Mitochondria-Directing Fluorogenic Probe: An Efficient Amyloid Marker for Imaging Lipid Metabolite-Induced Protein Aggregation in Live Cells and Caenorhabditis elegans.

The design and development of optical probes for sensing neurotoxic amyloid fibrils are active and important areas of research and are undergoing continuous advancements. In this paper, we have synthesized a red emissive styryl chromone-based fluorophore (SC1) for fluorescence-based detection of amyloid fibrils. SC1 records exceptional modulation in its photophysical properties in the presence of amyloid fibrils, which has been attributed to the extreme sensitivity of its photophysical properties toward the immediate microenvironment of the probe in the fibrillar matrix. SC1 also shows very high selectivity toward the amyloid-aggregated form of the protein as compared to its native form. The probe is also able to monitor the kinetic progression of the fibrillation process, with comparable efficiency as that of the most popular amyloid probe, Thioflavin-T. Moreover, the performance of SC1 is least sensitive to the ionic strength of the medium, which is an advantage over Thioflavin-T. In addition, the molecular level interaction forces between the probe and the fibrillar matrix have been interrogated by molecular docking calculations which suggest the binding of the probe to the exterior channel of the fibrils. The probe has also been demonstrated to sense protein aggregates from the Aß-40 protein, which is known to be responsible for Alzheimer's disease. Moreover, SC1 exhibited excellent biocompatibility and exclusive accumulation at mitochondria which allowed us to successfully demonstrate the applicability of this probe to detect mitochondrial-aggregated protein induced by an oxidative stress indicator molecule 4-hydroxy-2-nonenal (4-HNE) in A549 cell lines as well as in a simple animal model like Caenorhabditis elegans. Overall, the styryl chromone-based probe presents a potentially exciting alternative for the sensing of neurotoxic protein aggregation species both in vitro as well as in vivo.

Guest Binding with Sulfated Cyclodextrins: Does the Size of Cavity Matter?

Sulfated cyclodextrins have recently emerged as potential candidates for producing host-induced guest aggregation with properties better than p-sulfonatocalixarenes that have previously shown numerous applications involving the phenomena of host-induced guest aggregation. In the class of sulfated cyclodextrins (SCD), sulfated ß-cyclodextrin (ß-SCD) remains the most extensively investigated host molecule. Although it is assumed that the host-induced guest aggregation is predominantly an outcome of interaction of the guest molecule with the charges on the exterior of SCD cavity, it has not been deciphered whether the variation in the cavity size will make a difference in the efficiency of host-induced guest-aggregation process. In this investigation, we present a systematic study of host-induced guest aggregation of a cationic molecular rotor dye, Thioflavin T (ThT) with three different sulfated cyclodextrin molecules, α-SCD, ß-SCD and γ-SCD, which differ in their cavity size, using steady-state emission, ground-state absorption and time-resolved emission measurements. The obtained photophysical properties of ThT, upon interaction with different SCD molecules, indicate that the binding strength of ThT with different SCD molecules correlate with the cavity size of the host molecule, giving rise to the strongest complexation of ThT with the largest host molecule (γ-SCD). The binding affinity of ThT towards different host molecules has been supported by molecular docking calculations. The results obtained are further supported with the temperature and ionic strength dependent studies performed on the host-guest complex. Our results indicate that for host-induced guest aggregation, involving oppositely charged molecules, the size of the cavity also plays a crucial role beside the charge density on the exterior of host cavity.

Supramolecular Control on the Optical Properties of a Dye-Polyelectrolyte Assembly.

Control of fluorescent molecular assemblies is an exciting area of research with large potential for various important applications, such as, fluorescence sensing/probing, cell imaging and monitoring drug-delivery. In the present contribution, we have demonstrated control on the extent of aggregation of a dye-polyelectrolyte assembly using a macrocyclic host molecule, sulfobutylether-ß-cyclodextrin (SBE-ß-CD). Initially, a cationic molecular rotor based organic dye, Auramine-O (AuO), undergoes aggregation in the presence of an anionic polyelectrolyte, polystyrene sulfonate (PSS), and displays a broad intense new emission band along with large variation in its absorption features and excited-state lifetime. A manipulation of the monomer-aggregate equilibrium of the dye-polyelectrolyte assembly has been achieved by introducing a cyclodextrin based supramolecular host, SBE-ß-CD, which leads to relocation of AuO molecules from polyelectrolyte (PSS) to supramolecular host cavity, owing to the formation of a host-guest complex between AuO and SBE-ß-CD. A reversible control on this manipulation of monomer-aggregate equilibrium is further achieved by introducing a competitive guest for the host cavity i. e., 1-Adamantanol. Thus, we have demonstrated an interesting control on the dye-polyelectrolyte aggregate assembly using a supramolecular host molecule which open up exciting possibilities to construct responsive materials using a repertoire of various host-specific guest molecules.

Effect of counter-anions on the aggregation of Thioflavin-T.

The aggregation of small molecules in aqueous solution is known to be influenced by the ionic strength of the medium; however, the role played by the identity of salt in the phenomenon of small molecule aggregation is rarely investigated. In the present contribution, we have investigated the effect of counter-anions on the aggregation of a popular cationic amyloid sensing probe, Thioflavin-T (ThT), by taking six different anions, viz. chloride, bromide, acetate, iodide, tetrafluoroborate, and perchlorate. Our results clearly indicate that it is not the ionic strength of the medium which solely controls aggregation of small molecules but distinct ions behave distinctly with regard to the organization. In fact, distinct ion effects play a major role in the salt induced organization of fluorophores. Using detailed steady-state emission, time-resolved emission, and ground-state absorption measurements, the optical properties of salt induced aggregates of ThT have been characterized. We have rationalized our observations on the basis of the theory of matching water affinity, which suggests that the matching free hydration energy is a critical aspect for the formation of contact ion pairs, which eventually results in aggregation. In brief, a larger sized anion, perchlorate, has a lower free energy of hydration and forms a suitable contact ion pair, with a larger organic cation, ThT, having weaker hydration. This contact ion-pair formation subsequently leads to the formation of an aggregate assembly which is found to be emissive in nature. Therefore, it is possible to induce aggregation of ThT by selecting the right counterion with the appropriate size, which may help us to evaluate the false positive signals when high ionic strength and specific counterions are present in the sensing matrix.

Supramolecular tuning of thioflavin-T aggregation hosted by polystyrene sulfonate.

Tunable and controllable emission is an extremely desirable feature for advanced functional materials that finds usage in optoelectronic utilization, fluorescence probing/sensing, drug-delivery monitoring, etc. In the present contribution, we have employed a macrocyclic host molecule, sulfobutyl ether-ß-cyclodextrin (SBE-ß-CD), as a tuning agent for an intensely emissive aggregate assembly of a molecular rotor dye, thioflavin-T (ThT), in the presence of an anionic polyelectrolyte, polystyrene sulfonate (PSS). The macrocyclic host breaks the PSS templated ThT aggregates and leads to encapsulation of released ThT molecules, tailoring the emission response of the system in terms of intensity and wavelength. Utilizing the established selectivity of the cyclodextrin-adamantane system, reverse control of this tunable emission has been further achieved. The controllable fluorescence system has been extensively investigated using ground-state absorption, steady-state and time-resolved emission spectroscopy. This kind of supramolecular tailoring of self-assembled aggregate emission has enormous potential in the field of fluorescence sensors and probes, and imaging and tracking in biological systems.

A turn-on fluorescence sensor for detection of heparinase with heparin templated aggregation of tetracationic porphyrin derivative.

Heparinase is the only mammalian endoglycosidase that breaks down the commonly used blood-anticoagulant heparin into therapeutically relevant low-molecular-weight-heparin. Importantly, heparinase has been considered a malignant disease diagnostic marker. Thus, it is essential to develop detection scheme for heparinase. However, optical methods for heparinase determination are limited. In the present work, we report a turn-on fluorescence sensor for detection of heparinase that utilizes heparin-templated aggregation of a tetra-cationic porphyrin derivative, TMPyP4+, as a sensing framework. Heparinase cleaves the glycosidic linkage between hexosamine and uronic acid in the structure of heparin to destroy its polyelectrolytic nature that originally causes the aggregation of TMPyP4+. Thus, heparinase leads to dissociation of TMPyP4+ aggregates and generates an optical signal. This system leads to a sensitive and selective response towards heparinase with a Limit of Detection (LOD) of 0.3 pmol/L. Further, the same system is demonstrated to sense a trace amount of Oversulfated Chondrootin Sulphate (OSCS) in heparin, which is a heparin adulterant, by utilizing the fact that OSCS serves as an inhibitor for heparinase activity, which leads to reverse modulation in the photo-physical features of the monomer/aggregate equilibrium of the TMPyP4+-heparin-heparinase system. The sensing mechanism has been thoroughly demonstrated by ground-state absorption, steady-state emission, and time-resolved emission measurements. The selectivity of the sensor was tested using lysozyme, α-amylase, pepsin, trypsin, lipase, and glucose oxidase in the heparinase selectivity study and the method is also validated using another method reported in the literature. The study provides a new approach for the development of optical methods for the detection of heparinase and oversulfated chondroitin sulfate, which is currently limited.

Anionic Polyelectrolyte-Induced Aggregation of Basic Orange 21: A Clue toward Metachromasia.

The change in the color of chromophore upon being embedded in a biological tissue is known as metachromasia. Basic Orange 21 (BO21) is a cationic polymethine dye that has been implied as a supravital dye, which produces metachromasia in leukocytes. An improved differential counting of leukocytes has been achieved in the clinical setup based on characteristic metachromatic expressions of BO21 for different types of leukocytes. Although BO21 has been utilized as a chromatic indicator for leukocyte counting, there are limited number of investigations that focus on the factors that may be responsible for the spectral shift in absorption and emission spectra of BO21, which leads to its metachromatic behavior. In this work, we have investigated the effect of a synthetic anionic polyelectrolyte, polystyrene sulfonate (PSS), on the photophysical properties of BO21, using steady-state emission, ground-state absorption, and time-resolved emission measurements, to get an understanding of the factors that may be responsible for the spectral shift of BO21 in the cellular environment. PSS induces aggregation of BO21 molecules with large changes in its photophysical properties; this appears to be most likely the mechanism of spectral shift for BO21 reported in the cellular environment. The employment of external stimulus reveals BO21 aggregates to be significantly responsive toward external stimuli, for example, temperature and presence of salt in the medium, which further strengthens the proposal of aggregate formation. Further, we have also employed fluorescence upconversion spectroscopy with subpicosecond time resolution to estimate the excited-state lifetime of BO21.

A colorimetric and fluorometric based dual readout approach for effective heparin sensing.

Devising fluorescence-based turn-on probes for the specific and sensitive detection of Heparin is of utmost clinical importance. In this contribution, we have identified a molecular rotor based asymmetric cyanine probe, thiazole orange (TO), which enables an efficient colorimetric and fluorimetric detection of Heparin. TO undergoes the formation of emissive H-aggregates upon interaction with Heparin that display an impressive emission enhancement of ~22 fold together with drastic changes in the absorption spectra that yields a prominent colour change in the solution from orange to yellow. These seldom reported emissive H-aggregates of TO, serve as an efficient platform for Heparin detection with a LOD of 19 nM, fluorometrically and 34 nM, colorimetrically. The TO-Heparin complex is also accompanied by a large change in the excited-state lifetime. The TO-Heparin complex has been further utilized for the detection of Protamine, which is the only medically affirmed antitoxin of Heparin. Overall, our sensing system offers several advantages, such as, simple, dual read-out, economic and specific detection of Heparin with longer excitation and emission wavelength, rapid naked eye detection and utilizes an in-expensive commercially available fluoprophore, TO. Most importantly, our sensing system also displays a good performance in the biologically complex human serum matrix.