Macromolecular Crowding Promotes Re-entrant Liquid-Liquid Phase Separation of Human Serum Transferrin and Prevents Surface-Induced Fibrillation.

Protein aggregation and inactivation upon surface immobilization are major limiting factors for analytical applications in biotechnology-related fields. Protein immobilization on solid surfaces often requires multi-step surface passivation, which is time-consuming and inefficient. Herein, we have discovered that biomolecular condensates of biologically active human serum transferrin (Tf) can effectively prevent surface-induced fibrillation and preserve the native-like conformation of phase-separated Tf over a period of 30 days. It has been observed that macromolecular crowding promotes homotypic liquid-liquid phase separation (LLPS) of Tf through enthalpically driven multivalent hydrophobic interactions possibly via the involvement of its low-complexity domain (residues 3-20) containing hydrophobic amino acids. The present LLPS of Tf is a rare example of salt-mediated re-entrant phase separation in a broad range of salt concentrations (0-3 M) solely via the involvement of hydrophobic interactions. Notably, no liquid-to-solid-like phase transition has been observed over a period of 30 days, suggesting the intact conformational integrity of phase-separated Tf, as revealed from single droplet Raman, circular dichroism, and Fourier transform infrared spectroscopy measurements. More importantly, we discovered that the phase-separated condensates of Tf completely inhibit the surface-induced fibrillation of Tf, illustrating the protective role of these liquid-like condensates against denaturation and aggregation of biomolecules. The cell mimicking compact aqueous compartments of biomolecular condensates with a substantial amount of interfacial water preserve the structure and functionality of Tf. Our present study highlights an important functional aspect of biologically active protein condensates and may have wide-ranging implications in cell physiology and biotechnological applications.

Coacervate-Based Plexcitonic Assembly toward Peroxidase-like Activity and Ultrasensitive Glucose Sensing.

Inbuilt catalytic centers anchored inside the confined architecture of artificial nanoreactors have gained tremendous attention owing to their vast applicability in various catalytic transformations. However, designing homogeneously distributed catalytic units with exposed surfaces in confined environment is a challenging task. Here, we have utilized quantum dot (QD)-embedded coacervate droplets (QD-Ds) as a confined compartment for the in situ synthesis of gold nanoparticles (Au NPs) without any additional reducing agent. High-resolution transmission electron microscopy images reveal homogeneous distribution of 5.6 ± 0.2 nm-sized Au NPs inside the QD-Ds (Au@QD-Ds). The in situ synthesized Au NPs are found to be stable over a period of 28 days without any agglomeration. Control experiments reveal that the free surface carboxylic acid groups of embedded QDs simultaneously act as reducing and stabilizing agents for Au NPs. Notably, these Au@QD-Ds exhibit superior peroxidase-like activity compared to bulk aqueous Au NPs and Au@QDs under similar experimental conditions. The observed peroxidase-like activity follows the classical Michaelis-Menten model inside the Au@QD-Ds via the fast electron-transfer pathway. The enhanced peroxidase-like activity has been explained by considering confinement, mass action, and the ligand-free surface of embedded Au NPs. The present plexcitonic nanocomposites exhibit excellent recyclability over several consecutive cycles without any compromise in their catalytic activity. Finally, a cascade reaction with glucose oxidase (GOx)-loaded Au@QD-Ds have been utilized for colorimetric detection of glucose with a limit of detection of 272 nM in solution as well as on filter paper. The present work highlights a facile and robust methodology for the fabrication of optically active functional hybrid plexcitonic assemblies and may find importance in various fields including bioanalytical chemistry and optoelectronics.

Biomolecular Condensates Regulate Enzymatic Activity under a Crowded Milieu: Synchronization of Liquid-Liquid Phase Separation and Enzymatic Transformation.

Cellular crowding plays a key role in regulating the enzymatic reactivity in physiological conditions, which is challenging to realize in the dilute phase. Enzymes drive a wide range of complex metabolic reactions with high efficiency and selectivity under extremely heterogeneous and crowded cellular environments. However, the molecular interpretation behind the enhanced enzymatic reactivity under a crowded milieu is poorly understood. Herein, using the horseradish peroxidase (HRP) and glucose oxidase (GOx) cascade pair, we demonstrate for the first time that macromolecular crowding induces liquid-liquid phase separation (LLPS) via the formation of liquid-like condensates/droplets and thereby increases the intrinsic catalytic efficiencies of HRP and GOx. Both these enzymes undergo crowding induced homotypic LLPS via enthalpically driven multivalent electrostatic as well as hydrophobic interactions. Using a set of kinetic and microscopic experiments, we show that precise synchronization of spontaneous LLPS and enzymatic transformations is key to realize the enhanced enzymatic activity under the crowded environments. Our findings reveal an unprecedented enhancement (91- to 205-fold) in the catalytic efficiency (kcat/Km) of HRP at pH 4.0 within the droplet phase relative to that in the bulk aqueous phase in the presence of different crowders. In addition, we have shown that other enzymes also undergo spontaneous LLPS under macromolecular crowding, signifying the generality of this phenomenon under the crowded environments. More importantly, coalescence driven highly regulated GOx/HRP cascade reactions within the fused droplets have been demonstrated with enhanced activity and specificity under the crowded environments. The present discovery highlights the active role of membraneless condensates in regulating the enzymatic efficacy for complex metabolic reactions under the crowded cellular environments and may find significant importance in the field of biocatalysis.

Synthetic Protocell as Efficient Bioreactor: Enzymatic Superactivity and Ultrasensitive Glucose Sensing in Urine.

It is believed that membraneless cellular condensates play a critical role in accelerating various slow and thermodynamically unfavorable biochemical processes. However, the exact mechanisms behind the enhanced activity within biocondensates remain poorly understood. Here, we report the fabrication of a high-performance integrated cascade bioplatform based on synthetic droplets for ultrasensitive glucose sensing. Using a horseradish peroxidase (HRP) and glucose oxidase (GOx) cascade pair, we report an unprecedented enhancement in the catalytic activity of HRP inside the synthetic membraneless droplet. Liquidlike membraneless droplets have been prepared via multivalent electrostatic interactions between adenosine triphosphate (ATP) and poly(diallyldimethylammonium chloride) (PDADMAC) in an aqueous medium. Compartmentalized enzymes (GOx/HRP@Droplet) exhibit high encapsulation efficiency, low leakage, prolong retention of activity, and exceptional stability toward protease digestion. Using an HRP@Droplet composite, we have shown that the enzymatic reaction within the droplet follows the classical Michaelis-Menten model. Our findings reveal remarkable enhancement in the catalytic activity of up to 100- and 51-fold for HRP@Droplet and GOx/HRP@Droplet, respectively. These enhanced activities have been explained on the basis of increased local concentrations of enzymes and substrates, along with altered conformations of sequestered enzymes. Furthermore, we have utilized highly efficient and recyclable GOx/HRP@Droplet composite to demonstrate ultrasensitive glucose sensing with a limit of detection of 228 nM. Finally, the composite platform has been exploited to detect glucose in spiked urine samples in solution and filter paper. Our present study illustrates the unprecedented activity of the compartmentalized enzymes and paves the way for next-generation composite bioreactors for a wide range of applications.

Macromolecular Crowding-Induced Unusual Liquid-Liquid Phase Separation of Human Serum Albumin via Soft Protein-Protein Interactions.

Macromolecular crowding has a profound impact on the conformational dynamics and intermolecular interactions of biological macromolecules. In this context, the role of inert synthetic crowders in the protein-protein interactions of globular proteins is poorly understood. Here, using native human serum albumin (HSA) under physiological conditions, we show that macromolecular crowding induces liquid-liquid phase separation (LLPS) via liquid-like membrane-less droplet formation in a concentration- and time-dependent manner. Circular dichroism measurements reveal significant alteration in the secondary structure of HSA inside the droplet during aging. In contrast, at a high protein concentration, a liquid-to-solid-like phase transition has been observed upon maturation. Our findings reveal that the LLPS of HSA is mainly driven by enthalpically controlled intermolecular protein-protein interactions via hydrophobic contacts involving aromatic and/or nonaromatic residues. Moreover, modulation of LLPS of HSA has been demonstrated upon denaturation and ligand binding. This study highlights the importance of soft protein-protein interactions of globular proteins in a crowded cellular environment in driving the LLPS.

Kinetic and Mechanistic Insight into the Surfactant-Induced Aggregation of Gold Nanoparticles and Their Catalytic Efficacy: Importance of Surface Restructuring.

Understanding the fundamental interactions between plasmonic metal nanoparticles (MNPs) and small molecules is of utmost importance in various applications such as catalysis, sensing, drug delivery, optoelectronics, and surface-enhanced Raman spectroscopy. Herein, we have investigated the early stage of the aggregation pathway of citrate-stabilized Au NPs with surfactants and explored their catalytic efficacy. Our findings reveal that (17 ± 2)-nm-sized citrate-stabilized Au NPs undergo concentration and time-dependent aggregation with positively charged cetyltrimethylammonium bromide (CTAB). Kinetic analyses revealed the presence of two distinct kinds of aggregates, namely, smaller clusters and a larger branched network of Au nanochains. At longer times and in the presence of higher concentrations of CTAB, these branched networks of Au nanochains transform into dense compact globular aggregates. The catalytic efficacy of Au NPs, branched Au nanochains, and dense compact aggregates has been explored with respect to the reductive hydrogenation of 4-nitophenol in the presence of excess NaBH4. Our study revealed that the catalytic rate decreases in the order of Au NPs > branched Au nanochains > compact aggregates. Interestingly, pre-equilibrating different Au NP samples with excess NaBH4 prior to the onset of the reaction results in similar catalytic activity irrespective of the aggregation state of Au NPs. This observation has been explained by considering efficient surface restructuring via ligand exchange with H- ions and the subsequent disruption of CTAB-induced aggregates of Au NPs. Moreover, the aggregated Au NPs can be recycled over several consecutive cycles for the reductive hydrogenation of 4-NP upon ligand exchange with H- ions. Taken together, our present study highlights the early-stage aggregation kinetics of Au NPs with CTAB surfactants and demonstrates the importance of the surface restructuring of Au NPs on their catalytic efficacy.

Nanocatalysis under Nanoconfinement: A Metal-Free Hybrid Coacervate Nanodroplet as a Catalytic Nanoreactor for Efficient Redox and Photocatalytic Reactions.

Nature utilizes cellular and subcellular compartmentalization to efficiently drive various complex enzymatic transformations via spatiotemporal control. In this context, designing of artificial nanoreactors for efficient catalytic transformations finds tremendous importance in recent times. One key challenge remains the design of multiple catalytic centers within the confined space of a nanoreactor without unwanted agglomeration and accessibility barrier for reactants. Herein, we report a unique blend of nanoscience and chemical catalysis using a metal-free hybrid synthetic protocell as a catalytic nanoreactor for redox and photocatalytic transformations, which are otherwise incompatible in bulk aqueous medium. Hybrid coacervate nanodroplets (NDs) fabricated from 2.5 nm-sized carbon dots (CDs) and poly(diallyldimethyl)ammonium chloride have been utilized toward reductive hydrogenation of nitroarenes in the presence of sodium borohydride (NaBH4). It has been found that the reduction mechanism follows the classical Langmuir-Hinshelwood (LH) model at the surface of embedded CDs inside the NDs via the generation of reactive surface hydroxyl groups. These NDs show excellent recyclability without any compromise on reaction kinetics and conversion yield. Importantly, spatiotemporal control over the hydrogenation reaction has been achieved using two mixed populations of coacervates. Moreover, efficient visible light-induced photoredox conversion of ferricyanide to ferrocyanide and artificial peroxidase-like activity have also been demonstrated inside these catalytic NDs. Our findings indicate that the individual polymer-bound CD inside the NDs acts as the catalytic center for both the redox and photocatalytic reactions. The present study highlights the unprecedented catalytic activity of the metal-free CD-based coacervate NDs and paves the way for next-generation catalytic nanoreactors for a wide range of chemical and enzymatic transformations.

Highly Photostable and Two-Photon Active Quantum Dot-Polymer Multicolor Hybrid Coacervate Droplets.

Fabrication and precise control of the physicochemical properties of multifunctional organic-inorganic hybrid nanocomposites find great importance in various research fields. Herein, we report the fabrication of a new class of luminescent hybrid coacervate droplets from CdTe quantum dots (QDs) and a poly(diallyldimethylammonium chloride) (PDADMAC) aqueous mixture. The colloidal stability of these droplets has been explored over wide ranges of composition, pH, and ionic strength. Although these hybrid droplets are quite stable in a low-ionic-strength medium (<100 mM NaCl) and neutral/basic pH (pH >6.5), they are unstable in a higher-ionic-strength medium (>100 mM NaCl) and acidic pH (pH <5.5). Our findings indicate specific electrostatic interactions between negatively charged QDs and positively charged PDADMAC behind the observed coacervation. They exhibit the preferential sequestration of organic dyes and serum albumins. The intrinsic luminescent properties of these hybrid droplets have been explored using confocal laser scanning microscopy (CLSM) and epifluorescence microscopy. CLSM reveals the formation of intrinsically luminescent hybrid droplets. In addition, mixed two-color luminescent droplets have been fabricated by simultaneously mixing green- and red-emitting QDs with PDADMAC aqueous solution. Epifluorescence imaging reveals highly photostable and nonbleaching photoluminescence (PL) from individual droplets as a consequence of efficient surface passivation by polymeric chains of PDADMAC. Moreover, using two-photon (2P) confocal imaging we have shown that these hybrid droplets are ideal candidates for 2P confocal imaging applications. The present study can be easily extended to fabricate a wide range of hybrid droplets with various inorganic counterparts having unique optoelectronic properties, which will further expand their applicability in nanocatalysis, bioimaging, and biosensing.

Surfactant-Induced Self-Assembly of CdTe Quantum Dots into Multicolor Luminescent Hybrid Vesicles.

Here, we report the interaction of mercaptosuccinic acid (MSA)-capped CdTe quantum dots (QDs) with hexadecyltrimethylammonium bromide (CTAB) surfactant and subsequent formation of self-assembled multicolor luminescent vesicles in aqueous medium. A continuous phase sequence from clear (C1) to turbid (T1), precipitate (P), turbid (T2), and clear (C2) has been observed for QD solution upon increasing the concentration of positively charged CTAB, indicating dynamic equilibrium between various self-assembled supramolecular structures. In contrast, no such changes have been observed in the presence of negatively charged sodium dodecyl sulfate and neutral Triton X-100 surfactants, indicating specific electrostatic interactions behind the observed phase separation behavior. Epi-fluorescence imaging in the C1 and C2 regions reveals the presence of surfactant-induced aggregates of QD. The morphologies and photoluminescence properties of self-assembled supramolecular structures in the T1 and T2 region have been explored by using scanning electron microscopy (SEM), atomic force microscopy (AFM), and confocal laser scanning microscopy (CLSM). SEM and AFM images reveal distinct spherical vesicles in the T1 and T2 regions of the binary mixture. Moreover, CLSM results show that these spherical vesicles are inherently luminescent due to the presence of self-assembled QDs. Fabrication of multicolor luminescent vesicles has been demonstrated by tuning the size of CdTe QD. Using CLSM, we have further demonstrated efficient encapsulation of Rhodamine 6G dye into these self-assembled vesicles without any structural disruption. While these luminescent vesicles are quite stable in neutral and basic pH (pH = 6.5-11), they are unstable in acidic pH (pH = 4.5-5.5). Moreover, it has been observed that this pH-responsive structural change is totally reversible. The present findings of self-assembled luminescent vesicles from QD-CTAB binary mixture may open up new opportunities in various applications such as bioimaging, drug delivery, and sensing.

Synergistic Enhancement of Electron-Accepting and -Donating Ability of Nonconjugated Polymer Nanodot in Micellar Environment.

Understanding the fundamental electron-transfer dynamics in photoactive carbon nanoparticles (CNPs) is vitally important for their fruitful application in photovoltaics and photocatalysis. Herein, photoinduced electron transfer (PET) to and from the nonconjugated polymer nanodot (PND), a new class of luminescent CNP, has been investigated in the presence of N,N-dimethylaniline (DMA) and methyl viologen (MV2+) in homogeneous methanol and sodium dodecyl sulfate (SDS) micelles. It has been observed that both DMA and MV2+ interact with the photoexcited PND and quench the PL intensity as well as excited-state lifetime in bulk methanol. While in bulk methanol, purely diffusion-controlled PET from DMA to MV2+ via PND has been observed, the mechanism and dynamics differ significantly in SDS micelles. In contrast to homogeneous methanol medium, a distinct synergic effect has been observed in SDS micelles. The presence of both DMA and MV2+ enhances the electron-accepting and -donating abilities of PND in SDS micelles. Time-resolved photoluminescence (PL) measurements reveal that the PET process in SDS micelles is nondiffusive in nature mainly due to instantaneous electron transfer at the confined micellar surface. These results have been explained on the basis of heterogeneous microenvironments of SDS micelles which compartmentalize the donor and acceptor inside its micellar pseudo phase. The present findings provide valuable insights into the intrinsic relation between redox and PL properties of nonconjugated PND.

Tuning of resonance energy transfer from 4',6-diamidino-2-phenylindole to an ultrasmall silver nanocluster across the lipid bilayer.

Herein we have developed a simple liposome-based donor-acceptor system across the lipid bilayer using 4',6-diamidino-2-phenylindole (DAPI) as the donor and an ultrasmall ligand-capped silver nanocluster (Ag NC) as the acceptor. The process of Förster resonance energy transfer (FRET) between DAPI and the Ag NC across the liposome bilayer has been demonstrated using steady-state and time-resolved fluorescence spectroscopy. The synthesized Ag NCs with a majority of Ag4 and Ag5 cores have been characterized using FTIR, mass spectrometry, HRTEM, UV-Vis and PL spectroscopy. Scanning electron microscopy (SEM) and dynamic light scattering (DLS) measurements reveal that the synthesized liposomes are small unilamellar vesicles (SUV) with a mean hydrodynamic diameter of 86.91 ± 6.41 nm. By using two distinct synthetic methods, we have been able to identify two selective binding sites of DAPI with liposome namely, the liposome surface and hydrophilic aqueous core. Fluorescence confocal microscopy confirms the location of the donor DAPI within different locations of liposome. It has been observed that the association of DAPI at the surface of liposome results in less efficient energy transfer to Ag NCs compared to that in the bulk aqueous medium. Energy transfer efficiency decreases from a value of 0.76 in bulk aqueous medium to a value of 0.39 for surface-associated DAPI. On the other hand encapsulation of DAPI into the hydrophilic aqueous core of a liposome results in complete inhibition of the FRET process as a consequence of increased separation distance beyond the FRET range. Hence, our study illustrates that the present DAPI-Ag NC pair can be used as a FRET marker to explore various fundamental processes across the cell membrane.

Direct Evidence of Intrinsic Blue Fluorescence from Oligomeric Interfaces of Human Serum Albumin.

The molecular origin behind the concentration-dependent intrinsic blue fluorescence of human serum albumin (HSA) is not known yet. This unusual blue fluorescence is believed to be a characteristic feature of amyloid-like fibrils of protein/peptide and originates due to the delocalization of peptide bond electrons through the extended hydrogen bond networks of cross-ß-sheet structure. Herein, by combining the results of spectroscopy, size exclusion chromatography, native gel electrophoresis, and confocal microscopy, we have shown that the intrinsic blue fluorescence of HSA exclusively originates from oligomeric interfaces devoid of any amyloid-like fibrillar structure. Our study suggests that this low energy fluorescence band is not due to any particular residue/sequence, but rather it is a common feature of self-assembled peptide bonds. The present findings of intrinsic blue fluorescence from oligomeric interfaces pave the way for future applications of this unique visual phenomenon for early stage detection of various protein aggregation related human diseases.

Insights into the Thermodynamics of Polymer Nanodot-Human Serum Albumin Association: A Spectroscopic and Calorimetric Approach.

With the advent of newer luminescent nanoparticles for bioimaging applications, their complex interactions with individual biomolecules need to be understood in great detail, before their direct application into cellular environments. Here, we have presented a systematic and detailed study on the interaction between luminescent polymer nanodots (PNDs) and human serum albumin (HSA) in its free and ligand-bound state with the help of spectrophotometric and calorimetric techniques. At physiological pH (pH = 7.4), PNDs quench the intrinsic fluorescence of HSA as a consequence of ground-state complex formation. The binding stoichiometry and various thermodynamic parameters have been evaluated by using isothermal titration calorimetry and the van't Hoff equation. It has been found that the association of PNDs with HSA is spontaneous (ΔG0 = -32.48 ± 1.24 kJ mol-1) and is driven by a favorable negative standard enthalpy change (ΔH0 = -52.86 ± 2.12 kJ mol-1) and an unfavorable negative standard entropy change (ΔS0 = -68.38 ± 2.96 J mol-1 K-1). These results have been explained by considering hydrogen bonding interactions between amino and hydroxyl groups (-NH2 and -OH) of PNDs and carboxylate groups (-COO-) of glutamate (Glu) and aspartate (Asp) residues of HSA. The binding constant of PNDs with HSA is estimated to be 4.90 ± 0.19 × 105 M-1. Moreover, it has been observed that warfarin-bound HSA (war-HSA) shows a significantly lower binding affinity (Kb = 1.15 ± 0.19 × 105 M-1) toward PNDs, whereas ibuprofen-bound HSA (ibu-HSA) shows a slightly lower affinity (Kb = 3.47 ± 0.13 × 105 M-1) compared with the free HSA. In addition, our results revealed that PNDs displace warfarin from site I (subdomain IIA) of HSA because of the partial unfolding of war-HSA. We hope that the present study will be helpful to understand the fundamental interactions of these biocompatible PNDs with various biological macromolecules.

Resonant excitation energy transfer from carbon dots to different sized silver nanoparticles.

The influence of size on the efficiency of the nanometal surface energy transfer (NSET) process between excited donors and different sized metal nanoparticles (NPs) is poorly explored in the literature. Here we present a systematic study by correlating the size of silver nanoparticles (Ag NPs) with the efficiency of excitation energy transfer (EET) from carbon dots (CDs) to Ag NPs. Three different sized citrate-capped Ag NPs with a mean hydrodynamic diameter of 39.91 ± 1.03, 53.12 ± 0.31 and 61.84 ± 0.77 nm have been synthesized for the present study. The estimated zeta potential of the synthesized CD is -25.45 ± 1.23 mV while that for the smallest, medium and largest sized Ag NPs are -76.24 ± 3.92, -67.60 ± 4.40, and -58.01 ± 3.10 mV, respectively. The steady-state and time-resolved PL measurements reveal a significant PL quenching of CDs as a function of Ag NP size. A control experiment with Ag NPs having a LSPR at 398 nm shows a negligible amount of PL quenching of CDs as a consequence of inadequate spectral overlap. The origin behind this PL quenching of CDs has been rationalized on the basis of the increased nonradiative decay rate due to NSET from the CDs to the Ag NP surface. Various energy transfer related parameters have been estimated from the NSET theory and it has been observed that the NSET efficiency increases with the increase in the size of Ag NPs. This phenomenon has been explained by considering a larger spectral overlap and a shorter separation distance between the CDs and larger sized Ag NPs due to reduced electrostatic repulsion. Our present results reveal that the size of NPs plays an important role in the NSET process and this phenomenon can be easily utilized to tune the efficiency of energy transfer for various applications.

Effect of compartmentalization of donor and acceptor on the ultrafast resonance energy transfer from DAPI to silver nanoclusters.

The mechanism and dynamics of excitation energy transfer (EET) from photo-excited 4',6-diamidino-2-phenylindole (DAPI) to silver nanoclusters (Ag NCs) and its subsequent modulation in the presence of cationic polymer poly(diallyldimethylammonium chloride) (PDADMAC) and Calf Thymus DNA (CT-DNA) have been demonstrated using steady-state fluorescence and femtosecond fluorescence upconversion techniques. The synthesized Ag NCs were characterized using FTIR, mass spectrometry, XPS, HRTEM, DLS, UV-Vis and PL spectroscopy. Mass spectrometric analysis reveals the formation of ultrasmall Ag4 NCs with a small amount of Ag5 NCs. UV-Vis and PL spectra reveal distinct molecular-like optoelectronic behaviour of these ultrasmall Ag NCs. The dihydrolipoic acid-capped Ag NCs strongly quench the fluorescence of DAPI with concomitant increase in its photoluminescence (PL) intensity at 675 nm. This steady-state fluorescence quenching proceeds with a significant shortening of the fluorescence lifetime of DAPI in the presence of Ag NCs, signifying the nonradiative Förster resonance energy transfer (FRET) from DAPI to Ag NCs. Various energy transfer parameters have been estimated from FRET theory. The present FRET pair shows a characteristic Förster distance of 2.45 nm and can be utilized as a reporter of short-range distances in various FRET based applications. Moreover, this nonradiative FRET is completely suppressed in the presence of both 0.2 wt% PDADMAC and CT-DNA. Our results reveal selective compartmentalization of Ag NCs and DAPI in the presence of 0.2 wt% PDADMAC and CT-DNA, respectively. This selective compartmentalization of donor and acceptor and the subsequent modification of the FRET process may find application in various sensing, photovoltaic, and light harvesting applications.

Insights into the morphology of human serum albumin and sodium dodecyl sulfate complex: A spectroscopic and microscopic approach.

Exploring and understanding the fundamental interaction between protein and surfactant is utmost important for various pharmaceutical and biomedical applications. However, very less is known about the arrangement of individual negatively charged sodium dodecyl sulfate (SDS) molecules on the human serum albumin (HSA). Here, we have investigated the morphology and mechanistic insights of complexation between HSA and SDS by means of photoluminescence (PL) spectroscopy, circular dichroism (CD) and PL microscopy using amine-functionalized silicon quantum dot (Si QD) as an external luminescent marker. The present study is based on a unique and dynamic SDS-Si QD system. The synthesized allylamine-functionalized Si QDs show a distinct PL band centered at 455nm upon excitation at 375nm. At neutral pH, these Si QDs form ordered aggregates in the presence of 1mM SDS due to the hydrogen bonding interaction with the sulfate head groups of surfactants, which is manifested in the appearance of a large Stokes shifted luminescence band centered at 610nm. It has been observed that the PL intensity of SDS-Si QD aggregates at 610nm decreases gradually with concomitant increase in the PL intensity of monomeric Si QDs at 455nm upon increasing the concentration of HSA from 1 to 10µM. These observations combined with PL lifetime, PL microscopy and CD results reveal that SDS forms micelle-like aggregates on the partially unfolded HSA mainly via electrostatic interaction between negatively charged sulfate head groups and positively charged residues of partially unfolded HSA. For the present HSA-SDS system, our results fit a model with type I "necklace and bead"-like structures, where micelle-like SDS aggregates wrap around by the partially unfolded HSA backbone.

Luminescence turn-on/off sensing of biological iron by carbon dots in transferrin.

Iron is a key nutrient as well as a potential toxin for almost all living organisms. In mammalian cells, serum transferrin (Tf) is responsible for iron transport and its iron overload/deficiency causes various diseases. Therefore, closely regulated iron homeostasis is extremely essential for cellular metabolism. In the present article we report the pH-dependent luminescence turn-on/off sensing of bound Fe(3+) ions of serum Tf by carbon dots (CDs) with the help of photoluminescence (PL) spectroscopy, FTIR spectroscopy, dynamic light scattering (DLS), circular dichroism (CD) and PL imaging techniques. At physiological pH (7.4), the intrinsic luminescence of CDs gets quenched in the presence of Tf as a consequence of ground-state association, which is driven by favorable electrostatic interactions between negatively charged CDs (-25.45 ± 1.23 mV) and positively charged Fe(3+) ions of Tf. The estimated detection limit of Tf by CDs at physiological pH is found to be 1.82 µM (signal-to-noise ratio of 3), which is much lower than the in vivo plasma concentration of Tf (∼ 25-35 µM). Various thermodynamic parameters have been evaluated by using the van't Hoff equation. Importantly, the secondary structure of Tf remains unaltered upon association with CDs. However, at pH 3.5, no such luminescence quenching of CDs has been observed in the presence of Tf due to the lack of ground-state interactions between positively charged (+17.63 ± 0.84 mV) CDs and Tf. Furthermore, the results from UV-Vis and far-UV CD measurements revealed a significant conformational change of Tf at pH 3.5 relative to pH 7.4, which triggers the subsequent release of bound iron from Tf. PL microscopy of individual CD revealed significant luminescence quenching at the single particle level, which further supports the non-emissive ground-state complexation at pH 7.4. Our present results show that these chemically synthesized water-dispersed CDs have the ability to selectively sense the bound iron from released iron of Tf without any conformational perturbation and hence they can be used as potential biological iron sensors as well as luminescent markers for the detection of iron deficiency/overload in biological macromolecules.

Thermal luminescence quenching of amine-functionalized silicon quantum dots: a pH and wavelength-dependent study.

Understanding and resolving the mechanisms that affect the photoluminescence (PL) of Si QDs are of great importance because of their strong potential for optoelectronic and solar cell materials. In this article, the intrinsic exciton dynamics of water-dispersed allylamine-functionalized silicon quantum dots (Si QDs) have been explored as a function of temperature by means of steady-state and time-resolved PL spectroscopy. Significant PL quenching of Si QDs has been observed with increase in temperature from 278 K to 348 K. This thermal quenching is found to be a reversible process. The mechanism involves nonradiative reversible relaxation of conduction band electrons through the thermally-created temporary trap states. These temporary trap states arise due to the displacement of surface atoms from their regular positions at elevated temperature. Upon cooling, these surface irregularities relax back to their equilibrium positions with retrieval of the original PL intensity. It has been observed that the quenching mechanism is strongly influenced by the pH and excitation wavelength (λex). At pH 3.5, the quenching mechanism involves nonradiative relaxation of conduction band electrons through the thermally-created temporary trap states. However, at pH 7.4, the unprotonated surface amine groups introduce permanent nitrogen-related surface defects inside the bandgap of Si QDs. At elevated temperature, the conduction band electrons get trapped in these nitrogen-related surface defects through the involvement of thermally-created temporary trap states. Subsequent exciton recombination of these nitrogen-related defect states results in red-shifted green color luminescence. By using the Arrhenius equation we have estimated the activation energy of this nonradiative thermal relaxation process and it was found to be 138 and 139 meV at pH 3.5 and pH 7.4, respectively.

Size-dependent penetration of carbon dots inside the ferritin nanocages: evidence for the quantum confinement effect in carbon dots.

The origin of the excitation wavelength (λex)-dependent photoluminescence (PL) of carbon dots (CDs) is poorly understood and still remains obscured. This phenomenon is often explained on the basis of surface trap/defect states, while the effect of quantum confinement is highly neglected in the literature. Here, we have shown that the λex-dependent PL of CDs is mainly due to the inhomogeneous size distribution. We have demonstrated the λex-dependent PL quenching of CDs inside the ferritin nanocages through selective optical excitation of differently sized CDs. It has been observed that Fe(3+) ions of ferritin effectively quench the PL of CDs due to static electron transfer, which is driven by favorable electrostatic interactions. However, control experiment with aqueous Fe(3+) ions in bulk medium revealed λex-independent PL quenching of CDs. The λex-dependent PL quenching of CDs by Fe(3+) ions of ferritin has been rationalized on the basis of a different extent of accessibility of Fe(3+) ions by differently sized CDs through the funnel-shaped ferritin channels. PL microscopy of individual CDs has been performed to get further information about their inherent PL properties at single dot resolution. Our results have shown that these hydrophilic CDs can be used as potential iron sensors in biological macromolecules.

Concentration-dependent reversible self-oligomerization of serum albumins through intermolecular ß-sheet formation.

Proteins inside a cell remain in highly crowded environments, and this often affects their structure and activity. However, most of the earlier studies involving serum albumins were performed under dilute conditions, which lack biological relevance. The effect of protein-protein interactions on the structure and properties of serum albumins at physiological conditions have not yet been explored. Here, we report for the first time the effect of protein-protein and protein-crowder interactions on the structure and stability of two homologous serum albumins, namely, human serum albumin (HSA) and bovine serum albumin (BSA), at physiological conditions by using spectroscopic techniques and scanning electron microscopy (SEM). Concentration-dependent self-oligomerization and subsequent structural alteration of serum albumins have been explored by means of fluorescence and circular dichroism spectroscopy at pH 7.4. The excitation wavelength (λex) dependence of the intrinsic fluorescence and the corresponding excitation spectra at each emission wavelength indicate the presence of various ground state oligomers of serum albumins in the concentration range 10-150 µM. Circular dichroism and thioflavin T binding assay revealed formation of intermolecular ß-sheet rich interfaces at high protein concentration. Excellent correlations have been observed between ß-sheet content of both the albumins and fluorescence enhancement of ThT with protein concentrations. SEM images at a concentration of 150 µM revealed large dispersed self-oligomeric states with sizes vary from 330 to 924 nm and 260 to 520 nm for BSA and HSA, respectively. The self-oligomerization of serum albumins is found to be a reversible process; upon dilution, these oligomers dissociate into a native monomeric state. It has also been observed that synthetic macromolecular crowder polyethylene glycol (PEG 200) stabilizes the self-associated state of both the albumins which is contrary to expectations that the macromolecular crowding favors compact native state of proteins.