Xiaolong Luo

Chitosan: an integrative biomaterial for lab-on-a-chip devices

Chitosan is a naturally derived polymer with applications in a variety of industrial and biomedical fields. Recently, it has emerged as a promising material for biological functionalization of microelectromechanical systems (bioMEMS). Due to its unique chemical properties and film forming ability, chitosan serves as a matrix for the assembly of biomolecules, cells, nanoparticles, and other substances. The addition of these components to bioMEMS devices enables them to perform functions such as specific biorecognition, enzymatic catalysis, and controlled drug release. The chitosan film can be integrated in the device by several methods compatible with standard microfabrication technology, including solution casting, spin casting, electrodeposition, and nanoimprinting. This article surveys the usage of chitosan in bioMEMS to date. We discuss the common methods for fabrication, modification, and characterization of chitosan films, and we review a number of demonstrated chitosan-based microdevices. We also highlight the advantages of chitosan over some other functionalization materials for micro-scale devices.

In situ quantitative visualization and characterization of chitosan electrodeposition with paired sidewall electrodes

We report the first in situ quantitative visualization and characterization of electro-induced chitosan hydrogel growth in an aqueous environment. This was enabled with a pair of sidewall electrodes within a transparent fluidic system, which allowed us to resolve the electrogelling mechanism and interpret the dominant causes responsible for the formation and density distribution of the deposited hydrogel. The pH and the time-dependent growth profiles of the chitosan hydrogel were directly visualized, analyzed, and characterized. The results indicate that the gelation and immobilization of chitosan onto the cathode at a pH above its pKa value (∼6.3) are due to the electrochemically generated concentration gradient of reactant OH− ions, and their subsequent neutralization of the NH3+ groups of chitosan chains in solution near the cathode. The increased gel density around the fringes of the electrodes was demonstrated and correlated with the electrophoretic migration of chitosan cations during deposition. Simulation of the electric potential/field distribution, together with the corresponding dry film topography confirmed the non-uniform, electric field-dependent density distribution of deposited hydrogel. This report provides fundamental understanding towards the mechanism and the kinetics of the electro-induced chitosan gel formation. It also provides important guidelines for pursuing its application in bio-components integrated microsystems. The method in use exemplifies a simple, effective and non-destructive approach for in situ characterization of electro-responsive biopolymers in an aqueous environment.

Biocompatible multi-address 3D cell assembly in microfluidic devices using spatially programmable gel formation

Programmable 3D cell assembly under physiological pH conditions is achieved using electrodeposited stimuli-responsive alginate gels in a microfluidic device, with parallel sidewall electrodes enabling direct observation of the cell assembly. Electrically triggered assembly and subsequent viability of mammalian cells is demonstrated, along with spatially programmable, multi-address assembly of different strains of E. coli cells. Our approach enables in vitro study of dynamic cellular and inter-cellular processes, from cell growth and stimulus/response to inter-colony and inter-species signaling.

Mechanism of anodic electrodeposition of calcium alginate

Stimuli-responsive polysaccharides that can undergo a sol–gel transition in response to localized electrical signals provide a unique opportunity to electroaddress biological components at device interfaces. Most polysaccharide electroaddressing mechanisms use electrochemical reactions to generate pH gradients that can locally neutralize the polysaccharide and induce its reversible sol–gel transition to form a hydrogel film adjacent to the electrode surface. The calcium-responsive polysaccharide alginate is an exception; it may electrodeposit without requiring extreme pH gradients and thus may provide a means to electroaddress pH-sensitive biological components. Here, we use a novel device to characterize the mechanism for the anodic electrodeposition of a calcium alginate hydrogel. This device consists of a transparent fluidic channel with built-in sidewall electrodes that allows Ca–alginate electrodeposition to be directly measured by non-destructive optical and spectroscopic methods. We hypothesize a 3-step mechanism for calcium–alginate electrodeposition: (i) water is electrolyzed to locally generate protons (or hydronium ions); (ii) these protons are consumed by reacting with suspended CaCO3 particles and this “buffering” reaction generates a gradient in soluble Ca2+; and (iii) the locally generated Ca2+ ions interact with alginate to induce its sol–gel transition. We verified this electrodeposition mechanism using pH-responsive dyes to observe the local pH gradients during gel formation, Ca2+ indicator dyes to observe the Ca2+ gradient, and in situ Raman spectroscopy to demonstrate a strong interaction between soluble Ca2+ and alginate. Importantly, these results demonstrate electrodeposition without the need for a substantial pH excursion from neutrality. Thus, calcium alginate appears especially well-suited for electroaddressing labile biological components for applications in biosensors, biofabrication and BioMEMS.

Biofabrication: programmable assembly of polysaccharide hydrogels in microfluidics as biocompatible scaffolds

Because of their stimuli-responsiveness to chemical and pH gradients, polysaccharide hydrogels such as chitosan and alginate can be assembled as scaffolds for biomolecules or cells. Using the electrical and flow control available in microfluidic networks, in situ fabrication of 3D hydrogel scaffolds can be programmed in space and time to arrange biological components as an in vitro biochemically communicating system. Flexible in situ on-demand construction of a biocompatible scaffold within microfluidics holds promise for the assembly of biological components and systems for in vitro analysis and investigation. We foresee a wide spectrum applications ranging from replication of metabolic pathways as testbeds for drug discovery to identification of cell signaling mechanisms and observation of cellular response.

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 of chitosan?silver composite SERS substrates enabling quantification of adenine by a spectroscopic shift

Surface-enhanced Raman scattering (SERS) has grown dramatically as an analytical tool for the sensitive and selective detection of molecules adsorbed on nano-roughened noble metal structures. Quantification with SERS based on signal intensity remains challenging due to the complicated fabrication process to obtain well-dispersed nanoparticles and well-ordered substrates. We report a new biofabrication strategy of SERS substrates that enable quantification through a newly discovered spectroscopic shift resulting from the chitosan-analyte interactions in solution. We demonstrate this phenomenon by the quantification of adenine, which is an essential part of the nucleic acid structure and a key component in pathways which generate signal molecules for bacterial communications. The SERS substrates were fabricated simply by sequential electrodeposition of chitosan on patterned gold electrodes and electroplating of a silver nitrate solution through the chitosan scaffold to form a chitosan-silver nanoparticle composite. Active SERS signals of adenine solutions were obtained in real time from the chitosan-silver composite substrates with a significant concentration-dependent spectroscopic shift. The Lorentzian curve fitting of the dominant peaks suggests the presence of two separate peaks with a concentration-dependent area percentage of the separated peaks. The chitosan-mediated composite SERS substrates can be easily biofabricated on predefined electrodes within microfluidic channels for real-time detection in microsystems.

Design optimization for bioMEMS studies of enzyme-controlled metabolic pathways

Biological microelectromechanical systems (bioMEMS) provide an attractive approach to understanding and modifying enzymatic pathways by separating and interrogating individual reaction steps at localized sites in a microfluidic network. We have previously shown that electrodeposited chitosan enables immobilization of an enzyme at a specific site while maintaining its catalytic activity. While promising as a methodology to replicate metabolic pathways and search for inhibitors as drug candidates, these investigations also revealed unintended (or parasitic) effects, including products generated by the enzyme either (1) in the homogeneous phase (in the liquid), or (2) nonspecifically bound to microchannel surfaces. Here we report on bioMEMS designs which significantly suppress these parasitic effects. To reduce homogeneous reactions we have developed a new packaging and assembly strategy which eliminates fluid reservoirs that are commonly used for fluidic interconnects with external tubing. To suppress reactions by nonspecifically bound enzyme on microchannel walls we have implemented a cross-flow microfluidic network design so that enzyme flow for assembly and substrate/product for reaction share only the region where the enzyme is immobilized at the intended reaction site. Our results show that the signal-to-background ratio of sequential enzymatic reactions increases from 0.72 to 1.28 by eliminating the packaging reservoirs, and increases to 2.43 by separating the flow direction of enzymatic reaction from that of enzyme assembly step. These techniques can be easily applied to versatile microfluidic devices to minimize parasitic reactions in sequential biochemical reactions.

Conferring biological activity to native spider silk: A biofunctionalized protein-based microfiber

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.

Birefringence of flow-assembled chitosan membranes in microfluidics

Biopolymer membrane assembly in microfluidics offers precise spatial and temporal resolution for biomolecular and cellular interactions during and after assembly. Control over molecular transport across the biofabricated membranes requires microstructural characterization. This study investigates, for the first time, the birefringence of chitosan membranes assembled with flow in a microfluidic environment, and the effects of pH and flow rate on the membranes micro-alignment. The optical anisotropy of the formed membranes was quantified using a de Sénarmont compensator for transmitted quantitative polarized light microscopy. The chitosan membranes were biofabricated within a small aperture in a microfluidic network with various flow and pH conditions of chitosan and alginate solutions. The measured optical retardance and parallelism index clearly indicate that the microstructure of the flow-assembled membrane was well organized and aligned along the direction of chitosan flow. Optical retardance increased significantly with the pH of the alginate solution, but was less sensitive to the variation of the flow rates of the polymer solutions during the biofabrication process. It was also determined that the birefringence signal dropped significantly across the membrane growth direction regardless of the molecular density in the membrane. The mechanism of the micro-alignment was discussed, which was presumably due to the molecular un-wrapping by shear flow. We envision that the current study paves a path to further understand and actively manipulate the microstructure of flow-assembled membranes for broad lab-on-a-chip applications.