We describe a new class of photorheological (PR) fluids whose rheological properties can be reversibly tuned by light. The fluids were obtained by doping lecithin/sodium deoxycholate (SDC) reverse micelles with a photochromic spiropyran (SP) compound. Initially, the lecithin/SDC/SP mixtures formed highly viscoelastic fluids, reflecting the presence of long, wormlike reverse micelles. Under UV irradiation, the SP was isomerized to the open merocyanine (MC) form, causing the fluid viscosity to decrease 10-fold. When the UV irradiation was switched off, the MC reverted to the SP form, and the viscosity recovered its initial value. This cycle could be repeated several times without loss of response. The rheological transitions are believed to reflect changes in the lengths of the reverse worms. To our knowledge, this is the first example of a simple, reversible PR fluid that can be made entirely from commercially available components.

Abstract Upscaling is often applied to generate practical simulation models from highly detailed geocellular descriptions. In this paper we develop and evaluate a new upscaling procedure – a variable compact multipoint adaptive local-global technique (VCMP-ALG) – that is able to capture, accurately and efficiently, both full-tensor and global flow effects in the coarse model. The method successfully combines the positive attributes of its two underlying component procedures: the variable compact multipoint (VCMP) flux scheme, which provides coarse-scale transmissibilities that are appropriate for use in problems characterized by strong full-tensor permeabilities, and adaptive local-global (ALG) upscaling, which accounts for the effects of large-scale flow in the upscaling computations without solving any global fine-scale flow problems. The performance of the local-global VCMP upscaling technique is evaluated for multiple realizations of oriented variogram-based models and synthetic deltaic systems. Extensive numerical results for 2D cases demonstrate that the VCMP-ALG approach provides better overall accuracy than either of the underlying methods – an extended local VCMP technique and an adaptive local-global procedure based on two-point flux approximations – applied individually. The extension of the VCMP-ALG method to irregular quadrilateral grids is also accomplished. Finally, we present results for two-phase oil-water flows, for which the models based on the VCMP-ALG method again provide the best overall accuracy.

New transmissibility upscaling procedures designed to accurately capture full-tensor effects are developed and applied. These techniques are based on variable compact multipoint (VCMP) flux approximations. VCMP is extended to irregular grids. Two approaches for including global flow information within the VCMP upscaling procedure are considered—one in which the upscaled model is determined directly and one in which iteration of the coarse-scale model is used to minimize the mismatch between coarse-scale fluxes and integrated fine-scale fluxes. To guarantee monotonicity, the VCMP stencils are adapted to assure the coefficient matrix is an M-matrix whenever nonmonotone solutions are encountered. The new VCMP procedures are applied to multiple realizations of two-dimensional fine-scale permeability descriptions for coarse models defined on both Cartesian and irregular quadrilateral grids. Both log-normally distributed permeability fields with oriented layers and channelized models are considered. Six different upscaling techniques (extended local, direct global, and iterated global, each using both two-point and VCMP flux approximations) are assessed for four different sets of global boundary conditions. The global VCMP techniques consistently display high degrees of accuracy for total flow rate, L 2 flux error, and L 2 pressure error. For the oriented-layer cases, where full-tensor effects are important, the global VCMP methods are shown to provide clearly better overall accuracy than analogous methods based on two-point flux approximations. For channelized cases in which full-tensor effects are not significant, both types of methods provide high levels of accuracy. The selective M-fix procedure is also shown to lead to improved accuracy, which can be significant in some cases. In total, for the systems considered here, the new global VCMP upscaling techniques are observed to provide the best overall accuracy of any of the upscaling methods investigated.

An adhesive that cures under moist/wet conditions could facilitate surgical procedures for retinal reattachment. We are investigating an adhesive that mimics the factor XIIIa-mediated crosslinking of fibrin that occurs in the late stages of the blood coagulation cascade. Specifically, we use gelatin as the structural protein (in place of fibrin), and crosslink gelatin using a calcium-independent microbial transglutaminase (in place of the calcium-dependent transglutaminase factor XIIIa). Injection of gelatin and microbial transglutaminase (mTG) into the vitreous cavity of Sprague Dawley white rats did not elicit structural or cellular damage to the retina as evidenced from histological evaluation 2 weeks post-injection. Qualitative in vitro studies indicate that the gelatin-mTG adhesive binds to bovine retinal tissue under wet conditions. Quantitative lap-shear tests were performed with more robust bovine tissue from the choroid and sclera. The lap-shear strength of the biomimetic gelatin-mTG adhesive was independent of tissue-type and ranged from 15 to 45 kPa, which is comparable to the values reported for other soft-tissue adhesives. These studies suggest that the mTG-crosslinked gelatin may provide a simple, safe, and effective adhesive for ophthalmic applications.

Protein recovery is often achieved by a series of capture and release steps that often involve chromatographic binding and elution. We report an alternative, non-chromatographic, capture and release approach that employs enzymes and the stimuli-responsive polysaccharide chitosan. We capture our protein using the enzyme tyrosinase that oxidizes accessible tyrosine residues of the protein and "activates" these residues for covalent capture (i.e., conjugation) onto chitosan. Using fusions of green fluorescent protein (GFP) we observed that: (i) enzymatic activation is required for protein capture to chitosan; and (ii) capture is enhanced (approximately five-fold) by engineering the protein to have a penta-tyrosine fusion tag that provides additional accessible tyrosine residues for enzymatic activation. Because the fusion tag appears to be the primary site for capture, and capture requires activation, we designate penta-tyrosine as a "pro-tag." The captured GFP-chitosan conjugate possesses the pH-responsive solubility that is characteristic of chitosan. We exploit this pH-responsive solubility to facilitate purification of the captured protein. Two enzymatic methods were explored to release the captured GFP from the chitosan conjugate. The first method employs enterokinase (EK) to cleave the protein at an engineered EK-cleavage site. The second method employs chitosanase to hydrolyze the chitosan backbone. Using GFP as a model protein, we demonstrated that enzymatic capture and release provides a simple, non-chromatographic means to recover proteins directly from cell lysates.

Tyrosinase is a versatile enzyme that oxidizes a broad range of substrates that include low molecular weight phenols, peptides that contain tyrosine, and proteins with accessible tyrosine residues. The products of tyrosinase-catalyzed reactions are reactive o-quinones (or o-quinone residues) that can undergo uncatalyzed reactions with various nucleophiles. We are studying the use tyrosinase to generate quinones to undergo grafting reactions with the aminopolysaccharide chitosan. Initially, our goal was to demonstrate that tyrosinase could initiate the grafting of various plant phenols onto chitosan to create functional polymers from bio-based renewable resources. More recently, we are examining how tyrosinase can be enlisted to generate protein-chitosan conjugates with stimuli-responsive properties. Here we review our group's efforts over the last decade.

The effect of adding an associating biopolymer to surfactant vesicles and micelles is studied using rheology and small-angle neutron scattering (SANS). The associating polymer is obtained by randomly tethering hydrophobic alkyl chains to the backbone of the polysaccharide, chitosan. Adding this polymer to surfactant vesicles results in a gel; that is, the sample transforms from a Newtonian liquid to an elastic solid having frequency-independent dynamic shear moduli. SANS shows that the vesicles remain intact within the gel. The results suggest a gel structure in which the vesicles are connected by polymer chains into a three-dimensional network. Vesicle-polymer binding is expected to occur via the insertion of polymer hydrophobes into the vesicle bilayer. Each vesicle thus acts as a multifunctional junction in the network structure. Significantly, gel formation does not occur with the native chitosan that has no hydrophobes. Moreover, adding the hydrophobically modified chitosan to a viscous sample containing wormlike micelles increases the viscosity further but does not give rise to a gel-like response. Thus, the formation of a robust gel network requires both the presence of hydrophobes on the polymer and vesicles in solution.

Fibrin sealants are a type of soft tissue adhesive that employs biochemical reactions from the late stages of the blood coagulation cascade. Intrinsic to these adhesives are a structural protein and a transglutaminase crosslinking enzyme. We are investigating an alternative biomimetic adhesive based on gelatin and a calcium-independent microbial transglutaminase (mTG). Rheological measurements show that mTG catalyzes the conversion of gelatin solutions into hydrogels, and gel times are on the order of minutes depending on the gelatin type and concentration. Tensile static and dynamic loading of the adhesive hydrogels in bulk form demonstrated that the Young's modulus ranged from 15 to 120 kPa, and these bulk properties were comparable to those reported for hydrogels obtained from fibrin-based sealants. Lap-shear adhesion tests of porcine tissue were performed using a newly published American Society for Testing and Materials (ASTM) standard for tissue adhesives. The gelatin-mTG adhesive bound the opposing tissues together with ultimate adhesive strengths of 12-23 kPa which were significantly higher than the strength observed for fibrin sealants. Even after failure, strands of the gelatin-mTG adhesive remained attached to both of the opposing tissues. These results suggest that gelatin-mTG adhesives may offer the benefits of fibrin sealants without the need for blood products.

Tyrosinase was used to initiate the grafting of peptides onto the amine-containing polysaccharide chitosan. Chemical evidence for covalent grafting was obtained from electrospray mass spectrometry for products formed from reactions with glucosamine (the monomeric unit of chitosan) and the model dipeptide Tyr-Ala. When this model dipeptide was incubated with tyrosinase and chitosan, there was a marked increase in the viscosity of the solution. This viscosity increase provides physical evidence that tyrosinase can initiate peptide grafting onto the chitosan backbone. A peptide-modified chitosan derivative was generated by reacting chitosan (0.32 w/v%) with acid-hydrolyzed casein (0.5 w/v %) using tyrosinase. After reaction, the peptide-modified chitosan was partially purified and dissolved in an aqueous acetic acid solution. Low concentrations of this peptide-modified chitosan were observed to confer viscoelastic properties to the solutions. Specifically they conferred high viscosities and shear thinning properties to the solutions, and solutions containing only 1 w/w % of the peptide-modified chitosan behaved as weak gels. Thus, tyrosinase provides a simple and safe way to convert food-processing byproducts into environmentally friendly products that offer useful functional properties. The selectivity of tyrosinase and the relatively high reactivity of chitosan's amines allow grafting to be performed with uncharacterized peptide mixtures present in crude hydrolysates.

The need for environmentally-friendly processing has stimulated research on biocatalysis, while a growing interest in sustainability is stimulating interest in the use of renewable resources. We are currently examining an enzymatic approach to graft renewable phenols onto the biopolymer chitosan. In this approach, tyrosinase is used to convert phenols into reactive o-quinones which undergo non-enzymatic reactions that lead to grafting onto chitosan. As discussed, tyrosinase-catalyzed chitosan modification results in dramatic changes in functional properties.

Hydrogels are increasingly considered for creating three-dimensional structures in miniaturized devices, yet few techniques exist for creating such hydrogel structures. We report a new approach for creating hydrogels using the amine-containing polysaccharide chitosan. Specifically, electrodes are immersed into a slightly acidic chitosan solution and a voltage is applied to promote the proton-consuming hydrogen evolution reaction at the cathode surface. This reaction leads to a high localized pH in the vicinity of the cathode surface, and if this localized pH exceeds about 6.3, then chitosan becomes insoluble and deposits at the cathode surface. As the current density is increased, the region of high pH is expected to extend further from the cathode surface into the bulk solution. Using moderately high current densities (50 A/m(2)), we observed that chitosan deposited as a thick hydxogel. Measurements of the water content confirmed that the deposited chitosan was a hydrogel. To suggest the potential utility, we deposited a chitosan hydrogel on a patterned surface to create a channel. Because of chitosan's pH-dependent solubility, this channel could be "disassembled" by mild acid treatment. We envision that electrochemically-induced deposition of chitosan-based hydrogels may offer interesting opportunities for the integration of biological systems into miniaturized devices.

We report an enzyme-based method for the in situ entrapment of cells within a biopolymeric hydrogel matrix. Specifically, we used a calcium-independent microbial transglutaminase that is known to cross-link proteins and observed that it catalyzes the formation of gels from a pre-gel solution containing 10% gelatin and E. coli cells. Hydrogel formation occurs 2-3 h after adding transglutaminase, and no additional external intervention is required to initiate gel formation. The in situ entrapped cells grow rapidly and to high cell densities within the gelatin hydrogel. Additionally, the entrapped cells respond to isopropylthiogalactoside induction. The cross-linked gelatin network can be rapidly hydrolyzed (within 1 h) by the protease, proteinase K. Treatment of the network by this protease releases the entrapped E. coli cells. These cells appear unharmed by proteinase K; they can grow and be induced after protease treatment. The ability to in situ entrap, grow, and release cells under mild conditions provides unique opportunities for a range of applications and should be especially useful for microfluidic biosensor systems.

We compared the ability of two enzymes to catalyze the formation of gels from solutions of gelatin and chitosan. A microbial transglutaminase, currently under investigation for food applications, was observed to catalyze the formation of strong and permanent gels from gelatin solutions. Chitosan was not required for transglutaminase-catalyzed gel formation, although gel formation was faster, and the resulting gels were stronger if reactions were performed in the presence of this polysaccharide. Consistent with transglutaminase's ability to covalently crosslink proteins, we observed that the transglutaminase-catalyzed gelatin-chitosan gels lost the ability to undergo thermally reversible transitions (i.e. sol-gel transitions) characteristic of gelatin. Mushroom tyrosinase was also observed to catalyze gel formation for gelatin-chitosan blends. In contrast to transglutaminase, tyrosinase-catalyzed reactions did not lead to gel formation unless chitosan was present (i.e. chitosan is required for tyrosinase-catalyzed gel formation). Tyrosinase-catalyzed gelatin-chitosan gels were observed to be considerably weaker than transglutaminase-catalyzed gels. Tyrosinase-catalyzed gels were strengthened by cooling below gelatin's gel-point, which suggests that gelatin's ability to undergo a collagen-like coil-to-helix transition is unaffected by tyrosinase-catalyzed reactions. Further, tyrosinase-catalyzed gelatin-chitosan gels were transient as their strength (i.e. elastic modulus) peaked at about 5h after which the gels broke spontaneously over the course of 2 days. The strength of both transglutaminase-catalyzed and tyrosinase-catalyzed gels could be adjusted by altering the gelatin and chitosan compositions. Potential applications of these gels for in situ applications are discussed.

A protein's functional properties can be adjusted by conjugating it to other polymers. We used a nature-inspired route to create a protein-polysaccharide conjugate and examined the properties of this conjugate. Specifically, the enzyme tyrosinase was used to oxidize accessible tyrosine residues of the model protein green fluorescent protein (GFP). Oxidation yields quinone residues that are "activated" for the covalent conjugation of GFP to nucleophilic groups of the aminopolysaccharide chitosan. Conjugation to chitosan conferred distinct properties to GFP. The GFP-chitosan conjugate was observed to have pH-responsive, "smart" properties, and GFP could be conjugated onto a gel matrix. Additionally, the GFP-chitosan conjugate can be selectively deposited onto a micropatterned surface in response to an applied voltage. This nature-inspired method provides a simple and safe method to conjugate proteins to chitosan, and these conjugates can be readily assembled onto patterned surfaces.