Highly stable, fluorescent, artificial lipoprotein nanoparticles.

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°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.

Tailoring Synthetic Polypeptide Design for Directed Fibril Superstructure Formation and Enhanced Hydrogel Properties.

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.

Non-Covalent Assembly of Multiple Fluorophores in Edible Protein/Lipid Hydrogels for Applications in Multi-Step Light Harvesting and White-Light Emission.

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.

Modeling and Designing Particle-Regulated Amyloid-like Assembly of Synthetic Polypeptides in Aqueous Solution.

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.

One-step preparation of bioactive enzyme/inorganic materials.

Simultaneous exfoliation of crystalline α-zirconium phosphate (α-ZrP) nanosheets and enzyme binding, induced by shearing, without the addition of any toxic additives is reported here for the first time. These materials were thoroughly characterized and used for applications. The bulk α-ZrP material (20 mg mL-1) was exfoliated with low concentrations of a protein such as bovine serum albumin (BSA, 3 mg mL-1) in a shear reactor at 10k rpm for <80 minutes. Exfoliation was monitored by powder X-ray diffraction with samples displaying a gradual but complete loss of the 7.6 Å (002) peak, which is characteristic of bulk α-ZrP. The fully exfoliated sample loaded with the protein was characterized by transmission and scanning electron microscopy in addition to other biophysical methods. Lysozyme, glucose oxidase, met-hemoglobin, and ovalbumin also induced exfoliation and directly produced enzyme/ZrP biocatalysts. Thus, exfoliation, biophilization and enzyme binding are accomplished in a single step. Several factors contributed to the exfoliation kinetics, and the rate increased with α-ZrP and BSA concentrations and decreased with pH. However, the exfoliation efficiency inversely depended on the isoelectric point of the protein with ovalbumin (pI = 4.5) being the best and lysozyme (pI = 11.1) being the worst. A strong correlation between the protein size and exfoliation efficiency was noted, and the latter suggests the role of hydrodynamic factors in the process. Exfoliation was also achieved by simple stirring using a magnetic stirrer, under low volumes, and model enzymes, indicating 60-90% retention of bound enzymatic activities. The addition of BSA to enzymes as the diluent and stabilizing agent also prevents enzymes from the denaturing effect caused by stirring. This new method requires no pre-treatment of α-ZrP with toxic exfoliating agents such as tetrabutyl ammonium hydroxide and provides bioactive enzyme/inorganic materials in a single step. These protein-loaded biocompatible nanosheets may be useful for biocatalysis and biomedical applications.

Kinetic Study on Enzymatic Hydrolysis of Cellulose in an Open, Inhibition-Free System.

Due to the complexity of cellulases and the requirement of enzyme adsorption on cellulose prior to reactions, it is difficult to evaluate their reaction with a general mechanistic scheme. Nevertheless, it is of great interest to come up with an approximate analytic description of a valid model for the purpose of developing an intuitive understanding of these complex enzyme systems. Herein, we used the surface plasmonic resonance method to monitor the action of a cellobiohydrolase by itself, as well as its mixture with a synergetic endoglucanase, on the surface of a regenerated model cellulose film, under continuous flow conditions. We found a phenomenological approach by taking advantage of the long steady state of cellulose hydrolysis in the open, inhibition-free system. This provided a direct and reliable way to analyze the adsorption and reaction processes with a minimum number of fitting parameters. We investigated a generalized Langmuir-Michaelis-Menten model to describe a full set of kinetic results across a range of enzyme concentrations, compositions, and temperatures. The overall form of the equations describing the pseudo-steady-state kinetics of the flow-system shares some interesting similarities with the Michaelis-Menten equation. The use of familiar Michaelis-Menten parameters in the analysis provides a unifying framework to study cellulase kinetics. The strategy may provide a shortcut for approaching a quantitative while intuitive understanding of enzymatic degradation of cellulose from top to bottom. The open system approach and the kinetic analysis should be applicable to a variety of cellulases and reaction systems to accelerate the progress in the field.

Micelles Embedded in Multiphasic Protein Hydrogel Enable Efficient and Air-Tolerant Triplet Fusion Upconversion with Heavy-Atom and Spin-Orbit Charge-Transfer Sensitizers.

The applications of triplet-triplet annihilation-based photon upconversion (TTA-UC) in solar devices have been limited by the challenges in designing a TTA-UC system that is efficient under aerobic conditions. Efficient TTA-UC under aerobic conditions is typically accomplished by using soft matter or solid-state media, which succeed at protecting the triplet excited states of upconverters (sensitizer and annihilator) from quenching by molecular oxygen but fail at preserving their mobility, thus limiting the TTA-UC efficiency (ΦUC). We showcase a protein/lipid hydrogel that succeeded in doing both of the above due to its unique multiphasic design, with a high ΦUC of 19.0 ± 0.7% using a palladium octaethylporphyrin sensitizer. This hydrogel was made via an industrially compatible method using low-cost and eco-friendly materials: bovine serum albumin (BSA), sodium dodecyl sulfate (SDS), and water. A dense BSA network provided oxygen protection while the encapsulation of upconverters within a micellar SDS environment preserved upconverter mobility, ensuring near-unity triplet energy transfer efficiency. In addition to heavy atom-containing sensitizers, several completely organic, spin-orbit charge-transfer intersystem crossing (SOCT-ISC) Bodipy-based sensitizers were also studied; one of which achieved a ΦUC of 3.5 ± 0.2%, the only reported SOCT-ISC-sensitized ΦUC in soft matter to date. These high efficiencies showed that our multiphasic design was an excellent platform for air-tolerant TTA-UC and that it can be easily adapted to a variety of upconverters.

Exfoliated and water dispersible biocarbon nanotubes for enzymology applications.

In this chapter, we report a simple and facile method to armor enzymes with carbon nanotubes (CNTs) which are exfoliated, and debundled using bovine serum albumin (BSA). The fabricated CNT/BSA dispersions are biofriendly, biocompatible, defect-free, and highly stable solutions. BSA gives maximum exfoliation efficiency, exceeding the 4mg/mL of CNT concentration compared to any previous reports. Further, the produced bioCNT dispersions were characterized by UV-visible, Raman, circular dichroism spectroscopy, and scanning electron microscopy (SEM). Exfoliation and debundling of the bioCNT dispersions is possible due to the π-π interaction, hydrogen bonding, hydrophobic interaction, and electrostatic attractive forces driving the adsorption of BSA on CNTs surface. Protein adsorption then makes a highly stable suspension in water that can be stored for a prolonged period. CNT dispersions are stable over a wide range of pH from 3 to 10 and at 4°C or 25°C for more than 2 months. Here, we also report the facile, inexpensive and green-chemistry method to fabricate a buckypaper (CNT paper), composed of the high packing density, self-assembled and randomly oriented bioCNTs, and these assemblies could be used in many emerging applications like air and water purification, nanocomposites, energy storage, and biosensing. Moreover, the CNT dispersions stabilized by BSA were successfully used in enzyme binding and kinetic studies and bound enzyme retained substantial catalytic activity. The current approach may facilitate bulk production of water dispersed CNTs in both academic and industrial laboratories. This is done by a simple method of stirring, which provides new opportunities for a wider range of CNT applications.

Stirred Not Shaken: Facile Production of High-Quality, High-Concentration Graphene Aqueous Suspensions Assisted by a Protein.

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.

Engineering functional inorganic nanobiomaterials: controlling interactions between 2D-nanosheets and enzymes.

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 α-zirconium(iv) phosphate (α-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.

Interlocking Enzymes in Graphene-Coated Cellulose Paper for Increased Enzymatic Efficiency.

A simple method for interlocking glucose oxidase and horseradish peroxidase in a network of cellulose fibers coated with bovine serum albumin (BSA)-exfoliated graphene (biographene) is reported here. The resulting paper reactor is inexpensive and stable. Biographene is expected to function as an electron shuttle, making the reaction between the enzyme and the substrate more efficient, and this hypothesis is examined here. The BSA used to separate the sheets of graphene provides extra carboxylic acid groups and primary amines to help interlock the enzymes and the graphene in between the fibers. The decrease in entropy associated with interlocking the enzymes on a solid support is likely responsible for the increase in enzymatic stability/activity observed. Each cellulose disk contained 5.2mg of enzyme per gram of paper and 93% of the enzyme is retained after washing for 0.5-2h. This simple methodology provides a low cost, effective approach for achieving high enzymatic activity and good loadings on a benign, versatile support.

A Simple Flow Reactor for Continuous Synthesis of Biographene for Enzymology Studies.

The unique properties of graphene make it an intriguing platform for the attachment and enhancement of biological molecules, but it has yet to achieve its full potential in terms of biological applications. Single-layer graphene is expensive, making alternatives to this material highly desired for applications that require high-quality graphene in large quantities. In this context, we report a simple, environmentally friendly, nonlabor-intensive method for the synthesis of colloidal graphene suspensions of 3-5 layers, stabilized by bovine serum albumin, in water. The method involves a flow reactor designed to continually yield high-quality graphene colloids, synthesized, purified, and optimized all in one setup. The flow reactor is able to produce colloidal graphene sheets on a multigram scale, and these colloids were characterized by Raman spectroscopy, electron microscopy, and zeta potential studies. The average size of the sheets is 0.16µm2, each consisting of 3-5 layers of graphene with little or no sp3 defects. These graphene colloids stabilized by the protein were successfully used in protein kinetic studies as well as in surface plasmon resonance protein binding studies. The ease of synthesis of these high-quality graphene colloidal suspensions in water provides an exciting opportunity for biographene to be used on an industrial scale for electronic, thermal, and enzymology applications.

Tuning the chain length of new pyrene derivatives for site-selective photocleavage of avidin.

Rational design of photoreagents with systematic modifications of their structures can provide valuable information for a better understanding of the protein photocleavage mechanism by these reagents. Variation of the length of the linker connecting the photoactive moiety with the protein anchoring-group allowed us to investigate the control of the protein photocleavage site. A series of new photochemical reagents (PMA-1A, PMA-2A and PMA-3A) with increasing chain lengths is examined in the current study. Using avidin as a model system, we examined the interaction of these probes by UV-Vis, fluorescence spectroscopic methods, photocleavage and computational docking studies. Hypochromism of the absorption spectrum was observed for the binding of these new photochemical reagents with estimated binding constants (Kb) of 6.2 × 105, 6.7 × 105 and 4.6 × 105 M-1, respectively. No significant changes of Stern-Volmer quenching constant (Ksv) with Co(NH3)6Cl3 has been noted and the data indicated that the probes bind near the surface of the protein with sufficient exposure to the solvent. Photoexcitation of the probe-avidin complex, in the presence of Co(NH3)6Cl3, resulted in protein fragmentation, and the cleavage yield decreased with the increase in the linker length, and paralleled with the observed Ksv values. Amino acid sequencing of the photofragments indicated that avidin is cleaved between Thr77 and Val78, as a major cleavage site for all the three photoreagents. This site is proximate to the biotin binding site on avidin, and molecular docking studies indicated that the H-bonding interactions between the polar end-group of the photoreagents and hydrophilic amino acids of avidin were important in positioning the reagent on the protein. The major cleavage site, at residues 77-78, was within 5 Šof the pyrenyl moiety of the probe, and hence, molecular tuning of the linker provided a simple approach to position the photoreagent along the potential photocleavage site.

Epitope-Resolved Detection of Peanut-Specific IgE Antibodies by Surface Plasmon Resonance Imaging.

Peanut allergy can be life-threatening and is mediated by allergen-specific immunoglobulin E (IgE) antibodies. Investigation of IgE antibody binding to allergenic epitopes can identify specific interactions underlying the allergic response. Here, we report a surface plasmon resonance imaging (SPRi) immunoassay for differentiating IgE antibodies by epitope-resolved detection. IgE antibodies were first captured by magnetic beads bearing IgE ϵ-chain-specific antibodies and then introduced into an SPRi array immobilized with epitopes from the major peanut allergen glycoprotein Arachis hypogaea h2 (Ara h2). Differential epitope responses were achieved by establishing a binding environment that minimized cross-reactivity while maximizing analytical sensitivity. IgE antibody binding to each Ara h2 epitope was distinguished and quantified from patient serum samples (10 µL each) in a 45 min assay. Excellent correlation of Ara h2-specific IgE values was found between ImmunoCAP assays and the new SPRi method.

Tuning Enzyme/α-Zr(IV) Phosphate Nanoplate Interactions via Chemical Modification of Glucose Oxidase.

Using glucose oxidase (GOx) and α-Zr(IV) phosphate nanoplates (α-ZrP) as a model system, a generally applicable approach to control enzyme-solid interactions via chemical modification of amino acid side chains of the enzyme is demonstrated. Net charge on GOx was systematically tuned by appending different amounts of polyamine to the protein surface to produce chemically modified GOx(n), where n is the net charge on the enzyme after the modification and ranged from -62 to +95 electrostatic units in the system. The binding of GOx(n) with α-ZrP nanosheets was studied by isothermal titration calorimetry (ITC) as well as by surface plasmon resonance (SPR) spectroscopy. Pristine GOx showed no affinity for the α-ZrP nanosheets, but GOx(n) where n ≥ -20 showed binding affinities exceeding (2.1 ± 0.6) × 106 M-1, resulting from the charge modification of the enzyme. A plot of GOx(n) charge vs Gibbs free energy of binding (ΔG) for n = +20 to n = +65 indicated an overall increase in favorable interaction between GOx(n) and α-ZrP nanosheets. However, ΔG is less dependent on the net charge for n > +45, as evidenced by the decrease in the slope as charge increased further. All modified enzyme samples and enzyme/α-ZrP complexes retained a significant amount of folding structure (examined by circular dichroism) as well as enzymatic activities. Thus, strong control over enzyme-nanosheet interactions via modulating the net charge of enzymes may find potential applications in biosensing and biocatalysis.

Controlling the Graphene-Bio Interface: Dispersions in Animal Sera for Enhanced Stability and Reduced Toxicity.

Liquid phase exfoliation of graphite in six different animal sera and evaluation of its toxicity are reported here. Previously, we reported the exfoliation of graphene using proteins, and here we extend this approach to complex animal fluids. A kitchen blender with a high-turbulence flow gave high quality and maximum exfoliation efficiency in all sera tested, when compared to the values found with shear and ultrasonication methods. Raman spectra and electron microscopy confirmed the formation of three- or four-layer, submicrometer size graphene, independent of the serum used. Graphene prepared in serum was directly transferred to cell culture media without post-treatments. Contrary to many reports, a nanotoxicity study of this graphene fully dispersed to human embryonic kidney cells, human lung cancer cells, and nematodes (Caenorhabditis elegans) showed no acute toxicity for up to 7 days at various doses (50-500 µg/mL), but prolonged exposure at higher doses (300-500 µg/mL, 10-15 days) showed cytotoxicity to cells (∼95% death) and reproductive toxicity to C. elegans (5-10% reduction in brood size). The origin of toxicity was found to be due to the highly fragmented smaller graphene sheets (<200 nm), while the larger sheets were nontoxic (50-300 µg/mL dose). In contrast, graphene produced with sodium cholate as the mediator has been found to be cytotoxic to these cells at these dosages. We demonstrated the toxicity of liquid phase exfoliated graphene is attributed to highly fragmented fractions or nonbiocompatible exfoliating agents. Thus, low-toxicity graphene/serum suspensions are produced by a facile method in biological media, and this approach may accelerate the much-anticipated development of graphene for biological applications.

Ultrathin Graphene-Protein Supercapacitors for Miniaturized Bioelectronics.

Nearly all implantable bioelectronics are powered by bulky batteries which limit device miniaturization and lifespan. Moreover, batteries contain toxic materials and electrolytes that can be dangerous if leakage occurs. Herein, an approach to fabricate implantable protein-based bioelectrochemical capacitors (bECs) employing new nanocomposite heterostructures in which 2D reduced graphene oxide sheets are interlayered with chemically modified mammalian proteins, while utilizing biological fluids as electrolytes is described. This protein-modified reduced graphene oxide nanocomposite material shows no toxicity to mouse embryo fibroblasts and COS-7 cell cultures at a high concentration of 1600 µg mL-1 which is 160 times higher than those used in bECs, unlike the unmodified graphene oxide which caused toxic cell damage even at low doses of 10 µg mL-1. The bEC devices are 1 µm thick, fully flexible, and have high energy density comparable to that of lithium thin film batteries. COS-7 cell culture is not affected by long-term exposure to encapsulated bECs over 4 d of continuous charge/discharge cycles. These bECs are unique, protein-based devices, use serum as electrolyte, and have the potential to power a new generation of long-life, miniaturized implantable devices.