Virus-Based Separation of Rare Earth Elements.

The utilization of biomaterials for the separation of rare earth elements (REEs) has attracted considerable interest due to their inherent advantages, including diverse molecular structures for selective binding and the use of eco-friendly materials for sustainable systems. We present a pioneering methodology for developing a safe virus to selectively bind REEs and facilitate their release through pH modulation. We engineered the major coat protein of M13 bacteriophage (phage) to incorporate a lanthanide-binding peptide. The engineered lanthanide-binding phage (LBPh), presenting ∼3300 copies of the peptide, serves as an effective biological template for REE separation. Our findings demonstrate the LBPh's preferential binding for heavy REEs over light REEs. Moreover, the LBPh exhibits remarkable robustness with excellent recyclability and stability across multiple cycles of separations. This study underscores the potential of genetically integrating virus templates with selective binding motifs for REE separation, offering a promising avenue for environmentally friendly and energy-efficient separation processes.

Evolutionary Design of Self-Templated Supramolecular Fibrils Using M13 Bacteriophage for Tissue Engineering.

Biomaterials in nature form hierarchical structures and functions across various length scales through binding and assembly processes. Inspired by nature, we developed hierarchically organized tissue engineering materials through evolutionary screening and self-templating assembly. Leveraging the M13 bacteriophage (phage), we employed an evolutionary selection process against hydroxyapatite (HA) to isolate HA-binding phage (HAPh). The newly discovered phage exhibits a bimodal length, comprising 950 nm and 240 nm, where the synergistic effect of these dual lengths promotes the formation of supramolecular fibrils with periodic banded structures. The assembled HAPh fibrils show the capability of HA mineralization and the directional growth of osteoblast cells. When applied to a dentin surface, it induces the regeneration of dentin-like tissue structures, showcasing its potential applications as a scaffold in tissue engineering. The integration of evolutionary screening and self-templating assembly holds promise for the future development of hierarchically organized tissue engineering materials.

Elastic Fluorescent Protein-Based Down-Converting Optical Films for Flexible Display.

Protein-based material design provides great advantages to developing smart biomaterials with tunable structures and desired functions. They have been widely used in many biomedical applications including tissue engineering and drug delivery. However, protein-based materials are not yet widely used in optoelectronic materials despite their excellent optical and tunable mechanical properties. Here, we synthesized engineered fluorescent proteins (FPs) fused with elastic protein for the development of optoelectrical down-converting optical filters for flexible display materials. We synthesized sequence-specific FPs to tune blue, green, yellow, and red colors and fused them with elastic protein to tune mechanical properties. We fabricated flexible self-supporting film materials and characterized mechanical properties and down-converting optical properties. We also fabricated a hybrid light-emitting diode (LED) to down convert blue to desired green, red, and white colors. Furthermore, we constructed a flexible white LED using organic LED as a flexible substrate. Our modular synthesis approach of tunable bio-optoelectrical material approaches will be useful to design future biocompatible and flexible display materials and technologies.

M13 Virus Triboelectricity and Energy Harvesting.

Triboelectrification is a phenomenon that generates electric potential upon contact. Here, we report a viral particle capable of generating triboelectric potential. M13 bacteriophage is exploited to fabricate precisely defined chemical and physical structures. By genetically engineering the charged structures, we observe that more negatively charged phages can generate higher triboelectric potentials and can diffuse the electric charges faster than less negatively charged phages can. The computational results show that the glutamate-engineered phages lower the LUMO energy level so that they can easily accept electrons from other materials upon contact. A phage-based triboelectric nanogenerator is fabricated and it could produce ∼76 V and ∼5.1 µA, enough to power 30 light-emitting diodes upon a mechanical force application. Our biotechnological approach will be useful to understand the electrical behavior of biomaterials, harvest mechanical energy, and provide a novel modality to detect desired viruses in the future.

Elastin-Based Thermoresponsive Shape-Memory Hydrogels.

A shape-memory hydrogel is a programmable hydrogel material that can store specific shapes and execute functions in response to stimuli. In this report, we developed shape-memory hydrogels by creating double-network polymeric structures using a physically cross-linking elastin-like polypeptide (ELP) and a chemically cross-linking polyacrylamide (PAM). We synthesized the hydrogel matrix by polymerizing the acrylamide mixed in an ELP solution. We exploited the lower critical solution temperature transition of the ELP to enable the hydrogel to hold a new desired shape at an elevated temperature of 55 °C. The original shape of the hydrogel can then be recovered by lowering the temperature to 20 °C. The shape-memory hydrogels we developed exhibit ultrafast functionality and high repeatability. Taking advantage of the temperature-induced shape-memory capability, we also demonstrate practical functions such as gripping an object and connecting two tubes. Our materials with effective temperature-driven shape-memory functionality will be useful for developing novel materials for biomedical applications in the future.

Catechol-Functionalized Elastin-like Polypeptides as Tissue Adhesives.

Adhesives can potentially be used to achieve fast and efficient wound closure; however, current products show poor bonding on wet surfaces, undergo swelling, and lack adequate biocompatibility. We designed a hydrogel adhesive with recombinant elastin-like polypeptides (ELPs), which are flexible proteins that can be customized for biomedical needs. The adhesive proteins are synthesized by chemically modifying the ELPs with dopamine, which contain adhesive catechol moieties. The resulting catechol-functional ELPs or Cat-ELPs can form flexible hydrogels that show stable swelling in aqueous conditions at 37 °C. We demonstrate their flexibility and viscoelastic properties through rheology. We also show the advantage of using customizable recombinant proteins to improve the material biological properties by demonstrating improved fibroblast binding on Cat-ELP by adding ELP with "RGD" peptides. We further confirmed in vivo biocompatibility and biodegradation of Cat-ELP hydrogels by implanting them in mice and monitoring for 10 weeks. Finally, we tested the bonding strength of the adhesives on porcine skin through tensile pull-off and lap-shear testing, in which we found strengths of 37 and 39 kPa, respectively. We demonstrated the tensile bonding strength by suspending a 2 kg mass on a one square inch (6.5 cm2) skin sample. As our adhesives are developed further, our strategy combining recombinant protein engineering and chemical modification will help yield an ideal bioadhesive for wound closure.

Vertical Self-Assembly of Polarized Phage Nanostructure for Energy Harvesting.

Controlling the shape, geometry, density, and orientation of nanomaterials is critical to fabricate functional devices. However, there is limited control over the morphological and directional characteristics of presynthesized nanomaterials, which makes them unsuitable for developing devices for practical applications. Here, we address this challenge by demonstrating vertically aligned and polarized piezoelectric nanostructures from presynthesized biological piezoelectric nanofibers, M13 phage, with control over the orientation, polarization direction, microstructure morphology, and density using genetic engineering and template-assisted self-assembly process. The resulting vertically ordered structures exhibit strong unidirectional polarization with three times higher piezoelectric constant values than that of in-plane aligned structures, supported by second harmonic generation and piezoelectric force microscopy measurements. The resulting vertically self-assembled phage-based piezoelectric energy harvester (PEH) produces up to 2.8 V of potential, 120 nA of current, and 236 nW of power upon 17 N of force. In addition, five phage-based PEH integrated devices produce an output voltage of 12 V and an output current of 300 nA, simply by pressing with a finger. The resulting device can operate light-emitting diode backlights on a liquid crystal display. Our approach will be useful for assembling many other presynthesized nanomaterials into high-performance devices for various applications.

MoS2 Liquid Cell Electron Microscopy Through Clean and Fast Polymer-Free MoS2 Transfer.

Two dimensional (2D) materials have found various applications because of their unique physical properties. For example, graphene has been used as the electron transparent membrane for liquid cell transmission electron microscopy (TEM) due to its high mechanical strength and flexibility, single-atom thickness, chemical inertness, etc. Here, we report using 2D MoS2 as a functional substrate as well as the membrane window for liquid cell TEM, which is enabled by our facile and polymer-free MoS2 transfer process. This provides the opportunity to investigate the growth of Pt nanocrystals on MoS2 substrates, which elucidates the formation mechanisms of such heterostructured 2D materials. We find that Pt nanocrystals formed in MoS2 liquid cells have a strong tendency to align their crystal lattice with that of MoS2, suggesting a van der Waals epitaxial relationship. Importantly, we can study its impact on the kinetics of the nanocrystal formation. The development of MoS2 liquid cells will allow further study of various liquid phenomena on MoS2, and the polymer-free MoS2 transfer process will be implemented in a wide range of applications.

Experimental and numerical evaluation of a genetically engineered M13 bacteriophage with high sensitivity and selectivity for 2,4,6-trinitrotoluene.

Selective and sensitive detection of desired targets is very critical in sensor design. Here, we report a genetically engineered M13 bacteriophage-based sensor system evaluated by quantum mechanics (QM) calculations. Phage display is a facile way to develop the desired peptide sequences, but the resulting sequences can be imperfect peptides for binding of target molecules. A TNT binding peptide (WHW) carrying phage was self-assembled to fabricate thin films and tested for the sensitive and selective surface plasmon resonance-based detection of TNT molecules at the 500 femtomole level. SPR studies performed with the WHW peptide and control peptides (WAW, WHA, AHW) were well-matched with those of the QM calculations. Our combined method between phage engineering and QM calculation will significantly enhance our ability to design selective and sensitive sensors.

Growth of Au and ZnS nanostructures via engineered peptide and M13 bacteriophage templates.

We demonstrate directed nucleation of Au and ZnS patterns on templates comprised of functional peptides and an M13 bacteriophage. We discuss the control over nucleation in terms of the interplay between enhanced ion binding and reduced interfacial energy resulting from the presence of the templates.

Biomimetic self-templating supramolecular structures.

In nature, helical macromolecules such as collagen, chitin and cellulose are critical to the morphogenesis and functionality of various hierarchically structured materials. During tissue formation, these chiral macromolecules are secreted and undergo self-templating assembly, a process whereby multiple kinetic factors influence the assembly of the incoming building blocks to produce non-equilibrium structures. A single macromolecule can form diverse functional structures when self-templated under different conditions. Collagen type I, for instance, forms transparent corneal tissues from orthogonally aligned nematic fibres, distinctively coloured skin tissues from cholesteric phase fibre bundles, and mineralized tissues from hierarchically organized fibres. Nature's self-templated materials surpass the functional and structural complexity achievable by current top-down and bottom-up fabrication methods. However, self-templating has not been thoroughly explored for engineering synthetic materials. Here we demonstrate the biomimetic, self-templating assembly of chiral colloidal particles (M13 phage) into functional materials. A single-step process produces long-range-ordered, supramolecular films showing multiple levels of hierarchical organization and helical twist. Three distinct supramolecular structures are created by this approach: nematic orthogonal twists, cholesteric helical ribbons and smectic helicolidal nanofilaments. Both chiral liquid crystalline phase transitions and competing interfacial forces at the interface are found to be critical factors in determining the morphology of the templated structures during assembly. The resulting materials show distinctive optical and photonic properties, functioning as chiral reflector/filters and structural colour matrices. In addition, M13 phages with genetically incorporated bioactive peptide ligands direct both soft and hard tissue growth in a hierarchically organized manner. Our assembly approach provides insight into the complexities of hierarchical assembly in nature and could be expanded to other chiral molecules to engineer sophisticated functional helical-twisted structures.

Elastin-Based Rubber-Like Hydrogels.

We developed rubber-like elastomeric materials using a natural elastin derived sequence and genetic engineering to create precisely defined elastin-like polypeptides. The coiled elastin-like polypeptide chains, which behave like entropic springs, were cross-linked using an end-to-end tethering scheme to synthesize simple hydrogels with excellent extensibility and reversibility. Our hydrogels extend to strains as high as 1500% and remain highly resilient with elastic recovery as high as 94% even at 600% strain, significantly exceeding any other protein-based hydrogel. These materials are valuable as elastomeric hydrogels for designing extremely robust scaffolds useful for tissue engineering.

Self-Healing Elastin-Bioglass Hydrogels.

Tailorable hydrogels that are mechanically robust, injectable, and self-healable, are useful for many biomedical applications including tissue repair and drug delivery. Here we use biological and chemical engineering approaches to develop a novel in situ forming organic/inorganic composite hydrogel with dynamic aldimine cross-links using elastin-like polypeptides (ELP) and bioglass (BG). The resulting ELP/BG biocomposites exhibit tunable gelling behavior and mechanical characteristics in a composition and concentration dependent manner. We also demonstrate self-healing in the ELP/BG hydrogels by successfully reattaching severed pieces as well as through rheology. In addition, we show the strength of genetic engineering to easily customize ELP by fusing cell-stimulating "RGD" peptide motifs. We showed that the resulting composite materials are cytocompatible as they support the cellular growth and attachment. Our robust in situ forming ELP/BG composite hydrogels will be useful as injectable scaffolds for delivering cell and drug molecules to promote soft tissue regeneration in the future.

Biomimetic Self-Templated Hierarchical Structures of Collagen-Like Peptide Amphiphiles.

Developing hierarchically structured biomaterials with tunable chemical and physical properties like those found in nature is critically important to regenerative medicine and studies on tissue morphogenesis. Despite advances in materials synthesis and assembly processes, our ability to control hierarchical assembly using fibrillar biomolecules remains limited. Here, we developed a bioinspired approach to create collagen-like materials through directed evolutionary screening and directed self-assembly. We first synthesized peptide amphiphiles by coupling phage display-identified collagen-like peptides to long-chain fatty acids. We then assembled the amphiphiles into diverse, hierarchically organized, nanofibrous structures using directed self-assembly based on liquid crystal flow and its controlled deposition. The resulting structures sustained and directed the growth of bone cells and hydroxyapatite biominerals. We believe these self-assembling collagen-like amphiphiles could prove useful in the structural design of tissue regenerating materials.

Selective and Sensitive Sensing of Flame Retardant Chemicals Through Phage Display Discovered Recognition Peptide.

We report a highly selective and sensitive biosensor for the detection of an environmentally toxic molecule, decabrominated diphenyl ether (DBDE), one of the most common congeners of the polybrominated frame retardants (polybrominated diphenyl ether (PBDE)), using newly discovered DBDE peptide receptors integrated with carbon nanotube field-effect transistors (CNT-FET). The specific DBDE peptide receptor was identified using a high-throughput screening process of phage library display. The resulting binding peptide carries an interesting consensus binding pocket with two Trp-His/Asn-Trp repeats, which binds to the DBDE in a multivalent manner. We integrated the novel DBDE binding peptide onto the CNT-FET using polydiacetylene coating materials linked through cysteine-maleimide click chemistry. The resulting biosensor could detect the desired DBDE selectively with a 1 fM detection limit. Our combined approaches of selective receptor discovery, material nanocoating through click chemistry, and integration onto a sensitive CNT-FET electronic sensor for desired target chemicals will pave the way toward the rapid development of portable and easy-to-use biosensors for desired chemicals to protect our health and environment.

Cyclic RGD peptide incorporation on phage major coat proteins for improved internalization by HeLa cells.

Delivering therapeutic materials or imaging reagents into specific tumor tissues is critically important for development of novel cancer therapeutics and diagnostics. Genetically engineered phages possess promising structural features to develop cancer therapeutic materials. For cancer targeting purposes, we developed a novel engineered phage that expressed cyclic RGD (cRGD) peptides on the pVIII major coat protein using recombinant DNA technology. Using a type 88 phage engineering approach, which inserts a new gene to express additional major coat protein in the noncoding region of the phage genome, we incorporated an additional pVIII major coat protein with relatively bulky cRGD and assembled heterogeneous major coat proteins on the F88.4 phage surfaces. With IPTG control, we could tune different numbers of cRGD peptide displayed on the phage particles up to 140 copies. The resulting phage with cRGD on the recombinant pVIII protein exhibited enhanced internalization efficiency into HeLa cells in a ligand density and conformational structure dependent manner when comparing with the M13 phages modified with either linear RGD on pVIII or cRGD on pIII. Our cRGD peptide engineered phage could be useful for cancer therapy or diagnostic purposes after further modifying the phage with drug molecules or contrast reagents in the future.

Graphene-based materials functionalized with elastin-like polypeptides.

Graphene-based materials commonly require functionalization for biological applications in order to control their physical/colloidal properties and to introduce additional capabilities, such as stimuli-responsiveness and affinity to specific biomolecules. Here, we functionalized CVD-grown graphene and graphene oxide with a genetically engineered elastin-like polypeptide fused to a graphene binding peptide and then showed that the resulting hybrid materials exhibit thermo- and photoresponsive behaviors. Furthermore, we demonstrate that our genetic engineering strategy allows for the facile introduction of bioactivity to reduced graphene oxide. The stimuli-responsiveness and genetic tunability of our graphene-protein nanocomposites are attractive for addressing future biomedical applications.

Synthetic phage for tissue regeneration.

Controlling structural organization and signaling motif display is of great importance to design the functional tissue regenerating materials. Synthetic phage, genetically engineered M13 bacteriophage has been recently introduced as novel tissue regeneration materials to display a high density of cell-signaling peptides on their major coat proteins for tissue regeneration purposes. Structural advantages of their long-rod shape and monodispersity can be taken together to construct nanofibrous scaffolds which support cell proliferation and differentiation as well as direct orientation of their growth in two or three dimensions. This review demonstrated how functional synthetic phage is designed and subsequently utilized for tissue regeneration that offers potential cell therapy.

M13 bacteriophage production for large-scale applications.

Bacteriophage materials have the potential to revolutionize medicine, energy production and storage, agriculture, solar cells, optics and many other fields. To fulfill these needs, this study examined critical process parameters during phage propagation to increase phage production capability. A representative scale-down system was created in tube spin reactors to allow parallel experimentation with single- and multi-variable analysis. Temperature, harvest time, media composition, feed regime, bacteriophage, and bacteria concentration were analyzed in the scale-down system. Temperature, media composition, and feeding regimens were found to affect phage production more than other factors. Temperature affected bacterial growth and phage production inversely. Multi-variate analysis identified an optimal parameter space which provided a significant improvement over the base line method. This method should be useful in scaled production of bacteriophage for biotechnology.

Light-controlled graphene-elastin composite hydrogel actuators.

Hydrogels actuators (HAs) that can reversibly respond to stimuli have applications in diverse fields. However, faster response rates and improved control over actuation timing and location are required to fulfill their potential. To address these criteria, we synthesized near-infrared light-driven HAs by interfacing genetically engineered elastin-like polypeptides with reduced-graphene oxide sheets. The resulting nanocomposites exhibited rapid and tunable motions controlled by light position, intensity, and path, including finger-like flexing and crawling. This work demonstrates the ability of rationally designed proteins to be combined with synthetic nanoparticles for the creation of macroscale functional materials.