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.

Excited-state proton transfer (ESPT) of 2-(2'-pyridyl)benzimidazole (2PBI) in reverse micelles has been studied by steady-state and time-resolved fluorescence spectroscopy. The nanometer sized water pool in the n-heptane/Aerosol OT (AOT)/water microemulsion is found to promote tautomer emission of this probe, as is evident from the emergence of a Stokes shifted band at 450 nm at the expense of the normal emission band on increasing the water content of the system. In the nonaquous microemulsion with a methanol core, the normal emission is quenched but no tautomer emission is obtained. With an acetonitrile core, there is no change in emission properties. Similarly, there is no evidence of ESPT in Triton X-100 reverse micelles. This indicates the requirement of ESPT to occur in microheterogeneous media; the medium should be a ternary system comprised of water and a hydrophobic phase separated by a negatively charged interface. In the microemulsions with an aqueous core, the fluorescence decays of 2PBI at the red end exhibit rise times of 0.8 ns and the time-resolved area-normalized emission spectra (TRANES) exhibit an isoemissive point, indicating slow dynamics of the two-state ESPT of 2PBI in aqueous AOT reverse micelles. The origin of the selective enhancement in AOT microemulsions as well as the slow dynamics is explored using fluorescence spectroscopic techniques, with support from quantum chemical calculation.

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.

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.