Molecular hybrids consisting of hydrophobic, water-insoluble cobalt phthalocyanine (CoPC) complex and noncatalytic protein bovine serum albumin (BSA) have been developed as a green catalyst for stereoselective oxidations, including C=C epoxidation and C-H hydroxylation. Effective encapsulation of CoPC into the BSA scaffold (CoPC-BSA) was achieved through agitation of BSA and CoPC in an aqueous solution, resulting in a structurally intact, stable protein-cofactor hybrid. Upon activation with H2O2, CoPC-BSA catalyzed epoxidations of styrene and cyclooctene with yields of 87-99%, achieving >99% enantioselectivity for R-styrene oxide. CoPC-BSA also enabled selective C-H oxidation of propanoic acid and methyl phenylacetate under mild conditions. For propanoic acid, L-lactic acid was the sole product of oxidation of propanoic acid, while methyl phenylacetate showed temperature-dependent enantioselectivity. When immobilized on silica beads, R-methyl mandelate with an enantioselectivity >92% at 80 °C was produced at a conversion of ∼99%. These results demonstrate the potential of molecular hybrids for enantioselective oxidations and highlight the critical role of protein flexibility and dynamics in tuning catalytic outcomes.

Background: Live attenuated and inactivated virus vaccines are commonly used against infectious bronchitis virus (IBV) in chickens, but they have limitations such as mutation risks and short efficacy. This study explores cationic bovine serum albumin (BSA) polyamine nanoparticles (NPs) for delivering IBV spike protein mRNA, aiming to develop a safer and more effective vaccine. Methods: A BSA-based nanoparticle system was designed with positive surface charges and characterized using dynamic light scattering (DLS), Zetasizer, and transmission electron microscopy (TEM). Its cytotoxicity, cellular uptake, and ability to deliver IBV spike protein mRNA were evaluated in macrophage-like chicken cell lines (HD11), followed by immunogenicity studies in SPF chickens to assess immune responses. Results: The study demonstrated successful binding and transfection efficiency of the in vitro transcription (IVT)-mRNA complexed with the NPs, which was enhanced with chloroquine. Immunogenicity studies in SPF chickens showed a significant increase in antibody titers in chickens vaccinated with the mRNA vaccine compared to the PBS control, indicating an effective immune response against the IBV S protein. Furthermore, the neutralization index doubled after a higher-dose mRNA booster with chloroquine, and PBMCs from immunized chickens exhibited a threefold higher stimulation index than the PBS control. Conclusions: BSA-based NPs effectively deliver IBV spike protein mRNA, enhancing immune responses and offering a promising strategy for a safer, more effective IBV vaccine.

We herein report a design of artificial enzymes by incorporating a synthetic copper complex into noncatalytic bovine serum albumin (Cu-BSA) to carry out stereoselective oxidation. This Cu-BSA catalyst with stably bound Cu complex as a cofactor shows peroxidase-like activity to catalyze epoxidation of styrene with high chiral selectivity (>99%) to R-styrene epoxide. With the electrochemical conversion of Cu2+ to Cu+, Cu-BSA also exhibits oxidase-like activity to selectively reduce oxygen to hydrogen peroxide (H2O2), which can be combined with its peroxidase function to drive oxidation of C & boxH;C bonds using air. This artificial enzymatic system holds promise for chiral-selective transformations of non-natural substances and highlights the versatility of noncatalytic proteins in the design of artificial enzymes.

Here, we report a novel kind of protein nanoparticles of 11 nm in size, which have a central protein core surrounded by two layers of lipid. One layer of the lipid was covalently attached to the protein, while the other layer has been physically assembled around the protein core. Particle synthesis is highly modular, while both the size and charge of the protein nanoparticles are controlled in a predictable manner. Circular dichroism studies of the conjugate showed that the protein secondary structure is retained, while biophysical characterizations indicated the particle purity, size, and charge. The conjugate had a high thermal stability to steam sterilization conditions at 121 degrees C (17 psi). After labeling the protein core with few different fluorescent dyes, they were strongly fluorescent with the corresponding colors independent of their size, unlike quantum dots. They are readily digested by proteases, and these water-soluble, non-toxic, highly stable, biocompatible, and biodegradable conjugates are suitable for cell imaging and drug delivery applications. Covalent coupling of a short-chain fatty acid to the amine groups of serum albumin by carbodiimide chemistry and subsequent labeling of the product with a reactive fluorescent dye gave 11 nm lipid-protein fluorescent nanoparticles. These could be useful for imaging and sensing applications.image

The biological significance of self-assembled protein filament networks and their unique mechanical properties have sparked interest in the development of synthetic filament networks that mimic these attributes. Building on the recent advancement of autoaccelerated ring-opening polymerization of amino acid N-carboxyanhydrides (NCAs), this study strategically explores a series of random copolymers comprising multiple amino acids, aiming to elucidate the core principles governing gelation pathways of these purpose-designed copolypeptides. Utilizing glutamate (Glu) as the primary component of copolypeptides, two targeted pathways were pursued: first, achieving a fast fibrillation rate with lower interaction potential using serine (Ser) as a comonomer, facilitating the creation of homogeneous fibril networks; and second, creating more rigid networks of fibril clusters by incorporating alanine (Ala) and valine (Val) as comonomers. The selection of amino acids played a pivotal role in steering both the morphology of fibril superstructures and their assembly kinetics, subsequently determining their potential to form sample-spanning networks. Importantly, the viscoelastic properties of the resulting supramolecular hydrogels can be tailored according to the specific copolypeptide composition through modulations in filament densities and lengths. The findings enhance our understanding of directed self-assembly in high molecular weight synthetic copolypeptides, offering valuable insights for the development of synthetic fibrous networks and biomimetic supramolecular materials with custom-designed properties.

The design and production of biodegradable and sustainable non-toxic materials for solar-energy harvesting and conversion is a significant challenge. Here, our goal was to report the preparation of novel protein/lipid hydrogels and demonstrate their utility in two orthogonal fundamental studies-light harvesting and white-light emission. Our hydrogels contained up to 90% water, while also being self-standing and injectable with a syringe. In one application, we loaded these hydrogels with suitable organic donor-acceptor dyes and demonstrated the energy-transfer cascade among four different dyes, with the most red-emitting dye as the energy destination. We hypothesized that the dyes were embedded in the protein/lipid phase away from the water pools as monomeric entities and that the excitation of any of the four dyes resulted in intense emission from the lowest-energy acceptor. In contrast to the energy-transfer cascade, we demonstrate the use of these gels to form a white-light-emitting hydrogel dye assembly, in which excitation migration is severely constrained. By restricting the dye-to-dye energy transfer, the blue, green, and red dyes emit at their respective wavelengths, thereby producing the composite white-light emission. The CIE color coordinates of the emission were 0.336 and 0.339-nearly pure white-light emission. Thus, two related studies with opposite requirements could be accommodated in the same hydrogel, which was made from edible ingredients by a simple method. These gels are biodegradable when released into the environment, sustainable, and may be of interest for energy applications.

Graphene quantum dots (GQDs) are carbon-based, zero-dimensional nanomaterials and unique due to their astonishing optical, electronic, chemical, and biological properties. Chemical, photochemical, and biochemical properties of GQDs are intensely being explored for bioimaging, biosensing, and drug delivery. The synthesis of GQDs by top-down and bottom-up approaches, their chemical functionalization, bandgap engineering, and biomedical applications are reviewed here. Current challenges and future perspectives of GQDs are also presented.

In cells, actin and tubulin polymerization is regulated by nucleation factors, which promote the nucleation and subsequent growth of protein filaments in a controlled manner. Mimicking this natural mechanism to control the supramolecular polymerization of macromolecular monomers by artificially created nucleation factors remains a largely unmet challenge. Biological nucleation factors act as molecular scaffolds to boost the local concentrations of protein monomers and facilitate the required conformational changes to accelerate the nucleation and subsequent polymerization. An accelerated assembly of synthetic poly(L-glutamic acid) into amyloid fibrils catalyzed by cationic silica nanoparticle clusters (NPCs) as artificial nucleation factors is demonstrated here and modeled as supramolecular polymerization with a surface-induced heterogeneous nucleation pathway. Kinetic studies of fibril growth coupled with mechanistic analysis demonstrate that the artificial nucleators predictably accelerate the supramolecular polymerization process by orders of magnitude (e.g., shortening the assembly time by more than 10 times) when compared to the uncatalyzed reaction, under otherwise identical conditions. Amyloid-like fibrillation was supported by a variety of standard characterization methods. Nucleation followed a Michaelis-Menten-like scheme for the cationic silica NPCs, while the corresponding anionic or neutral nanoparticles had no effect on fibrillation. This approach shows the effectiveness of charge-charge interactions and surface functionalities in facilitating the conformational change of macromolecular monomers and controlling the rates of nucleation for fibril growth. Molecular design approaches like these inspire the development of novel materials via biomimetic supramolecular polymerizations.

This chapter reports a single-step preparation of nanoarmored bi-enzyme systems assembled on 1-D and 2-D nanomaterials, with glucose oxidase and peroxidase enzymes as model systems for cascade bio-catalysis. This is a simple and facile method to both exfoliate the bulk 1D (carbon nanotubes, CNT) and 2D nanomaterials (α-Zirconium phosphate, α-ZrP) and bind the enzymes in a single step. Exfoliation of the bulk material enhances the accessible surface area of the materials for the enzyme binding, and it also boosts the diffusion of reagents from the bulk phase to the active sites of the bio-catalysts. For example, a mixture of horseradish peroxidase, glucose oxidase, and bovine serum albumin (BSA) were adsorbed on the surfaces of the α-ZrP nanoplates or carbon nanotubes (CNT) as the bulk materials are exfoliated simultaneously, in a one-step process. The resulting bio-catalysts were thoroughly characterized by powder X-ray diffraction, electron microscopy, biochemical and biophysical methods, while enzyme activity studies proved successful binding of enzymes with retention of activities or even enhancements in their specific activities. For example, GOx/HRP/BSA/CNT displayed 6 times the activity of a mixture of GOx/HRP/BSA, under otherwise identical conditions. Similarly, GOx/HRP/BSA/ZrP had 3.5 times the activity of the corresponding mixture of GOx/HRP/BSA, in the absence of the nanoplates. These robust nano-dispersions worked extraordinarily well as active bio-catalysts. These two kinds of fabricated biocatalyst dispersions are also highly stable.

Graphene is the most studied and debated material over the last two decades. The optoelectronic and mechanical properties of graphene and its derivatives led graphene research to several scientific fields. This chapter aims to provide an introduction to graphene, starting with the historic developments, all the way to nanotoxicity. A major emphasis on synthesis routes to graphene is discussed and examples relevant to composite science are provided. Subsequent sections describe classifications, nomenclature and characterizations with specific examples of graphene literature. Classification of graphene composites are discussed next as an introduction to the major theme of the book. Finally, the nanotoxicity concerns and considerations are described. We are hopeful that the above-mentioned topics provide necessary foundation for readers to dwell into the following chapters.

A simple method to produce record concentrations (up to 10 mg mL-1) of high-quality aqueous graphene suspensions by using an ordinary benchtop magnetic stirrer is reported. The shear rates employed here are almost 10 times less than those in previous reports, and graphene is efficiently separated from unexfoliated graphite during the synthesis. Systematic optimization of synthesis parameters, such as pH, protein concentration, temperature, stirrer speed, and volume of solution, afforded efficient conversion (100%) of graphite to graphene-aqueous suspensions. The synthesis is readily scaled-up with a continuous flow reactor where the graphene is produced and separated 24/7, with little or no human intervention. Raman spectroscopy confirmed little to no sp3 or oxidative defects, and that the graphene nanosheets consisted of three to five layers. The graphene suspensions were coated on aluminum and tested for thermal conductivity applications. The thermal conductivity of our graphene sample was calculated to be 684 W m-1 K-1, a value greater than that of a commercial sample. The activation energy measured for shear exfoliation by stirring was found to be over 45 billion times smaller than the corresponding thermal activation energy, affording physical insight into the process. We hypothesize that stirring selectively populates translational states that are necessary for exfoliation and thus requires far less energy than conventional exfoliation methods, where the energy is uniformly distributed among all available modes. Therefore, an efficient, convenient, and inexpensive method for graphene production in limited-resource settings is reported here.

A better understanding of the enzyme-nanosheet interface is imperative for the design of functional, robust inorganic nanobiomaterials and biodevices, now more than ever, for use in a broad spectrum of applications. This feature article discusses recent advances in controlling the enzyme-nanosheet interface with regards to alpha-zirconium(iv) phosphate (alpha-ZrP), graphene oxide (GO), graphene, and MoS2 nanosheets. Specific focus will be placed on understanding the mechanisms with which these materials interact with enzymes and elaborate on particular ways to engineer and control these interactions. Our main discoveries include: (1) upon adsorption to the nanosheet surface, a decrease in the entropy of the enzyme's denatured state enhances stability; (2) proteins are used to create biophilic landing pads for increased enzyme stability on many different types of nanosheets; (3) proteins and enzymes are used as exfoliants by shear force to produce biofunctionalized nanosheet suspensions; and (4) bionfunctionalized nanosheets exhibit no acute toxicity. Recognizing proper methods to engineer the interface between enzymes and 2D-nanosheets, therefore, is an important step towards making green, sustainable, and environmentally conscious inorganic bionanomaterials for sensing, catalysis and drug delivery applications, as well as towards the successful manipulation of enzymes for advanced applications.

Most of the killed, inactivated, or live-attenuated pathogen vaccines are now replaced by modern vaccines containing isolated, highly purified antigenic protein subunits which are safer than live viruses or deactivated viruses. Another strategy is the development of nanoparticles which can mimic the repetitiveness, geometry, size and shape of the host-pathogen surface and provide improved stability and long lasting immunogenicity, as well as serve as vehicles to deliver multiple copies of the antigens to the target cells. Several interesting advances have been made recently in the area of protein nanotechnology and here, we provide a concise review of one such novel nanoparticle called FluoDot which may be effectively used as a delivery system in some veterinary medicine applications.

Japanese encephalitis (JE) is a re-emerging mosquito-borne zoonotic flaviviral disease. Swine sero-convert 2-3 weeks before infection occurs in humans and thus serves as a suitable sentinel for JE surveillance and outbreak prediction in human population. The present study was conducted with the objective of developing a lateral flow assay (LFA) for detecting JEV antibodies in swine sera. Three different formats were tried using recombinant NS1 protein as antigen in order to select the best format. In format I, gold nanoparticles were conjugated with antigen followed by spotting of antigen on NCM as test line and anti-antigen IgG on NCM as control line. In format II, gold nanoparticles were conjugated with antigen followed by spotting of staphylococcal protein A as test line and anti-antigen IgG as control line. Format III used gold nanoparticles conjugated with goat anti-pig IgG followed by spotting of antigen as test line and pig IgG as control line. Amongst the three formats, format II was found to be superior with 100% relative diagnostic sensitivity and 100% relative diagnostic specificity during monsoon and post-monsoon period. A panel of 500 field swine serum samples was tested using format II which revealed sero-positivity of 15.6%, and the format was found suitable to screen swine serum samples during monsoon and post-monsoon period.

The present study was conducted to assess the impact of different land-use systems on major nutrient status viz: Nitrogen, Phosphorus and Potassium in soils of central part of Western ghat, Karnataka. The land use types include both manmade systems (Agriculture: paddy; Horticulture: coffee, are canut, tea and banana); forest plantation (accacia and teak) and natural systems (Evergreen, semi-evergreen and grassland). In each land use systems, samples were collected from two depths (0-15 cm and 15-30 cm) at 20 locations during pre-monsoon and post monsoon period. Soil samples were analyzed for available nitrogen, available phosphorus and available potassium. The results revealed that available nitrogen content in surface soil was significantly higher (394.55 kg ha-1) than that of subsurface soil (330.57 kg ha-1). Among manmade land use systems, available nitrogen content was significantly higher in coffee (435.82 kg ha-1) followed by in soils under banana 404.40 kg ha- 1. Similarly, natural system the available-N content was in the range of 294.97 to 376.55 kg ha-1 with minimum in grassland and maximum in semi-evergreen forest. Available-P varied significantly among different land use systems. The highest available-P content was recorded in soils under coffee (29.31 kg ha-1) followed by evergreen forest (28.37 kg ha-1), semi-evergreen (27.26 kg ha-1) and other systems. The available-P was least in grassland (20.28 kg ha-1). The available K2O content in soils under paddy was 299.42 kg ha-1 and it was significantly lower than other systems but was at par with those observed in grassland (311.01 kg ha-1), acacia (321.01 kg ha-1) and teak system (329.86 kg ha-1). These results are indicated that well-managed horticultural system in general and coffee in particular had better soil fertility which is comparable to that found in natural forest system.

A facile methodology to prepare N-heterocyclic carbene (NHC)-terminated polymers as surface ligands to functionalize gold nanoparticles (AuNPs) is reported.

Cytochrome c–poly(acrylic acid) conjugates with 34-fold enhanced peroxidase activity due to acidification of enzyme microenvironment and suppression of wasteful intermediates.

This chapter provides general methods for the synthesis and characterization of the photoreagents and associated methods. Absorption and fluorescence titrations, for example, and methods for the photocleavage of proteins are described here. We also included some examples of N-terminal amino acid sequencing of the photofragments and quantum yield determinations. We hope that a novice practitioner might benefit from these methods in implementing these methodologies with some ease. As always, proper safety methods are to be applied at each and every step of the way as necessary. We caution the users to follow these methods at their own discretion and we are not responsible for any issues that might arise in following these recommended methods, and hold us harmless for any consequences.

The role of the linker connecting the hydrophobic and hydrophilic moieties of the reagent in the photocleavage of the protein is examined in this chapter. One to three amino acid residues were used in the linker and their influence on the binding, photochemistry and cleavage selectivity have been examined. The length and nature of the linker has a major influence on the protein-binding/photocleavage properties of the probe. The binding constants of various probes with particular linkers toward bovine serum albumin (BSA) were in the range of 10(5)-10(7)/M, demonstrating the ability to tune the affinity over two orders of magnitude by simply changing the linker. Thus, the pyrenyl moiety might dominate the affinity but the linker plays an important role in controlling the binding affinities with BSA. Probes with short linkers, Py-Gly and Py-L-Phe, indicated hypochromism and high binding constants. Probes with medium length linkers (Py-Gly-Phe, Py-Gly-Tyr, Py-Gly-Trp and Py-Gly-His) indicated hyperchromism and binding constants dropped with increased hydrophilicity of the linker residues. For the tripeptide probes, Py-Gly-Gly-Phe and Py-Phe-Gly-Gly, a new broad emission was observed at 466 nm when bound to BSA, as observed with Py-L-Phe, but no such emission was observed with any of the other probes and BSA. The circular dichroism (CD) spectra provided fingerprints of the proteins and no two CD spectra of the probe-protein complexes were superimposable. Fluorescence quenching experiments using cobalt(III)hexammine (CoHA) as the quencher indicated that the tripeptide probes were protected better than the dipeptide probes but reasons for this difference have not been established. The distance of separation between the carboxyl function and the pyrenyl chromophore had substantial effect on the accessibility of the protein-bound probe, which influenced the photocleavage efficiencies. Photoexcitation of the probe-protein complexes, in the presence of CoHA, resulted in protein photocleavage with all the above probes, except that the linker containing either tyrosine or tryptophan residues did not give any photofragmentation. This is likely due to the interception of the excited states of these probes by the electron-rich side chains of these residues. This also implies that such residues at the binding site may interfere with the photoreaction. Flash photolysis studies of the probe-protein mixtures, in support of this conclusion, indicated that the initially produced pyrene cation radical was strongly quenched by tyrosine and tryptophan residues. Probes with longer linkers caused photocleavage further down the protein sequence, thus providing a strong handle to tune the cleavage site by a rational approach. A strong modulation of the photoreactivities of the probes by specific residues of the probe side chains provided specific avenues to improve the photocleavage yields. The linker played a very important role in the binding and cleavage properties and these details are enumerated here.