Bridging the bio-electronic interface with biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern. Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods. Alginate films and their entrapment of live cells will also be addressed. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.     

基于壳聚糖的生物组装技术及应用研究进展

壳聚糖是一种具有良好生物相容性、独特pH值响应性、易改性和易成膜特性的高分子材料。在生物组装技术中,壳聚糖可作为多功能活性介体,与生物组分和微加工装置连接,制成高选择性、高灵敏度的生物微机电系统(BioMEMS)。以下介绍了基于壳聚糖的3种生物组装技术——定向组装、酶促组装、自组装的制备原理和过程,评述了基于壳聚糖的BioMEMS在生物、医学、环境领域中的应用现状,并展望了今后的研究方向。     

Integrated biofabrication for electro-addressed in-film bioprocessing

Many recent advances in bioprocessing have been enabled by developments in miniaturization and microfluidics. A continuing challenge, however, is integrating multiple unit operations that require distinct spatial boundaries, especially with included labile biological components. We have suggested "biofabrication" as a means for organizing cells and biomolecules in complex configurations while preserving function of individual components. Polysaccharide films of chitosan and alginate that are assembled on-chip by electrodeposition are "smart" configurable interfaces that mediate communication between the biological systems and microfabricated devices. Here, we demonstrate the scalable performance of a production address, where incubated cells secrete antibodies, and a capture address, where secreted antibody is retained with specificity and subsequently assayed. The antibody exchange from one electro-address to another exemplifies integrated in-film bioprocessing, facilitated by the integrated biofabrication techniques used. This in-film approach enables complex processes without need for microfluidics and valving. Finally, we have shown scalability by reducing electrode sizes to a 1 mm scale without compromising film biofabrication or bioprocessing performance. The in situ reversible deposition of viable cells, productivity characterization, and capture of secreted antibodies could find use in bioprocessing applications such as clonal selection, run-to-run monitoring, initial scale-up, and areas including drug screening and biopsy analysis.     

Biofabrication: using biological materials and biocatalysts to construct nanostructured assemblies

Emerging opportunities are placing greater demands on device fabrication: next-generation microelectronics will need minimum features of less than 100 nm, high-throughput drug screening will require facile methods to incorporate sensitive biological components into microelectromechanical systems (MEMS), and implantable devices will need to be built from biocompatible materials. Increasingly, these emerging demands are being addressed by combining traditional microfabrication methods with 'biofabrication': namely, the use of biologically derived materials and biocatalysts. Recent fabrication techniques are using biological construction materials as process aids or structural components, and enzymes are being considered for their potential to fabricate devices with high selectivity under mild conditions. If incompatibilities between biology and microfabrication can be eliminated, then biofabrication will be poised to emerge as the standard for nanoscale construction.     

Biopolymer-based materials: the nanoscale components and their hierarchical assembly

Protein and nucleic acid biopolymers are well appreciated for their high-performance capabilities for molecular recognition, catalysis and information storage. Increasingly, these biopolymers are being examined for materials applications. Less tractable are polysaccharides and polymers of phenols, which, despite being nature's most abundant macromolecules, remain largely ignored for advanced materials applications. In our opinion, it seems certain that biology will contribute two major capabilities for materials biofabrication - the means to generate biopolymeric components with nanoscale precision, and the mechanisms for the hierarchical assembly of nanocomponents. These capabilities will enable unprecedented control of materials structure and provide exciting opportunities at the convergence of molecular biology and macromolecular science.     

Fabrication of hierarchical hybrid structures using bio-enabled layer-by-layer self-assembly

Development of versatile and flexible assembly systems for fabrication of functional hybrid nanomaterials with well-defined hierarchical and spatial organization is of a significant importance in practical nanobiotechnology applications. Here we demonstrate a bio-enabled self-assembly technique for fabrication of multi-layered protein and nanometallic assemblies utilizing a modular gold-binding (AuBP1) fusion tag. To accomplish the bottom-up assembly we first genetically fused the AuBP1 peptide sequence to the C'-terminus of maltose-binding protein (MBP) using two different linkers to produce MBP-AuBP1 hetero-functional constructs. Using various spectroscopic techniques, surface plasmon resonance (SPR) and localized surface plasmon resonance (LSPR), we verified the exceptional binding and self-assembly characteristics of AuBP1 peptide. The AuBP1 peptide tag can direct the organization of recombinant MBP protein on various gold surfaces through an efficient control of the organic-inorganic interface at the molecular level. Furthermore using a combination of soft-lithography, self-assembly techniques and advanced AuBP1 peptide tag technology, we produced spatially and hierarchically controlled protein multi-layered assemblies on gold nanoparticle arrays with high molecular packing density and pattering efficiency in simple, reproducible steps. This model system offers layer-by-layer assembly capability based on specific AuBP1 peptide tag and constitutes novel biological routes for biofabrication of various protein arrays, plasmon-active nanometallic assemblies and devices with controlled organization, packing density and architecture.     

Biofabrication of antibodies and antigens via IgG‐binding domain engineered with activatable pentatyrosine pro‐tag

We report the assembly of seven different antibodies (and two antigens) into functional supramolecular structures that are specifically designed to facilitate integration into devices using entirely biologically based bottom‐up fabrication. This is enabled by the creation of an engineered IgG‐binding domain (HG3T) with an N‐terminal hexahistidine tag that facilitates purification and a C‐terminal enzyme‐activatable pentatyrosine “pro‐tag” that facilitates covalent coupling to the pH stimuli‐responsive polysaccharide, chitosan. Because we confer pH‐stimuli responsiveness to the IgG‐binding domain, it can be electrodeposited or otherwise assembled into many configurations. Importantly, we demonstrate the loading of both HG3T and antibodies can be achieved in a linear fashion so that quantitative assessment of antibodies and antigens is feasible. Our demonstration formats include: conventional multiwell plates, micropatterned electrodes, and fiber networks. We believe biologically based fabrication (i.e., biofabrication) provides bottom‐up hierarchical assembly of a variety of nanoscale components for applications that range from point‐of‐care diagnostics to smart fabrics. Biotechnol. Bioeng. 2009;103: 231–240. © 2008 Wiley Periodicals, Inc.     

Eugene--a domain specific language for specifying and constraining synthetic biological parts, devices, and systems

Background

Synthetic biological systems are currently created by an ad-hoc, iterative process of specification, design, and assembly. These systems would greatly benefit from a more formalized and rigorous specification of the desired system components as well as constraints on their composition. Therefore, the creation of robust and efficient design flows and tools is imperative. We present a human readable language (Eugene) that allows for the specification of synthetic biological designs based on biological parts, as well as provides a very expressive constraint system to drive the automatic creation of composite Parts (Devices) from a collection of individual Parts.

Results

We illustrate Eugene''s capabilities in three different areas: Device specification, design space exploration, and assembly and simulation integration. These results highlight Eugene''s ability to create combinatorial design spaces and prune these spaces for simulation or physical assembly. Eugene creates functional designs quickly and cost-effectively.

Conclusions

Eugene is intended for forward engineering of DNA-based devices, and through its data types and execution semantics, reflects the desired abstraction hierarchy in synthetic biology. Eugene provides a powerful constraint system which can be used to drive the creation of new devices at runtime. It accomplishes all of this while being part of a larger tool chain which includes support for design, simulation, and physical device assembly.     

Micromachined biomimetic artificial haircell sensors

The biological haircell is a modular building block of a rich variety of biological sensors. Using micro- and nanofabrication technology, an engineering equivalent artificial haircell sensor can be developed, imitating the structure and transfer function of the biological haircell. The artificial haircells can be made of hybrid semiconductor, metal and polymers. This paper discusses a number of strategies, using representative material systems, for building artificial haircell sensors and briefly outlines their fabrication method and performance. The motivation for imitating the biological haircell is also discussed to provide a background for this work.     

Whole-cell biocomputing.

The ability to manipulate systems on the molecular scale naturally leads to speculation about the rational design of molecular-scale machines. Cells might be the ultimate molecular-scale machines and our ability to engineer them is relatively advanced when compared with our ability to control the synthesis and direct the assembly of man-made materials. Indeed, engineered whole cells deployed in biosensors can be considered one of the practical successes of molecular-scale devices. However, these devices explore only a small portion of cellular functionality. Individual cells or self-organized groups of cells perform extremely complex functions that include sensing, communication, navigation, cooperation and even fabrication of synthetic nanoscopic materials. In natural systems, these capabilities are controlled by complex genetic regulatory circuits, which are only partially understood and not readily accessible for use in engineered systems. Here, we focus on efforts to mimic the functionality of man-made information-processing systems within whole cells.     

Assembly of multimeric phage nanostructures through leucine zipper interactions

One barrier to the construction of nanoscale devices is the ability to place materials into 2D- and 3D-ordered arrays by controlling the assembly and ordering of connections between nanomaterials. Ordered assembly of nanoscale materials may potentially be achieved using biological tools that direct specific connections between individual components. Recently, viruses were successfully employed as scaffolds for the nucleation of nanoparticles and nanowires (Mao et al., 2004); however, there is a paucity of methods for the higher order assembly of phage-templated materials. Here we describe a general strategy for the assembly of filamentous bacteriophages into long, wire-like or into tripod-like structures. To prepare the linear phage assemblies, dimeric leucine zipper protein domains, fused to the p3 and p9 proteins of M13 bacteriophage, were employed to direct the specific end-to-end self-association of the bacteriophage particles. Electron microscopy revealed that up to 90% of the phage displaying complementary leucine zipper domains formed linear multi-phage assemblies, composed of up to 30 phage in length. To prepare tripod-like assemblies, phage were engineered to express trimeric leucine zippers as p3 fusion proteins. This resulted in 3D assembly with three individual phages attached at a single point. These ordered phage structures should provide a foundation for self-assembly of virally templated nanomaterials into useful devices.     

Recent advances in the development of biosensor for phenol: a review

Phenol and its derivatives are widespread contaminants whose sources are both natural and industrial. Phenol is massively produced and used as a starting material for synthetic polymers and fibers. Although phenolic compounds play important biochemical and physiological roles in living systems, their accumulation in the environment as a result of intensive human activity may result in drastic ecological problem. Various analytical techniques are available for the detection of phenol in environmental samples. But they need complex sample pre-treatment so as are time consuming, costly and use heavy devices. On the other hand a biosensor is a device that gives rapid detection, cost effective and easy. A review study was carried out to accumulate the possible biosensors for the detection of phenolic compounds in environmental samples. A number of biological components including microorganisms, enzymes, antibodies, antigens, nucleic acids etc. can be used for the construction of biosensors that was found to detect phenolic compounds. Of all type of biological components microorganisms and enzymes are mostly used. The microorganisms are Pseudomonas, Moraxella, Arthrobacter, Rhodococcus, and Trichosporon. The most used enzymes are tyrosinase, peroxidase, laccase, glucose dehydrogenase, cellobiose dehydrogenase etc. Antibody sensors can detect a very trace level. The biorecognition of DNA biosensors occur by hybridization of DNA. Biosensors are found to work well when the biological sensing element is immobilized. A variety of immobilization techniques were found to use as adsorption, covalent binding, entrapment, cross-linking etc. For immobilization the matrices used was polyvinyl alcohol, Osmium complex, nafion/sol?Cgel silicate, chitosan, silica gel etc.     

Fabrication,Densification, and Replica Molding of 3D Carbon Nanotube Microstructures

The introduction of new materials and processes to microfabrication has, in large part, enabled many important advances in microsystems, lab-on-a-chip devices, and their applications. In particular, capabilities for cost-effective fabrication of polymer microstructures were transformed by the advent of soft lithography and other micromolding techniques ^{1, 2}, and this led a revolution in applications of microfabrication to biomedical engineering and biology. Nevertheless, it remains challenging to fabricate microstructures with well-defined nanoscale surface textures, and to fabricate arbitrary 3D shapes at the micro-scale. Robustness of master molds and maintenance of shape integrity is especially important to achieve high fidelity replication of complex structures and preserving their nanoscale surface texture. The combination of hierarchical textures, and heterogeneous shapes, is a profound challenge to existing microfabrication methods that largely rely upon top-down etching using fixed mask templates. On the other hand, the bottom-up synthesis of nanostructures such as nanotubes and nanowires can offer new capabilities to microfabrication, in particular by taking advantage of the collective self-organization of nanostructures, and local control of their growth behavior with respect to microfabricated patterns. Our goal is to introduce vertically aligned carbon nanotubes (CNTs), which we refer to as CNT "forests", as a new microfabrication material. We present details of a suite of related methods recently developed by our group: fabrication of CNT forest microstructures by thermal CVD from lithographically patterned catalyst thin films; self-directed elastocapillary densification of CNT microstructures; and replica molding of polymer microstructures using CNT composite master molds. In particular, our work shows that self-directed capillary densification ("capillary forming"), which is performed by condensation of a solvent onto the substrate with CNT microstructures, significantly increases the packing density of CNTs. This process enables directed transformation of vertical CNT microstructures into straight, inclined, and twisted shapes, which have robust mechanical properties exceeding those of typical microfabrication polymers. This in turn enables formation of nanocomposite CNT master molds by capillary-driven infiltration of polymers. The replica structures exhibit the anisotropic nanoscale texture of the aligned CNTs, and can have walls with sub-micron thickness and aspect ratios exceeding 50:1. Integration of CNT microstructures in fabrication offers further opportunity to exploit the electrical and thermal properties of CNTs, and diverse capabilities for chemical and biochemical functionalization ³.     

Liquid–Solid Interfacial Assemblies of Soft Materials for Functional Freestanding Layered Membrane–Based Devices toward Electrochemical Energy Systems

Freestanding layered membrane–based devices have broad applications in highly efficient energy‐storage/conversion systems. The liquid–solid interface is considered as a unique yet versatile interface for constructing such layered membrane–based devices. In this review, the authors outline recent developments in the fabrication of soft materials to functionalize layered devices from the aspect of liquid–solid interfacial assembly and engineering arts. Seven liquid–solid interfacial assembly strategies, including flow‐directed, superlattice, solvent‐casting, evaporation‐induced, dip‐coating, spinning, and electrospinning assemblies, are comprehensively highlighted with a focus on their synthetic pathways, formation mechanisms, and interface engineering strategies. Meanwhile, recent representative works on layered membrane–based devices for electrochemical energy applications are presented. Finally, challenges and opportunities of this research area are highlighted in order to stimulate future developments. This review not only offers comprehensive and practical approaches to assemble liquid–solid interfaces with soft materials for various important layered electrochemical energy devices but also sheds lights on fundamental insights by thoughtful discussions on performance enhancement mechanisms of these electrochemical energy systems.     

Multifunctional Luminescent Down‐Shifting Fluoropolymer Coatings: A Straightforward Strategy to Improve the UV‐Light Harvesting Ability and Long‐Term Outdoor Stability of Organic Dye‐Sensitized Solar Cells

A new multifunctional coating for photovoltaic cells incorporating light‐management, UV‐protection, and easy‐cleaning capabilities is presented. Such coating consists of a new photocurable fluorinated polymer embedding a luminescent europium complex that acts as luminescent down‐shifting (LDS) material converting UV photons into visible light. The combination of this system with ruthenium‐free organic dye‐sensitized solar cells (DSSCs) gives a 70% relative increase in power conversion efficiency as compared with control uncoated devices, which is the highest efficiency enhancement reported to date on organic DSSC systems by means of a polymeric LDS layer. Long‐term (>2000 h) weathering tests in real outdoor conditions reveal the excellent stabilizing effect of the new coating on DSSC devices, which fully preserve their initial performance. This excellent outdoor stability is attributed to the combined action of the luminescent material that acts as UV‐screen and the highly photostable, hydrophobic fluoropolymeric carrier that further prevents photochemical and physical degradation of the solar cell components. The straightforward approach presented to simultaneously improve performance and outdoor stability of DSSC devices may be readily extended to a large variety of sensitizer/luminophore combinations, thus enabling the fabrication of highly efficient and extremely stable DSSCs in an easy and versatile fashion.     

Delivery of Human Adipose Stem Cells Spheroids into Lockyballs

Adipose stem cells (ASCs) spheroids show enhanced regenerative effects compared to single cells. Also, spheroids have been recently introduced as building blocks in directed self-assembly strategy. Recent efforts aim to improve long-term cell retention and integration by the use of microencapsulation delivery systems that can rapidly integrate in the implantation site. Interlockable solid synthetic microscaffolds, so called lockyballs, were recently designed with hooks and loops to enhance cell retention and integration at the implantation site as well as to support spheroids aggregation after transplantation. Here we present an efficient methodology for human ASCs spheroids biofabrication and lockyballs cellularization using micro-molded non-adhesive agarose hydrogel. Lockyballs were produced using two-photon polymerization with an estimated mechanical strength. The Young’s modulus was calculated at level 0.1362 +/-0.009 MPa. Interlocking in vitro test demonstrates high level of loading induced interlockability of fabricated lockyballs. Diameter measurements and elongation coefficient calculation revealed that human ASCs spheroids biofabricated in resections of micro-molded non-adhesive hydrogel had a more regular size distribution and shape than spheroids biofabricated in hanging drops. Cellularization of lockyballs using human ASCs spheroids did not alter the level of cells viability (p › 0,999) and gene fold expression for SOX-9 and RUNX2 (p › 0,195). The biofabrication of ASCs spheroids into lockyballs represents an innovative strategy in regenerative medicine, which combines solid scaffold-based and directed self-assembly approaches, fostering opportunities for rapid in situ biofabrication of 3D building-blocks.     

Nanotechnology in vascular tissue engineering: from nanoscaffolding towards rapid vessel biofabrication

The existing methods of biofabrication for vascular tissue engineering are still bioreactor-based, extremely expensive, laborious and time consuming and, furthermore, not automated, which would be essential for an economically successful large-scale commercialization. The advances in nanotechnology can bring additional functionality to vascular scaffolds, optimize internal vascular graft surface and even help to direct the differentiation of stem cells into the vascular cell phenotype. The development of rapid nanotechnology-based methods of vascular tissue biofabrication represents one of most important recent technological breakthroughs in vascular tissue engineering because it dramatically accelerates vascular tissue assembly and, importantly, also eliminates the need for a bioreactor-based scaffold cellularization process.     

Microbeads on microposts: an inverted architecture for bead microarrays

The rapid development of genomics and proteomics requires accelerated improvement of the microarrays density, multiplexing, readout capabilities and cost-effectiveness. The bead arrays are increasingly attractive because of their self-assembly-based fabrication, which alleviates many problems of top-down microfabrication. Here we present a simple, reliable, robust and modular technique for the fabrication of bead microarrays, which combines the directed assembling of beads in microstructures and PDMS-based replica molding. The beads are first self-assembled in pyramidal microwells fabricated by anisotropic etching of silicon substrates, then transferred on the apex of PDMS pyramids that replicate the silicon microstructures. The arrays are chemically and biochemically robust; they are spatially addressable and have the potential for being informationally addressable; and they appear to offer better readout capabilities than the classical microarrays.     

面向重要实质器官的生物制造技术

在体外制造可修复人体受损组织与器官功能的活性替代物一直是人类的梦想.制造、材料与生命科学的交叉与融合发展,为生物组织与器官的体外制造提供了必要的技术、材料与生物学基础,从而实现了皮肤、骨、膀胱等简单活性组织的临床应用,但人体重要实质器官如肝脏、肺等的再造研究至今未取得突破性进展.重要实质器官内部复杂的微观结构系统及多细胞体系的构建是实现其体外制造的关键,也是当前生物组织与器官制造技术所面临的巨大挑战.从生物制造的角度,综述国内外在重要实质器官复杂微结构制造领域的主要技术方法及最新研究进展,通过分析与评价,对未来重要实质器官的生物制造技术发展进行展望.     

Minimizing prion risk without compromising the microbial composition of biofilms grown in vivo in a human plaque model

AIMS: To determine whether the stringency of sterilization procedures for biological components of in vivo dental plaque-generating devices based on enamel can be increased to minimize prion risk without compromising natural biofilm composition. METHODS AND RESULTS: The composition of in vitro biofilms, grown on hypochlorite-treated and untreated autoclaved enamel surfaces, was determined using culture-based methods and checkerboard DNA: DNA hybridization analysis. No differences were found between biofilms recovered from either substrate. SIGNIFICANCE: Several in situ models allow generation of plaque in the oral cavity, followed by recovery of intact biofilms for experimentation. Approaches allowing plaque formation on natural tooth surfaces are most valuable, but present a possible infection risk to volunteers wearing plaque-collecting devices, particularly with respect to prions. Hypochlorite treatment of biological material, as an adjunct to autoclaving, reduces infection risk without compromising biofilm composition and should be adopted in all future studies using plaque-generating devices incorporating enamel, where there is a potential prion threat, and further investigated in other biological hard tissues.