Patterning of Protein-Sequestered Liquid-Crystal Droplets Using Acoustic Wave Trapping.

Development of spatially organized structures and understanding their role in controlling kinetics of multistep chemical reactions are essential for the successful design of efficient systems and devices. While studies that showcase different types of methodologies for the spatial organization of various colloidal systems are known, design and development of well-defined hierarchical assemblies of liquid-crystal (LC) droplets and subsequent demonstration of biological reactions using such assemblies still remain elusive. Here, we show reversible and reconfigurable one-dimensional (1D) assemblies of protein-bioconjugate-sequestered monodisperse LC droplets by combining microfluidics with noninvasive acoustic wave trapping technology. Tunable spatial geometries and lattice dimensions can be achieved in an aqueous medium comprising ≈19 or 62 µm LC droplets. Different assemblies of a mixed population of larger and smaller droplets sequestered with glucose oxidase (GOx) and horseradish peroxidase (HRP), respectively, exhibit spatially localized enzyme kinetics with higher initial rates of reaction compared with GOx/HRP cascades implemented in the absence of an acoustic field. This can be attributed to the direct substrate transfer/channeling between the two complementary enzymes in close proximity. Therefore, our study provides an initial step toward the fabrication of LC-based devices for biosensing applications.

Light-responsive self-assembled microstructures of branched polyethyleneimine at low pH.

Soft materials comprising polyethyleneimine (PEI), which integrate low pH-stimulated higher-order assemblies (fibres and sheets) with light responsiveness, have been shown. Excitation wavelength light-driven interactions enable the formation of bead-necklace-type structures in fibres or volume collapse of sheets. This work can have significant implications for transfection or encapsulation through PEI-based complexes.

Ultraslow Biological Water-Like Dynamics in Waterless Liquid Protein.

Fluorescence correlation spectroscopy and time-dependent fluorescence Stokes shift have been employed to elucidate dynamics in different time scales, ranging from picoseconds to nanoseconds, for human serum albumin, in its native and cationized forms as well as in the self-assembled complex of the cationized protein with the polymer surfactant (PS) glycolic acid ethoxylate lauryl ether. The effect of crowding in this complex, especially in the waterless condition, is of prime importance in this context. Excellent correlation of the dynamics with the structures, obtained by circular dichroism and Fourier transform infrared spectroscopy, has been observed. Slow solvation, associated classically with biological water, has been observed in these systems, even in the waterless condition. This apparently intriguing observation has been rationalized by the relaxation of segments of the protein and the PS in the microenvironment of the fluorescent probe.

Polymer-Surfactant Driven Interactions and the Resultant Microstructure in Protein-Containing Liquid Crystal Droplets.

Integration of molecular liquid crystals (LCs) with functional proteins can provide new class of materials for potential applications in optical biosensing. However, hydrophobic nematic LCs (length ∼ 1-2 nm) and hydrophilic proteins, size ∼ O (nm), do not intermix without chemical modification of at least one of them. Bioconjugation of proteins with a polyethylene glycol-based polymeric surfactant (PS) can provide a core-shell system that is sequestered within nonaqueous LC (4-cyano-4'-pentylbiphenyl) microdroplets. However, the nature of interactions between the components and detailed understanding of the resultant hybrid microstructure remains unclear. Here, using a combination of isothermal titration calorimetry (ITC), fluorescence microscopy, and infrared-imaging spectroscopy, we show that strong hydrophobic interactions between the LC and PS drives the sequestration of a myoglobin-PS (Mb-PS; dispersed in the aqueous phase) into the LC spherical microdroplets or even into a bulk LC phase. The average values of both, the binding constant and the standard molar enthalpy change, are increased by approximately a factor of 2.5 times when the unmodified Mb is conjugated to the PS. Small-angle X-ray scattering studies reveal that LC molecules act as a solvent for the Mb-PS conjugate; furthermore, the LC long-range order is disturbed due to mixing, as exemplified by the change in its coherence length from 8.9 to 5.7 nm. Detailed all-atomistic molecular dynamic simulations for a three-component PS-water-LC system show a change in interaction energy of -144 kJ mol-1 PS-1 upon the contact of PS chains (initially dispersed in water) with LC and agree with the ITC experiments.

Composite Porous Liquid for Recyclable Sequestration, Storage and In†Situ Catalytic Conversion of Carbon Dioxide at Room Temperature.

Permanent pores combined with fluidity renders flow processability to porous liquids otherwise not seen in porous solids. Although porous liquids have been utilized for sequestration of different gases and their separation, there is still a dearth of studies for deploying in situ chemical reactions to convert adsorbed gases into utility chemicals. Here, we show the design and development of a new type of solvent-less and hybrid (meso-)porous liquid composite, which, as demonstrated for the first time, can be used for in situ carbon mineralization of adsorbed CO2 . The recyclable porous liquid composite comprising polymer-surfactant modified hollow silica nanorods and carbonic anhydrase enzyme not only sequesters (5.5 cm3 g-1 at 273 K and 1 atm) and stores CO2 but is also capable of driving an in situ enzymatic reaction for hydration of CO2 to HCO3 - ion, subsequently converting it to CaCO3 due to reaction with pre-dissolved Ca2+ . Light and electron microscopy combined with X-ray diffraction reveals the nucleation and growth of calcite and aragonite crystals. Moreover, the liquid-like property of the porous composite material can be harnessed by executing the same reaction via diffusion of complimentary Ca2+ and HCO3 - ions through different compartments separated by an interfacial channel. These studies provide a proof of concept of deploying chemical reactions within porous liquids for developing utility chemical from adsorbed molecules.

Dispersion and Interaction of Charged Fluorescent Dyes in Protein-Polymer Surfactant-based Non-Aqueous Liquid.

Viscous, non-aqueous liquid comprising stoichiometric conjugates of polymer surfactant-bovine serum albumin (PSpBSA) is used as a host matrix for the dispersion of chemically distinct hydrophilic dyes. Using a combination of bright field polarized optical microscopy and fluorescence spectroscopy, we investigate the dispersion of dry and powdered cationic (Rhodamine 6G; Rh6G) and anionic (Fluorescein; FL) dyes in the PSpBSA liquid at room temperature. As the dyes disperse and dissolve in the PSpBSA liquid, it results in a pronounced increase in emission intensity of the former. Interestingly, a shift from 571 to 582 nm is observed in the emission maxima of Rh6G as it disperses in the PSpBSA solvent. Whilst no such red shift is found for the Rh6G dispersion in the aqueous solutions of either native BSA or polymer-surfactant conjugated BSA, a similar shift occurs when Rh6G is dispersed in neat polymer-surfactant (PS), suggesting the interaction of the dye with the PS chains. In the case of anionic FL, no shift is observed in its emission maximum as it disperses in the PSpBSA liquid. Furthermore, within 120 minutes of FL dispersion in the PSpBSA liquid, we observe a ≈26 % decrease in the tryptophan emission intensity (λexc. =285 nm; λemi. =330 nm) of BSA, which could be attributed to both static and dynamic quenching. Our findings provide a proof of concept of an alternative non-aqueous solvent matrix which can dissolve and disperse charged fluorescent dyes, provide suitable binding sites, and show substantial photoluminescence. Thus, it can be envisaged for utilization as an alternative solvent medium for lasing dyes and related applications.

Neat Ionic liquid and α-Chymotrypsin-Polymer Surfactant Conjugate-Based Biocatalytic Solvent.

Performing biocatalysis in nonaqueous solvents is advantageous as it imparts enhanced solubility to hydrophobic substrates and an ability to increase the temperature for shifting reaction equilibrium in the forward direction. In this work, we show the design and development of another class of nonaqueous composite solvent obtained by mixing surface modified enzyme and neat ionic liquid (IL). We systematically probe the interaction and solubility of industrially relevant α-chymotrypsin in its native or surface-bound polymer-surfactant bioconjugated form, with neat protic (N-methyl-2-pyrrolidonium trifluoromethanesulfonate; [NMP][OTf]), or aprotic (1-methyl-3-(4-sulfobutyl)-1H-imidazol-3-ium trifluoromethanesulfonate; [HO3S(CH2)4MIm][OTf]), ILs. Polarized optical micrographs show that the lyophilized powder of native α-chymotrypsin, nCT, does not disperse in either of the neat ILs, however, its polymer surfactant (PS)-coated bioconjugate counterparts, PScCT, in the waterless state, can be well-dispersed and solubilized in the neat [HO3S(CH2)4MIm][OTf]. The solubilization of waterless bioconjugates of PScCT in neat aprotic IL provides a composite liquid, WL-ImPScCT (WL: waterless, Im: [HO3S(CH2)4MIm][OTf]), having a viscosity of 69.6 Pa·s at 25 °C with a shear-thinning behavior, ≈ 15 w/w % α-chymotrypsin, and ≈ 1.2 w/w % residual water content. Detailed secondary structural analysis using circular dichroism and Fourier self-deconvolution on the ATR-FTIR data of WL-ImPScCT liquid reveals retention of the near native secondary structure of α-chymotrypsin. Further, using a combination of fluorescence spectroscopy and electron spray ionization mass spectrometry, we show that scattering of dry and powdered bovine serum albumin (BSA) protein on the WL-ImPScCT composite liquid results in the solubilization of the former, followed by limited proteolysis of BSA by the α-chymotrypsin. Our results, therefore, show the stabilization of α-chymotrypsin in a neat aprotic IL environment to yield a composite liquid, which not only acts as a nonaqueous, nonvolatile, and environmentally benign solvent, but also provides a biocatalytic platform capable of carrying out reactions relevant for biotransformations, food processing, drug delivery, and various other applications.

α-Synuclein Spontaneously Adopts Stable and Reversible α-Helical Structure in Water-Less Environment.

A highly stable, spontaneous, and reversible α-helical-structure formation in recombinant and chemically modified α-synuclein protein is demonstrated for the first time in a water-less (1.5 % w/w H2 O) polymer surfactant environment. Using a combination of circular dichroism and ATR-FTIR spectroscopy, we show that whilst native α-synuclein in aqueous solution shows a predominant unordered conformation (≈64 %), mixing with polyethylene glycol based anionic polymer surfactant (PS) and removing water reveals a 25 % unordered, 25 % α-helical, and 27 % ß-sheet structure. Interestingly, bioconjugation of native α-synuclein with a diamine molecule, to increase the positive charge on the protein chain, and subsequent electrostatic coupling with the PS forms a conjugate with a retained unordered structure. Removal of water from this system provides a highly stable α-helical (≈74 %) water-less liquid system. Surprisingly, the α-helical-to-unordered state transition is completely reversible and is achieved at ≈25-30 w/w% of water in the system. Moreover, the α-helix shows an extraordinary temporal stability (>6 months) in a waterless environment.

A solvent-free porous liquid comprising hollow nanorod-polymer surfactant conjugates.

Liquids having permanent porosity can offer significant processing advantages over their solid counterparts. This has recently led to tremendous activity towards the design and development of intrinsic pores in the liquid phase, predominantly for studies involving gas sequestration. We show here the development of a solvent-free mesoporous liquid material based on anisotropic "hollow-core and silica-shell" nanorods conjugated with polymer surfactant chains, which can sequester CO2 gaseous molecules at 0 °C. Hollow silica nanorods (SiNRs) with average aspect ratios of 2.5, 8, and 11 (as obtained by transmission electron microscopy (TEM) and small angle X-ray scattering) were synthesized using a surfactant-templating methodology, and fluidity/flow processability were imparted by a three-step process involving covalent coupling of an organosilane (OS) canopy to form OS@SiNR, followed by electrostatic grafting of polymer surfactant (PS) chains to the organosilane, and subsequent removal of solvent to provide a solvent-free composite, PS-OS@SiNR. Differential scanning calorimetric and frequency sweep rheological measurements of PS-OS@SiNR indicated melting transition between 15 and 20 °C, while thermal gravimetric analysis showed ca. 20 w/w% silica content (i.e. 9.5% volume fraction of silica and containing ca. 3% volume fraction as voids). As observed using TEM, the surface modification of the nanorods resulting in the formation of PS-OS@SiNR does not lead to blockage of the hollow core. We show that whilst N2 adsorption in the porous liquid is hindered due to the glassy polymer-surfactant layer at -196 °C, CO2 adsorption at 0 °C showed 3.3-4.8 w/w% gas uptake. Overall we demonstrate the synthesis of an anisotropic porous liquid which not only sequesters CO2 but also has the ability to flow like a liquid.

Neat Protein-Polymer Surfactant Bioconjugates as Universal Solvents.

Solvents, particularly those having low volatility, are of great interest for the biocatalytic synthesis of utility chemicals and fuels. We show novel and universal solvent-like properties of a neat and water-less polymer surfactant-bovine serum albumin (BSA) conjugated material (WL-PS pBSA). This highly viscous, nonvolatile material behaves as a liquid above its solid-liquid transition temperature (∼25-27 °C) and can be used as a solvent for variety of completely dry solutes of different sizes and surface chemistries. We show using a combination of optical microscopy and steady -state and time-resolved fluorescence spectroscopy that dry and powdered dyes (hydrophobic Coumarin 153 (C153)), enzymes (α-chymotrypsin (α-Chy)), or even 1 µm microparticles (diffusion coefficient ca. three orders slower than C153), can be solubilized and completely dispersed in the WL-PS pBSA solvent above 25 °C. While C153, irrespective of its mode of addition to WL-PS pBSA, binds similarly to BSA, α-Chy can be stabilized and activated to perform its hydrolysis function, even at 100 °C. This work therefore provides insights into the form of universal solvent characteristic property for this new class of nonaqueous, nonvolatile, biodegradable protein-polymer surfactant-based conjugated materials and suggests potential new avenues that can have a huge impact on biocatalysis, bionanotechnology, drug delivery, and other applications.

Mechanoresponsive and recyclable biocatalytic sponges from enzyme-polymer surfactant conjugates and nanoparticles.

The development of multifunctional hybrid biomaterials is an important area of focus in tissue engineering, drug delivery, biocatalysis, and biosensing applications. Combining bioconjugation methodology with ice templating technique, we show here the development of a new class of multifunctional and biocatalytic scaffold-like spongy material fabricated from an aqueous solution of enzyme-polymer surfactant (enzyme-PS) core-shell conjugates, and polyethyleneimine (PEI) coated silica/silk nanoparticles. The generality of this process is demonstrated by the fabrication of biocatalytic sponges comprising PEI coated nanoparticles and core-shell conjugates of alkaline phosphatase (ALP-PS), or glucose oxidase (GOx-PS), and horseradish peroxidase (HRP-PS). We show that ALP-PS conjugate driven biocatalytic transformations can be simply achieved by saturating the highly porous, and manoeuvrable sponges with the p-nitrophenyl phosphate substrate solution. Subsequently, the compressible and elastic property of the sponge can be utilized for the extrusion of the product, p-nitrophenol, by applying controlled and normal mechanical stress. Further, the sponges can be washed and recycled upto ten times, with approximately 67% retention of initial biocatalytic activity. Interestingly, the ALP-PS conjugate based sponges exhibit mechanoresponsive catalytic behaviour; the amount of product obtained over 25 minutes of reaction time can be increased by approx. 8 times by compressing-decompressing the sponge after every 15 seconds. This is attributed to the change in mass transfer and diffusion of the substrate within the porous channels of the sponge. We also highlight the importance of bioconjugation of enzymes for fabricating such sponges; our results show that, whilst the native enzymes either denature or are leached away during the fabrication/biocatalytic usage, their enzyme-PS conjugate counterparts integrate efficiently to form sturdy, robust, highly catalytic, and recyclable sponge material.

Multi-enzyme cascade reactions using protein-polymer surfactant self-standing films.

Hierarchical self-assembly is used to fabricate bio-catalytically active, self-supporting protein-polymer surfactant films capable of sustaining two- or three-enzyme cascade reactions.

Light-induced dynamic shaping and self-division of multipodal polyelectrolyte-surfactant microarchitectures via azobenzene photomechanics.

Light-induced shape transformations represent a fundamental step towards the emergence of adaptive materials exhibiting photomechanical behaviours. Although a range of covalent azobenzene-based photoactive materials has been demonstrated, the use of dynamic photoisomerization in mesostructured soft solids involving non-covalent co-assembly has received little attention. Here we prepare discrete micrometre-sized hydrated particles of a hexagonally ordered polyelectrolyte-surfactant mesophase based on the electrostatically induced co-assembly of poly(sodium acrylate) (PAA) and trans-azobenzene trimethylammonium bromide (trans-azoTAB), and demonstrate unusual non-equilibrium substrate-mediated shape transformations to complex multipodal microarchitectures under continuous blue light. The microparticles spontaneously sequester molecular dyes, functional enzymes and oligonucleotides, and undergo self-division when transformed to the cis state under UV irradiation. Our results illustrate that weak bonding interactions in polyelectrolyte-azobenzene surfactant mesophases can be exploited for photo-induced long-range molecular motion, and highlight how dynamic shape transformations and autonomous division can be activated by spatially confining azobenzene photomechanics in condensed microparticulate materials.

Dynamic Behavior in Enzyme-Polymer Surfactant Hydrogel Films.

Dynamic protein-polymer surfactant films are highly hydrophilic and show a soft solid to hydrogel transition upon hydration to produce a swollen hydrogel. An unusual reversible autospreading/self-folding response is observed when the water-saturated films are transferred from water into air.

Enzyme activity in liquid lipase melts as a step towards solvent-free biology at 150 °C.

Water molecules play a number of critical roles in enzyme catalysis, including mass transfer of substrates and products, nucleophilicity and proton transfer at the active site, and solvent shell-mediated dynamics for accessing catalytically competent conformations. The pervasiveness of water in enzymolysis therefore raises the question concerning whether biocatalysis can be undertaken in the absence of a protein hydration shell. Lipase-mediated catalysis has been undertaken with reagent-based solvents and lyophilized powders, but there are no examples of molecularly dispersed enzymes that catalyse reactions at sub-solvation levels within solvent-free melts. Here we describe the synthesis, properties and enzyme activity of self-contained reactive biofluids based on solvent-free melts of lipase-polymer surfactant nanoconjugates. Desiccated substrates in liquid (p-nitrophenyl butyrate) or solid (p-nitrophenyl palmitate) form can be mixed or solubilized, respectively, into the enzyme biofluids, and hydrolysed in the solvent-free state. Significantly, the efficiency of product formation increases as the temperature is raised to 150 °C.

Self-organization of glucose oxidase-polymer surfactant nanoconstructs in solvent-free soft solids and liquids.

An anisotropic glucose oxidase-polymer surfactant nanoconjugate is synthesized and shown to exhibit complex temperature-dependent phase behavior in the solvent-free state. At close to room temperature, the nanoconjugate crystallizes as a mesolamellar soft solid with an expanded interlayer spacing of ca. 12 nm and interchain correlation lengths consistent with alkyl tail-tail and PEO-PEO ordering. The soft solid displays a birefringent spherulitic texture and melts at 40 °C to produce a solvent-free liquid protein without loss of enzyme secondary structure. The nanoconjugate melt exhibits a birefringent dendritic texture below the conformation transition temperature (Tc) of glucose oxidase (58 °C) and retains interchain PEO-PEO ordering. Our results indicate that the shape anisotropy of the protein-polymer surfactant globular building block plays a key role in directing mesolamellar formation in the solvent-free solid and suggests that the microstructure observed in the solvent-free liquid protein below Tc is associated with restrictions in the intramolecular motions of the protein core of the nanoconjugate.

Redox transitions in an electrolyte-free myoglobin fluid.

Redox responses associated with the heme prosthetic group in a myoglobin-polymer surfactant solvent-free liquid are investigated for the first time in the absence of an electrolyte solution. Cyclic voltammograms from the biofluid exhibit responses that are consistent with planar diffusion of mobile charges in the melt. Temperature-dependent dynamic electrochemical and rheological responses are rationalized in terms of the effective electron hopping rate between heme centers and the transport of intrinsic ionic species in the viscous protein liquid.

Exclusion from hexagonal mesophase surfactant domains drives end-to-end enchainment of rod-like particles.

Anisotropic rod-like particles assemble end-to-end when the surfactant/water matrix in which they are dispersed is cooled from the isotropic to the lyotropic hexagonal phase. We demonstrate the formation of such end-to-end assemblies for gold nanorods, which are tens of nanometers in size, as well as for micrometer-sized ellipsoidal polystyrene particles. In both cases, the particles are well-dispersed in the low-viscosity surfactant/water phase above the isotropic-H1 transition temperature. On cooling into the H1 phase, mesophase domains form and the particles are expelled to the isotropic phase. As the H1 domains grow and finally impinge, the particles are localized at the domain boundaries where they reorient and assemble end-to-end. Remarkably, we observe the formation of end-to-end assemblies of gold nanorods even for volume fractions as low as 2 × 10(-6) in the initially dispersed state. The extent of particle "enchainment" increases with the particle concentration and with the aspect ratio of the particles.

Isolation of a highly reactive ß-sheet-rich intermediate of lysozyme in a solvent-free liquid phase.

The thermal denaturation of solvent-free liquid lysozyme at temperatures in excess of 200 °C was studied by synchrotron radiation circular dichroism spectroscopy. Temperature-dependent changes in the secondary structure were used to map the equilibrium denaturation pathway and characterize a reactive ß-sheet-rich unfolding intermediate that was stable in the solvent-free liquid phase under anhydrous conditions but which underwent irreversible aggregation in the presence of water. The unfolding intermediate had a transition temperature of 78 °C and was extremely stable to temperature, eventually reaching the fully denatured state at 178 °C. We propose that the three-stage denaturation pathway arises from the decreased stability of the native state due to the absence of any appreciable hydrophobic effect, along with an entropically derived stabilization of the reactive intermediate associated with molecular crowding in the solvent-free liquid.

Enzymatically active self-standing protein-polymer surfactant films prepared by hierarchical self-assembly.

Cross-linked protein-polymer surfactant films consisting of enzymatically active hybrid nanoclusters are prepared using a novel approach based on electrostatically mediated hierarchical self-assembly. The free-standing films are structurally robust, highly hydrophilic, and exhibit sustained fluorescence or recyclable enzymatic phosphatase or oxido-reductase behavior.