Li-Qun Wu

Biomimetic sealant based on gelatin and microbial transglutaminase: an initial in vivo investigation.

The potential of an in situ gel-forming adhesive was examined as a hemostatic surgical sealant. The gel-forming mechanism for this adhesive mimics the last stages of blood coagulation but uses nonblood proteins. Specifically, gelatin is used as the structural protein and a calcium-independent microbial transglutaminase (mTG) is used as the crosslinking enzyme. In vitro burst pressure tests with porcine skin demonstrate that the gelatin-mTG adhesive forms a gel within 30 min under moist conditions and this gel can restrain pressures of 200 mmHg. In vivo tests with a rat liver wound model showed that the gelatin-mTG adhesive achieves complete hemostasis in 2.5 min and the gel (i.e., the biomimetic clot) offers substantial adhesive and cohesive strength. Complete hemostasis was also observed in 2.5 min after the gelatin-mTG adhesive was applied to a briskly bleeding rat femoral artery wound. In a large animal porcine model, a femoral artery wound that resulted in extensive bleeding was sealed in 4 min by (i) clamping the artery for temporary hemostasis, (ii) removing excess blood, and (iii) applying the gelatin-mTG adhesive. Thus, the biomimetic gelatin-mTG adhesive may provide a simple, safe, and cost-effective surgical sealant.

Mechano-transduction of DNA hybridization and dopamine oxidation through electrodeposited chitosan network.

While microcantilevers offer exciting opportunities for mechano-detection, they often suffer from limitations in either sensitivity or selectivity. To address these limitations, we electrodeposited a chitosan film onto a cantilever surface and mechano-transduced detection events through the chitosan network. Our first demonstration was the detection of nucleic acid hybridization. In this instance, we electrodeposited the chitosan film onto the cantilever, biofunctionalized the film with oligonucleotide probe, and detected target DNA hybridization by cantilever bending in solution (static mode) or resonant frequency shifts in air (dynamic mode). In both detection modes, we observed a two-order of magnitude increase in sensitivity compared to values reported in literature for DNA immobilized on self-assembled monolayers. In our second demonstration, we coupled electrochemical and mechanical modes to selectively detect the neurotransmitter dopamine. A chitosan-coated cantilever was biased to electrochemically oxidize dopamine solution. Dopamines oxidation products react with the chitosan film and create a tensile stress of approximately 1.7 MPa, causing substantial cantilever bending. A control experiment was performed with ascorbic acid solution. It was shown that the electrochemical oxidation of ascorbic acid does not lead to reactions with chitosan and does not change cantilever bending. These results suggest that chitosan can confer increased sensitivity and selectivity to microcantilever sensors.

Biomimetic fabrication of information-rich phenolic-chitosan films

Phenolics appear to be everywhere: they dominate the organic content of our landscape (humics and lignins); they impart color to plants and animals (flavonoids and melanins); they enable biology to harvest energy (quinone redox couples in respiration and photosynthesis); and they may enhance our health (dietary antioxidants). Biology enlists the diverse physicochemical properties of phenolics to perform a variety of functions that are not yet fully understood or appreciated. In this review, the synthesis, structures and functions of biological phenols is briefly sketched while an array of examples is provided to illustrate their diverse functions. The focus of this review however is recent studies which show that the biomimetic incorporation of phenolics into thin chitosan films can controllably impart mechanical, optical and redox properties. These studies demonstrate the potential for accessing natures rich diversity of phenolics to impart functionality to soft matter.

Chitosan biotinylation and electrodeposition for selective protein assembly.

An alternative route to protein assembly at surfaces based on using the unique capabilities of biological materials for the spatially selective assembly of proteins is described. Specifically, the stimuli-responsive properties of aminopolysaccharide chitosan are combined with the molecular-recognition capabilities of biotin-streptavidin binding. Biotinylated chitosan retains its stimuli-responsive properties and is capable of electrodepositing at specific electrode addresses. Once deposited, it is capable of binding streptavidin, which can mediate the subsequent assembly of biotinylated proteins. Spatially selective protein assembly using biotinylated Protein A and fluorescently-labeled antibodies is demonstrated.

Biofabrication with biopolymers and enzymes: Potential for constructing scaffolds from soft matter

Purpose Regenerative medicine will benefit from technologies capable of fabricating soft matter to have appropriate architectures and that provide the necessary physical, chemical and biological cues to recruit cells and guide their development. The goal of this report is to review an emerging set of biofabrication techniques and suggest how these techniques could be applied for the fabrication of scaffolds for tissue engineering. Methods Electrical potentials are applied to submerged electrodes to perform cathodic and anodic reactions that direct stimuli-responsive film-forming polysaccharides to assemble into hydrogel films. Standard methods are used to microfabricate electrode surfaces to allow the electrical signals to be applied with spatial and temporal control. The enzymes mushroom tyrosinase and microbial transglutaminase are used to catalyze macromolecular grafting and crosslinking of proteins. Results Electrodeposition of the polysaccharides chitosan and alginate allow hydrogel films to be formed in response to localized electrical signals. Co-deposition of various components (e.g., proteins, vesicles and cells), and subsequent electrochemical processing allow the physical, chemical and biological activities of these films to be tailored. Enzymatic processing allows for the generation of stimuli-responsive protein conjugates that can also be directed to assemble in response to imposed electrical signals. Further, enzyme-catalyzed crosslinking of gelatin allows replica molding of soft matter to create hydrogel films with topological structure. Conclusions Biofabrication with biological materials and mechanisms provides new approaches for soft matter construction. These methods may enable the formation of tissue engineering scaffolds with appropriate architectures, assembled cells, and spatially organized physical, chemical and biological cues.

Chitosan as a functional interface between biology and microsystems

We report the use of the amino polysaccharide chitosan for the immobilization and patterning of biomolecules on microfabricated surfaces. Chitosan is a biocompatible and biodegradable substance derived from chitin, which is the structural material in the exoskeleton of crustaceans. Chitosan has two key properties of interest to biologists and engineers alike. First, it has an abundance of primary amine groups which can be covalently coupled to various biomolecules. Second, it possesses pH-dependent network-forming properties. Below pH of 6.5, chitosans amine groups become protonated and positively charged, making it soluble in acidic conditions. At pH above 6.5 the amines become deprotonated, and chitosan forms an insoluble polymer network. This allows a film of chitosan to be deposited from solution onto a negatively charged electrode due to a localized region of high pH established near the electrode surface. This electrodeposition is a simple yet robust method of patterning biomolecules with significant advantages over traditional patterning techniques such as microcontact printing. We have demonstrated electrodeposited chitosan as an immobilization agent for DNA and several proteins. Amine-labeled DNA are coupled to chitosan by glutaraldehyde crosslinking and proteins are coupled by enzyme-activated genetically engineered tyrosyl residues. These coupling chemistries can be extended to other biomolecules as well. In addition, the biomolecule attachment can be reversed or completely blocked by passivating chitosan. With its electrodeposition property and its easily accessible amine groups, chitosan is an attractive material from both the microfabrication and biology perspective. Chitosan can be used for a wide range of devices as a spatially controllable interface between organic and inorganic components. The possible applications of chitosan include biosensors, drug delivery devices, and labs on a chip. In our work, we have successfully used chitosan for three different BioMEMS applications. One is an optical biosensor, in which chitosan is used to immobilize fluorescently labeled biomolecules on the facets of waveguides. Another is a micromechanical biosensor, in which chitosan immobilizes DNA on the surface of a microcantilever. The third one is a microfluidic device, in which chitosan is used to pattern biomolecules inside sealed microchannels. These devices demonstrate the simplicity and flexibility of chitosan-based biomolecular patterning