This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.
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 1,2,3, 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 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. 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 6,7. 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 9,10. Alginate films and their entrapment of live cells will also be addressed 11. 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 12,13. 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.
Our procedures demonstrate the electrodeposition and functionalization of biopolymer films, a process we term biofabrication. Through functionalization with cells and biomolecules we create biological surfaces capable of interacting with each other and the electrode address they are assembled upon. The first step, electrodeposition, takes place through the triggered self-assembly of biopolymers, alginate and chitosan in our studies, in response to an electrical signal. As stated earlier a pH gradient is generated which can be controlled by the current density and deposition time, providing additional control over the film dimensions and properties 6,17. We have found that a variety of current density and deposition time combinations can be used for the electrodes indicated in Table 1. While use of other electrodes is feasible, adjustments to the procedure would be necessary. Compared with other techniques of film formation the process of electrodeposition is simple, rapid and reagentless. There is no need for an extensive repertoire of expensive equipment and laborious preparations. Importantly, the process can withstand minor experimental deviations and can be easily started over if a problem occurs.
Chitosan is capable of responding to a high cathodic pH gradient due to important functional properties conferred to it by a high content of primary amines. At high pH (greater than its pKa of ~6.3) the amines are deprotonated and chitosan becomes insoluble, allowing for film formation. Following deposition, the films will remain attached to the electrode. However, the ability exists to delaminate them if desired. The films will remain stable as long as the pH of the solution does not drop below the pKa. Acidic solutions protonate the amines and the subsequent electrostatic repulsions swell the gel until it dissolves 18. That is, the assembly/disassembly process is reversible on demand and allows for removal of deposited films and reuse of electrodes. Conveniently, the pH range at which the sol-gel transition takes place is close to that in which most biological components function optimally. This makes the process ideal for the retention of functionality during assembly6.
Alginate film formation is facilitated by the anodic electrolysis of water as well as the presence of calcium carbonate 7. The localized low pH at the anode solubilizes the calcium carbonate leading to the release calcium cations. These ions are chelated by alginate, forming a crosslinked network on the electrode surface. Alginate films are notably reversible by competition for calcium ions from other chelating compounds such as citrate or EDTA, which can be used to dissolve the films, allowing for the re-use of the underlying electrodes. Thus, alginate films are relatively fragile when subjected to physiological conditions because calcium ions are easily scavenged from the gel matrix, weakening its structure and promoting film delamination or redissolution. To overcome this limitation, we have included an incubation step for the film in 1 M CaCl2 to strengthen the gel. Additionally, we recommend that the film’s incubation solution (cell media, etc.) be supplemented with CaCl2 at a concentration of 500 μM-3 mM.
The second major procedure is the functionalization of the deposited film with relevant biological components. This can be achieved in two ways, the first being electrochemical conjugation, a strategy that allows for rapid, reagentless assembly of proteins with exceptional spatial control 10. However, functionalization in this manner is limited by the diffusion of Cl– ions through the film to the electrode as well as the diffusion of HOCl, the generated reactive intermediate, back out into solution. The ability of electrochemically active molecules to pass through the film allows for the transduction of chemical and biological signals into easy-to-read electrical signals 15. We have shown tyrosinase-mediated coupling as a second strategy for enzyme functionalization to chitosan, demonstrated by covalently attaching AI-2 Synthase. This strategy allows the functionalization process to be controlled and selective – dependent on a specific reagent, tyrosinase, which acts discriminately on proteins containing a tyrosine tag 9.
We show the usefulness and biocompatibility of multi-address systems by replicating natural pathways on a chip. First we organized two cell populations (i.e. “senders” and “receivers”) at distinct addresses, and showed that they interacted across adjacent electrodes to deliver AI-2 and generate a fluorescence response. This concept has also been demonstrated by Cheng et al. in a microfluidic chip 14. We also mimicked the interaction, but instead used an enzyme to synthesize AI-2 for delivery. In this way, a synthetic intracellular pathway, AI-2 synthesis, was replicated through biofabrication and functioned much as it would in solution.
In both cases, assembly of multiple addresses presents the challenge of avoiding non-specific binding between addresses because each deposition solution must be introduced to the entire electrode array, even though electrodeposition is only intended at one address. Gentle yet thorough washing can remove the majority of residual solution from non-biased electrodes; the use of flow in microfluidic channels may further minimize non-specificity. Particularly for the adjacent biofabrication of chitosan and alginate addresses, we recommend depositing the chitosan film first, following this with biofunctionalization steps, and after this, electrodepositing alginate. Although we have not done so here, we have found that blocking the chitosan film with inert proteins (such as milk, BSA, etc.) greatly diminishes non-specific binding of unwanted molecules to chitosan’s aminated surface.
We have found utility in establishing patterned electrodes, often found in bioMEMS devices, as the “blueprints” for a complex arrangement of cells and biomolecules. The uses of electrodeposited chitosan in bioMEMS devices can go well beyond the examples mentioned here 19. Chitosan can be deposited on various microscale geometries – such as in microchannels and on non-planar surfaces 20,15. The films can also be modified with other polymers and a variety of proteins, DNA, nanoparticles, and redox-active molecules for novel properties 21,22,23. In bioMEMS devices, chitosan films have been used for drug delivery, redox and small molecule detection, biocatalysis, and cell studies 20,23,24,25. Similarly, alginate is widely used as a cell-entrapment matrix and has been explored for reversible fluidic containment of cell populations and in-film immunoanalysis 26,27,28. Composite films for tissue engineering applications have been fabricated using alginate electrodeposition, with components such as with hydroxyapatite for orthopedic implants 29.
In our demonstrations of biofabrication, we have shown both the interactions between biological components and across the bio-electronic interface to be equally applicable; this brings into reach the prospect of integrating all varieties of interactions for sophisticated performance in on-chip signal transmission. Accordingly, biofabrication may facilitate the fabrication of devices with reduced “minimum feature sizes” as a direct follow-on to rapid developments in microfabrication, as often motivated by consumer electronics. That is, next next-generation devices might in fact include labile biological components that offer nature’s exquisite assembly and recognition capabilities at even smaller length scales than man-made systems. We envision near-term applications in analytical instrumentation, environmental sensors, and even biocompatible implantable devices.
The authors have nothing to disclose.
We acknowledge funding from DTRA for support of this manuscript and from ONR, DTRA, and NSF for partial support of underlying research.
Name of the component | Company | Catalogue number |
Power Supply | Keithley | SourceMeter 2400 |
Three electrode potentiostat | CH Instruments | Potentiostat/Galvanostat 600D |
RE-5B Ag/AgCl Reference Electrode with Flexible Connector | BASi | MF-2052 |
Gold coated silicon wafer, 500um Si, 12nM Cr, 120nM Au, SiO2 for insulation | custom fabricated | |
Indium Tin oxide coated glass slide, rectangular, 8-12 ohm resist | Sigma-Aldrich | 578274 |
Platinum sheet/foil (0.002 in) | Surepure Chemetals | 1897 |
Slim Line 2″ Alligator Clips | RadioShack | 270-346 |
Multi-Stacking Banana Plug Patch Cord | TSElectronic | B-36-02 B-24-02 |
SYLGARD 184 silicone elastomer kit | Dow Corning | NC9020938 From Fischer |
Fluorescecence stereomicroscope | Olympus | MVX10 MacroView |
cellSens Standard | Olympus | version 1.3 |
Table 1. Electrodeposition and fluorescence visualization equipment.
Name of the reagent | Company | Catalogue number |
Chitosan, medium molecular weight | Sigma-Aldrich | 448877 |
Hydrochloric Acid, ARISTAR. ACS, NF, FCC Grade | VWR | BDH3030 |
Sodium Hydroxide, Solution. 10.00N | VWR | VW3247 |
Alginic acid, sodium salt | Sigma-Aldrich | 180947 |
Multifex-MM Precipitated Calcium Carbonate, 70nm particles |
Speciality Minerals Inc. |
100-3630-3 |
Table 2. Chitosan and alginate solution reagents.
Name of the reagent | Company | Catalogue number |
Calcium chloride, dihydrate | J.T. Baker | 0504 |
Sodium Chloride, Certified ACS crystalline |
Fischer Scientific |
S271 |
Potassium Phosphate Monobasic, anhydrous | Sigma-Aldrich | P9791 |
Potassium Phosphate Dibasic, anhydrous | Sigma- Aldrich | P3786 |
Phosphate Buffered Saline | Sigma- Aldrich |
P4417 |
Table 3. Other solution components and buffer reagents.
Name of the reagent | Company/Source | Catalogue number |
Glucose oxidase from aspergillus niger | Sigma-Aldrich | G2133 |
Tyrosinase from mushroom | Sigma-Aldrich | T3824 |
LB broth, Miller (granulated) | Fischer Scientific | BP9723-2 |
“AI2-Synthase” (HGLPT) | Lab stock 16 | |
W3110 wildtype cells | Lab stock 30 | |
MDAI2 + pCT6-lsrR–ampr + pET-dsRed–kanr cells | Lab stock 30 | |
FluoroSpheres: 1μm diameter, Ex/Em: 505/515 | Invitrogen | F8765 |
5-(and-6)-carboxyrhodamine 6G succinimidyl ester, Ex/Em: 525/560 | Invitrogen | C-6157 |
DyLight antibody labeling kit, 405 | Thermo Scientific | PI-53020 |
Table 4. Enzymes, cells, and other functionalization reagents.