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.

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.