Department of Chemistry
Permanent URI for this collectionhttp://localhost:4000/handle/123456789/1924
Browse
8 results
Search Results
Item Mechanistic insights into the C-myc G-quadruplex and berberine binding inside an aqueous two-phase system mimicking biomolecular condensates(ACS, 2024-08) Patra, SatyajitWe investigated the binding between the c-MYC G-quadruplex (GQ) and berberine chloride (BCl) in an aqueous two-phase system (ATPS) with 12.3 wt % polyethylene glycol and 5.6 wt % dextran, mimicking the highly crowded intracellular biomolecular condensates formed via liquid–liquid phase separation. We found that in the ATPS, complex formation is significantly altered, leading to an increase in affinity and a change in the stoichiometry of the complex with respect to neat buffer conditions. Thermodynamic studies reveal that binding becomes more thermodynamically favorable in the ATPS due to entropic effects, as the strong excluded volume effect inside ATPS droplets reduces the entropic penalty associated with binding. Finally, the binding affinity of BCl for the c-MYC GQ is higher than those for other DNA structures, indicating potential specific interactions. Overall, these findings will be helpful in the design of potential drugs targeting the c-MYC GQ structures in cancer-related biocondensates.Item Long-Range Single-Molecule Förster Resonance Energy Transfer between Alexa Dyes in Zero-Mode Waveguides(ACS, 2020-03) Patra, SatyajitZero-mode waveguide (ZMW) nano-apertures milled in metal films were proposed to improve the Förster resonance energy transfer (FRET) efficiency and enable single-molecule FRET detection beyond the 10 nm barrier, overcoming the restrictions of diffraction-limited detection in a homogeneous medium. However, the earlier ZMW demonstrations were limited to the Atto 550–Atto 647N fluorophore pair, asking the question whether the FRET enhancement observation was an artifact related to this specific set of fluorescent dyes. Here, we use Alexa Fluor 546 and Alexa Fluor 647 to investigate single-molecule FRET at large donor–acceptor separations exceeding 10 nm inside ZMWs. These Alexa fluorescent dyes feature a markedly different chemical structure, surface charge, and hydrophobicity as compared to their Atto counterparts. Our single molecule data on Alexa 546–Alexa 647 demonstrate enhanced FRET efficiencies at large separations exceeding 10 nm, extending the spatial range available for FRET and confirming the earlier conclusions. By showing that the FRET enhancement inside a ZMW does not depend on the set of fluorescent dyes, this report is an important step to establish the relevance of ZMWs to extend the sensitivity and detection range of FRET, while preserving its ability to work on regular fluorescent dye pairs.Item A Fluorescence Correlation Spectroscopy, Steady-State, and Time-Resolved Fluorescence Study of the Modulation of Photophysical Properties of Mercaptopropionic Acid Capped CdTe Quantum Dots upon Exposure to Light(ACS, 2013-10) Patra, SatyajitLight-induced modulation of the fluorescence behavior of mercaptopropionic acid (MPA) capped CdTe quantum dots (QDs) in aqueous solution is studied by a combination of fluorescence correlation spectroscopy (FCS) and steady-state and time-resolved fluorescence techniques. These investigations reveal a dramatic variation in the fluorescence properties of the QDs under exposure to light. In the FCS measurement, a large decrease in amplitude and change in shape of the correlation curves are observed with increasing excitation power. The change in the shape of the correlation curves, particularly at short lag time, e.g., a faster relaxation at high excitation power, is attributed to the increasing contribution of the off state of the QDs. Interestingly, despite this increasing contribution of the off state, which reduces the effective number of emitters in the observation volume and hence should increase the amplitude of the correlation curve, the latter actually decreases at high excitation power. This apparent contradiction is resolved by considering light-induced transformation of the dark QDs to bright QDs due to surface passivation of the QDs with increasing excitation power. Enhancement of the steady-state fluorescence intensity under light irradiation, both in aerated and deaerated environments, supports the mechanism of passivation of the surface trap states by photoadsorption of water molecules. Fluorescence lifetime data is also shown to be consistent with this light-induced surface passivation mechanism.Item Effect of Capping Agent and Medium on Light-Induced Variation of the Luminescence Properties of CdTe Quantum Dots: A Study Based on Fluorescence Correlation Spectroscopy, Steady State and Time-Resolved Fluorescence Techniques(ACS, 2014-07) Patra, SatyajitThe influence of ligand and solvent on light-induced modulation of the emission behavior of the quantum dots (QDs) has been studied for CdTe QDs capped with hexadecylamine (HDA), mercaptopropionic acid (MPA), and 1-(1-undecanethiol)-3-methyl imidazolium bromide (SMIM) in CHCl3, H2O, and [bmim][PF6] ionic liquid, respectively, using steady state and time-resolved fluorescence and fluorescence correlation spectroscopy techniques. While an aqueous solution of CdTe/MPA QDs exhibits fluorescence enhancement and a small blue shift of the emission peak (λmaxem) in the early stages of the light irradiation, such enhancement could not be observed in the case of CHCl3 solution of CdTe/HDA and [bmim][PF6] solution of CdTe/SMIM. Instead, exposure to light leads to a rapid reduction in luminescence intensity and large blue shift of λmaxem in the case of CHCl3 solution of CdTe/HDA and a very slow decrease of luminescence intensity with negligible shift of λmaxem in the case of an ionic liquid solution of CdTe/SMIM. The time-resolved fluorescence behavior of the QDs is found to be consistent with the steady state results. Fluorescence correlation spectroscopy measurements on the other hand reveal a large decrease of the amplitude of correlation at time zero [G(0)] for the aqueous solution of CdTe/MPA and negligible change in the G(0) value for the ionic liquid solution of CdTe/SMIM with increasing excitation power. The mechanism of these light-induced changes of the luminescence behavior of the QDs is investigated.Item Diffusion of organic dyes in bovine serum albumin solution studied by fluorescence correlation spectroscopy(RSC, 2012-05) Patra, SatyajitThe understanding of the transport of drugs and naturally occurring molecules in living cells and tissues requires a thorough knowledge of the diffusion behaviour of the molecular systems in these media. In this work, we studied the translational diffusion of three fluorescent molecules, electrically neutral coumarin 102 (C102), cationic rhodamine 6G (R6G) and anionic fluorescein (FL) in phosphate-buffered (pH 7) aqueous solutions of bovine serum albumin (BSA) protein in the absence and presence of common salt and urea using fluorescence correlation spectroscopy (FCS) by monitoring the fluorescence intensity fluctuations in a small confocal observation volume. The diffusion due to both free and BSA-bound molecules is observed in the case of the C102-BSA system. While no exchange between the bound and free states of the molecule is observed in this case, a rapid exchange between the two states is observed in the case of electrically charged hydrophilic dyes R6G and FL. This molecular picture, which is the first of its kind, is a reflection of a weaker binding of R6G and FL compared to C102 with the protein molecule. The binding sites of the probe molecules in BSA were identified based on the urea-induced change of diffusion of the probes in BSA.Item Deep Ultraviolet Plasmonic Enhancement of Single Protein Autofluorescence in Zero-Mode Waveguides(ACS, 2019-09) Patra, SatyajitSingle molecule detection provides detailed information about molecular structures and functions but it generally requires the presence of a fluorescent marker which can interfere with the activity of the target molecule or complicate the sample production. Detecting a single protein with its natural UV autofluorescence is an attractive approach to avoid all the issues related to fluorescence labeling. However, the UV autofluorescence signal from a single protein is generally extremely weak. Here, we use aluminum plasmonics to enhance the tryptophan autofluorescence emission of single proteins in the UV range. Zero-mode waveguide nanoapertures enable the observation of the UV fluorescence of single label-free β-galactosidase proteins with increased brightness, microsecond transit times, and operation at micromolar concentrations. We demonstrate quantitative measurements of the local concentration, diffusion coefficient, and hydrodynamic radius of the label-free protein over a broad range of zero-mode waveguide diameters. Although the plasmonic fluorescence enhancement has generated a tremendous interest in the visible and near-infrared parts of the spectrum, this work pushes further the limits of plasmonic-enhanced single molecule detection into the UV range and constitutes a major step forward in our ability to interrogate single proteins in their native state at physiological concentrations.Item Extending Single-Molecule Förster Resonance Energy Transfer (FRET) Range beyond 10 Nanometers in Zero-Mode Waveguides(ACS, 2019-07) Patra, SatyajitSingle-molecule Förster resonance energy transfer (smFRET) is widely used to monitor conformations and interaction dynamics at the molecular level. However, conventional smFRET measurements are ineffective at donor–acceptor distances exceeding 10 nm, impeding the studies on biomolecules of larger size. Here, we show that zero-mode waveguide (ZMW) apertures can be used to overcome the 10 nm barrier in smFRET. Using an optimized ZMW structure, we demonstrate smFRET between standard commercial fluorophores up to 13.6 nm distance with a significantly improved FRET efficiency. To further break into the classical FRET range limit, ZMWs are combined with molecular constructs featuring multiple acceptor dyes to achieve high FRET efficiencies together with high fluorescence count rates. As we discuss general guidelines for quantitative smFRET measurements inside ZMWs, the technique can be readily applied for monitoring conformations and interactions on large molecular complexes with enhanced brightness.Item Fluorescence Blinking and Photoactivation of All-Inorganic Perovskite Nanocrystals CsPbBr3 and CsPbBr2I(ACS, 2016-01) Patra, SatyajitStudy of the emission behavior of all-inorganic perovskite nanocrystals CsPbBr3 and CsPbBr2I as a function of the excitation power employing fluorescence correlation spectroscopy and conventional techniques reveals fluorescence blinking in the microsecond time scale and photoinduced emission enhancement. The observation provides insight into the radiative and nonradiative deactivation pathways of these promising substances. Because both blinking and photoactivation processes are intimately linked to the charge separation efficiency and dynamics of the nanocrystals, these key findings are likely to be helpful in realizing the true potential of these substances in photovoltaic and optoelectronic applications.