unique and robust platform to screen for modulators of the first steps of ataxin-3 aggregation—a starting point for further studies with cell and animal models of SCA3. [ABSTRACT FROM AUTHOR]

Amyloids, proteinous aggregates with β-sheet-rich fibrils, are involved in several neurodegenerative diseases such as Alzheimer's disease; thus, their detection is critically important. The most common fluorescent dye for amyloid detection is thioflavin-T (ThT), which shows on/off fluorescence upon amyloid binding. We previously reported that an engineered globular protein with a flat β-sheet, peptide self-assembly mimic (PSAM), can be used as an amyloid binding model. In this study, we further explored the residue-specific properties of ThT-binding to the flat β-sheet by introducing systematic mutations. We found that site-specific mutations at the ThT-binding channel enhanced affinity. We also evaluated the binding of a ThT-based photocatalyst, which showed the photooxygenation activity on the amyloid fibril upon light radiation. Upon binding of the photocatalyst to the PSAM variant, singlet oxygen-generating activity was observed. The results of this study expand our understanding of the detailed binding mechanism of amyloid-specific molecules. [ABSTRACT FROM AUTHOR]

The anti-oncogenic protein p53 is a transcription factor that prevents tumorigenesis by inducing gene repair proteins or apoptosis under DNA damage. Since the DNA-binding domain of p53 (p53C) is aggregation-prone, the anti-oncogenic function of p53 is often lost in cancer cells. This tendency is rather severe in some tumor-related p53 mutants, such as R175H. In this study, we examined the effect of salts, including KCl and sugars, on the aggregation of p53C by monitoring two distinct aggregates: amorphous-like and amyloid-like. The amorphous aggregates are detectable with 8-(phenylamino)-1-naphthalenesulfonic acid (ANS) fluorescence, whereas the amyloid aggregates are sensitive to thioflavin-T (ThT) fluorescence. We found that KCl inhibited the formation of amorphous aggregates but promoted the formation of amyloid aggregates in a p53C R175H mutant. The salts exhibited different effects against the wild-type and R175H mutants of p53C. However, the ratio of ANS/ThT fluorescence for the wild-type and R175H mutant remained constant. KCl also suppressed the structural transition and loss of the DNA-binding function of p53C. These observations indicate the existence of multiple steps of p53C aggregation, probably coupled with the dissociation of Zn. Notably, amorphous aggregates and amyloid aggregates have distinct properties that could be discriminated by various small additives upon aggregation.

Amyloid deposition underlies a broad range of diseases including multiple neurodegenerative diseases, systemic amyloidosis and type-2 diabetes. Amyloid sensitive dyes, particularly thioflavin-T, are widely used to detect ex-vivo amyloid deposits, to monitor amyloid formation in vitro and to follow the kinetics of amyloid self-assembly. We show that the dye SYPRO-orange binds to amyloid fibrils formed by human amylin, the polypeptide responsible for islet amyloid formation in type-2 diabetes. No fluorescence enhancement is observed in the presence of pre-fibrillar species or in the presence of non-amyloidogenic rat amylin. The kinetics of human amylin amyloid formation can be monitored by SYPRO-orange fluorescence and match the time course determined with thioflavin-T assays. Thus, SYPRO-orange offers an alternative to thioflavin-T assays of amylin amyloid formation. The implications for the interpretation of SYPRO-orange-based assays of protein stability and protein-ligand interactions are discussed.

In this work, the potential application of the fluorescence dye Thioflavin-T (ThT), which can specifically bind to amyloid, as a powerful tool for monitoring secondary structural transitions of silk fibroin (SF) induced by pH in low solution concentrations was examined. Results showed that ThT emission intensities substantially increased when pH decreased from 6.8 to 4.8. This increase may be ascribed to conformational transitions from random coil to β-sheet. The morphology and secondary structure of SF were also investigated via TEM, AFM and circular dichroism spectroscopy. The information obtained herein can be utilized not only for the development of convenient and efficient noninvasive method for monitoring the assembly behavior of SF in aqueous solution but also for in vitro fluorescence imaging. [ABSTRACT FROM AUTHOR]

Drusen deposition on sub-retinal pigment epithelium is the causal factor for age-related macular degeneration for the old-aged individuals. These deposits contain hydroxyapatite–cholesterol spherules on which several proteins and lipids accumulate to cover the retina and choroid, causing blurred vision and blindness. Amyloid-β, the known culprit in Alzheimer's disease, is one among the few major proteins known to occur in these deposits. In the present article, we report preliminary analyses of interactions between amyloid-β and hydroxyapatite–cholesterol composites using Thioflavin-T binding kinetics, solid-state NMR and transmission electron microscopy (TEM). Thioflavin-T fluorescence kinetics shows that amyloid-β (1–42) aggregates only under certain conditions of concentration of cholesterol in the hydroxyapatite–cholesterol composites prepared by two different methods. These results were confirmed by 1D 13C CPMAS solid-state NMR. TEM imaging revealed that there is an exposure of the cholesterol surface in the composites prepared by sonication method. These imaging experiments explain the dependence of aggregation kinetics on the exposure and availability of cholesterol surface in the composites to a certain extent. [ABSTRACT FROM AUTHOR]

Human serum albumin (HSA) is an abundant carrier protein present in the blood plasma manifesting affinity for drugs and ligands. The bindings of several drugs can cause changes in the structural conformation of HSA that may affect its function. HSA is also known to in vitro form amyloid-like aggregates with fibrillar morphology as observed under TEM. Earlier, we showed that the HSA amyloid-like aggregates display self-seeding potential and detergent stability and the dimers of HSA, which are preferable for clinical applications due to their longer circulatory life, can also form amyloid-like aggregates. As aspirin, a commonly prescribed drug, was previously documented to acetylate HSA at one of its lysine residues, here, we examined if acetylation has any effect on the in vitro amyloid-like aggregation of HSA. We show that HSA acetylated in vitro using acetylsalicylic acid manifests relatively reduced levels of amyloid-specific properties such as turbidity, Thioflavin-T-positive aggregation, ß-sheet content and stability against an ionic detergent. Also, TEM imaging shows that the acetylated HSA forms relatively less aggregates and with smaller sizes whereas, the aggregates of HSA are more abundant and larger in sizes with fibrillar morphology which further supports that acetylation can attenuate the amyloid-like aggregation of HSA. [ABSTRACT FROM AUTHOR]

[Display omitted] • Amyloid assembly in Alzheimer's disease may be influenced by the lipid rich brain. • Anionic phospholipids accelerate fibril assembly by primary nucleation. • Micromolar levels of any phospholipid impacts fibril surface catalysed nucleation. • Physiological, cellular derived exosomes, accelerate Aβ fibril formation. • Alzheimer's related changes in lipid metabolism accelerate fibril formation. Alzheimer's disease (AD) is linked with the self-association of the amyloid-β peptide (Aβ) into oligomers and fibrils. The brain is a lipid rich environment for Aβ to assemble, while the brain membrane composition varies in an age dependent manner, we have therefore monitored the influence of lipid bilayer composition on the kinetics of Aβ40 fibril assembly. Using global-fitting models of fibril formation kinetics, we show that the microscopic rate constant for primary nucleation is influenced by variations in phospholipid composition. Anionic phospholipids and particularly those with smaller headgroups shorten fibril formation lag-times, while zwitterionic phospholipids tend to extend them. Using a physiological vesicle model, we show cellular derived exosomes accelerate Aβ40 and Aβ42 fibril formation. Two distinct effects are observed, the presence of even small amounts of any phospholipid will impact the slope of the fibril growth curve. While subsequent additions of phospholipids only affect primary nucleation with the associated change in lag-times. Heightened anionic phospholipids and cholesterol levels are associated with aging and AD respectively, both these membrane components strongly accelerate primary nucleation during Aβ assembly, making a link between disrupted lipid metabolism and Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of a glutamine repeat in the protein ataxin-3, which is deposited as intracellular aggregates in affected brain regions. Despite the controversial role of ataxin-3 amyloid structures in SCA3 pathology, the identification of molecules with the capacity to prevent aberrant self-assembly and stabilize functional conformation(s) of ataxin-3 is a key to the development of therapeutic solutions. Amyloid-specific kinetic assays are routinely used to measure rates of protein self-assembly in vitro and are employed during screening for fibrillation inhibitors. The high tendency of ataxin-3 to assemble into oligomeric structures implies that minor changes in experimental conditions can modify ataxin-3 amyloid assembly kinetics. Here, we determine the self-association rates of ataxin-3 and present a detailed study of the aggregation of normal and pathogenic ataxin-3, highlighting the experimental conditions that should be considered when implementing and validating ataxin-3 amyloid progress curves in different settings and in the presence of ataxin-3 interactors. This assay provides a unique and robust platform to screen for modulators of the first steps of ataxin-3 aggregation—a starting point for further studies with cell and animal models of SCA3.

The aggregation of proteins is considered to be the main cause of several neurodegenerative diseases. Despite much progress in amyloid research, the process of fibrillization is still not fully understood, which is one of the main reasons why there are still very few effective treatments available. When the aggregation of insulin, a model amyloidogenic protein, is tracked using thioflavin-T (ThT), an amyloid specific dye, there is an anomalous occurrence of double-sigmoidal aggregation kinetics. Such an event is likely related to the formation of ThT-positive intermediates, which may affect the outcome of both aggregation kinetic data, as well as final fibril structure. In this work we explore insulin fibrillization under conditions, where both normal and double-sigmoidal kinetics are observed and show that, despite their dye-binding properties and random occurrence, the ThT-positive intermediates do not significantly alter the overall aggregation process.

The aggregation of proteins is considered to be the main cause of several neurodegen-erative diseases. Despite much progress in amyloid research, the process of fibrillization is still not fully understood, which is one of the main reasons why there are still very few effective treatments available. When the aggregation of insulin, a model amyloidogenic protein, is tracked using thioflavin-T (ThT), an amyloid specific dye, there is an anomalous occurrence of double-sigmoidal aggregation kinetics. Such an event is likely related to the formation of ThT-positive intermediates, which may affect the outcome of both aggregation kinetic data, as well as final fibril structure. In this work we explore insulin fibrillization under conditions, where both normal and double-sigmoidal kinetics are observed and show that, despite their dye-binding properties and random occurrence, the ThT-positive intermediates do not significantly alter the overall aggregation process. [ABSTRACT FROM AUTHOR]

Although N-acetylaspartate (NAA) has long been recognized as the most abundant amino acid in neurons by far, its primary role has remained a mystery. Based on its unique tertiary structure, we explored the potential of NAA to modulate aggregation of amyloid-beta (Aβ) peptide 1-42 via multiple corroborating aggregation assays along with electron microscopy. Thioflavin-T fluorescence assay demonstrated that at physiological concentrations, NAA substantially inhibited the initiation of Aβ fibril formation. In addition, NAA added after 25 min of Aβ aggregation was shown to break up preformed fibrils. Electron microscopy analysis confirmed the absence of mature fibrils following NAA treatment. Furthermore, fluorescence correlation spectroscopy and dynamic light scattering measurements confirmed significant reductions in Aβ fibril hydrodynamic radius following treatment with NAA. These results suggest that physiological levels of NAA could play an important role in controlling Aβ aggregation in vivo where they are both found in the same neuronal compartments.

In this work, we have synthesized gold nanoparticles (AuNPs) using flavonoids of Scutellaria barbata as reducing and stabilizing agent. The prepared AuNPs were studied by using various spectroscopic and microscopic techniques. TEM analysis showed the formation of 20 nm sized spherical nanoparticles. XRD analysis confirmed the formation of crystalline face centred cubic structured particles. Further, we studied the as prepared AuNPs for their efficiency to act as a novel class of amyloid-β (Aβ) inhibitors. The results showed that several small and relatively amorphous aggregates validated the exceptional efficiency of AuNPs in inhibiting Aβ40 aggregation. Therefore, the prepared AuNPs has future prosperous towards the treatment of Alzheimer's disease. [ABSTRACT FROM AUTHOR]

A cyclodextrin-based fluorescence light-up and ratiometric sensor is reported for highly selective and sensitive recognition of glutathione over cystein and homocystein. The sensing scheme developed builds up on a supramolecular assembly formed between a molecular rotor dye (ThT) and a polyanionic supramolecular host (sulfated-β-cyclodextrin, SCD). The detection scheme is accomplished as follows: firstly, the bivalent Cu2+ quenches the emission from ThT-SCD assembly by causing the dissociation of ThT molecules from SCD surface. Secondly, when GSH is added to the copper-quenched system, owing to specific interaction between Cu2+ and GSH, Cu2+ is removed from the SCD which again allows the formation of ThT-SCD assembly. Indeed, this scheme of disassembly and reassembly successively caused by Cu2+ and GSH in the aqueous solution empowers our sensor framework to work as a good ratiometric sensor for the detection of GSH. The sensor scheme shows a linear response in the range 0–250 μM with a LOD of 2.4 ± 0.2 μM in aqueous solution and 13.6 ± 0.5 μM in diluted human serum sample. The sensor system is excited at 410 nm and the emission signal is plotted as a ratio of intensity at 545 nm (aggregate band) and 490 nm (monomer band). This ratiometric sensor system is highly selective to glutathione over cystein, homocystein, and other amino acids. Additionally, response of the sensor system towards GSH in complex biological media of serum samples demonstrates its potential for practical utility. [ABSTRACT FROM AUTHOR]

Fluorescence of thioflavin T (ThT), a universal marker for amyloid fibril investigations, is shown to be highly dependent on fibrils' surface charge. This feature sheds new light on the relationship between ThT fluorescence and amyloid peptide fibrillation. Thioflavin T (ThT), a benzothiazole-based fluorophore, is a prominent dye widely employed for monitoring amyloid fibril assembly. Despite the near-universal presumption that ThT binds to β-sheet domains upon fibrillar surface via hydrophobic forces, the contribution of the positive charge of ThT to fibril binding and concomitant fluorescence enhancement have not been thoroughly assessed. Here we demonstrate a considerable interdependence between ThT fluorescence and electrostatic charges of peptide fibrils. Specifically, by analyzing both fibril-forming synthetic peptides and prominent natural fibrillar peptides, we demonstrate pronounced modulations of ThT fluorescence signal that were solely dependent upon electrostatic interactions between ThT and peptide surface. The results further attest to the fact that fibril ζ-potential rather than pH-dependent assembly of the fibrils constitute the primary factor affecting ThT binding and fluorescence. This study provides the first quantitative assessment of electrostatically driven ThT fluorescence upon adsorption to amyloid fibrils. [ABSTRACT FROM AUTHOR]

Protein aggregation and amyloid formation are associated with multiple human diseases, but are also a problem in protein production. Understanding how aggregation can be modulated is therefore of importance in both medical and industrial contexts. We have used bovine insulin as a model protein to explore how amyloid formation is affected by buffer pH and by the addition of short-chain alcohols. We find that bovine insulin forms amyloid fibrils, albeit with different rates and resulting fibril morphologies, across a wide pH range (2–7). At pH 4.0, bovine insulin displayed relatively low aggregation propensity in combination with high solubility; this condition was therefore chosen as basis for further exploration of how bovine insulin's native state can be stabilized in the presence of short-chain alcohols that are relevant because of their common use as eluents in industrial-scale chromatography purification. We found that ethanol and isopropanol are efficient modulators of bovine insulin aggregation, providing a three to four times retardation of the aggregation kinetics at 30–35% (vol/vol) concentration; we attribute this to the formation of oligomers, which we detected by AFM. We discuss this effect in terms of reduced solvent polarity and show, by circular dichroism recordings, that a concomitant change in α-helical packing of the insulin monomer occurs in ethanol. Our results extend current knowledge of how insulin aggregates, and may, although bovine insulin serves as a simplistic model, provide insights into how buffers and additives can be fine-tuned in industrial production of proteins in general and pharmaceutical insulin in particular. [ABSTRACT FROM AUTHOR]

The infection cycle of phage λ terminates in lysis mediated by three types of lysis proteins, each disrupting a layer in the bacterial envelope: the S105 holin, the R endolysin, and the Rz/Rz1 spanin complex targeting the inner membrane, cell wall or peptidoglycan, and the outer membrane, respectively. Video microscopy has shown that in most infections, lysis occurs as a sudden, explosive event at a cell pole, such that the initial product is a less refractile ghost that retains rod-shaped morphology. Here, we investigate the molecular basis of polar lysis using time-lapse fluorescence microscopy. The results indicate that the holin determines the morphology of lysis by suddenly forming two-dimensional rafts at the poles about 100 s prior to lysis. Given the physiological and biochemical similarities between the lambda holin and other class I holins, dynamic redistribution and sudden concentration may be common features of holins, probably reflecting the fitness advantage of all-or-nothing lysis regulation.IMPORTANCEIn this study, we use fluorescent video microscopy to track -green fluorescent protein (GFP)-labeled holin in the minutes prior to phage lysis. Our work contextualizes prior genetic and biochemical data, showing when hole formation starts and where holin oligomers form in relation to the site of lytic rupture. Furthermore, prior work showed that the morphology of lambda-infected cells is characterized by an explosive event starting at the cell pole; however, the basis for this was not clear. This study shows that holin most often oligomerizes at cell poles and that the site of the oligomerization is spatially correlated with the site of lytic blowout. Therefore, the holin is the key contributor to polar lysis morphology for phage lambda., Competing Interests: The authors declare no conflict of interest.

Prion diseases are neurodegenerative disorders caused by misfolding of the prion protein (PrP) from a normal cellular protein (PrPC) to a protease-resistant isoform (PrPSc). However, the aggregation mechanism is not entirely understood because of the physical properties of PrP, such as its solubility or aggregation in vitro and conformational or mutation diversity. Recently, we reported the physical and physiological properties of a synthetic fragment peptide. In the present study, we assessed the importance of a point mutation at the C-terminal region of PrP in structural conversion and aggregation and evaluated the physical and physiological properties of the point-mutated human-PrP180-192 V180I (hPrP180-192 V180I) using circular dichroism spectra, high-performance liquid chromatography, Affinix QNμ, and thioflavin-T staining, including the effects of Cu2+. The secondary structure of hPrP180-192 V180I changed from a random coil to a β-sheet in Cu2+ free buffer. In addition, we observed molecular interactions in hPrP180-192 V180I and aggregation with itself, which were inhibited by Cu2+. We conclude that the point mutation in the C-terminal region of PrP, including hPrP180-192 V180I, and Cu2+ may play an important role in the conversion of PrPC to PrPSc. • hPrP 180-192 V180I switched from a random coil to a β-sheet structure. • Conformational change of hPrP180-192 V180I was inhibited with Cu2+. • Insoluble form of hPrP180-192 V180I did not to revert to soluble form. • Fragment peptide is useful for studying the effect of each mutation in PrP. • Cu2+ may not have effect to the solubility of aggregated mutated PrPC. [ABSTRACT FROM AUTHOR]

The formation of amyloid fibrils is linked to multiple neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease. Despite years of research and countless studies on the topic of such aggregate formation, as well as their resulting structure, the current knowledge is still fairly limited. One of the main aspects prohibiting effective aggregation tracking is the environment’s effect on amyloid-specific dyes, namely thioflavin-T (ThT). Currently, there are only a few studies hinting at ionic strength being one of the factors that modulate the dye’s binding affinity and fluorescence intensity. In this work we explore this effect under a range of ionic strength conditions, using insulin, lysozyme, mouse prion protein, and α-synuclein fibrils. We show that ionic strength is an extremely important factor affecting both the binding affinity, as well as the fluorescence intensity of ThT.

Kidney disorders are a rising global health issue, necessitating early diagnosis for effective treatment. Creatinine, a metabolic waste product from muscles, serves as an ideal biomarker for kidney damage. The existing optical methods for creatinine detection often involve labor-intensive synthesis processes and present challenges with the aqueous solubility and sensitivity to experimental variations. In this study, we introduce a straightforward fluorescence "turn-on" ratiometric sensor system for creatinine detection in aqueous media with a limit of detection of 0.5 μM. The sensor is based on sulfated-β-cyclodextrin (SCD)-templated H-aggregate of a commercially available, ultrafast rotor dye thioflavin-T (ThT). The Al 3+ ion-induced dissociation of ThT-SCD aggregates, followed by reassociation upon creatinine addition, generates a detectable signal. The modulation of monomer/aggregate equilibrium due to the disassembly/reassembly of the ThT-SCD system under Al 3+ /creatinine influence serves as the optimal strategy for ratiometric creatinine detection in aqueous media. Our sensor framework offers several advantages: utilization of the readily available dye ThT, which eliminates the need for a laborious synthesis of custom fluorescent probes; ratiometric sensing, which improves quantitative analysis accuracy; and compatibility with complex aqueous media. The sensor's practical utility has been successfully demonstrated in artificial urine samples. In summary, our sensor system represents a significant advancement in the rapid, selective, and sensitive detection of the clinically crucial bioanalyte creatinine, offering potential benefits for the early diagnosis and management of kidney disorders.

Although N -acetylaspartate (NAA) has long been recognized as the most abundant amino acid in neurons by far, its primary role has remained a mystery. Based on its unique tertiary structure, we explored the potential of NAA to modulate aggregation of amyloid-beta (Aβ) peptide 1-42 via multiple corroborating aggregation assays along with electron microscopy. Thioflavin-T fluorescence assay demonstrated that at physiological concentrations, NAA substantially inhibited the initiation of Aβ fibril formation. In addition, NAA added after 25 min of Aβ aggregation was shown to break up preformed fibrils. Electron microscopy analysis confirmed the absence of mature fibrils following NAA treatment. Furthermore, fluorescence correlation spectroscopy and dynamic light scattering measurements confirmed significant reductions in Aβ fibril hydrodynamic radius following treatment with NAA. These results suggest that physiological levels of NAA could play an important role in controlling Aβ aggregation in vivo where they are both found in the same neuronal compartments. [ABSTRACT FROM AUTHOR]

Thioflavin-T (ThT) is the most commonly used fluorescent dye for following amyloid formation semi-quantitatively in vitro, specifically probing the fibrillar cross-β-sheet content. In recent years, structural polymorphism of amyloid fibrils has been shown to be an important aspect of amyloid formation, both in vitro and in neurodegenerative diseases. Therefore, understanding ThT-amyloid interactions in the context of structural polymorphism of amyloids is necessary for correct interpretation of ThT fluorescence data. Here we study the influence of fibril morphology on ThT fluorescence and ThT binding sites, with two morphologically distinct but chemically identical α-synuclein polymorphs. In ThT fluorescence assays the two polymorphs show type-specific fluorescence intensity behaviour although their β-sheet content has been shown to be similar. Further, fluorescence lifetime measurements of fibril-bound ThT reveal the presence of at least two qualitatively different ThT binding sites on the polymorphs. The relative distributions of the binding sites on the fibril surfaces appear to be morphology dependent, thus determining the observed polymorph-specific ThT fluorescence intensities. These results, highlighting the role of fibril morphology in ThT-based amyloid studies, underline the relevance of polymorphs in ThT-amyloid interaction and can explain the variability often observed in ThT amyloid binding assays. [ABSTRACT FROM AUTHOR]

The pathological Aβ aggregates associated with Alzheimer's disease follow a nucleation-dependent path of formation. A nucleus represents an oligomeric assembly of Aβ peptides that acts as a template for subsequent incorporation of monomers to form a fibrillar structure. Nuclei can form de novo or via surface-catalyzed secondary nucleation, and the combined rates of elongation and nucleation control the overall rate of fibril formation. Transthyretin (TTR) obstructs Aβ fibril formation in favor of alternative non-fibrillar assemblies, but the mechanism behind this activity is not fully understood. This study shows that TTR does not significantly disturb fibril elongation; rather, it effectively interferes with the formation of oligomeric nuclei. We demonstrate that this interference can be modulated by altering the relative contribution of elongation and nucleation, and we show how TTR's effects can range from being essentially ineffective to almost complete inhibition of fibril formation without changing the concentration of TTR or monomeric Aβ. [ABSTRACT FROM AUTHOR]

Fibril formation of the amyloid-β peptide (Aβ) follows a nucleation-dependent polymerization process and is associated with Alzheimer's disease. Several different lengths of Aβ are observed in vivo, but Aβ1–40 and Aβ1–42 are the dominant forms. The fibril architectures of Aβ1–40 and Aβ1–42 differ and Aβ1–42 assemblies are generally considered more pathogenic. We show here that monomeric Aβ1–42 can be cross-templated and incorporated into the ends of Aβ1–40 fibrils, while incorporation of Aβ1–40 monomers into Aβ1–42 fibrils is very poor. We also show that via cross-templating incorporated Aβ monomers acquire the properties of the parental fibrils. The suppressed ability of Aβ1–40 to incorporate into the ends of Aβ1–42 fibrils and the capacity of Aβ1–42 monomers to adopt the properties of Aβ1–40 fibrils may thus represent two mechanisms reducing the total load of fibrils having the intrinsic, and possibly pathogenic, features of Aβ1–42 fibrils in vivo. We also show that the transfer of fibrillar properties is restricted to fibril-end templating and does not apply to cross-nucleation via the recently described path of surface-catalyzed secondary nucleation, which instead generates similar structures to those acquired via de novo primary nucleation in the absence of catalyzing seeds. Taken together these results uncover an intrinsic barrier that prevents Aβ1–40 from adopting the fibrillar properties of Aβ1–42 and exposes that the transfer of properties between amyloid-β fibrils are determined by their path of formation. [ABSTRACT FROM AUTHOR]

The inevitable importance of basic amino acids, arginine and lysine, in human health and metabolism demands construction of efficient sensor systems for them. However, there are only limited reports on the ‘ratiometric’ detection of basic amino acids which is further restricted by the use of chemically complex sensor molecules, which impedes their prospect for practical applications. Herein, we report a ratiometric sensor system build on simple mechanism of disassociation of novel emissive Thioflavin-T H-aggregates from heparin surface, when subjected to interaction with basic amino acids. The strong and selective electrostatic and hydrogen bonding interaction of basic amino acids with heparin leads to large alteration in photophysical attributes of heparin bound Thioflavin-T, which forms a highly sensitive sensor platform for detection of basic amino acids in aqueous solution. These selective interactions between basic amino acids and heparin allow our sensor system to discriminate arginine and lysine from other amino acids. This unique mechanism of dissociation of Thioflavin-T aggregates from heparin surface provides ratiometric response on both fluorimetric and colorimetric outputs for detection of arginine and lysine, and thus it holds a significant advantage over other developed sensor systems which are restricted to single wavelength detection. Apart from the sensitivity and selectivity, our system also provides the advantage of simplicity, dual mode of sensing, and more importantly, it employs an inexpensive commercially available probe molecule, which is a significant advantage over other developed sensor systems that uses tedious synthesis protocol for the employed probe in the detection scheme, an impediment for practical applications. Additionally, our sensor system also shows response in complex biological media of serum samples. [ABSTRACT FROM AUTHOR]

We analysed specificity and sensitivity of confocal laser microscopy (CLSM) on tissue sections for a diagnosis of amyloidosis, in an attempt to reduce technical errors and better standardise pathological diagnosis. We first set up a protocol for the use of CLSM on this type of specimen, using a group of 20 amyloid negative and 20 positive samples. Of all specimens, 2, 4 and 8-μm sections were cut. Sections were stained with Congo red (CR) and thioflavin-T (ThT) and observed by cross-polarised light microscopy (CR-PL), epifluorescence microscopy (CRF-epiFM and ThT-epiFM) and CLSM (CRF-CLSM and ThT-CLSM). To validate the method in a diagnostic setting, we examined tissue samples from 116 consecutive patients with clinical suspicion of amyloidosis, selected from the period 2005 to 2014 from the database of the Pathology Unit of the University of Padua. The results were compared with those of transmission electron microscopy (TEM), which we consider as reference. We found that with CRF-CLSM, the false negative rate was reduced from 17 to 5%, while the sensitivity of detection increased to 12%. The results were in complete agreement with those of TEM ThT-CLSM; both sensitivity and specificity were 100%. Finally, ThT-CLSM results did not vary with section thickness, and small amounts of amyloid could even be detected in 2-μm sections. In conclusion, we found ThT-CLSM to be more sensitive as a screening method for amyloidosis than CR and ThT epifluorescence optical imaging. The method was easier to standardise, provided images with better resolution and resulted in more consistent pathologist diagnoses. [ABSTRACT FROM AUTHOR]

BACKGROUND Persistent hyperglycaemia causes increased advanced glycation end products ( AGEs), which contribute to the pathogenesis of diabetic complication. Therefore, effect of black gram milled by-product ( BGBP) extract on inhibition of AGE formation in a bovine serum albumin ( BSA)/glucose system was investigated. RESULTS BGBP extract had a total polyphenol content of 82 mg GAE g−1 and flavonoid content of 46 mg CE g−1. Ferulic acid, protocatechuic acid, gallic acid, gentisic acid, isovitexin, vitexin and epicatechin were the major bioactives in the extract. BGBP extract exhibited an effective Fe2+ chelating activity. Size exclusion-high-performance liquid chromatographic studies indicated that upon BSA-AGE formation the BSA monomer content was 38%; however, in the presence of BGBP extract at 50 and 100 µg levels, the monomer content increased and it was found to be 48% and 73%, respectively. BGBP extract at 50 and 100 µg levels decreased the protein carbonyl and fructosamine contents, and quenched the fluorescence intensity of glycated BSA in a dose-dependent manner. Further, fluorescence and transmission electron microscopic studies confirmed the decrease in formation of AGEs by BGBP extract. CONCLUSION As BGBP extract inhibited the formation of AGEs, the extract can be used as a nutraceutical or it can be incorporated into food products to obtain functional foods. © 2016 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

• The identified compounds could inhibit the growth of fibrils and oligomers of Aβ. • ZINC53276039 is a dual inhibitor of BACE1 and Aβ aggregation. • Dihydromyricetin could inhibit the Aβ fibrils. Aggregates of β-amyloid peptide are found to occur in brains of AD patients and are formed upon sequential cleavage of the amyloid precursor protein by BACE1 and γ-secretase. Strategies inhibiting either peptide aggregation or the rate limiting enzyme BACE1 have been in demand for its implication in AD therapeutics. The present study is undertaken to mine compounds with dual ability. In this context, some natural compounds that were already predicted as BACE1 inhibitors by our group, were further tested for their activity as aggregation inhibitors. A pharmacophore model built with known antiamyloidogenic compounds was then applied for screening the natural compounds previously predicted as BACE1 inhibitors. Subsequently experimental validation by Thioflavin-T and Aβ-GFP assay filtered four compounds genistein, syringetin, tamarixetin and ZINC53276039. Out of them, ZINC53276039 showed promising antiamyloidogenic activity to act as a potent inhibitor of aggregation. Interestingly, our previous study revealed syringetin and ZINC53276039 to be good BACE1 inhibitors while tamarixetin to be a moderate BACE1 inhibitor. These good to moderate BACE1 inhibitors with moderate to reasonable antiamyloidogenic activity might show potency in reducing the amyloid load of AD brains. [ABSTRACT FROM AUTHOR]

[Display omitted] • We report the first fluorescence 'Turn-On' probe for HMP detection. • Thioflavin-T displays enhanced fluorescence in the presence of HMP. • The method records a LOD of 116 nM for HMP in linear concentration range of 0–6 μM. • The enhanced fluorescence arises due to HMP induced aggregation of Thioflavin-T. • The methods shows high selectivity for HMP detection over other phosphate ions. Sodium hexa -meta-phosphate (HMP) is an important quality improver in the food industry. However, an excessive use of HMP not only deteriorates the nutrition value of the food but also causes ailments, such as, skeletal fluorosis, bone sclerosis, tumor lesions, osteoporosis etc. In this work, a simple, convenient, sensitive, rapid and selective 'Turn-On' method has been developed to monitor sodium hexa -meta-phosphate by using a molecular rotor probe Thioflavin-T (ThT). The sensing principle is based on aggregation induced emission of ThT by HMP due to charge neutralisation of ThT via electrostatic interactions between mono-cationic dye ThT and hexa -anionic hexa -meta-phosphate to form a fluorescent ThT-HMP aggregate complex. The ThT-HMP aggregate system has been thoroughly investigated by using steady-state fluorescence, ground-state absorption, and time resolved fluorescence spectroscopy at different conditions, such as, salt, pH, temperature etc. Further, zeta potential measurements have also been performed to support the results. Also, the selectivity of ThT-HMP system has been tested against other common phosphate ions. The sensing method records a LOD of 116 nM for HMP over a wide linear concentration range of 0–6 μM. The sensing probe is highly selective for HMP over other common phosphate ions present in aqueous solution. Finally, the potential use of the probe has also been demonstrated in real water sample collected after rinsing the frozen green peas. Thus, the current sensing method is a sensitive fluorescence 'Turn-On' probe to detect HMP in real samples. [ABSTRACT FROM AUTHOR]

[Display omitted] • We report a fluorescence-based ratiometric sensor for Al3+ detection. • Thioflavin-T displays monomer-aggregate equilibrium in the presence of poly(acrylic) acid. • Al3+ modulated monomer-aggregate equilibrium of Thioflavin-T and generates a ratiometric response. • The method records a LOD of 0.9 μM for Al3+ in linear concentration range of 0–14 μM. • The methods shows high selectivity for Al3+ and capability to detect Al3+ in tap water. Aluminium ions play an important role in various biological processes and in the development of methods for its detection has received significant attention recently. Among the reported analytical methods for aluminium ions, fluorescence based detection is considered very advantageous because of its extreme sensitivity and simplicity. However, a large majority of these reported sensor systems for aluminium detection are either based on synthetically involved fluoregenic probes or perform sensing operation via single wavelength based response which makes them susceptible to error due to minute variations in analyte-independent experimental variables. Thus, in the present contribution, we describe a fluorescence ratiometric sensor for the sensitive and selective detection of Al3+ which is based on Al3+ ion dependent modulation of aggregate-monomer equilibrium of Thioflavin- T -Polyacrylic acid system. The molecular rotor dye, Thioflavin-T (ThT), undergoes efficient aggregation, with a distinct emission band, in the presence of a carboxyl laced polyelectrolyte, poly (acrylic acid), PAA. This ThT-PAA monomer-aggregate system undergoes significant modulation in the presence of Al3+ ion due to selective interaction of hard Al3+ ions with hard donors like oxygen containing carboxylate groups. This modulation provides a fluorescence ratiometric response for Al3+ originating from distinct spectroscopic features of ThT monomers and aggregates. Overall, the sensor system provides a selective and sensitive response towards detection of Al3+ with a LOD of 0.9 μM. Importantly, the sensor system is composed of commercially available components and provides freedom from the reliance on time-consuming and tedious protocols required for producing the probe molecules. The ratiometric response provides a robustness to the analytical performance as compared to the system that operates through single wavelength based measurements. We have also demonstrated the sensor system response in real water samples from tap water. [ABSTRACT FROM AUTHOR]

Early detection of amyloid fibrils is very important for the timely diagnosis of several neurological diseases. Thioflavin-T (ThT) is a gold standard fluorescent probe for amyloid fibrils and has been used for the last few decades. However, due to its positive charge, ThT is incapable of crossing the blood-brain barrier and cannot be used for in vivo imaging of fibrils. In the present work, we synthesized a neutral ThT derivative, 2-[2'-Me,4'-(dimethylamino)phenyl]benzothiazole (2Me-DABT), which showed a strong affinity towards the amyloid fibrils. On association with the amyloid fibrils, 2Me-DABT not only showed a large increase in its emission intensity, but also, unlike ThT, a large blueshift in its emission spectrum was observed. Thus, unlike ThT, 2Me-DABT is a potential candidate for the ratiometric sensor of the amyloid fibrils. Detailed photophysical properties of 2Me-DABT in amyloid fibrils and different solvent media were studied to understand its sensory activity. Fluorescence resonance energy transfer (FRET) studies suggested that the sites of localization for ThT and 2Me-DABT in amyloid fibrils are not same and their average distance of separation in amyloid fibrils was determined. The experimental data was nicely supported by molecular docking studies, which confirmed the binding of 2Me-DABT in the inner core of the amyloid fibrils. [ABSTRACT FROM AUTHOR]

Most macrocyclic host molecules, including cyclodextrins, usually prevent self-aggregation of the guest organic molecules, by exploiting inclusion complexation of the guest with the host. In this work, it was found that a negatively charged β-cylcodextrin derivative induces aggregation of a well-known amyloid sensing dye, Thioflavin-T, and leads to an unprecedented formation of the rarely observed emissive H-type aggregates of the dye. [ABSTRACT FROM AUTHOR]

Identification of the chemical and biological properties of amyloid fibrils is important for understanding their roles in human diseases and to clarify the mechanisms that govern their formation. In pursuit of these goals, small molecule fluorescent dyes have received increasing attention as probes of amyloid conformations. In this study, we report on the ability of YOYO-1, a homodimeric derivative of oxazole yellow, to detect fibrils formed by the Alzheimer's disease related Aβ(1–42) peptide. We find that YOYO-1 binds to Aβ(1–42) fibrils with the long axes of its oxazole yellow moieties parallel to the fibril axis, resulting in a 200x emission enhancement; a result that shows that YOYO-1 is a sensitive amyloid probe. Further, YOYO-1 exhibits characteristic absorption shifts upon binding to the Aβ(1–42) fibrils that we attribute to a self-stacking to non-stacking transition in its homodimer configuration; herein we show how this phenomenon can be exploited to estimate the degree of dye binding. Furthermore, we show that YOYO-1 can be used to monitor the kinetics of amyloid formation reactions. Taken together, our results show that YOYO-1 is a sensitive amyloid probe that can operate with both absorption and fluorescence read-outs, and this suggests that this commercially available dye could become a useful complement to thioflavin-T for in vitro amyloid-sensing applications. [ABSTRACT FROM AUTHOR]

CRISPR/Cas12a, as a powerful and programmable biosensing tool, has brought great convenience in visual detection of the target in a sequence-specific mode. Though fluorescent output is the predominant way of present visual methods, this strategy highly depends on cleaving fluorophore-/quencher-labeled oligonucleotides. Few research focuses on realizing fluorescent visual detection without DNA-labeling. Herein, we have proposed a CRISPR/Cas12a-based label-free fluorescent visual detection strategy. The basic principle of this strategy is that G-quadruplex can enhance the fluorescent emission of fluorescent ligands while dsDNA target-activated Cas12a make them impotent by disrupting the higher-ordered structure. After comparing four kinds of G-quadruplex-specific ligands, we selected Thioflavin T (ThT) to achieve the visual observation goal. The application feasibility was confirmed by combining with loop-mediated isothermal amplification (LAMP) for detection of Vibrio parahaemolyticus (V. parahaemolyticus), and it demonstrated a high sensitivity (1.36 × 102 copies) and specificity (among 12 kinds of bacteria). The method displayed similar performance as the real-time PCR for detection of real samples yet had lower requirement on the instrument. The signal output format could also distinguish Salmo salar from other seven kinds of sequence-similar Salmonidae. The findings and strategy proposed in this manuscript will expand the application of CRISPR/Cas12a-based label-free fluorescent analysis. • A label-free and visible fluorescent reporter of CRISPR/Cas12a has been developed. • CRISPR/Cas12a makes G-quadruplex impotent to enhance the fluorescence of ThT. • ThT is better than NMM to visually probe the cleavage of G-quadruplex. • Combined with LAMP, V. parahaemolyticus was detected specifically and sensitively. [ABSTRACT FROM AUTHOR]

The premelanosomal protein (Pmel17) is a human functional amyloid that promotes pigmentation by serving as a scaffold for melanin polymerization. This occurs within the melanosome, where Pmel17 is first proteolyzed into smaller domain(s) that are responsible for fibril formation. Our work has shown that the Pmel17 repeat domain (RPT, residues 315-444) forms amyloid fibrils in vitro under acidic conditions similar to those found in melanosomes. Mechanistically, this is driven by the protonation of acidic residues, resulting in charge neutralization and subsequent aggregation. Interestingly, the deprotonation of acidic residues leads to rapid disaggregation, highlighting a reversible mechanism of fibril formation and dissolution thus far only observed for functional amyloid proteins. In this chapter, we describe how to monitor pH-dependent RPT aggregation and disaggregation using extrinsic thioflavin-T and intrinsic tryptophan fluorescence, respectively. These methods can also be adapted more broadly to investigate the reversibility of other amyloid systems, both functional and pathogenic., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Growing evidence indicates that liquid-liquid phase separation (LLPS), a phenomenon whereby transient, weak interactions can facilitate self-assembly of proteins into liquid-like droplets and can contribute to the formation of amyloid fibrils. Such an observation has posited that LLPS and the associated formation of membrane-less organelles in the cell can contribute to protein aggregation in neurodegenerative disease. In this chapter, we describe methods for performing biophysical studies on the transactive response DNA-binding protein of 43 kDa (TDP-43), a protein that forms aggregates in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We describe purification of the disordered low-complexity domain (LCD) of TDP-43 and provide a methodology for studying the protein's behavior using site-directed spin labeling coupled with electron paramagnetic resonance. We additionally discuss visualization of TDP-43 LCD liquid droplets and methods for quantifying LLPS and aggregation into amyloid fibrils., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Thioflavin-T (ThT) is one of the most commonly used dyes for amyloid detection, but the origin of its fluorescence enhancement is not fully understood. Herein we have characterised the ThT fluorescence response upon binding to the Aβ(1-40) and Aβ(1-42) variants of the Alzheimer's-related peptide amyloid-β, in order to explore how the photophysical properties of this dye relates to structural and morphological properties of two amyloid fibril types formed by peptides with a high degree of sequence homology. We show that the steady-state ThT fluorescence is 1.7 times more intense with Aβ(1-40) compared to Aβ(1-42) fibrils in concentration matched samples prepared under quiescent conditions. By measuring the excited state lifetime of bound ThT, we also demonstrate a distinct difference between the two fibril isoforms, with Aβ(1-42) fibrils producing a longer ThT fluorescence lifetime compared to Aβ(1-40). The substantial steady-state intensity difference is therefore not explained by differences in fluorescence quantum yield. Further, we find that the ThT fluorescence intensity, but not the fluorescence lifetime, is dependent on the fibril preparation method (quiescent versus agitated conditions). We therefore propose that the fluorescence lifetime is inherent to each isoform and sensitively reports on fibril microstructure in the protofilament whereas the total fluorescence intensity relates to the amount of exposed β-sheet in the mature Aβ fibrils and hence to differences in their morphology. Our results highlight the complexity of ThT fluorescence, and demonstrate its extended use in amyloid fibril characterisation. [ABSTRACT FROM AUTHOR]

Using sub-picosecond fluorescence upconversion spectroscopy, the effect of supramolecular confinement on the ultrafast excited state torsional dynamics of an amyloid fibril sensing dye, Thioflavin-T (ThT), is investigated in a novel cyclodextrin derivative, Sulphobutylether β-cyclodextrin (SBE-β-CD). The encapsulation of ThT inside the nanocavity of SBE-β-CD results in a significant increase in the emission intensity and the excited state lifetime of ThT when compared with native β-CD. Detailed analysis of the time-resolved emission spectra (TRES) shows that the time dependent changes in the integrated area, mean frequency and the width of the emission spectra are appreciably slower as compared with bulk water and native β-CD. These features suggest a significant confinement effect on the ultrafast torsional dynamics of ThT. The ionic strength of the medium significantly affects the complexation of ThT with SBE-β-CD, which indicates that the interaction between the host and the guest is predominantly electrostatic in nature. The results further suggest that the hydrophobic interaction in SBE-β-CD, although minor in contribution in the present case, is comparatively stronger than the native β-CD. [ABSTRACT FROM AUTHOR]

A unique supramolecular assembly based fluorescence turn-on and ratiometric detection for a common food adulterant, Melamine, has been achieved. [Display omitted] • Melamine is a life-threatening adulterant for food and dairy products. • A supramolecular assembly is formed consisting of sulfated cyclodextrin (SCD), Ag+ and melamine. • The supramolecular assembly encapsulates Thioflavin-T to generate a turn-on fluorescence response. • The system shows a LOD of 1.5 μM with very high selectivity towards melamine. • The performance in the commercial milk sample has been demonstrated. Nitrogen-rich melamine has been often used by fraudulent merchants as an adulterant to food and dairy products which exhibit toxic effects, leading to life-threatening diseases. This has triggered enormous activity on developing methods for the recognition of melamine in various food items for protecting public health and food safety. In recent times, fluorescence methods have emerged as powerful analytical tools for sensor development. The previously reported fluorescence-based methods for melamine detection, either require tedious synthesis procedures or operate through single wavelength-based signal output, making their accuracy susceptible to minute changes in the experimental environment. Herein, to overcome these limitations, we report the recognition of melamine which is achieved through the formation of a serendipitously discovered unique supramolecular assembly. The assembly consists of sulfated cyclodextrin, silver ion and melamine which, in turn, encapsulates a molecular rotor dye, Thioflavin-T, to yield a fluorescence turn-on response towards melamine. Importantly, the supramolecular assembly is responsive at dual wavelengths and is builtup with all commercially available economical molecules. Overall, the present system offers a simple, sensitive, label-free and highly selective detection for melamine. Importantly, the melamine containing supramolecular ensemble is also found to perform in commercial milk sample promising its future application prospect. [ABSTRACT FROM AUTHOR]