Designing Configurable Soft Microgelsomes as a Smart Biomimetic Protocell.

Self-assembly is an intriguing aspect of primitive cells. The construction of a semipermeable compartment with a robust framework of soft material capable of housing an array of functional components for chemical changes is essential for the fabrication of synthetic protocells. Microgels, loosely cross-linked polymer networks, are suitable building blocks for protocell capsule generation due to their porous structure, tunable properties, and assembly at the emulsion interface. Here, we present an interfacial assembly of microgel-based microcompartments (microgelsomes, MGC) that are defined by a semipermeable, temperature-responsive elastic membrane formed by densely packed microgels in a monolayer. The water-dispersible microgelsomes can thermally shuttle between 10 and 95 °C while retaining their structural integrity. Importantly, the microgelsomes exhibited distinct properties of protocells, such as cargo encapsulation, semipermeable membrane, DNA amplification, and membrane-gated compartmentalized enzymatic cascade reaction. This versatile approach for the construction of biomimetic microcompartments augments the protocell library and paves the way for programmable synthetic cells.

Advances in paper based isothermal nucleic acid amplification tests for water-related infectious diseases.

Water-associated or water-related infectious disease outbreaks are caused by pathogens such as bacteria, viruses, and protozoa, which can be transmitted through contaminated water sources, poor sanitation practices, or insect vectors. Low- and middle-income countries bear the major burden of these infections due to inadequate hygiene and subpar laboratory facilities, making it challenging to monitor and detect infections in a timely manner. However, even developed countries are not immune to these diseases, as inadequate wastewater management and contaminated drinking water supplies can also contribute to disease outbreaks. Nucleic acid amplification tests have proven to be effective for early disease intervention and surveillance of both new and existing diseases. In recent years, paper-based diagnostic devices have made significant progress and become an essential tool in detecting and managing water-associated infectious diseases. In this review, we have highlighted the importance of paper and its variants as a diagnostic tool and discussed the properties, designs, modifications, and various paper-based device formats developed and used for detecting water-associated pathogens.

Bactericidal and biocatalytic temperature responsive microgel based self-cleaning membranes for water purification.

The present study focuses on creating an antimicrobial and biocatalytic smart gating membrane by synthesizing unique core-shell microgels. The core-shell microgels are synthesized by grafting short chains of poly(ethylenimine) (PEI) onto a poly((N-isopropyl acrylamide)-co-glycidyl methacrylate)) (P(NIPAm-co-GMA)) core. Subsequently, the produced microgels are utilized as a substrate for synthesizing and stabilizing silver nanoparticles (Ag NPs) through an in-situ approach. These Ag NPs immobilized microgels are then suction filtered over a polyethylene terephthalate (PET) track-etched support to create cross-linked composite microgel membranes (CMMs). After structural and permeation characterization of the prepared CMMs, the laccase enzyme is then covalently grafted to the surface of the membrane and tested for its effectiveness in degrading Reactive red-120 dye. The laccase immobilized biocatalytic CMMs show effective degradation of the Reactive red-120 by 71%, 48%, and 34% at pH 3, 4, and 5, respectively. Furthermore, the immobilized laccase enzyme showed better activity and stability in terms of thermal, pH, and storage compared to the free laccase, leading to increased reusability. The unique combination of Ag NPs and laccase on a thermoresponsive microgel support resulted in a responsive self-cleaning membrane with excellent antimicrobial and dye degradation capabilities for environmentally friendly separation technology.

Biocatalytic self-assembled synthetic vesicles and coacervates: From single compartment to artificial cells.

Compartmentalization is an intrinsic feature of living cells that allows spatiotemporal control over the biochemical pathways expressed in them. Over the years, a library of compartmentalized systems has been generated, which includes nano to micrometer sized biomimetic vesicles derived from lipids, amphiphilic block copolymers, peptides, and nanoparticles. Biocatalytic vesicles have been developed using a simple bag containing enzyme design of liposomes to multienzymes immobilized multi-vesicular compartments for artificial cell generation. Additionally, enzymes were also entrapped in membrane-less coacervate droplets to mimic the cytoplasmic macromolecular crowding mechanisms. Here, we have discussed different types of single and multicompartment systems, emphasizing their recent developments as biocatalytic self-assembled structures using recent examples. Importantly, we have summarized the strategies in the development of the self-assembled structure to improvise their adaptivity and flexibility for enzyme immobilization. Finally, we have presented the use of biocatalytic assemblies in mimicking different aspects of living cells, which further carves the path for the engineering of a minimal cell.

Zwitterionic silica nanogel-modified polysulfone nanoporous membranes formed by in-situ method for water treatment.

We report a simple methodology to prepare nano-porous polysulfone membranes using zwitterionic functionalized silica nanogels with high BSA protein rejection and antifouling properties. The zwitterionic silica precursor was prepared by reacting 1,3-propane sultone with 3-aminopropyl triethoxysilane under an inert atmosphere. The precursor was in situ hydrolyzed and condensed in the polysulfone nanoporous membrane network by one-pot acidic phase inversion. The prepared membranes were characterized to establish their physicochemical nature, morphology, and basic membrane properties such as permeation, rejection, and recovery. The zwitterionic membranes showed improved hydrophilicity, membrane water uptake (∼83.5%), water permeation, BSA protein rejection (>95%), and dye rejection (congo red: >52% (∼6-fold increase); methylene blue: ∼15% (∼2-fold increase)) were improved without compromising the membrane flux and fouling resistance. Overall, we report an easy fabrication method of efficient nanocomposite zwitterionic ultrafilter membranes for water treatment with excellent flux, protein separation, filtration efficiency, and antifouling behavior.

Nature Inspired Multienzyme Immobilization: Strategies and Concepts.

In a biological system, the spatiotemporal arrangement of enzymes in a dense cellular milieu, subcellular compartments, membrane-associated enzyme complexes on cell surfaces, scaffold-organized proteins, protein clusters, and modular enzymes have presented many paradigms for possible multienzyme immobilization designs that were adapted artificially. In metabolic channeling, the catalytic sites of participating enzymes are close enough to channelize the transient compound, creating a high local concentration of the metabolite and minimizing the interference of a competing pathway for the same precursor. Over the years, these phenomena had motivated researchers to make their immobilization approach naturally realistic by generating multienzyme fusion, cluster formation via affinity domain-ligand binding, cross-linking, conjugation on/in the biomolecular scaffold of the protein and nucleic acids, and self-assembly of amphiphilic molecules. This review begins with the discussion of substrate channeling strategies and recent empirical efforts to build it synthetically. After that, an elaborate discussion covering prevalent concepts related to the enhancement of immobilized enzymes' catalytic performance is presented. Further, the central part of the review summarizes the progress in nature motivated multienzyme assembly over the past decade. In this section, special attention has been rendered by classifying the nature-inspired strategies into three main categories: (i) multienzyme/domain complex mimic (scaffold-free), (ii) immobilization on the biomolecular scaffold, and (iii) compartmentalization. In particular, a detailed overview is correlated to the natural counterpart with advances made in the field. We have then discussed the beneficial account of coassembly of multienzymes and provided a synopsis of the essential parameters in the rational coimmobilization design.

Bienzymatic Sequential Reaction on Microgel Particles and Their Cofactor Dependent Applications.

We report, the preparation and characterization of bioconjugates, wherein enzymes pyruvate kinase (Pk) and l-lactic dehydrogenase (Ldh) were covalently bound to poly(N-isopropylacrylamide)-poly(ethylenimine) (PNIPAm-PEI) microgel support using glutaraldehyde (GA) as the cross-linker. The effects of different arrangements of enzymes on the microgels were investigated for the enzymatic behavior and to obtain maximum Pk-Ldh sequential reaction. The dual enzyme bioconjugates prepared by simultaneous addition of both the enzymes immobilized on the same microgel particles (PL), and PiLi, that is, dual enzyme bioconjugate obtained by combining single-enzyme bioconjugates (immobilized pyruvate kinase (Pi) and immobilized lactate dehydrogenase (Li)), were used to study the effect of the assembly of dual enzymes systems on the microgels. The kinetic parameters (Km, kcat), reaction parameters (temperature, pH), stability (thermal and storage), and cofactor dependent applications were studied for the dual enzymes conjugates. The kinetic results indicated an improved turn over number (kcat) for PL, while the kcat and catalytic efficiency was significantly decreased in case of PiLi. For cofactor dependent application, in which the ability of ADP monitoring and ATP synthesis by the conjugates were studied, the activity of PL was found to be nearly 2-fold better than that of PiLi. These results indicated that the influence of spacing between the enzymes is an important factor in optimization of multienzyme immobilization on the support.

Smart core-shell microgel support for acetyl coenzyme A synthetase: a step toward efficient synthesis of polyketide-based drugs.

The flexibility in tuning the structure and charge properties of PNIPAm microgels during their synthesis makes them a suitable choice for various biological applications. Two-step free radical polymerization, a common method employed for synthesis of core-shell microgel has been well adopted to obtain cationic poly(N-isopropylacrylamide-aminoethyl methacrylate) (PNIPAm-AEMA) shell and PNIPAm core. Scanning electron microscopy (SEM), dynamic light scattering (DLS), zeta potential, and ninhydrin assay suggests nearly monodispersed particles of cationic nature. Amino groups on the microgel provides suitable attachment point for covalent immobilization of acetyl coenzyme A synthetase (Acs) via 1-ethyl-3-(3-N,N- dimethylaminopropyl) carbodiimide (EDC) chemistry. On immobilization, 61.55% of initial activity of Acs has been retained, while Michaelis-Menten kinetics of the immobilized Acs indicates identical K(m) (Michaelis constant) but decrease in the V(max) (maximum substrate conversion rate) compared to free enzyme. Immobilized Acs shows an improvement in activity at wide temperature and pH range and also demonstrates good thermal, storage, and operational stability. The Acs-microgel bioconjugate has been successfully reused for four consecutive operation cycles with more than 50% initial activity.

Functional polyelectrolyte multilayer membranes for water purification applications.

A diverse set of supported multilayer assemblies with controllable surface charge, hydrophilicity, and permeability to water and solute was fabricated by pressure driven permeation of poly(sodium 4-styrenesulfonate) (PSS) and poly(diallyldimethylammonium chloride) (PDDA) solution through poly(ethylene terephthalate) (PET) track-etched membranes. The polyelectrolyte multilayer fabrication was confirmed by means of FTIR, SEM, AFM, ellipsometry, zetapotential, and contact angle characterization. The prepared membranes were characterized in terms of their pure water permeability, flux recovery, and resistance to organic and biofouling properties. The antifouling behavior of the membranes was assessed in terms of protein adsorption and antibacterial behavior. Finally, the membranes were tested for rejection of selected water soluble dyes to establish their usefulness for organic contaminant removal from water. The membranes were highly selective and capable of nearly complete rejection of congo red with sufficiently high fluxes. The feasibility of regenerating the prepared membranes fouled by protein was also demonstrated and good flux recovery was obtained. In summary, the multilayer approach to surface and pore modification was shown to enable the design of membranes with the unique combination of desirable separation characteristics, regenerability of the separation layer, and antifouling behavior.

Antifouling and antibiofouling pH responsive block copolymer based membranes by selective surface modification.

Antifouling and antibacterial membranes are prepared by selective surface modification of pH responsive polystyrene-b-poly(4-vinylpyridine) (PS-P4VP) diblock copolymers by quaternization and zwitterionization reactions on a P4VP moiety. Nanoporous membranes based on the self-assembly of 2-(4'-hydroxybenzeneazo) benzoic acid (HABA)-PS-P4VP supramolecular complexes and nonsolvent induced phase separation are first prepared and the surfaces are functionalized by crosslinking with diiodobutane vapors and reacting with propane sultone vapors at moderate temperature and under vacuum conditions. Selective functionalization of surfaces is carried out to enhance the antifouling and antibiofouling properties of the membrane and to retain its pH switching behavior. The membranes are thoroughly characterized by various instrumental techniques such as Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, quartz crystal microbalance, contact angle, etc. Antifouling and antibacterial properties are proven by analyzing the adsorption of bovine serum albumin protein and bacterial cell attachment and killing efficiency. The actual membrane performance is assessed in terms of water flux under different pressures and pHs and Congo red dye rejection efficiency.

Bifunctionalized organic-inorganic charged nanocomposite membrane for pervaporation dehydration of ethanol.

Chitosan was modified into N-p-carboxy benzyl chitosan (NCBC) by introducing an aromatic ring grafted with acidic -COOH group and highly stable and cross-linked nanostructured NCBC-silica composite membranes were prepared for pervaporation dehydration of water-ethanol mixture. These membranes were tailored to comprise three regions namely: hydrophobic region, highly charged region and selective region, in which weak acidic group (-COOH) was grafted at organic segment while strong acidic group (-SO(3)H) was grafted at inorganic segment to achieve high stability and less swelling in water-ethanol mixture. Cross-linking density and NCBC-silica content in membrane matrix has been systematically optimized to control the nanostructure of the developed polymer matrix for studying the effects of molecular structure on the swelling, and PV performance. Among prepared membranes, nanocomposite membrane with 3h cross-linking time and 90% (w/w) of NCBC-silica content (PCS-3-3) exhibited 1.66×10(-4)cm(3)(STP) cm/cm(2) s cmHg water permeability (P(W)), while 1.35×10(-7) cm(3)(STP) cm/cm(2) s cmHg ethanol permeability (P(EtOH)) of developed membrane and 1231 PV selectivity factor at 30 °C for separating water from 90% (w/w) ethanol mixture.

Crosslinked chitosan/polyvinyl alcohol blend beads for removal and recovery of Cd(II) from wastewater.

Crosslinked chitosan/poly(vinyl alcohol) (PVA) beads were prepared by suspension of chitosan-PVA aqueous solution in a mixture of toluene and chlorobenzene. Some quantity of the water was distilled out as an azeotrope along with toluene-chlorobenzene and the droplets were chemically crosslinked by adding glutaraldehyde. The prepared crosslinked beads were characterized by FTIR, X-ray diffraction (XRD), and scanning electron microscopy (SEM). The developed beads were used as an adsorbent for the adsorption of Cd(II) from wastewater. Effect of time, temperature, pH, adsorbent dose and adsorbate concentration on the adsorption of Cd(II) were investigated in batch process and pseudo-first and pseudo-second-order kinetic models were also evaluated. The equilibrium adsorption obeyed Langmuir and Freundlich isotherms as well as the thermodynamic parameters such as DeltaG degrees , DeltaH degrees and DeltaS degrees were calculated. From thermodynamic data, it was found that the adsorption process was endothermic and spontaneous. The maximum adsorption of Cd(II) ions was found to be 73.75% at pH 6 and indicated that developed material could be effectively utilized for the removal of Cd(II) ions from wastewater.

3-[[3-(Triethoxysilyl)propyl]amino]propane-1-sulfonic acid-poly(vinyl alcohol) cross-linked zwitterionic polymer electrolyte membranes for direct methanol fuel cell applications.

Recently, organic-inorganic nanocomposite zwitterionic polymer electrolyte membranes (PEMs) have attracted remarkable interest for application to the direct methanol fuel cell (DMFC) operated at intermediate temperature (100-200 degrees C). In this paper, we report the synthesis of an organic-inorganic hybrid zwitterionomer silica precursor with ammonium and sulfonic acid functionality by the ring-opening of 3-propanesultone under mild heating conditions and the preparation procedure of a proton-conductive and stable organic-inorganic zwitterion-poly(vinyl alcohol) (PVA) cross-linked PEM by sol-gel in aqueous media. Developed PEMs were extensively characterized by studying their physicochemical and electrochemical properties under DMFC operating conditions. These membranes were designed to possess all of the required properties of a proton-conductive membrane, namely, reasonable swelling, good mechanical, dimensional, and oxidative strength, flexibility, and low methanol permeability along with reasonable proton conductivity (4.85 x 10(-2) S cm(-1)) due to zwitterionic functionality. Moreover, from the selectivity parameter among all developed membranes, ZI-70 [zwitterionomer membrane with 70 wt % of PVA of 3-[[3-(triethoxysilyl)propyl]amino]propane-1-sulfonic acid in the membrane matrix], exhibited the best results in comparison to the Nafion117 membrane for DMFC applications.

Membrane-based techniques for the separation and purification of proteins: an overview.

Membrane processes are increasingly reported for various applications in both upstream and downstream technology, such as microfiltration, ultrafiltration, emerging processes as membrane chromatography, high performance tangential flow filtration and electrophoretic membrane contactor. Membrane-based processes are playing critical role in the field of separation/purification of biotechnological products. Membranes became an integral part of biotechnology and improvements in membrane technology are now focused on high resolution of bioproduct. In bioseparation, applications of membrane technologies include protein production/purification, protein-virus separation. This manuscript provides an overview of recent developments and published literature in membrane technology, focusing on special characteristics of the membranes and membrane-based processes that are now used for the production and purification of proteins.

An improved process for separation of proteins using modified chitosan-silica cross-linked charged ultrafilter membranes under coupled driving forces: isoelectric separation of proteins.

Functionalized chitosan namely as N-methylene phosphonic chitosan (PC) and quaternized chitosan (QC) silica composite charged ultrafilter membranes were prepared by acid catalyzed sol-gel method in the aqueous media and gelated in methanol for tailoring their pore structure. These membranes were employed for developing a simple membrane process for pH sensitive protein fractionation under coupled driving forces (pressure and electric gradient). Protein transmission (selectivity) and membrane throughput across both membranes were studied using binary mixture of protein under different gradients at pH points: 2.0, 4.8, 10.7, and 13.0. It was concluded that separation from the binary mixture of BSA-LYS, separation LYS at pH 4.8 (pI of BSA) using negatively charged PC-Si membrane or separation BSA at pH 10.7 (pI of LYS) using positively charged QC-Si membrane, was possible with high selectivity. Also in all cases, due to coupling of driving forces, filtrate flux and selectivity were enhanced by several folds. Furthermore, applied electric gradient progressively increased the separation factor values, which was close to 10 for PC-Si and 15 for QC-Si membranes. Relatively high separation value of individual protein from binary mixture and filtrate velocity suggests the practical usefulness of this novel process and biopolymer membranes.

Functionalized organic-inorganic nanostructured N-p-carboxy benzyl chitosan-silica-PVA hybrid polyelectrolyte complex as proton exchange membrane for DMFC applications.

Chitosan was modified into N-p-carboxy benzyl chitosan (NCBC) by introducing an aromatic ring grafted with carboxylic acid as the proton conducting group. A preparation procedure of highly conductive and stable organic-inorganic nanostructured NCBC-silica-poly(vinyl alcohol) (PVA), proton exchange membrane (PEM) for direct methanol fuel cell (DMFC), by the sol-gel method in aqueous media has been reported. These PEMs were developed by cross-linking and designed to consist of weak proton conducting (-COOH) groups at organic segments and strong proton conducting (-SO3H) groups at inorganic segments to achieve high charge density and stabilities. Cross-linking density and NCBC-silica content in the membrane matrix were systematically optimized to control their nanostructure, thermal, mechanical, and chemical stabilities, as well as proton and fuel transport properties. Developed PEMs were extensively characterized by studying their physicochemical and electrochemical properties under DMFC operating conditions. As these PEMs were well processed as self-supporting film, they showed high stabilities and proton conductivity and low methanol permeability. Moreover, among all synthesized membranes, PCS-3-3 hybrid PEM exhibited quite a high selectivity parameter in comparison to Nafion117 membrane for DMFC applications.

Sulfonated poly(styrene-co-maleic anhydride)-poly(ethylene glycol)-silica nanocomposite polyelectrolyte membranes for fuel cell applications.

A method for the preparation of highly conductive and stable organic-inorganic nanocomposite polyelectrolyte membranes with controlled spacing between inorganic segment and covalently bound sulfonic acid functional groups has been established. These polyelectrolyte membranes were prepared by condensation polymerization of the silica precursor (tetraethylorthosilicate) in dimethylacetamide in the presence of poly(ethylene glycol) (PEG) of desired molecular weight, and sulfonated poly(styrene-co-maleic anhydride) was attached to the polymeric backbone by hydrogen bonding. Molecular weight of PEG has been systematically changed to control the nanostructure of the developed polymer matrix for studying the effects of molecular structure on the thermal as well as conductive properties. These polyelectrolyte membranes were extensively characterized by studying their thermo-gravimetric analysis (TGA), ion-exchange capacity (IEC), water content, conductivity, methanol permeability, and current-voltage polarization curves under direct methanol fuel cell (DMFC) operating conditions as a function of silica content and molecular weight of PEG used for membrane preparation. Moreover, from these studies and estimation of selectivity parameter among all synthesized membranes, 30% silica content and 400 Da molecular weight of PEG resulted in the best nanocomposite polyelectrolyte membranes, which exhibited performance comparable to that of the Nafion 117 membrane for DMFC applications.

SPEEK-zirconium hydrogen phosphate composite membranes with low methanol permeability prepared by electro-migration and in situ precipitation.

Sulfonated poly(ether ether ketone) (SPK)-zirconium hydrogen phosphate (ZrP) composite membranes were prepared by electro-driven migration of Zr(4+) and simultaneous in situ precipitation of ZrP using phosphoric acid under different electrical gradient, in order to avoid loss in its mechanical stability. Degree of sulfonation was estimated from (1)H NMR and ion-exchange capacity study that was found to be 61% and 57%, respectively. In this method Zr(4+) and HPO(4)(2-) were allowed to diffuse within the pores/channels of the preformed SPK membrane under given electrical gradient and ZrP was precipitated within the membrane matrix. ZrP loading density was measured as a function of applied electrical gradient for a definite reaction time (4 h) and electrolytic environment. Membranes with varied ZrP loading densities were characterized for their thermal and mechanical stabilities, physicochemical and electrochemical properties using thermogravimetric analysis (TGA), dynamic mechanical analysis (DMA), scanning electron microscopy (SEM), water content, proton conductivity and methanol permeability. No loss in thermal and mechanical stability of membranes was observed due to incorporation of inorganic component (ZrP) in the membrane matrix. Although the composite membranes exhibited low proton conductivity in comparison to SPK membrane at room temperature, but the presence of the inorganic particles led to an improvement in high temperature conductivity. Selectivity parameter of these composite membranes was estimated at two temperatures namely 30 and 70 degrees C, in latter case it was found significantly higher than that for Nafion membrane (0.79 x 10(5) S s cm(-3)) under similar experimental conditions.