RESUMO
Microbial cells have for many years been engineered to facilitate efficient production of biologics, chemicals, and other compounds. As the "metabolic" burden of synthetic genetic components can impair cell performance, microbial consortia are being developed to piece together specialized subpopulations that collectively produce desired products. Their use, however, has been limited by the inability to control their composition and function. One approach to leverage advantages of the division of labor within consortia is to link microbial subpopulations together through quorum sensing (QS) molecules. Previously, we directed the assembly of "quantized quorums," microbial subpopulations that are parsed through QS activation, by the exogenous addition of QS signal molecules to QS synthase mutants. In this work, we develop a more facile and general platform for creating "quantized quorums." Moreover, the methodology is not restricted to QS-mutant populations. We constructed quorum quenching capsules that partition QS-mediated phenotypes into discrete subpopulations. This compartmentalization guides QS subpopulations in a dose-dependent manner, parsing cell populations into activated or deactivated groups. The capsular "devices" consist of polyelectrolyte alginate-chitosan beads that encapsulate high-efficiency (HE) "controller cells" that, in turn, provide rapid uptake of the QS signal molecule AI-2 from culture fluids. In this methodology, instead of adding AI-2 to parse QS-mutants into subpopulations, we engineered cells to encapsulate them into compartments, and they serve to deplete AI-2 from wild-type populations. These encapsulated bacteria therefore, provide orthogonal control of population composition while allowing only minimal interaction with the product-producing cell population or consortia. We envision that compartmentalized control of QS should have applications in both metabolic engineering and human disease. Biotechnol. Bioeng. 2017;114: 407-415. © 2016 Wiley Periodicals, Inc.
Assuntos
Bactérias , Engenharia Metabólica/métodos , Consórcios Microbianos/fisiologia , Modelos Biológicos , Percepção de Quorum/fisiologia , Bactérias/citologia , Bactérias/metabolismo , FenótipoRESUMO
Spider silk is an extraordinary material with physical properties comparable to the best scaffolding/structural materials, and as a fiber it can be manipulated with ease into a variety of configurations. Our work here demonstrates that natural spider silk fibers can also be used to organize biological components on and in devices through rapid and simple means. Micron scale spider silk fibers (5-10 µm in diameter) were surface modified with a variety of biological entities engineered with pentaglutamine tags via microbial transglutaminase (mTG). Enzymes, enzyme pathways, antibodies, and fluorescent proteins were all assembled onto spider silk fibers using this biomolecular engineering/biofabrication process. Additionally, arrangement of biofunctionalized fiber should in of itself generate a secondary level of biomolecular organization. Toward this end, as proofs of principle, spatially defined arrangement of biofunctionalized spider silk fiber was shown to generate effects specific to silk position in two cases. In one instance, arrangement perpendicular to a flow produced selective head and neck carcinoma cell capture on silk with antibodies complexed to conjugated protein G. In a second scenario, asymmetric bacterial chemotaxis arose from asymmetric conjugation of enzymes to arranged silk. Overall, the biofabrication processes used here were rapid, required no complex chemistries, were biologically benign, and also the resulting engineered silk microfibers were flexible, readily manipulated and functionally active. Deployed here in microfluidic environments, biofunctional spider silk fiber provides a means to convey complex biological functions over a range of scales, further extending its potential as a biomaterial in biotechnological settings. Biotechnol. Bioeng. 2017;114: 83-95. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteínas Recombinantes de Fusão , Seda , Animais , Anticorpos/química , Anticorpos/metabolismo , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Biotecnologia , Linhagem Celular Tumoral , Separação Celular/métodos , Feminino , Engenharia Genética , Humanos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Seda/química , Seda/genética , Seda/metabolismo , Aranhas , Transglutaminases/química , Transglutaminases/genética , Transglutaminases/metabolismoRESUMO
Microfabricated devices have increasingly incorporated bacterial cells for microscale studies and exploiting cell-based functions in situ. However, the role of surface interactions in controlling the bacterial cell behavior is not well understood. In this study, microfluidic substrates of varied bacterial-binding affinity were used to probe the interaction-driven behavior of filamentous Escherichia coli. In particular, cell alignment under controlled shear flow as well as subsequent orientation and filamentation were compared between cells presenting distinct outer membrane phenotypes. We demonstrated that filaments retained position under flow, which allowed for dynamic single-cell monitoring with in situ elongation of over 100 µm for adherent cells. This maximum was not reached by planktonic cells and was, therefore, adhesion-dependent. The bound filaments initially aligned with flow under a range of flow rates and their continual elongation was traced in terms of length and growth path; analysis demonstrated that fimbriae-mediated adhesion increased growth rate, increased terminal length, as well as dramatically changed the adherent geometry, particularly buckling behavior. The effects to filament length and buckling were further exaggerated by the strongest, specificity-driven adhesion tested. Such surface-guided control of the elongation process may be valuable to yield interesting "living" filamentous structures in microdevices. In addition, this work may offer a biomedically relevant platform for further elucidation of filamentation as an immune-resistant morphology. Overall, this work should inspire broader exploration of microfabricated devices for the study and application of single bacterial cells.
Assuntos
Escherichia coli/fisiologia , Microfluídica/instrumentação , Aderência Bacteriana , Microfluídica/métodos , Estresse Mecânico , Propriedades de SuperfícieRESUMO
Escherichia coli were genetically modified to enable programmed motility, sensing, and actuation based on the density of features on nearby surfaces. Then, based on calculated feature density, these cells expressed marker proteins to indicate phenotypic response. Specifically, site-specific synthesis of bacterial quorum sensing autoinducer-2 (AI-2) is used to initiate and recruit motile cells. In our model system, we rewired E. coli's AI-2 signaling pathway to direct bacteria to a squamous cancer cell line of head and neck (SCCHN), where they initiate synthesis of a reporter (drug surrogate) based on a threshold density of epidermal growth factor receptor (EGFR). This represents a new type of controller for targeted drug delivery as actuation (synthesis and delivery) depends on a receptor density marking the diseased cell. The ability to survey local surfaces and initiate gene expression based on feature density represents a new area-based switch in synthetic biology that will find use beyond the proposed cancer model here.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Receptores ErbB/metabolismo , Escherichia coli/genética , Neoplasias de Cabeça e Pescoço/genética , Homosserina/análogos & derivados , Lactonas/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Engenharia Genética/métodos , Neoplasias de Cabeça e Pescoço/patologia , Homosserina/genética , Homosserina/metabolismo , Humanos , Nanotecnologia , Percepção de QuorumRESUMO
We demonstrate that "nanofactory"-loaded biopolymer capsules placed in the midst of a bacterial population can direct bacterial communication. Quorum sensing (QS) is a process by which bacteria communicate through small-molecules, such as autoinducer-2 (AI-2), leading to collective behaviors such as virulence and biofilm formation. In our approach, a "nanofactory" construct is created, which comprises an antibody complexed with a fusion protein that produces AI-2. These nanofactories are entrapped within capsules formed by electrostatic complexation of cationic (chitosan) and anionic (sodium alginate) biopolymers. The chitosan capsule shell is crosslinked by tripolyphosphate (TPP) to confer structural integrity. The capsule shell is impermeable to the encapsulated nanofactories, but freely permeable to small molecules. In turn, the capsules are able to take in substrates from the external medium via diffusion, and convert these via the nanofactories into AI-2, which then diffuses out. The exported AI-2 is shown to stimulate QS responses in vicinal Escherichia coli. Directing bacterial population behavior has potential applications in next-generation antimicrobial therapy and pathogen detection. We also envision such capsules to be akin to artificial "cells" that can participate in native biological signaling and communicate in real-time with the human microbiome. Through such interaction capabilities, these "cells" may sense the health of the microbiome, and direct its function in a desired, host-friendly manner.
Assuntos
Bioengenharia/métodos , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Nanocápsulas , Nanotecnologia/métodos , Percepção de Quorum/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Alginatos/química , Animais , Anticorpos , Bovinos , Quitosana/química , Ácido Glucurônico/química , Proteínas de Fluorescência Verde/química , Ácidos Hexurônicos/química , Homosserina/análogos & derivados , Homosserina/química , Homosserina/farmacologia , Lactonas/química , Lactonas/farmacologia , Polifosfatos , Proteínas Recombinantes de Fusão/química , S-Adenosil-Homocisteína , Soroalbumina Bovina/químicaRESUMO
We developed a bioelectronic communication system that is enabled by a redox signal transduction modality to exchange information between a living cell-embedded bioelectronics interface and an engineered microbial network. A naturally communicating three-member microbial network is 'plugged into' an external electronic system that interrogates and controls biological function in real time. First, electrode-generated redox molecules are programmed to activate gene expression in an engineered population of electrode-attached bacterial cells, effectively creating a living transducer electrode. These cells interpret and translate electronic signals and then transmit this information biologically by producing quorum sensing molecules that are, in turn, interpreted by a planktonic coculture. The propagated molecular communication drives expression and secretion of a therapeutic peptide from one strain and simultaneously enables direct electronic feedback from the second strain, thus enabling real-time electronic verification of biological signal propagation. Overall, we show how this multifunctional bioelectronic platform, termed a BioLAN, reliably facilitates on-demand bioelectronic communication and concurrently performs programmed tasks.
Assuntos
Eletrônica/métodos , Escherichia coli/metabolismo , Microrganismos Geneticamente Modificados/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Células Imobilizadas/química , Eletrodos , Desenho de Equipamento , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Regulação Bacteriana da Expressão Gênica , Ouro/química , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Peróxido de Hidrogênio/metabolismo , Microbiota , Microrganismos Geneticamente Modificados/genética , Oxirredução , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , beta-Galactosidase/metabolismoRESUMO
In this study, naturally derived cellulose nanofibrils (CNFs), a renewable and easily modified nanomaterial with low cytotoxicity, were rendered bioactive via one-step functionalization with mannopyranoside (CNFs-mannose) for use as a new glyconanomaterial platform for control of bacterial pathogenesis. The recognition affinity of the bioactive surfaces toward fimbriated Escherichia coli was assessed using genetically engineered strains as well as wild-type (WT) MG1655 bacteria. The results revealed high surface coverages of FimH+ (with overexpressed FimH) and WT bacteria on the films of CNFs-mannose due to specific interaction between prevalent mannose on nanofibrils and FimH receptors on E. coli fimbriae. The CNFs-mannose nanofibrils were capable of capturing E. coli from a liquid suspension, as demonstrated either by the nanofibril clusters or by the cellulose filter papers impregnated with CNFs-mannose. More importantly, CNFs-mannose efficiently inhibited adhesion of both FimH+ and WT E. coli to mannosylated surfaces even at a very low concentration, resulting in over 95% reduction of bacterial adhesion. Furthermore, the bioactive nanofibrils showed effective disruption of nonspecific binding of bacteria to abiotic surfaces in flow channel tests. These findings highlight the potential of cellulose nanofibrils as a biocompatible polyvalent nanoscale scaffold and exemplify sugar grafted nanofibrils as novel and effective tools in control of bacterial pathogenesis, bacterial removal, as well as in many other applications.
RESUMO
A hydrogel-based dual film coating is electrofabricated for transducing bio-relevant chemical information into electronical output. The outer film has a synthetic biology construct that recognizes an external molecular signal and transduces this input into the expression of an enzyme that converts redox-inactive substrate into a redox-active intermediate, which is detected through an amplification mechanism of the inner redox-capacitor film.
Assuntos
Eletrônica , Hidrogéis/química , Membranas Artificiais , Biologia Sintética , OxirreduçãoRESUMO
Eukaryotic cells have an architecture consisting of multiple inner compartments (organelles) such as the nucleus, mitochondria, and lysosomes. Each organelle is surrounded by a distinct membrane and has unique internal contents; consequently, each organelle has a distinct function within the cell. In this study, we create biopolymer microcapsules having a compartmentalized architecture as in eukaryotic cells. To make these capsules, we present a biocompatible method that solely uses aqueous media (i.e., avoids the use of oil phases), requires no sacrificial templates, and employs a minimal number of steps. Our approach exploits the electrostatic complexation of oppositely charged polymers dissolved in aqueous media. Specifically, droplets of an anionic biopolymer are generated using a simple microcapillary device, with the droplets being sheared off the capillary tip by pulses of gas (air or nitrogen). The liquid droplets are then introduced into a reservoir whereupon they encounter multivalent cations as well as a cationic biopolymer; thereby, a solid shell is formed around each droplet by electrostatic interactions between the polymers while the core is ionically cross-linked into a gel. In the next step, a discrete number of these capsules are encapsulated within a larger outer capsule by repeating the same process with a wider capillary. Our approach allows us to control the overall diameter of these multicompartment capsules (MCCs) (â¼300-500 µm), the diameters of the inner compartments (â¼100-300 µm), and the number of inner compartments in an MCC (1 to >5). More importantly, we can encapsulate different payloads in each of the inner compartments, including colloidal particles, enzymes, and microbial cells, in all cases preserving their native functions. A hallmark of biological cells is the existence of cascade processes, where products created in one organelle are transported and used in another. As an initial demonstration of the capabilities afforded by our MCCs, we study a simple cascade process involving two strains of bacteria (E. coli), which communicate through small molecules known as autoinducers. In one compartment of the MCC, we cultivate E. coli that produces autoinducer 2 (AI-2) in the presence of growth media. The AI-2 then diffuses into an adjacent compartment within the MCC wherein a reporter strain of E. coli is cultivated. The reporter E. coli imbibes the AI-2 and in turn, produces a fluorescence response. Thus, the action (AI-2 production) and response (fluorescence signal) are localized within different compartments in the same MCC. We believe this study is an important advance in the path towards an artificial cell.
RESUMO
The potential advantages of cell-based biohybrid devices over conventional nonliving systems drive the interest to control the behavior of the underlying biological cells in microdevices. Here, the authors studied how shear influenced the geometry and elongation of fimbriated filaments on affinity substrates. The cells were engineered to express FimH, which binds to mannose with a high affinity. A microfluidic channel was functionalized with RNAse B, which is rich in mannose residues, and the device was used to control the hydrodynamic force on live Escherichia coli under filamentous growth. It was discovered that filamentous E. coli cells adopt buckled geometry when the shear rate is low, but assume an extended geometry at high shear and align with the flow direction. The extension moves from bidirectional to preferentially downstream as the shear rate increases. Furthermore, living filaments slide easily on the substrate, and detach from the substrates at a rate nearly ten times greater than unfilamented live E. coli at high shear conditions (1000-4000 s-1). The hydrodynamic force and binding force experienced by the cells are further analyzed by COMSOL simulation and atomic force microscopy measurements, respectively, to explore the mechanism behind the living cell dynamics. Knowledge from this work helps guide design of interfacial properties and shear environments to control the geometry of living filamentous bacteria.
Assuntos
Adesinas de Escherichia coli , Engenharia Celular , Escherichia coli , Proteínas de Fímbrias , Hidrodinâmica , Resistência ao Cisalhamento , Adesinas de Escherichia coli/química , Adesinas de Escherichia coli/genética , Adesinas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fímbrias/química , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Ribonucleases/química , Ribonucleases/genética , Ribonucleases/metabolismoRESUMO
Biology and electronics are both expert at for accessing, analyzing, and responding to information. Biology uses ions, small molecules, and macromolecules to receive, analyze, store, and transmit information, whereas electronic devices receive input in the form of electromagnetic radiation, process the information using electrons, and then transmit output as electromagnetic waves. Generating the capabilities to connect biology-electronic modalities offers exciting opportunities to shape the future of biosensors, point-of-care medicine, and wearable/implantable devices. Redox reactions offer unique opportunities for bio-device communication that spans the molecular modalities of biology and electrical modality of devices. Here, an approach to search for redox information through an interactive electrochemical probing that is analogous to sonar is adopted. The capabilities of this approach to access global chemical information as well as information of specific redox-active chemical entities are illustrated using recent examples. An example of the use of synthetic biology to recognize external molecular information, process this information through intracellular signal transduction pathways, and generate output responses that can be detected by electrical modalities is also provided. Finally, exciting results in the use of redox reactions to actuate biology are provided to illustrate that synthetic biology offers the potential to guide biological response through electrical cues.
Assuntos
Biologia , Eletrônica , Oxirredução , Técnicas Biossensoriais , Materiais Revestidos Biocompatíveis/química , Galvanoplastia , Expressão Gênica , Humanos , Hidrogéis/química , Esquizofrenia/diagnóstico , Transdução de Sinais , Biologia SintéticaRESUMO
Many reports have elucidated the mechanisms and consequences of bacterial quorum sensing (QS), a molecular communication system by which bacterial cells enumerate their cell density and organize collective behavior. In few cases, however, the numbers of bacteria exhibiting this collective behavior have been reported, either as a number concentration or a fraction of the whole. Not all cells in the population, for example, take on the collective phenotype. Thus, the specific attribution of the postulated benefit can remain obscure. This is partly due to our inability to independently assemble a defined quorum, for natural and most artificial systems the quorum itself is a consequence of the biological context (niche and signaling mechanisms). Here, we describe the intentional assembly of quantized quorums. These are made possible by independently engineering the autoinducer signal transduction cascade of Escherichia coli (E. coli) and the sensitivity of detector cells so that upon encountering a particular autoinducer level, a discretized sub-population of cells emerges with the desired phenotype. In our case, the emergent cells all express an equivalent amount of marker protein, DsRed, as an indicator of a specific QS-mediated activity. The process is robust, as detector cells are engineered to target both large and small quorums. The process takes about 6 h, irrespective of quorum level. We demonstrate sensitive detection of autoinducer-2 (AI-2) as an application stemming from quantized quorums. We then demonstrate sub-population partitioning in that AI-2-secreting cells can 'call' groups neighboring cells that 'travel' and establish a QS-mediated phenotype upon reaching the new locale.
Assuntos
Escherichia coli/fisiologia , Percepção de Quorum , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Homosserina/análogos & derivados , Homosserina/metabolismo , Lactonas/metabolismo , Transdução de SinaisRESUMO
Advances in nanotechnology have provided unprecedented physical means to sample molecular space. Living cells provide additional capability in that they identify molecules within complex environments and actuate function. We have merged cells with nanotechnology for an integrated molecular processing network. Here we show that an engineered cell consortium autonomously generates feedback to chemical cues. Moreover, abiotic components are readily assembled onto cells, enabling amplified and 'binned' responses. Specifically, engineered cell populations are triggered by a quorum sensing (QS) signal molecule, autoinducer-2, to express surface-displayed fusions consisting of a fluorescent marker and an affinity peptide. The latter provides means for attaching magnetic nanoparticles to fluorescently activated subpopulations for coalescence into colour-indexed output. The resultant nano-guided cell network assesses QS activity and conveys molecular information as a 'bio-litmus' in a manner read by simple optical means.
Assuntos
Proteínas de Transporte/metabolismo , Engenharia Celular , Homosserina/análogos & derivados , Lactonas/metabolismo , Nanotecnologia , Percepção de Quorum , Retroalimentação , Proteínas de Fluorescência Verde , Homosserina/metabolismo , Nanopartículas de MagnetitaRESUMO
A magnetic nanocomposite film with the capability of reversibly collecting functionalized magnetic particles was fabricated by simultaneously imposing two orthogonal stimuli (electrical and magnetic). We demonstrate that cathodic codeposition of chitosan and Fe3O4 nanoparticles while simultaneously applying a magnetic field during codeposition can (i) organize structure, (ii) confer magnetic properties, and (iii) yield magnetic films that can perform reversible collection/assembly functions. The magnetic field triggered the self-assembly of Fe3O4 nanoparticles into hierarchical "chains" and "fibers" in the chitosan film. For controlled magnetic properties, the Fe3O4-chitosan film was electrodeposited in the presence of various strength magnetic fields and different deposition times. The magnetic properties of the resulting films should enable broad applications in complex devices. As a proof of concept, we demonstrate the reversible capture and release of green fluorescent protein (EGFP)-conjugated magnetic microparticles by the magnetic chitosan film. Moreover, antibody-functionalized magnetic microparticles were applied to capture cells from a sample, and these cells were collected, analyzed, and released by the magnetic chitosan film, paving the way for applications such as reusable biosensor interfaces (e.g., for pathogen detection). To our knowledge, this is the first report to apply a magnetic field during the electrodeposition of a hydrogel to generate magnetic soft matter. Importantly, the simple, rapid, and reagentless fabrication methodologies demonstrated here are valuable features for creating a magnetic device interface.
Assuntos
Técnicas Biossensoriais/instrumentação , Galvanoplastia , Hidrogéis/química , Nanopartículas de Magnetita/química , Cristalização/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Hidrogéis/efeitos da radiação , Campos Magnéticos , Nanopartículas de Magnetita/efeitos da radiação , Nanopartículas de Magnetita/ultraestrutura , Teste de MateriaisRESUMO
The information age was enabled by advances in microfabrication and communication theory that allowed information to be processed by electrons and transmitted by electromagnetic radiation. Despite immense capabilities, microelectronics has limited abilities to access and participate in the molecular-based communication that characterizes our biological world. Here, we use biological materials and methods to create components and fabricate devices to perform simple molecular communication functions based on bacterial quorum sensing (QS). Components were created by protein engineering to generate a multidomain fusion protein capable of sending a molecular QS signal, and by synthetic biology to engineer E. coli to receive and report this QS signal. The device matrix was formed using stimuli-responsive hydrogel-forming biopolymers (alginate and gelatin). Assembly of the components within the device matrix was achieved by physically entrapping the cell-based components, and covalently conjugating the protein-based components using the enzyme microbial transglutaminase. We demonstrate simple devices that can send or receive a molecular QS signal to/from the surrounding medium, and a two-component device in which one component generates the signal (i.e., issues a command) that is acted upon by the second component. These studies illustrate the broad potential of biofabrication to generate molecular communication devices.
RESUMO
Previous efforts to control cellular behaviour have largely relied upon various forms of genetic engineering. Once the genetic content of a living cell is modified, the behaviour of that cell typically changes as well. However, other methods of cellular control are possible. All cells sense and respond to their environment. Therefore, artificial, non-living cellular mimics could be engineered to activate or repress already existing natural sensory pathways of living cells through chemical communication. Here we describe the construction of such a system. The artificial cells expand the senses of Escherichia coli by translating a chemical message that E. coli cannot sense on its own to a molecule that activates a natural cellular response. This methodology could open new opportunities in engineering cellular behaviour without exploiting genetically modified organisms.
Assuntos
Células Artificiais/metabolismo , Engenharia Celular/métodos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Riboswitch/genética , Células Artificiais/efeitos dos fármacos , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/metabolismo , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/efeitos dos fármacos , Proteínas Hemolisinas/efeitos dos fármacos , Isopropiltiogalactosídeo/metabolismo , Antagonistas de Receptores Purinérgicos P1/farmacologia , Riboswitch/efeitos dos fármacos , Teofilina/farmacologiaRESUMO
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.
Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Dispositivos Lab-On-A-Chip , Técnicas Biossensoriais/instrumentação , Quitosana/química , Materiais Revestidos Biocompatíveis/química , Técnicas Eletroquímicas/instrumentação , Eletrodos , Escherichia coli/química , Glucose Oxidase/químicaRESUMO
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.