Molecular Rotor Dye Research Articles

Cy3-Based Nanoviscosity Determination of Mucus: Effect of Mucus Collection Methods and Antibiotics Treatment.

The integrity of the protective mucus layer as a primary defense against pathogen invasion and microbial leakage into the intestinal epithelium can be compromised by the effects of antibiotics on the commensal microbiome. Changes in mucus integrity directly affect the solvent viscosity in the immediate vicinity of the mucin network, that is, the nanoviscosity, which in turn affects both biochemical reactions and selective transport. To assess mucus nanoviscosity, a reliable readout via the viscosity-dependent fluorescence lifetime of the molecular rotor dye cyanine 3 is established and nanoviscosities from porcine and murine ex vivo mucus are determined. To account for different mucin concentrations due to the removal of digestive residues during mucus collection, the power law dependence of mucin concentration on viscosity is used. The impact of antibiotics combinations (meropenem/vancomycin, gentamycin/ampicillin) on ex vivo intestinal mucus nanoviscosity is presented. The significant increase in viscosity of murine intestinal mucus after treatment suggests an effect of antibiotics on the microbiota that affects mucus integrity. This method will be a useful tool to assess how drugs, directly or indirectly, affect mucus integrity. Additionally, the method can be utilized to analyze the role of mucus nanoviscosity in health and disease, as well as in drug development.

Thioflavin-T: A Quantum Yield-Based Molecular Viscometer for Glycerol-Monohydroxy Alcohol Mixtures.

Molecular rotor dye thioflavin T (ThT) is almost nonfluorescent in low-viscosity solvents but highly fluorescent when bound to amyloid fibrils. This unique property arises from the rotation of the dimethylaniline moiety relative to the benzothiazole moiety in the excited state, which drives the dye from an emissive locally excited state to a twisted intramolecular charge-transfer state. This process is viscosity-controlled, and therefore, we can use the quantum yield of ThT to assess the viscosity of the environment. In this study, we have investigated the quantum yield of ThT (φThT) in various compositions of six alcoholic solvent mixtures of glycerol with methanol, ethanol, n-propanol, iso-propanol, n-butanol, and tert-butanol. We have proposed an empirical model using φThT as a function of the mole fraction of glycerol to estimate the interaction parameters between the components of the solvent mixtures. This analysis allowed us to predict the extent of nonideality of the solvent mixtures. The Förster-Hoffmann- and Loutfy-Arnold-type power law relationship was established between the quantum yield of ThT and bulk viscosity for solvent mixtures of methanol, ethanol, n-butanol, and tert-butanol with glycerol, and it was found to be similar in nature in all the four mixtures. Applying this knowledge, we proposed a methodology to quantify and predict the bulk viscosity coefficient values of several compositions of n-propanol-glycerol and iso-propanol-glycerol mixtures which have not been previously documented.

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 β-cyclodextrin (β-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, β-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.

A molecular rotor based ratiometric detection scheme for aluminium ions in 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.

A unique supramolecular assembly between sulfated cyclodextrin, silver and melamine: Towards a fluorescence based dual wavelength detection approach for melamine

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.

A Heparin based dual ratiometric sensor for Thrombin

Thrombin is an important enzyme that plays a pivotal role in the blood clotting pathways. An imbalance in the activity of this enzyme is clinically known to be associated with various diseases, such as thrombosis, inflammation, atherosclerosis, and haemophilia, suggesting the need to devise sensors for Thrombin detection. However, the majority of the fluorescence-based Thrombin assays rely on fluorescence labelling assays or Thrombin specific recognition biomolecules, such as, aptamers or antibody which requires sophisticated techniques and makes it very expensive. Herein, we report a simple, selective, sensitive and label-free fluorescence detection scheme for Thrombin which is based on the interaction between Thrombin and a fluorescent complex of Heparin with a molecular rotor dye, Thioflavin-T. The detection scheme exploits selective interaction between cationic Thrombin and anionic Heparin to modulate the monomer-aggregate equilibrium of the Thioflavin-T–Heparin system. Importantly, the present system offers a ratiometric response that has the ability for robust quantification of Thrombin concentration even in complex medium. The involvement of all commercially available components is a crucial advantage of this detection scheme. Further, the detection scheme also shows reasonable response in diluted serum matrix.

A novel supramolecule-based fluorescence turn-on and ratiometric sensor for highly selective detection of glutathione over cystein and homocystein.

Acyclodextrin-based fluorescence light-up and ratiometric sensor is reportedfor 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 successivelycaused 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 0-250μM with a LOD of 2.4 ± 0.2μM in aqueous solution and 13.6 ± 0.5μM in diluted human serum sample. The sensor system is excited at 410nm and the emission signal is plotted as a ratio of intensity at 545nm (aggregate band) and 490nm (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. Graphical abstract.

An anionic polyelectrolyte induced aggregate assembly of Thioflavin-T: A prospective platform for Protamine sensing

Protamine, a polycation, is biologically and medically relevant protein. Protamine exhibits a wide array of functions in biological processes like gene transfer, tissue and organogenesis, cell reproduction, etc. Medically, Protamine is the only clinically approved antidote for Heparin and is routinely used in various surgical interventions, and hence controlling Protamine dosing in patients is very crucial. Taking into account the medical significance of Protamine, designing simple, reliable and sensitive fluorescence sensors is highly desirable. In this work, we propose one such sensitive and reliable fluorescent sensor which is based on a template of dye-polyelectrolyte assembly constituting a molecular rotor dye, Thioflavin-T and an anionic synthetic polyelectrolyte, polystyrene sulfonate. The addition of Protamine, prompts drastic modulations in spectral features of dye-polyelectrolyte assembly which enables sensitive detection of Protamine in aqueous solution. Apart from sensitive detection, our sensing platform aids in highly selective sensing of Protamine compared to other proteins. Moreover, our sensor system is constructed on label-free, inexpensive, commercially available molecules posing as an advantage over other sensor systems which involve laborious synthesis protocols. Most importantly, our sensor template is able to sense Protamine in diluted serum sample, indicating the potential practical utility of our sensor system.

High-Throughput Image-Based Aggresome Quantification.

Aggresomes are subcellular perinuclear structures where misfolded proteins accumulate by retrograde transport on microtubules. Different methods are available to monitor aggresome formation, but they are often laborious, time-consuming, and not quantitative. Proteostat is a red fluorescent molecular rotor dye, which becomes brightly fluorescent when it binds to protein aggregates. As this reagent was previously validated to detect aggresomes, we have miniaturized its use in 384-well plates and developed a method for high-throughput imaging and quantification of aggresomes. Two different image analysis methods, including one with machine learning, were evaluated. They lead to similar robust data to quantify cells having aggresome, with satisfactory Z' factor values and reproducible EC50 values for compounds known to induce aggresome formation, like proteasome inhibitors. We demonstrated the relevance of this phenotypic assay by screening a chemical library of 1280 compounds to find aggresome modulators. We obtained hits that present similarities in their structural and physicochemical properties. Interestingly, some of them were previously described to modulate autophagy, which could explain their effect on aggresome structures. In summary, we have optimized and validated the Proteostat detection reagent to easily measure aggresome formation in a miniaturized, automated, quantitative, and high-content assay. This assay can be used at low, middle, or high throughput to quantify changes in aggresome formation that could help in the understanding of chemical compound activity in pathologies such as protein misfolding disorders or cancer.

A molecular rotor based dual ratiometric sensor for heparinase

Heparinase is a mammalian endoglycosidase which specifically degrades Heparin, the widely used blood-anticoagulant, into smaller fragments and produces therapeutically important low molecular weight Heparin. More importantly, the increased expression of Heparinase was found to correlate with malignancy of tumour and its ability to metastasize. Considering the various crucial biological roles played by Heparinase, it is important to develop detection scheme for Heparinase. However, very few reports exist for detection of Heparinase, especially from the viewpoint of ratiometric detection. Herein, we report a selective and sensitive fluorescence ratiometric sensor for the detection of Heparinase that has been developed by utilizing a fluorescent complex of a molecular rotor dye, Thioflavin T with Heparin. Heparinase specifically degrades Heparin into smaller fragments which leads to the release of the Thioflavin-T aggregates from the surface of Heparin and leads to large modulations in the fluorescence properties of Thioflavin T-Heparin complex. The sensing mechanism has been demonstrated by utilizing a variety of spectroscopic techniques such as ground-state absorption, steady-state and time-resolved emission and circular-dichroism spectroscopy. The sensing scheme provides a simple, sensitive, selective, and label-free approach for detection of Heparinase. Importantly, the sensing scheme involves all commercially available molecules which provides a significant advantage in terms of relief from extreme dependence on time-consuming and tedious synthetic pathways. More importantly, the current sensing scheme also shows response towards Heparinase in serum matrix.

Rotationally Gated Fluorescence: Common Theme Behind Molecular Rotors, Photoactivation and Förster Resonance Energy Transfer (FRET)

About the common features behind the mechanism of molecular rotors, photoswitching of engineered fluorescent proteins (GFPs), and Forster resonance energy transfer (FRET). Besides explining the common mechanism, we also offer a new method for increasing sensitivity of viscosity determination even in living cells, by the combination of polarization energy transfer (polFRET) method and a molecular rotor dye either as an energy donor or acceptor.

Chromatography process development aided by a dye‐based assay

AbstractBACKGROUNDThe lifetime of chromatography resins typically averages between 10 and 300 cycles for the manufacture of a therapeutic protein. Developing and establishing the robustness of the method for each separation process represents a significant challenge and is subject to extensive regulatory oversight. This paper presents a novel fluorescence‐based assay for residual aggregated proteins to aid the evaluation of the extent of resin regeneration.RESULTSThe versatility of this method was demonstrated by using strong anion and cation exchange agarose resins Praesto Q and SP in conjunction with bovine serum albumin and monoclonal antibody feed materials. The assay entails applying a molecular rotor dye to a sample of free resin and measuring the fluorescence intensity using a plate reader or visualizing under a confocal laser scanning microscope to gain a more detailed characterization. Following five consecutive chromatography cycles, both methods revealed a 10‐fold increase in fluorescence intensity along with a proportional reduction in dynamic binding capacity. Furthermore, the use of the assay suggested that fouling was dependent on spatial bead position in the column, bead channel structure and cleaning conditions.CONCLUSIONThis work presents a simple assay suitable for use in resin lifetime studies to enhance process understanding. © 2019 Society of Chemical Industry

Rapid Discovery of Illuminating Peptides for Instant Detection of Opioids in Blood and Body Fluids.

The United States is currently experiencing an opioid crisis, with more than 47,000 deaths in 2017 due to opioid overdoses. Current approaches for opioid identification and quantification in body fluids include immunoassays and chromatographic methods (e.g., LC-MS, GC-MS), which require expensive instrumentation and extensive sample preparation. Our aim was to develop a portable point-of-care device that can be used for the instant detection of opioids in body fluids. Here, we reported the development of a morphine-sensitive fluorescence-based sensor chip to sensitively detect morphine in the blood using a homogeneous immunoassay without any washing steps. Morphine-sensitive illuminating peptides were identified using a high throughput one-bead one-compound (OBOC) combinatorial peptide library approach. The OBOC libraries contain a large number of random peptides with a molecular rotor dye, malachite green (MG), that are coupled to the amino group on the side chain of lysine at different positions of the peptides. The OBOC libraries were then screened for fluorescent activation under a confocal microscope, using an anti-morphine monoclonal antibody as the screening probe, in the presence and absence of free morphine. Using this novel three-step fluorescent screening assay, we were able to identify the peptide-beads that fluoresce in the presence of an anti-morphine antibody, but lost fluorescence when the free morphine was present. After the positive beads were decoded using automatic Edman microsequencing, the morphine-sensitive illuminating peptides were then synthesized in soluble form, functionalized with an azido group, and immobilized onto microfabricated PEG-array spots on a glass slide. The sensor chip was then evaluated for the detection of morphine in plasma. We demonstrated that this proof-of-concept platform can be used to develop fluorescence-based sensors against morphine. More importantly, this technology can also be applied to the discovery of other novel illuminating peptidic sensors for the detection of illicit drugs and cancer biomarkers in body fluids.

An Ultrafast Molecular-Rotor-Based Fluorescent Turn-On Sensor for the Perrhenate Anion in Aqueous Solution.

Devising sensors for the perrhenate anion in aqueous media is extremely challenging, and has seldom been reported in the literature. Herein, we report a fluorescence turn-on sensor for the perrhenate anion in aqueous media based on the aggregation-induced emission of a popular ultrafast molecular rotor dye, Thioflavin-T. The selective response towards the perrhenate anion has been rationalized in terms of matching water affinity, with the weakly hydrated perrhenate anion spontaneously forming a contact ion pair with the weakly hydrated ultrafast molecule-rotor-based organic cation, Thioflavin-T, which in turn leads to an aggregate assembly that provides a fluorescence turn-on response towards perrhenate. The sensing response of Thioflavin-T has been found to be quite selective towards the perrhenate anion when tested against anions that are ubiquitously present in the environment, such as chloride, nitrate, and sulfate anions. The formation of self-assembled Thioflavin-T aggregates has also been investigated by time-resolved emission and temperature-dependent measurements.

Ratiometric fluorescence turn-on sensing of perrhenate anion, a non-radioactive surrogate of hazardous pertechnetate, in aqueous solution

Fluorescent sensor for the perrhenate/pertechnetate anions in aqueous media are rarely reported in literature as their large size and low charge density poses great challenge to their detection. Herein, we report the first fluorescent turn-on ratiometric sensor for perrhenate anion, exploiting its weak hydration property which leads to an advantageous formation of a contact ion-pair of perrhenate anion with a well-known cationic ultrafast molecular rotor dye, Auramine O. This ion-pair formation, in turn, leads to a fluorescent aggregate assembly that provides an excellent opportunity to detect perrhenate anion in aqueous solution.

Supramolecular Dye Aggregate Assembly Enables Ratiometric Detection and Discrimination of Lysine and Arginine in Aqueous Solution.

Constructing sensor systems for rapid and selective detection of small biomolecules such as amino acids is a major area of focus in bioanalytical chemistry. Considering the biological relevance of arginine and lysine, significant efforts have been directed to develop fluorescent sensors for their detection. However, these developed sensors suffer from certain disadvantages such as poor aqueous solubility, technically demanding and time-consuming synthetic protocols, and more importantly, most of them operate through single wavelength measurements, making their performance prone to small variations in experimental conditions. Herein, we report a ratiometric sensor that operates through lysine- and arginine-induced dissociation of a supramolecular assembly consisting of emissive H-aggregates of a molecular rotor dye, thioflavin-T (ThT), on the surface of a polyanionic supramolecular host, sulfated β-cyclodextrin. This disassembly brings out the modulation of monomer–aggregate equilibrium in the system which acts as an ideal scheme for the ratiometric detection of lysine and arginine in the aqueous solution. Besides facile framework of our sensor system, it employs a commercially available inexpensive probe molecule, ThT, which provides an added advantage over other sensor systems that employ synthetically demanding probe molecules. Importantly, the distinctive feature of the ratiometric detection of arginine and lysine provides an inherent advantage of increased accuracy in quantitative analysis. Interestingly, we have also demonstrated that arginine displays a multiwavelength distinctive recognition pattern which distinguishes it from lysine, using a single supramolecular ensemble. Furthermore, our sensor system also shows response in heterogeneous, biologically complex media of serum samples, thus extending its possible use in real-life applications.

Ultrafast excited state deactivation channel of thioflavin T adsorbed on SDS micelle: A combined femtosecond fluorescence and transient absorption study

Abstract In this contribution, we have presented the dynamics of molecular rotor dye thioflavin T (ThT) in Stern layer of sodium dodecyl sulfate (SDS) micelle using femtosecond fluorescence up-conversion and broadband transient absorption spectroscopy. ThT being a positively charged molecule is expected to remain in the Stern layer of micelle due to columbic attraction with the negatively charged surfactant head groups. The restriction imposed by the environment slows down the rotation of molecular fragments, which increases its quantum yield ∼8 times compared to pure water. We have confirmed the dual relaxation channel of ThT (LE → TICT1 and LE → TICT2) and found that the lifetime of the three states namely LE, TICT1 and TICT2 are 0.5, 3.1 and 15.7 ps respectively in 40 mM SDS.

Emissive H-Aggregates of an Ultrafast Molecular Rotor: A Promising Platform for Sensing Heparin.

Constructing "turn on" fluorescent probes for heparin, a most widely used anticoagulant in clinics, from commercially available materials is of great importance, but remains challenging. Here, we report the formation of a rarely observed emissive H-aggregate of an ultrafast molecular rotor dye, Thioflavin-T, in the presence of heparin, which provides an excellent platform for simple, economic and rapid fluorescence turn-on sensing of heparin. Generally, H-aggregates are considered as serious problem in the field of biomolecular sensing, owing to their poorly emissive nature resulting from excitonic interaction. To the best of our knowledge, this is the first report, where contrastingly, the turn-on emission from the H-aggregates has been utilized in the biomolecule sensing scheme, and enables a very efficient and selective detection of a vital biomolecule and a drug with its extensive medical applications, i.e., heparin. Our sensor system offers several advantages including, emission in the biologically advantageous red-region, dual sensing, i.e., both by fluorimetry and colorimetry, and most importantly constructed from in-expensive commercially available dye molecule, which is expected to impart a large impact on the sensing field of heparin. Our system displays good performance in complex biological media of serum samples. The novel Thioflavin-T aggregate emission could be also used to probe the interaction of heparin with its only clinically approved antidote, Protamine.

Simultaneous High-Throughput Conformational and Colloidal Stability Screening Using a Fluorescent Molecular Rotor Dye, 4-(4-(Dimethylamino)styryl)-N-Methylpyridinium Iodide (DASPMI)

Technologies to improve the throughput for screening protein formulations are continuously evolving. The purpose of this article is to highlight novel applications of a molecular rotor dye, 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (DASPMI) in screening for the conformational stability, colloidal stability, and subtle pretransition dynamics of protein structures during early formulation development. The measurement of the apparent unfolding temperature (Tm) for a monoclonal antibody in the presence of Tween 80 was conducted and data were compared to the results of differential scanning calorimetry (DSC) measurements. Additionally, measuring the fluorescence intensity of DASPMI as a function of protein concentration shows consistent correlation to the diffusion interaction parameter (kD) for two distinct monoclonal antibody formulations measured by DLS. Lastly, due to the sensitivity of the molecular rotor dye to changes in microviscosity (ηmicro), subtle pretransition dynamics were discernable for two monoclonal antibody formulations that correlate with findings by red-edge excitation shift (REES) experiments. This novel application of molecular rotor dyes offers a valuable and promising approach for streamlining the early formulation development process due to low material consumption and rapid analysis time in a 96-well plate format.

Colorimetric fluorescence response to carbon dioxide using charge transfer dye and molecular rotor dye in smart solvent system

Combination of a charge transfer dye and a molecular rotor dye in a smart solvent system resulted in a remarkable colorimetric fluorescence (FL) response to carbon dioxide (CO2). Nile Red and allyl-2-cyano-3-(1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinolin-9-yl)acrylate (ACHQA) were used as the charge transfer dye and molecular rotor dye, respectively, and an equivalent molar mixture of 1,8-diazabicyclo-[5.4.0]-undec-7-ene (DBU) and 1-propanol was chosen as the smart solvent system. Nile Red and ACHQA in the DBU/1-propanol solvent mixture emitted red light mainly due to Nile Red, whereas the FL color gradually turned to green with the increasing amount of CO2 injected. The FL color reverted into the initial state by simultaneously bubbling N2 gas and heating at 50 °C.