Department of Chemistry

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pH controlled synthesis of end to end linked Au nanorod dimer in an aqueous solution for plasmon enhanced spectroscopic applications
(RSC, 2024-11) Patra, Satyajit
End-to-end linked nanorod dimer nanogap antennas exhibit superior plasmonic enhancement compared to monomers due to the coupling of localized surface plasmon resonances (LSPR) of individual nanorods. However, controlling the assembly to stop at the dimer stage is challenging. Here, we report a pH-controlled synthesis of Au nanorod dimer nanogap antennas in an aqueous solution using 1,4-dithiothreitol (DTT) as a linker. Neutral to acidic pH (4.0 to 7.0) favors dimer formation, while higher pH decreases dimer yield, stopping completely at pH 11.0. The reaction can also be halted in neutral and acidic solutions by abruptly increasing the pH to 11.0 or higher. At basic pH, both thiol groups of DTT deprotonate and acquire a negative charge, causing both thiolate ends to adsorb onto the positively charged cetyltrimethylammonium bromide (CTAB) micellar layer on the transverse surface of the Au nanorod, preventing dimer formation. TEM images confirm nanorod dimers, showing a good conversion yield (∼80%) from monomers to dimers. Overall, out of all the DTT induced NR assemblies, 70% are found to be dimers. The majority of these dimers (>90%) are end-to-end linked dimers, with a gap distance of ∼1 nm, exhibiting exceptional stability and remaining intact for over two weeks. FDTD simulations demonstrate a significant enhancement of the light E field in the nanogap, ∼80 times higher than in a homogeneous water environment and 11 times higher than in nanorod monomers. Simulations also show that E field enhancement varies with the angular separation of monomeric nanorods, being highest for end-to-end dimers (180°) and lower for side-to-side dimers (0°). Overall, we present an inexpensive method to design and control nanorod dimer nanogap antennas in aqueous solution, useful for plasmon-enhanced spectroscopic applications such as biosensing, chemical sensing, and biomedical devices.
Mechanistic insights into the C-myc G-quadruplex and berberine binding inside an aqueous two-phase system mimicking biomolecular condensates
(ACS, 2024-08) Patra, Satyajit
We 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.
Effect of Pressure on the Conformational Landscape of a Large Loop DNA Hairpin in the Presence of Salts and Osmolytes
(Cell Press, 2018-02) Patra, Satyajit
The effect of pressure on the conformational landscape of a DNA hairpin has been investigated in the absence and presence of salts and osmolytes using both ensemble and single-molecule Förster resonance energy transfer (FRET) techniques.1 We use monovalent (K+), divalent (Mg2+) and trivalent (Co3+) salts and urea as well as TMAO as osmolytes. Unlike the canonical DNA duplex structures of similar melting points, this large loop DNA hairpin is rather sensitive to pressure.2 The transition volume upon unfolding is found to be −17 cm3 mol−1 in neat buffer solution. We found that the stabilizing effect of salt follows the order Co3+ > Mg2+ > K+. Above 1 mM Mg2+ and 0.3 mM Co3+, pressure has a negligible effect on the conformation of the DNA hairpin, which is due to the compensation of the negative charge density of the phosphate backbone, favoring the formation of loops by base pairing of complementary sequences, and thus the closed conformation. The compatible osmolyte TMAO also stabilizes the closed conformation even at high pressures and temperatures, while urea destabilizes the closed conformation synergistically with pressure and temperature. Intermediate states are populated by urea and high temperatures, indicating that the conformational landscape of the DNA hairpin is in fact a rugged one. The results obtained are interpreted in terms of preferential hydration and interaction effects of the cations and osmolytes.
Fluorescence Brightness, Photostability, and Energy Transfer Enhancement of Immobilized Single Molecules in Zero-Mode Waveguide Nanoapertures
(ACS, 2022-05) Patra, Satyajit
Zero-mode waveguide (ZMW) nanoapertures are widely used to monitor single molecules beyond the range accessible to normal microscopes. However, several aspects of the ZMW influence on the photophysics of fluorophores remain inadequately documented and sometimes controversial. Here, we thoroughly investigate the ZMW influence on the fluorescence of single immobilized Cy3B and Alexa 647 molecules, detailing the interplays between brightness, lifetime, photobleaching time, the total number of emitted photons, and Förster resonance energy transfer (FRET). Despite the plasmonic-enhanced excitation intensity in the ZMW, we find that the photostability is preserved with similar photobleaching times as on the glass reference. Both the fluorescence brightness and the total number of photons detected before photobleaching are increased, with an impressive gain of nearly five times that found for Alexa 647 dyes. Finally, the single-molecule data importantly allow a loophole-free characterization of the ZMW influence on the FRET process. We show that the FRET rate constant is enhanced by 50%, demonstrating that nanophotonics can mediate the energy transfer. These results deepen our understanding of the fluorescence enhancement in ZMWs and are of immediate relevance for single-molecule biophysical applications.
Effect of Controlled Deposition of ZnS Shell on the Photostability of CdTe Quantum Dots as Studied by Conventional Fluorescence and FCS Techniques
(Wiley, 2015-10) Patra, Satyajit
The effect of one and two monolayers of ZnS shells on the photostability of CdTe quantum dots (QDs) in aqueous and nonaqueous media has been studied by monitoring the fluorescence behavior of the QDs under ensemble and single-molecule conditions. ZnS capping of the CdTe QDs leads to significant enhancement of the fluorescence brightness of these QDs. Considerable enhancement of the photostability of the shell-protected QDs, including the suppression of photoactivation, is also observed. Fluorescence correlation spectroscopy measurements reveal an increase in the number of particles undergoing reversible fluorescent on–off transitions in the volume under observation with increasing excitation power; this effect is found to be more pronounced in the case of core-only QDs than for core–shell QDs.
Probing Colocalization of N-Ras and K-Ras4B Lipoproteins in Model Biomembranes
(Wiley, 2019-01) Patra, Satyajit
Signaling of N-Ras and K-Ras4B proteins depends strongly on their correct localization in the cell membrane. In vivo studies suggest that intermolecular interactions foster the self-association of both N-Ras and K-Ras4B and the formation of nanoclusters in the cell membrane. As sites for effector binding, nanocluster formation is thought to be essential for effective signal transmission of both N-Ras and K-Ras4B. To shed more light on the spatial arrangement and mechanism underlying the proposed cross-talk between spatially segregated Ras proteins, the simultaneous localization of N-Ras and K-Ras4B and their effect on the lateral organization of a heterogeneous model biomembrane has been studied by using AFM and FRET methodology. It is shown that, owing to the different natures of their membrane anchor systems, N-Ras and K-Ras4B not only avoid assembly in bulk solution and do not colocalize, but rather form individual nanoclusters that diffuse independently in the fluid membrane plane.
Long-Range Single-Molecule Förster Resonance Energy Transfer between Alexa Dyes in Zero-Mode Waveguides
(ACS, 2020-03) Patra, Satyajit
Zero-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.
Translational Dynamics of Lipidated Ras Proteins in the Presence of Crowding Agents and Compatible Osmolytes
(Wiley, 2016-03) Patra, Satyajit
Ras proteins are small GTPases and are involved in transmitting signals that control cell growth, differentiation, and proliferation. Since the cell cytoplasm is crowded with different macromolecules, understanding the translational dynamics of Ras proteins in crowded environments is crucial to yielding deeper insight into their reactivity and function. Herein, the translational dynamics of lipidated N-Ras and K-Ras4B is studied in the bulk and in the presence of a macromolecular crowder (Ficoll) and the compatible osmolyte and microcrowder sucrose by fluorescence correlation spectroscopy. The results reveal that N-Ras forms dimers due to the presence of its lipid moiety in the hypervariable region, whereas K-Ras4B remains in its monomeric form in the bulk. Addition of a macromolecular crowding agent gradually favors clustering of the Ras proteins. In 20 wt % Ficoll N-Ras forms trimers and K-Ras4B dimers. Concentrations of sucrose up to 10 wt % foster formation of N-Ras trimers and K-Ras dimers as well. The results can be rationalized in terms of the excluded-volume effect, which enhances the association of the proteins, and, for the higher concentrations, by limited-hydration conditions. The results of this study shed new light on the association state of these proteins in a crowded environment. This is of particular interest for the Ras proteins, because their solution state—monomeric or clustered—influences their membrane-partitioning behavior and their interplay with cytosolic interaction partners
Fast interaction dynamics of G-quadruplex and RGG-rich peptides unveiled in zero-mode waveguides
(OUP, 2021-12) Patra, Satyajit
G-quadruplexes (GQs), a non-canonical form of DNA, are receiving a huge interest as target sites for potential applications in antiviral and anticancer drug treatments. The biological functions of GQs can be controlled by specifically binding proteins known as GQs binding proteins. Some of the GQs binding proteins contain an arginine and glycine-rich sequence known as RGG peptide. Despite the important role of RGG, the GQs-RGG interaction remains poorly understood. By single molecule measurements, the interaction dynamics can be determined in principle. However, the RGG–GQs interaction occurs at micromolar concentrations, making conventional single-molecule experiments impossible with a diffraction-limited confocal microscope. Here, we use a 120 nm zero-mode waveguide (ZMW) nanoaperture to overcome the diffraction limit. The combination of dual-color fluorescence cross-correlation spectroscopy (FCCS) with FRET is used to unveil the interaction dynamics and measure the association and dissociation rates. Our data show that the RGG–GQs interaction is predominantly driven by electrostatics but that a specific affinity between the RGG sequence and the GQs structure is preserved. The single molecule approach at micromolar concentration is the key to improve our understanding of GQs function and develop its therapeutic applications by screening a large library of GQs-targeting peptides and proteins.
Zero-mode waveguides can be made better: fluorescence enhancement with rectangular aluminum nanoapertures from the visible to the deep ultraviolet
(RSC, 2020-07) Patra, Satyajit
Nanoapertures milled in metallic films called zero-mode waveguides (ZMWs) overcome the limitations of classical confocal microscopes by enabling single molecule analysis at micromolar concentrations with improved fluorescence brightness. While the ZMWs have found many applications in single molecule fluorescence studies, their shape has been mainly limited to be circular. Owing to the large parameter space to explore and the lack of guidelines, earlier attempts using more elaborate shapes have led to unclear conclusions whether or not the performance was improved as compared to a circular ZMW. Here, we comparatively analyze the performance of rectangular-shaped nanoapertures milled in aluminum to enhance the fluorescence emission rate of single molecules from the near infrared to the deep ultraviolet. Our new design is based on rational principles taking maximum advantage of the laser linear polarization. While the long edge of the nanorectangle is set to meet the cut-off size for the propagation of light into the nanoaperture, the short edge is reduced to 30 nm to accelerate the photodynamics while maintaining bright fluorescence rates. Our results show that both in the red and in the ultraviolet, the nanorectangles provide 50% brighter photon count rates as compared to the best performing circular ZMWs and achieve fluorescence lifetimes shorter than 300 ps. These findings can be readily used to improve the performance of ZMWs, especially for fast biomolecular dynamics, bright single-photon sources, and ultraviolet plasmonics.

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