Symposium S.SM07—Bioinspired Synthesis and Manufacturing of Materials

The fabrication of nanoscale devices requires architectural templates on which to align functional molecules in complex arrangements. Nature has met this challenge of nanofabrication by exploiting the remarkable ability of proteins to self-assemble into ordered and intricate nanostructures. Protein assemblies in nature can also serve as inspiration to build novel material templates with defined size and symmetry. Furthermore, control over assembly and disassembly processes of protein nanostructures will enable dynamic protein devices to be created that can sense and respond to specific input signals. This presentation will highlight recent engineering of modular protein subunits whose assembly and disassembly can be controlled to create novel biomaterials, including electrically conductive protein nanowires.

The central protein building block in our approach is gamma-prefoldin (gPFD), a filamentous protein chaperone from a hyperthermophilic archaeon. The protein-protein interfaces of gPFD were redesigned with helical sequences to create unique subunits that assemble into filaments through orthogonal dimeric coiled coils. Subsequently, these multicomponent filaments could be used to position and align functional enzymes. To gain switch-like control over the assembly process, post-translational modification systems were exploited to regulate the binding kinetics of coiled coils. By placing protein kinase recognition motifs in the interface of synthetic coiled coils of varied sequences, we could in vitro phosphorylate the coiled coils and thereby modulate electrostatic interactions. These engineerable protein interfaces are now being applied to control the assembly and disassembly of protein nanostructures. In one example, we aligned cytochrome c proteins on gPFD filaments to create metalloprotein nanowires. Electrochemical transport measurements indicated the nanowires could conduct current between electrodes at the redox potential of the cytochromes, and subsequently be used to interface with enzymes. The ability to control protein interactions will allow the design of smart protein devices capable of sensing inputs such as enzymatic activity, for applications in the disassembly of nanocages for drug delivery and new types of biosensors.

The nanomaterials field is rapidly evolving as scientists and engineers attempt to solve increasingly complex problems. Researchers are focused on the synthesis of new materials, assembly into higher order structures, improving consistency of materials properties, and synthesis at large scales. One area of increasing interest for creating new materials is biotemplating, an approach that uses a biological molecules, such as DNA or viruses, as scaffolds for deposition of materials of interest. This strategy has several notable advantages, including the exploitation of unique morphologies, the possibility of genetic engineering, and rapid and sustainable production at large scales. The M13 bacteriophage, in particular, is an attractive template for synthesizing nanomaterials and facilitating self-assembly.

Bacteriophages, or phages, are viruses that infect bacteria and replicate by coopting the cellular machinery. The phage genome can be genetically engineered to induce the display of specific surface proteins that may promote binding of a wide variety of molecules. This programmable functionalization makes phages an attractive template for nanotechnology applications ranging from batteries to vaccine carriers. The M13 filamentous phage is of particular interest due to its nanofiber-like shape and the high copy number (~2,700) of the pVIII major coat protein present along the 900 nm length, presenting many locations for material interactions. This particular phage is well studied and characterized, and can be easily manipulated on the genomic level due to its simplicity.

We have designed a novel, biorenewable, bacteriophage-templated, carbon-based material with a very high surface area (up to 2000 m²g^-1) and tunable porosity. The M13 filamentous phage was first used to create a resin-like material by adding resorcinol and formaldehyde. The incorporation of benzoxazine chemistry into this process results in the formation of uniform resorcinol-formaldehyde (RF) nanofibers and allows for the incorporation of heteroatoms and functional groups that can promote material binding. RF resins can serve as precursors for mesoporous carbon aerogels with controllable porosity and particle size. Carbon materials have been explored for many applications, including filtration of small molecules and gas storage, due to their high surface area and microporous structures. To create a material with these features, resin-coated phage were burned at a high temperature, resulting in “carbonized” nanofibers, termed BioCNFs. The desirable structure and surface area of the BioCNFs were confirmed by transmission electron microscopy and nitrogen isotherms, respectively. The material properties of BioCNFs could be tailored to specific applications by adjusting the precursor composition and carbonization temperature. Micro-breakthrough testing of BioCNFs revealed a high adsorption capacity for and neutralization activity against a range of chemical warfare agents (CWAs) and toxic industrial chemicals (TICs). Remarkably, this performance either met or exceeded that of current state-of-the-art materials such as activated carbon and metal organic frameworks. This capability can be harnessed for military or industrial applications in part due to the ability of BioCNFs to be integrated into a variety of materials ranging from decontamination wipes to gas masks.

Biosynthesis is on track to revolutionize the way new materials are developed. The biotemplating method is gaining traction because a biological material can be manipulated to promote certain properties and binding capabilities. BioCNFs, created from the biotemplating of phage, can be grown in a biorenewable manner and can be easily engineered to address emerging chemical and biological threats. Future studies with this material will focus on further optimization for chemical filtration and development of gas storage capabilities.

In nature, biominerals (e.g. bones and teeth) are outstanding examples of hierarchically-structured hybrid materials whose formation and functions are controlled over multiple length scales by high information content biomacromolecules. Inspired by these feats of nature, many biomimetic approaches have been developed for the preparation of nanostructured hybrid materials.¹ These approaches are attractive because they generate complex, functional nanomaterials under mild aqueous synthetic conditions. Despite the advances in developing bioinspired materials synthesis approaches, the rules of designing sequence-defined molecules that lead to the predictable synthesis of hybrid materials are unknown.

One of the most advanced classes of sequence-defined protein-mimetics are peptoids.² They offer unique opportunities for producing complex architectures with tunable functions based solely on sidechain chemistry. In this presentation, two peptoid-based approaches for controlled synthesis of hierarchically-structured hybrid materials will be discussed. The first involves the design and synthesis of surfacant-like amphiphilic peptoids for controlling formation and morphogenesis of inorganic nanomaterials. The second approach exploits self-assembling peptoids^3-5 for controlled synthesis of hierarchically structured hybrid materials. A combination of in situ imaging and molecular simulations were used to elucidate the principles underlying peptoid-controlled synthesis of hybrid materials with the ultimate goal of enabling predictive materials synthesis across scales.

References
(1) Chen, C. L.; Rosi, N. L. Peptide-based methods for the preparation of nanostructured inorganic materials. Angew. Chem., Int. Ed. 2010, 49, 1924-1942.
(2) Sun, J.; Zuckermann, R. N. Peptoid polymers: a highly designable bioinspired material. ACS Nano 2013, 7, 4715-4732.
(3) Jin, H.; Ding, Y.-H.; Wang, M.; Song, Y.; Liao, Z.; Newcomb, C. J.; Wu, X.; Tang, X.-Q.; Li, Z.; Lin, Y.; Yan, F.; Jian, T.; Mu, P.; Chen, C.-L. Designable and dynamic single-walled stiff nanotubes assembled from sequence-defined peptoids. Nat. Commun. 2018, 9, 270.
(4) Ma, X.; Zhang, S.; Jiao, F.; Newcomb, C. J.; Zhang, Y.; Prakash, A.; Liao, Z.; Baer, M. D.; Mundy, C. J.; Pfaendtner, J.; Noy, A.; Chen, C.-L.; De Yoreo, J. J. Tuning crystallization pathways through sequence engineering of biomimetic polymers. Nat. Mater. 2017, 16, 767-775.
(5) Jin, H.; Jiao, F.; Daily, M. D.; Chen, Y.; Yan, F.; Ding, Y.-H.; Zhang, X.; Robertson, E. J.; Baer, M. D.; Chen, C.-L. Highly stable and self-repairing membrane-mimetic 2D nanomaterials assembled from lipid-like peptoids. Nat. Commun. 2016, 7, 12252.

We are interested in how functionality emerges from sequence in ensembles of very short peptides, and subsequently how these functions can be incorporated into functional materials. Instead of using sequences known in biological systems, we use unbiased computational and experimental approaches to search and map the peptide sequence space, which has provided new families of functional short peptides. The talk will focus on our latest results in three areas. First, we will demonstrate how to program molecular order and disorder in tripeptides, and how the conformations adopted by these peptides can be exploited to regulate assembly properties, and give rise to tunable emission in the visible range. Second, we will demonstrate how dynamic exchange of peptide sequences can form adaptive libraries that provide insights into peptide sequences that can complex ligands. Finally, we discuss peptide-based melanin mimics with tunable chromophoric properties that are achieved through oxidative incorporation of amino acids.

While natural functional materials offer exceptional mechanical properties and reversibly respond to various stimuli, engineering their analogs with tunable properties remains challenging mainly due to complexity. Here we take advantage of bottom-up assembly to develop tunable peptide based materials that reversibly change their structure in response to applied stimulus. To do so we propose the use of simple tripeptide crystals as biocompatible, cheap and tunable materials which have significant responses to changes in relative humidity (RH). We selected tri-peptide sequences that contain hydrophobic - YF dyad, and charged amino acid. Using RH controlled Atomic Force Microscopy (AFM), Fourier-Transform Infrared Spectroscopy (FTIR), Powder X-ray Diffraction (PXRD) and Molecular Dynamics Simulations (MD) we observed that these supramolecular tri-peptide assemblies form three-dimensional, porous crystal networks that undergo reversible shape change in response to applied RH.
We demonstrated sequence-dependent performance allowing for tuning such properties as stiffness (0.5-2GPa), strain (3-25%), energy density (10-100kJ/m³) or pore size. The modulation of peptide-sequence dependent properties help us identify key parameters that contribute to functionality during transition processes, such as dual network domains, the importance of structured water and strengthening of hydrogen bonds, hierarchical order, order/disorder domains and intrinsic porosity. Due to their intrinsic order, short-peptide crystals are an excellent platform to investigate the fundamental understanding of the relationship between function and structure. We believe that these findings open up a magnitude of possibilities for programing simple, bioinspired peptide materials.

Recent advances in nanotechnology of two-dimensional (2D) layered materials combined with parallel improvements in biotechnology and synthetic biology demonstrated that more complex composites materials with properties engineered precisely to optimize performance could be achieved. We propose to create functional programmable materials with user defined physical properties from composites of 2D-layered materials and polymeric proteins [1]. Our approach is based on an ultra-fast microscopy technique to screen molecular morphology of polymeric proteins [2]. These proteins have several advantages as programmable materials [3]: (i) their chain length, sequence, and stereochemistry can be easily controlled, (ii) their molecular structure and morphology is well-defined, (iii) they provide a variety of functional chemistries for conjugation to 2D materials, and (iv) they can be designed to exhibit a variety of physical properties. The variability of the amino-acid sequences in the polymeric proteins, which will dictate the degree of crystallinity and alignment of the protein layers, are used to control the interactions at the 2D material/protein interface, ultimately dictating the functional physical properties (e.g., electrical resistivity and thermal conductivity) of novel materials and devices . References: [1] Demirel et al., Advanced Functional Materials 28 (27), 1704990, 2017; [2] Tomko et al., Nature nanotechnology 13 (10), 959, 2018; [3] Jung et al., Proceedings of the National Academy of Sciences 113 (23), 6478-6483, 2016.

Proteins reflect one fascinating class of natural polymers with huge potential for technical as well as biomedical applications. One well-known example is spider silk, a protein fiber with excellent mechanical properties such as strength and toughness. We have developed biotechnological methods using bacteria as production hosts which produce structural proteins mimicking the natural ones. Besides the recombinant protein fabrication, we analyzed the natural assembly processes and we have developed spinning techniques to produce protein threads closely resembling natural silk fibers. In addition to fibers, we employ silk proteins in other application forms such as hydrogels, particles or films with tailored properties, which can be employed especially for biomaterials applications.
Our bio-inspired approach serves as a basis for new materials in a variety of technical as well as biomedical applications.

Spider silk fibers show excellent mechanical properties such as a combination of strength and elasticity yielding a so far unreached fiber toughness. Such fibers can be used e.g. in various textile applications. In biomedical applications the performance of materials largely depend on their surfaces and is further strictly related to the materials biocompatibility. Often the appearance of unwanted side effects hampers the applicability of biomaterials including foreign body responses and inflammation, and interaction of cells with a material’s surface, for example cell adhesion. In case of implants or catheters cell adhesion plays a crucial role for the overall function of the to-be-used material. To change the properties of in-use polymers and to adopt their biocompatibility, we established coatings based on engineered spider silk proteins. All kinds of polymers (polyurethane, polytetrafluoroethylene, silicone) have so far be stably coated with recombinant spider silk proteins, which can themselves be functionalized. Several parameters of the silk proteins can be easily adopted to the intended application, including the surface net charge, hydrophilicity etc. Several cell types, including HaCaT keratinocytes (epidermal cells), B50 neuronal cells, C2C12 myoblasts (muscle cells) and BALB/3T3 fibroblasts (connective tissue), exhibit low or no adhesion on the silk-coated materials. In an vivo study in Sprague-Dawley rats, silk coatings diminished the risk of side effects of silicone breast implants such as fibrosis.

Drug delivery systems allow tissue / cell specific targeting of drugs in order to reduce total drug amounts administered to an organism and potential side effects upon systemic drug delivery. Spider silk proteins represent a new class of (bio)polymers that can be used as drug depots or drug delivery systems. A recombinant polyanionic spider silk protein, which can be processed into different morphologies such as particles, films, or hydrogels, has been shown to fulfil most criteria necessary for its use as biomaterial. Further, such particles have been shown to be well-suited as drug carriers for polycationic or neutral drugs, but cellular uptake of such particles is low.

Spider silk hydrogels can also be employed as new bioinks for biofabrication. Their elastic behavior dominate over the viscous behavior over the whole angular frequency range with a low viscosity flow behavior and good form stability. No structural changes occur during the printing process, and the hydrogels solidify immediately after printing by robotic dispensing. Due to the form stability it was possible to directly print multiple layers on top of each other without structural collapse. Cell-loaded spider silk constructs can be easily printed without the need of additional cross-linkers or thickeners for mechanical stabilization. Encapsulated cells show good viability in such spider silk hydrogels.

Traditionally, ocean salinity and temperature are measured using Conductivity, Temperature and Depth (CTD) sensors. CTDs are relatively large, rigid devices that are negatively buoyant. These CTDs are expensive and prone to marine biofouling (esp. for the conductivity probes), which greatly reduces the durability of these systems. Velellas are a subclass of jellyfish that live on the ocean surface. Velellas are often also called sailors-by-the-wind since their biological sails mimic conventional boat sails. Inspired by the Velellas, we proposed to develop a unique Velella Sensor system based on biodegradable hydrogels that can measure ocean surface temperature, salinity and indirectly, the winds at the ocean surface via Global Positioning System (GPS)-tracking.

Here, we report a cost effective approach for making the biodegradable hydrogels for this application. Our approach uses click-chemistry to tailor the molecular structure of polyethylene glycol (PEG) hydrogel networks in order to control the mechanical properties and degradation time. Oligomer chains were built by base catalyzed thiol-Michael addition reactions between acrylates and thiols, where the end groups are controlled by the molar rations between the reactants. The molecular weight of oligomer chains of varying monomer ratios and monomer weights is measured by dynamic light scattering, and the ester group content is measured by Fourier Transform Infrared Spectroscopy. The oligomer chains were crosslinked by radical polymerization, yielding either thiol-ene networks from thiol-capped chains or acrylate networks form acrylate-capped chains. Tensile tests were used to determine the effect of varying these chemistries on mechanical properties, and degradation tests were carried out in both deionized water and artificial seawater. The method of crosslinking affects the degradation profile of the networks though the oligomer compositions are nearly identical. Importantly, the size and composition of the degradation products are characterized by dynamic light scattering and nuclear magnetic resonance to confirm sufficient degradation occurs and safe byproducts are released. Effects of molecular weight between crosslinks on the lower critical solution temperature of the hydrogels and the impacts on degradation were also addressed. Further research will include deployment of these hydrogels in ocean environments to determine the susceptibility to biofouling and the actual ocean lifetime.

Differential dynamic microscopy (DDM) is a micro-rheology technique that enables parameter-free estimation of the mean-squared displacement of tracer particles embedded in a test medium. The mean-squared displacement of tracer particles is a bridge to important rheological information including the complex shear modulus and thus DDM provides an avenue to truly automated and potentially high-throughput micro-rheology.
In this presentation, we discuss an automated pipeline based on DDM which incorporates robotic liquid handling and machine learning. We present the utility of the pipeline in two case studies. First, we show how a complex, multi-parameter gel point formulation space of silk hydrogels can be objectively navigated and characterized. Second, we present results of high-throughput viscosity measurement of pre-polymers and polymer melts in non-aqueous solvents. We discuss challenges to automation including the mitigation of bulk drift in samples prepared using liquid handling robots, quality control in the face of numerous automation failure modes, and issues encountered in generalizing hardware and analysis over broad time scales and sample chemistries.

The COVID-19 pandemic has highlighted the need for rapid, high-throughput, and robust therapeutic antibody screening platforms. Advances in both synthetic selections and antibody repertoire mining have enabled the rapid identification of hundreds of candidate binders in a single experiment. However, the expression and evaluation of these candidate antibodies still remains a major bottleneck in the discovery pipeline due to labor intensive steps and process throughput mismatches. In this work, we present a newly developed workflow that leverages cell-free protein synthesis (CFPS) and an Echo® 525 acoustic liquid handler to enable the expression and evaluation of hundreds of antibodies in a single experiment. Our workflow consists of a cell-free DNA assembly step to generate antibody expression templates, a modified E. coli CFPS system to express antibodies, and the AlphaLISA protein-protein interaction assay to evaluate binding. Each step of this workflow can be carried out entirely within 384-well plates without ever going into cells, making it highly suitable for automation. The resulting platform is capable of evaluating more than an order of magnitude more antibody candidates in less than half the time when compared to state-of-the-art antibody discovery pipelines. To validate the platform, we expressed 13 different human antibodies, 12 of which neutralize the SARS-CoV-2 virus. We evaluated these antibodies by mapping their target epitopes and evaluating their ability to compete with ACE2, the SARS-CoV-2 target human receptor. Our results are largely consistent with published literature on these antibodies, indicating our workflow is suitable for identifying therapeutic candidates. We expect that these advancements will accelerate antibody discovery and development not only for viral diseases like COVID-19, but for the entire antibody industry.

We demonstrated synthetic use of biology cell-free expression chassis for the design and assembly of an entirely man-made energy transformation nano-bio hybrid assembly. Similarly to a natural light-driven proton pump bR from H. salinarium, the pump bRsyn in artificial purple nano-membranes was integrated with TiO2 semiconductor nanoparticles, yielding a catalytic assembly for photon-to-hydrogen
conversion. The system produces H2 at a turnover rate of 240μmol of H2 (μmol protein)−1 h−1 under green and 17.74 mmol of H2 (μmol protein)−1 h−1 under white light at ambient conditions, in water at neutral pH with methanol as a sacrificial electron donor. While the cell-free expression technique has been successfully developed as a handy approach for rapid high fidelity production of membrane proteins for fundamental structure−functional studies, cell-free methodology can become a useful flexible platform for on-demand expression
of natural and designed light-responsive membrane architectures with precisely controllable structure, nanoscale dimensions,
and photochemical properties. Such biological building blocks can be consequently integrated with semiconductor
nanoparticles via systemic manipulation at the nanoparticle−bio interface toward directed evolution of energy nanomaterials
and nanosystems.
ACS Nano 11, 6739−6745 (2019)
US Patent 10,220,378B2 (2019)

Genetic engineering enables new engineered living materials (ELMs) that harness the remarkable properties of nature to sense and respond to their environment. Bacterial cellulose (BC) is a natural biological material with impressive physical properties, high natural yield and low cost of production that is an attractive substrate for ELMs. Here, inspired by the ‘symbiotic culture of bacteria and yeast’ (SCOBY) used to make fermented kombucha tea, we describe a variety of novel BC-based ELMs containing yeast cells programmed to perform chosen functions. This is achieved via a synthetic SCOBY (Syn-SCOBY) approach that uses a stable co-culture of the model yeast Saccharomyces cerevisiae with BC-producing bacteria Komagataeibacter rhaeticus. We show that co-cultured yeast can be engineered to secrete enzymes into BC, generating autonomously grown catalytic materials and enabling DNA-encoded modification of BC bulk material properties. We further developed a method for incorporating S. cerevisiae within the growing cellulose matrix, creating living materials that can sense chemical and optical inputs. This enabled the growth of living sensor materials that can detect and respond to environmental pollutants, as well as living films that grow images based on projected patterns. Starting with only engineered cells and simple culture media, this novel and robust Syn-SCOBY system empowers the sustainable production of BC-based ELMs with genetically programmable properties under mild conditions.

Photoenzymes are the specialized component in photosynthetic organisms able to perform light transduction into charge separated states. As natural photoconverters that harvest light by photoactive antennas, generating electron-hole pairs and tunnelling electrons in precise biochemical pathways, they can be exploited for biodevices scaffolds [1-2]. In this frame, the engineering of bioelectronic frameworks have the upmarket asset to be considerate as an eco-friendly and scalability technology, using one of the greenest energy source available to us, sunlight, to gain other forms of useful energy. Therefore, the possibility of taking advantage of this unmatched photoconversion efficiency to create functional nanomaterials and bio-hybrid devices is very attractive[3].
The implementation of these unique biological systems into nanostructures or anchoring on devices electrode surfaces require the development of suitable chemical manipulation, because an efficient interfacing with electrodes for electronic applications still represents an issue. To overcome this problem, which limits the performance and applicability of photoenzymes-based technology, several attempts have been undertaken, focusing on the deployment of soft organic materials that can boost the bio-electrode interface. With this aim photoenzymes have been embedded in liposomes [4], giant vescicles [5] and polymersomes [6]. Moving forward, soft material with conductive and tunable features have been tested [7-8] to improve the energy extraction by photosynthetic proteins and the communication between the biological and electronic components in hybrid devices, that will be addressed in this presentation.
[1] F. Milano, A. Punzi, R. Ragni, M. Trotta, G. M. Farinola, Adv. Funct. Mater., 29, 1805521, (2019).
[2] F. Milano, F. Ciriaco, M.Trotta, D. Chirizzi, V. De Leo, A. Agostiano, L.Valli, L.Giotta, M.R.Guascito, Electrochim. Acta, 293, 105-115, (2019).
[3] A. Operamolla, R. Ragni, F. Milano, R. Tangorra, A. Antonucci, A. Agostiano, M. Trotta & G. M. Farinola, J. Mater. Chem. C, 3 (25), 6471-6478 (2015).
[4] F. Mavelli, M.Trotta, F. Ciriaco, A. Agostiano, L. Giotta, F. Italiano, F. Milano, Eur Biophys J, 43, 6-7, 301-315. (2014).
[5] E. Altamura, F. Milano, M. Trotta, P. Stano, F. Mavelli, Advances in Bionanomaterials, Lecture Notes in Bioengineering, Springer, 97-109 (2018).
[6] R. R. Tangorra, A. Operamolla, F. Milano, O. Hassan Omar, J. Henrard, R. Comparelli, F. Italiano, A. Agostiano, V. De Leo, R. Marotta, A. Falqui, G.M. Farinola, Trotta M., Photochem Photobiol Sci.,14(10):1844-52 (2015).
[7] M. Ambrico, P. F. Ambrico, T. Ligonzo, A. Cardone, S. R. Cicco, M. d'Ischia, G.M. Farinola, J. Mater. Chem. C, 3(25) 6413-6423, (2015).
[8] M. Grattieri, S. Patterson, J. Copeland, K. Klunder, S. Minteer, ChemSusChem., Accepted Author Manuscript. doi:10.1002/cssc.201902116.

Photosynthesis is the most important metabolic process taking place on Earth. It converts solar energy in many other forms of energy that are used to fuel virtually all other forms of life on our planet. Two forms of photosynthesis are known: oxygenic, developed more recently on Earth and widespread on the entire globe, and anoxygenic, very ancient and limited to small ecological niches. Notwithstanding what would resemble a drawback for its exploitation, anoxygenic bacteria are sturdy, very adaptable and thrive in environmental conditions that would not be accessible to other photosynthetic organisms [1,3].
Anoxygenic photosynthetic bacteria have often been used in combination with several soft materials, including biocompatible polymers[4] and conductive hydrogels[5], for various applications in the field of energy conversion [6].
The most recent results relative to the intimate interface between the whole photosynthetic bacteria, in particular the species Rhodobacter sphaeroides, its mutant strains, or portion of the bacteria will be addressed in this presentation.

[1] Giotta L. et al, Heavy metal ion influence on the photosynthetic growth of Rhodobacter sphaeroides, 2006 Chemosphere 62(9) 1490-1499.
[2] Asztalos E. et al., Early detection of mercury contamination by fluorescence induction of photosynthetic bacteria, 2010 Photochemical & Photobiological Sciences, 9(9), 1218-1223.
[3] Calvano C.D. et al., The lipidome of the photosynthetic bacterium Rhodobacter sphaeroides R26 is affected by cobalt and chromate ions stress, 2014 Biometals 27(1) 65-73
[4] Ambrico M. et al. From commercial tyrosine polymers to a tailored polydopamine platform: concepts, issues and challenges en route to melanin based bioelectronics. 2015 J. Mater. Chem. C 3(25) 6413-6423
[5] Grattieri M. et al., Purple bacteria & 3D redox hydrogels for bioinspired photobioelectrocatalysis, 2019 ChemSusChem in press.
[6] Milano F. et al., Photonics and optoelectronics with bacteria: making materials from photosynthetic microorganisms, 2019 Advanced Functional Materials 29(21) 1805521.

The ability to control the interactions between bacteria and materials is fundamental for creating living hybrid systems as well as pathogen anti-adhesion. Herein we discuss our work in tuning the abio-bio interfaces for binding nanomaterials to living bacteria either through genetic engineering or through material functionalization. In the first study, living bacteria/nanoparticle hybrids were prepared by genetically controlling binding peptide displayed on bacterial surfaces. Escherichia coli (E. coli) was engineered with inducible gene circuits to control display of peptides on bacteria with desired sequences. Driven by metal-peptide affinity, nanoparticles such as gold or magnetic nanoparticles could self-assemble onto the bacteria with programmed peptides. Peptide-mediated binding of gold nanoparticles to E. coli showed enhanced microbial fuel cell power generation. In the second study, cellulose nanofibrils, a biocompatible and easily modified nanomaterial platform, were chemically functionalized with mannose derivative to be used as a new tool in the control of bacterial pathogenesis. The functionalized nanofibrils were able to regulate fimbriated E. coli association due to strong affinity between mannose grafted on nanofibrils and FimH receptor on E. coli. These bioactive nanofibrils demonstrated the capability of capturing fimbriated E. coli as well as significant inhibition of E. coli adherence to mannosylated surfaces.

Nano-scale modification of macro-scale 3-dimensional implantable devices is gaining more attention as the cell attachment/detachment mechanisms at the bio-interfaces are profoundly affected by the nanoscale interfacial interactions [1]. Titanium-based biomaterials are widely used for dental prostheses, orthopedic devices, and cardiac pacemakers. Titanium and its alloys are most favored for hard tissue replacement due to their excellent mechanical properties and surface characteristics promoting biocompatibility due to the spontaneous formation of a thick oxide layer in the presence of an oxidizer. This study focuses on the development of a three-dimensional chemical mechanical nano-structuring (CMNS) process to induce smoothness or controlled nano-roughness on the bio-implant surfaces, particularly for an application on the dental implants. CMNS is an extension of the chemical mechanical polishing process. CMP is utilized in microelectronics manufacturing for planarizing the wafer surfaces to enable photolithography and multilayer metallization. In biomaterials applications, it has been shown that the same approach can be utilized to induce controlled surface nano-structure on 3-D implants to promote or demote cell attachment [2]. By tuning the polishing slurry particle size, solids loading and the chemical composition, both the chemical nature and the surface topography can be modified to make the surface very smooth or rough at the nano-scale. This new technique helps produce implant surfaces that are cleaned from potentially contaminated surface layers by removing a nano-scale top layer while simultaneously creating a protective oxide film on the surface to limit any further contamination to minimize the risk of infection [3]. As a synergistic method of nano-structuring on the implant surfaces, CMNS also modifies the mechanical properties on the implant surfaces and makes the titanium surface more adaptable for the bio-compatible coatings as well as the cells and tissue growth as demonstrated by the mechanical, electrochemical and biological evaluations. The ability of manipulating the surface nanostructure is also essential to understand the fibroblast and osteoblast cell attachment/detachment mechanisms on the implant surfaces to enable functionality of the implants whether the biocompatibility is a function of the cell attachment such as in the dental or prosthetic implants or limited attachment is needed for the functionality such as for the cardiac valves.

Staruch, R, Griffin, M, Butler, P. Nanoscale surface modifications of orthopedic implants: state of the art and perspectives. Open Orthop J 2016; 10: 920–938.
Ozdemir, Z., Ozdemir, A., Basim, G.B. “Biomedical Applications of Chemical Mechanical Polishing”, Materials Science and Engineering - Part C., 68 (1), P 383-396, 2016.
Ozdemir, Basim, G.B. "Effect of Chemical Mechanical Polishing on Surface Nature of Titanium Implants FT-IR and Wettability Data of Titanium Implants Surface After Chemical Mechanical Polishing Implementation," Data, in Brief, 10, P 20-25, 2017.

Physical signals have been used to control the degree of swelling or to induce a shape-change in hydrogels.^1,2,3 Taking in view potential future application of soft materials in life sciences more specific responsivities to biological signals are desirable. Chemical and physical sensors for glucose have been explored extensively motivated by device developments for diabetes treatment.⁴ Here we aim at integrating glucose sensitive chemical moieties into hydrophilic polymer networks in order to create a bio-induced shape-memory effect.
We hypothesized that glucose-sensitive shape-memory hydrogels can be created by minimizing the volume change upon stimulation by a porous cryogel structure and by implementing temporary netpoints which are cleavable by glucose.
Our concept for the glucose sensitive bases on a semi-interpenetrating network (semi-IPN) architecture. A primary copolymer network poly(AAm-co-AATris-co-BIS) from acrylamide (AAM) and N-[Tris(hydroxymethyl) methyl]acrylamide (AATris) and the non-glucose-sensitive crosslinker methylenebisacrylamide (BIS) was created. In this primary network linear copolymer poly(AAm-co-AAPBA) from AAm and m-acrylamido phenylboronic acid (AAPBA) was immobilized to form the semi-IPN. The boronic ester bonds, which can be reversibly formed between the PBA groups on linear chains and the triol groups provided by AATris act as the reversible crosslinks to fix the temporary shape.
In an alkaline media (pH = 10), the swelling ratio was ~ 35 independent of C_glu varied between 0 and 300 mg^.dL^-1. In rheological measurements it could be shown that G′ decreases remarkably from approximately 3800 Pa to approximately 2200 Pa when C_glu was raised from 0 to 90 mg^.dL^-1. In bending experiments shape fixity (R_f) and shape recovery (R_r) was determined. After 5 programming /recovery cycles a R_f ≈ 80% and a R_r ≈ 100% could be measured. R_r was a function of C_glu in the range from 0 to 300 mg^.dL^-1 which accords with the fluctuation range of C_glu in human blood, and in this way, the SMC could be used to determine C_glu.
The presented hydrogels could play a role in future Diabetes treatment options that are able to determine the blood glucose level and release insulin according to the detected glucose level automatically and in this way provide painless treatment for diabetes without insulin injection.

1. C. Löwenberg, M. Balk, C. Wischke, M. Behl, A. Lendlein Acc. Chem. Res. 50 (4), 723-732 (2017).
2. A. Kiriliova, L. Ionov. Mater. Chem. B 7, 1597-1624 (2019).
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Air Force requires wearable sensors to enhance performance and protect warfighters in operational environments. Volatile organic compounds (VOCs) found in exhaled breath are key biomarkers for estimating human physical/physiological statuses. Biorecognition elements (BREs) are biological materials specifically binding to the biomarker of interest. Short peptides (normally composed of 5-15 amino acid residuals) are promising BREs for the selective detection of VOCs. They provide chemical stability, straightforward design and engineering capability, and controlled affinity to a target. Carbon nanotube (CNT) field-effect transistors (FETs) have been generally accepted as a suitable platform for sensor miniaturization and wearable applications. Previous CNT FETs for VOC detection have measured explosives or food quality-related molecules. Selective detection of VOCs in breath, meanwhile, still remains a technical challenge since many VOCs share similar chemical characteristics such as polarity and partition coefficient. To facilitate discovery of peptides selective towards target VOCs, it is important to investigate a wide range of molecular properties relating to the peptides and VOCs. Here, we present an in-operando approach to investigate peptides’ affinity towards VOCs of interest (isopropyl alcohol, acetone, isoprene, and toluene) on a CNT FET platform. CNTs were functionalized by a number of peptides, and binding events between peptide and VOC have been directly observed by measuring changes in the electrical properties of the CNT FET. We tested three groups of peptides (14 total) that were obtained from phage display and/or in-silico modeling. The results showed not only decisive discrimination of the four different breath-related VOCs, but also possible binding affinity of amino acids towards target VOCs. The proposed BRE investigations have potential for selective breath monitoring sensors facilitating real-time human performance assessments.

The synthesis of materials with complex structures and well-defined properties is a central focus of the 'Directed assembly of extended structures with targeted properties' grand challenge, given their importance to economic growth and role in addressing key societal challenges. In materials produced by nature, i.e. biominerals; proteins, lipids and carbohydrates act as agents to control both the formation and the physicochemical properties of the composite materials that result. Taking inspiration from nature we extend the ideas of molecular recognition, adsorption/desorption to the formation of some commercially relevant materials. Using examples of MOFS (Metal-organic framework materials) and composites of ZnO and gold we showcase how peptides can be used to tailor the structures formed.

Cephalopods possess adaptive camouflage and signaling capabilities that are unrivaled among both artificial and natural systems. Such capabilities are enabled by the cephalopods’ unique skin morphology, wherein transparent dermal layers contain neurally-triggered, pigment-based color-changing organs called chromatophores; narrow-band reflective cells that act like Bragg stacks called iridocytes; and broadband reflective cells that act like diffuse reflectors called leucocytes. The optical functionalities of these organs and cells critically rely upon subcellular ultrastructures from unusual proteins known as reflectins. We have used these architectures and proteins as sources of inspiration to develop conductive materials for applications in biocompatible devices with a unique combination of electrical and optical capabilities. More recently, we have demonstrated the production of reflectin-based architectures for interfacing with various biological systems. In particular, we have discovered that reflectins can be used as a platform for controlling biological processes, such as cellular adhesion and differentiation. Altogether, our findings will facilitate the further development of cephalopod-based bio-electronic technologies that can control cell fate.

What if we could design materials that integrate powerful concepts of living organisms - self-organization, the ability to self-heal, tunability, and an amazing flexibility to create astounding material properties from abundant and inexpensive raw materials? This talk will present a review of bottom-up analysis and design of materials for various purposes - as structural materials such as bone in our body or for lightweight composites. These new materials are designed from the bottom up and through a close coupling of experiment and powerful computation as we assemble structures, atom by atom. We review case studies of joint experimental-computational work of biomimetic materials design, manufacturing and testing for the development of strong, tough and smart mutable materials for applications as protective coatings, cables and structural materials.

Modeling matter as resonating systems, this talk will then discuss the interface of material and sound, and present how we can transcend scales in space and time to make the invisible accessible to our senses and to manipulate matter from different vantage points, using innovative agents such as AI interacting with human creativity. The impact of this work is the design and making of new materials, art and music, and a deep mathematical understanding of the functional underpinnings of disparate manifestations of hierarchical systems. Building on these concepts, using AI, we explore a new interface of human expression with learned behavior to better understand the physiology and disease etiology due to the misfolding of proteins, explore it as the basis to generative algorithms, and present musical compositions based on the natural soundings of amino acids and proteins.

Using sets of harmonic oscillations as a unifying description, model of disparate hierarchical systems are developed, and then used to illustrate competing concepts of order and disorder and how they are the basis to create functional cross-scale relationships. The insights from this theory explain practically relevant issues such as the strength of silk or the emergence of disease, and the creation of new art. The translation from various hierarchical systems into one another presents a paradigm to understand the emergence of properties in materials, language, visual art, music, and similar systems.

Over the past years, significant efforts have been made towards manufacturing innovative sustainable materials. One of the most promising avenues is utilizing the remarkable efficiency and diversity of natural synthesis processes. In this direction, we present here a fabrication platform based on plant cells dehydration, which delivers bulk, biopolymer nanocomposite materials. Plant cells naturally synthesize intricate and hierarchical biopolymer composite structures that comprise their cell walls. Here we demonstrate a modified compression molding method that allows the preservation of cell walls upon dehydration. The final product is a bulk, hierarchical, lamellated biopolymer nanocomposite that is entirely biodegradable. We report the mechanical properties of the pure biopolymer composite, which are superior to other biological matrix materials, such as mycelium or yeast-based composites. Additionally, we demonstrate that filler additives can be incorporated in the fabrication process in order to introduce new properties or tune the native properties of the nanocomposite.

The ability to organize nano-components into the desired architectures with targeted properties is one of the major objectives of nanotechnology. Our efforts are focused on establishing a broadly applicable DNA-based platform for programmable assembly of nano-components into desired and structurally defined organizations. We explore the use of DNA-encoded nano-components and designed DNA constructs to guide self-assembly process towards the desired lattices and arbitrary designed architectures. Through the development of effective assembly strategies, revealing the principles governing these DNA-programmable systems and understanding the role of geometry, interactions and pathways, we develop methods for creation of well-defined three-dimensional lattices, two-dimensional arrays and finite-sized cluster architectures from the inorganic and biomolecular nano-components. Our resent advances in ability to form the desired inorganic, organic and hybrid nanoscale architectures and their applications for nano-optics and biomaterials will be discussed.

Biological systems have provided us much inspiration to design and engineering new materials. For example, nature has utilized biomolecules for the synthesis of complex inorganic structure in a process collective termed biomineralization. In-vitro biomineralization methods are capable of synthesizing nanoscale materials, for instance, SiO₂, TiO_2, and etc. with high surface area and disordered structure occurring under mild reaction conditions. Based on these unique material characteristics, in-vitro biomineralization has great potential for a variety of functional applications, particularly in catalysis wherein access to disordered surface structures is beneficial. As such, in-vitro biomineralization can bring an innovative strategy for synthesizing nanoscale metal oxides which can serve as catalysts and catalyst support materials.
In this work, a biomimetic analogue, protamine, is used to enable a cost-effective, larger scale metal oxide catalyst synthesis approach. The protamine based biomineralization technique was adopted to synthesize a range of mixed metal oxide nanoparticles for a series of reactions. This technique brings many advantageous features for catalytic materials such as nanoscale, high specific surface area, low preparation cost, and stable thermal properties. For example, biomineralized SiO₂-TiO₂ composite metal oxides were implemented for the CO₂ hydrogenation reaction. The modularity of the synthetic techniques enabled surface area increase and additional defect engineering strategies, that lead to the inducing of extra defects on the material which served as active sites to further enhance the catalytic performance. In addition, we have created Zn-Sn mixed metal oxides for catalysts in the electrochemical CO₂ reduction reaction. All materials were thoroughly characterized using a suite of synchrotron characterization methods to establish structure/function relationships. Through these techniques, we have discovered that most materials are largely disordered and change in local structure depending on the synthetic conditions applied. Through these efforts, we have demonstrated a new material manufacturing strategy based on in-vitro biomineralization for nanoscale metal oxide catalysts.

The mechanical performance of cellulose nanocrystal (CNC) is typically enhanced through crosslinking, the addition of strong filler materials or mixing with soft matrices. However, these modifications that can lead to higher mechanical strength tend to disrupt the original helical organization of the chiral phases and destroy the corresponding vivid iridescence. Herein, we demonstrate a unique case of enhanced and tunable structural colors combined with much improved strength of CNC composite materials through the addition of amorphous wood-derived polymers of similar chemical composition. Adding these polysaccharides, pullulan, dextran, and xylan, promotes seamless integration into the original helicoidal organization with full chain intercalation, even at very high loading, a unique behavior for composites from components with different structural organization, amorphous and chiral nematic in our case. CNC-polymer composites demonstrate enhanced mechanical performance, nearly a two-fold increase in toughness. A systematic red-shift in selective light reflection is observed for all polymer composites due to the gradual increase in pitch length because of the intercalation of natural polymer backbones into helicoidal organization without disturbing long-range ordering. The utilization of wood-derived amorphous polymers with similar composition, hydrogen bonding capabilities, and mechanical properties, instead of foreign synthetic components provides a sustainable method of significant mechanical enhancement of chiral composite films without disrupting the original helicoidal organization and vivid iridescence with controlled color appearance.

Nanostructured materials producing structural colors have a great potential in replacing toxic metals or organic pigments. Electrophoretic deposition (EPD) is a promising method for producing these materials quickly on a large scale. However, the colors reported so far lack brightness, saturation, and mechanical stability. Herein we use EPD assembly to co-deposit silica (SiO₂) nanoparticles with precursors of synthetic melanin, polydopamine (PDA), to produce mechanically robust, wide-angle structurally colored coatings. We use spectrophotometry to show that PDA precursors enhance saturation of structural colors and nanoscratch testing to demonstrate that they increase the mechanical stability of the EPD coatings. Stabilization by PDA precursors allows us to coat flexible substrates that can sustain bending stresses, opening an avenue for electro-printing on flexible materials.

The emerging field of synthetic biology provides opportunity and new capabilities for the precision synthesis, assembly, and function of materials. Currently, there are significant challenges for the integration of bio-derived and bio-based technologies in the defense-oriented materials space. Army platforms must reliably operate over wide temperature ranges (e.g. -40 to 75 deg C), extremes in humidity, exhibit durability to be fielded for long periods of time (e.g. 5+ years) and endure strain rate deformations from low strain, high frequency vibration to high strain impact events. We are making initial forays into the synthesis and assembly of materials, and are developing protocols to probe performance metrics, production repeatability, and aging. Materials under evaluation include bio-derived organics and bio-templated inorganics, and integration into materials systems has been undertaken using both traditional processes and bio-synthetic processes. This poster will give an overview of current projects employing the tools of Synthetic Biology and highlight the key performance targets of these materials.

Approaches to control the microstructure of hydrogels are critical for the control of cell-material interactions. Herein, we report a versatile approach for the synthesis of anisotropic polyacrylamide hydrogels by lyotropic chromonic liquid crystal (LCLC) templating. Specifically, polyacrylamide hydrogels with aligned porosity can be obtained by polymerizing the network in the presence of an aligned chromonic phase. By varying crosslink density, the resulting hydrogels have tunable pore size and distinct mechanical anisotropy. For example, the elastic moduli measured parallel and perpendicular to the LCLC order are 124.6 kPa ± 7.9 kPa and 17.4 kPa ± 1.1 kPa and the hydrogels have a 4-fold larger swelling normal to the LCLC director than along the LCLC director. This anisotropy can be patterned by using surface anchoring to locally control the nematic director, which in turn patterns the polymer network. The director can be patterned both within the plane and through the thickness of the hydrogels. Fibroblasts cultured on the resulting hydrogels align along the pore walls, which in turn provides a powerful approach to control cell orientation. This new strategy to make anisotropic hydrogels can have potential applications for patternable tissue scaffolds, soft robotics, or microfluidic devices.

Author for Correspondence: [email protected]

Shape-transformation is a prevalent function observed in living systems, from the blooming of flowers to muscle actuation. While current shape changing synthetic materials are capable of sensing non-specific stimuli such as pH, light, temperature or ultrasound, new materials are needed that detect specific biochemical cues and respond mechanically in a programmed manner. Materials that respond to such subtle cues could enable biosensors or drug-delivery devices. Recently, we demonstrated that synthetic materials can be designed to host genetically engineered yeast cells capable of responding to very specific and pre-determined stimuli (e.g. biogenic amines). Specifically, we presented a new method to create programmable shape-morphing composites using polyacrylamide hydrogels embed with Saccharomyces cerevisiae. Cellular proliferation within these composites can be spatio-temporally controlled to generate large volume expansions of up to 600% of the composite. Here, we report the design of living composites with shear-thinning properties based on cellulose nanocrystals (CNC) and acrylamide monomers using direct-write printing. CNC concentrations at 11 wt% and 22 wt% enabled active (yeast-embedding) and passive (cell-free) bioinks respectively to be printed. By developing a hydrogel-based printable bioink, engineered yeast strains can be patterned into three-dimensional (3D) structures with defined geometry, organization and porosity. Using this control, we spatially pattern cellular proliferation within a monolith to induce complex shape transformations and demonstrate specificity to single amino acids. Finally, we will discuss the 3D printing of a living composite capsule for in vitro biomolecular detection and subsequent drug delivery in a model of the gastrointestinal tract. In this device, genetically-engineered probiotic yeast only proliferate in the presence of a particular biomolecule, such as heme, which leads to expansion of one portion of the capsule. This expansion ruptures the device and releases a hydrophobic compound to the surrounding environment. Our study may enable new opportunities to develop drug-delivery devices for the diagnosis and treatment of gastrointestinal disorders.

Under laboratory conditions, the micron-scale dimensions and gliding motility of some prokaryotes, including Cellulophaga lytica, facilitate the self-assembly of discrete cells into iridescent biofilms. This is in sharp contrast to structural coloration in eukaryotic systems (e.g. lepidoptera, coleoptera), where multiple cell types, complex architectures and fixed structures are frequently involved. We propose using biofilms of C. lytica as a platform for material synthesis since properties derived from their exquisite ordering can be tuned using ‘simpler’ bacterial genetics. Importantly, the sequence of C. lytica strain 7489 is known, making it amenable to future synthetic biology approaches for tailoring. Though facile growth of its biofilms is possible, we report nuances of growth conditions which impact iridescence. Toward a fundamental understanding of the strategies employed in self-assembly, the current study also examines community organization and cellular morphology. Specifically, iridescent and non-iridescent regions of the biofilms are probed using complementary imaging techniques including confocal, electron and atomic force microscopies. These multi-scaled microscopies have provided surprising insights into the biofilm structure. Specifically, we report that differences in morphology and packing correspond to changes in the wavelength of reflected light. To facilitate these studies, sample preparation strategies which allow cells to be imaged in context were developed. In addition to establishing the structure-property relationship derived from the biofilm’s organization, our findings suggest that novel mechanisms may be involved in assembly and that a comprehensive investigation to identify cellular components contributing to assembly is indeed warranted.

Over the past few decades, cancer and other fatal diseases have become more prevalent affecting large populations around the world. Earlier detection could improve prognosis for the patient. A possible solution includes using photonic crystal-based sensors to detect biomarkers in exosomes shed by cancerous cells. Photonic crystals have become increasingly important for biosensor applications due to the need for a label-free alternative to detect biomarkers. Surface enhanced Raman scattering (SERS) using gold nanoparticles is a sensitive analytical technique, which has been exploited in chemical, molecule and environmental monitoring. In this study, a photonic crystal SERS substrate was designed and synthesized for the early detection of changes which occur in the exosomes shed by the cancerous cells. Optical signal amplification due to gold nanoparticles and photonic components, leads to the enhanced interactions between the analyte and the sensor. Electromagnetic enhancement using photonic crystal substrate can greatly enhance the SERS detection when compared to traditional methods. The two-dimensional photonic structural organization in the comb structures of the ctenophore species, Mnemiopsis Leidyi and Beroe Cucumis was used as the inspiration to design the photonic crystal substrate used in this study. The structural organization of the cilia in the comb rows consists of arrays of hexagonally packed cilia with a central microtubule pair. Transmission electron microscopy was used to study the arrangements of the comb structures. Reverse engineering of these submicron structures aids in designing a novel photonic template. The design of the comb structures was recreated using AutoCAD and Raith software. Photonic nanostructures were synthesized using the direct-write e-beam lithography technique. The structures were characterized with scanning electron microscope (SEM), and reflectance measurements. Gold nanoparticles will be used to in conjugation with the photonic crystal structure and Raman signatures measured, both with and without gold molecules. Finally, these gold nanoparticle coated photonic templates were utilized as a SERS active surface for exosomal detection. Some advantages of utilizing photonic crystals include point of care diagnostics and their response to a multitude of external stimuli including light.

The delivery of nanoparticles, as vectors in biological environments, depends on the interaction and stability in the environment where it is delivered. The functional groups are given by surfactants that gives stability to the particles, avoiding the aggregation between themselves aside from chemical associations not only with the environment but also with other molecules depending on the surface charge. Recent studies have shown that the use of carbodiimides has been helpful in achieving changes in the behavior in nanoparticle solution, from organic to aqueous phase [1-3].
There is an extensively research field on surfactants, called "green surfactants", based on the integration of amino acid and proteins, due to the functional groups that compose them, such as carboxyl and amino groups, that can be chemically adhered to the surface through a possible nucleophilic substitution achieving amphiphilic conformations in the form of micelles or enaniometers and dependent on the change of pH for the deliberation of the vehicle in the biological system [4–8].
This research aims iron carbide@iron oxide nanoparticles (ICIONPs) from organic nature[9], coated of amino acids through the conjugation of oleic acid with L-Arginine and L-Asparagine performing by N,N′-dicyclohexylcarbodiimide (DCC) with N-Hydrxysuccinimide (NHS) as a coupling reagents, forming behavioral structures, known as polypeptides, with an organic center and aqueous surface arrangement for encapsulation of nanoparticles as possible interaction with other biological molecules and their application in bioseparation processes.
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Nanotechnology has studied applications in biomedical areas, for which several nanomaterials have been synthesized that present limitations such as toxicity and instability in organisms, so green methodologies have been developed that help reduce this problem. In this work we report the green synthesis of AuNPs using as a reducing agent lemon extract (Citrus limon), and the multiwave ultrasonic technique as an excitation element. As the precursor reagent, trihydrated chlorouric acid (HAuCl4.3H2O, Sigma Aldrich SKU 520918) was used, and as a reducing agent the lemon juice extract. The reaction was carried out in an ultrasonic bath at a frequency of 40kHz, at 55 ° C for one hour (Branson 2800 series). The final solution obtained showed a purple coloration, and was characterized by UV-VIS spectrophotometry and transmission electron microscopy (JEOL JEM 1010). Absorbance peak resonant at 530 nm was observed and particle diameters between 12 and 16 nm were obtained. The reported results could be of interest to carry out fusion of nanometric inorganic materials by the ultrasonic technique, and by controlling the temperature and frequency, metal structures with different morphology and properties could be generated.