Gregory F. Payne

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.

Enzymatic grafting of a natural product onto chitosan to confer water solubility under basic conditions

Chitosan is a natural biopolymer whose rich amine functionality confers water solubility at low pH. At higher pHs (greater than 6. 5), the amines are deprotonated and chitosan is insoluble. To attain water solubility under basic conditions we enzymatically grafted the hydrophilic compound chlorogenic acid onto chitosan. Despite its name, chlorogenic acid is a nonchlorinated phenolic natural product that has carboxylic acid and hydroxyl functionality. The enzyme in this study was tyrosinase, which converts a wide range of phenolic substrates into electrophilic o-quinones. The o-quinones are freely diffusible and can undergo reaction with the nucleophilic amino groups of chitosan. Using slightly acidic conditions (pH = 6), it was possible to modify chitosan under homogeneous conditions. When the amount of chlorogenic acid used in the modification reaction exceeded 30% relative to chitosans amino groups, the modified chitosan was observed to be soluble under both acidic and basic conditions, and to have a pH window of insolubility at near neutral pH. 1H NMR spectra confirmed that chitosan was chemically modified, although the degree of modification was low. Copyright 1999 John Wiley & Sons, Inc.

Chitosan: a soft interconnect for hierarchical assembly of nano-scale components

Traditional microfabrication has tremendous capabilities for imparting order to hard materials (e.g., silicon wafers) over a range of length scales. However, conventional microfabrication does not provide the means to assemble pre-formed nano-scale components into higher-ordered structures. We believe the aminopolysaccharide chitosan possesses a unique set of properties that enable it to serve as a length-scale interconnect for the hierarchical assembly of nano-scale components into macro-scale systems. The primary amines (atomic length scale) of the glucosamine repeating units (molecular length scale) provide sites to connect pre-formed or self-assembled nano-scale components to the polysaccharide backbone (macromolecular length scale). Connections to the backbone can be formed by exploiting the electrostatic, nucleophilic, or metal-binding capabilities of the glucosamine residues. Chitosans film-forming properties provide the means for assembly at micron-to-centimetre lengths (supramolecular length scales). In addition to interconnecting length scales, chitosans capabilities may also be uniquely-suited as a soft component-hard device interconnect. In particular, chitosans film formation can be induced under mild aqueous conditions in response to localized electrical signals that can be imposed from microfabricated surfaces. This capability allows chitosan to assemble soft nano-scale components (e.g., proteins, vesicles, and virus particles) at specific electrode addresses on chips and in microfluidic devices. Thus, we envision the potential that chitosan may emerge as an integral material for soft matter (bio)fabrication.

Enzymatic grafting of hexyloxyphenol onto chitosan to alter surface and rheological properties

An enzymatic method to graft hexyloxyphenol onto the biopolymer chitosan was studied. The method employs tyrosinase to convert the phenol into a reactive o-quinone, which undergoes subsequent nonenzymatic reaction with chitosan. Reactions were conducted under heterogeneous conditions using chitosan films and also under homogeneous conditions using aqueous methanolic mixtures capable of dissolving both hexyloxyphenol and chitosan. Tyrosinase was shown to catalyze the oxidation of hexyloxyphenol in such aqueous methanolic solutions. Chemical evidence for covalent grafting onto chitosan was provided by three independent spectroscopic approaches. Specifically, enzymatic modification resulted in (1) the appearance of broad absorbance in the 350-nm region of the UV/vis spectra for chitosan films; (2) changes in the NH bending and stretching regions of chitosans IR spectra; and (3) a base-soluble material with (1)H-NMR signals characteristic of both chitosan and the alkyl groups of hexyloxyphenol. Hexyloxyphenol modification resulted in dramatic changes in chitosans functional properties. On the basis of contact angle measurements, heterogeneous modification of a chitosan film yielded a hydrophobic surface. Homogeneously modified chitosan offered rheological properties characteristic of associating water-soluble polymers.

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.

Enzymatic modification of the synthetic polymer polyhydroxystyrene

Abstract In aqueous-methanolic solutions, mushroom tyrosinase was observed to catalyze the oxidation of phenolic moieties of the synthetic polymer poly(4-hydroxystyrene) (PHS). Although oxidation is rapid, on the order of minutes, it seems that only a small number of phenolic moieties of the PHS polymer (1 to 2%) undergo oxidation. Enzymatically oxidized PHS was observed to undergo a subsequent nonenzymatic reaction with aniline yielding an aniline-modified PHS polymer with a low degree-of-substitution. To gain insights into linkages between aniline and oxidized PHS, we characterized products resulting from tyrosinase-catalyzed reactions of p-cresol and aniline. A major product from these small-molecule studies was the Michael’s-type adduct, 4-anilino-5-methyl-1,2-benzoquinone. Based on similarities in ultraviolet-visible and 1H nuclear magnetic resonance spectra, we believe Michael’s-type adducts are present in the aniline-modified PHS polymer. To illustrate the potential utility of this enzymatic approach for grafting PHS onto other polymers, we contacted enzymatically oxidized PHS with the amine-containing biopolymer chitosan. Ultraviolet spectra of this chitosan film suggests oxidized PHS is grafted onto chitosan.

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.

Coupling Electrodeposition with Layer‐by‐Layer Assembly to Address Proteins within Microfluidic Channels

Two thin-film assembly methods are coupled to address proteins. Electrodeposition confers programmability and generates a template for layer-by-layer (LbL) assembly. LbL enables precise control of film thickness and the incorporation of labile biological components. The capabilities are demonstrated using glucose oxidase (GOx) based electrochemical biosensing within a microfabricated fluidic device.

Peroxidase catalyzed grafting of gallate esters onto the polysaccharide chitosan

Peroxidases are believed to play a role in various natural polymerization processes and it may be possible to exploit peroxidases for environmentally-friendly industrial polymer processing. We examined the potential for using horseradish peroxidase to graft the phenolic substrate dodecyl gallate (DDG) onto the polysaccharide chitosan. Several analytical approaches were used to provide evidence that DDG was grafted onto chitosan. Compared to unmodified chitosan, DDG-modified chitosan had significantly increased absorbance in the UV-visible region, and in the C-H and carbonyl-stretching regions of the IR spectra. Also, the 1 H NMR spectrum of a soluble fraction of DDG-chitosan had broad peaks near 1.2 ppm consistent with the grafting of DDG onto the polymer. Additional evidence for DDG grafting was obtained in two studies in which the DDG-modified chitosan was subjected to nitrous acid hydrolysis. First, a highly modified and insoluble DDG-chitosan was suspended in 10% acetic acid. After partial hydrolysis, peaks associated with the sugar and dodecyl gallate moieties were observed to appear in the solution phase 1 H NMR spectrum. Finally, the DDG-modified chitosan was hydrolyzed and fractions were separated by HPLC. One fraction showing both UV absorbance (characteristic of the phenolic) and carbohydrate reactivity to anthrone was purified by two chromatographic steps. This fraction was analyzed by both FAB-MS and electrospray-MS, and observed to have a molecular weight of 371 Da. These results provide evidence that peroxidases can be used to graft phenolic moieties onto the polysaccharide chitosan.