The traditional motivation for integrating biological components into microfabricated devices has been to create biosensors that meld the molecular recognition capabilities of biology with the signal processing capabilities of electronic devices. However, a different motivation is emerging; biological components are being explored to radically change how fabrication is achieved at the micro- and nanoscales. Here we review biofabrication, the use of biological materials for fabrication, and focus on three specific biofabrication approaches: directed assembly, where localized external stimuli are employed to guide assembly; enzymatic assembly, where selective biocatalysts are enlisted to build macromolecular structure; and self-assembly, where information internal to the biological material guides its own assembly. Also reviewed are recent results with the aminopolysaccharide chitosan, a material that offers a combination of properties uniquely suited for biofabrication. In particular, chitosan can be directed to assemble in response to locally applied electrical signals, and the chitosan backbone provides sites that can be employed for the assembly of proteins, nucleic acids, and virus particles.
We examined the assembly of the amine-rich polysaccharide chitosan from solution onto electrode surfaces as a result of voltage bias on the electrode. Chitosan is positively charged and water soluble under mildly acidic conditions and is uncharged and insoluble under basic conditions. We observed that chitosan is deposited from acidic solution onto the surface of a negative electrode and the thickness of the deposited layer is on the order of a micron. The thickness of the deposited layer was observed to be dependent upon the deposition time, the applied voltage, and the chitosan concentration. No deposition was observed on the positive electrode or on an “electrode” that had no applied voltage. Once deposited and neutralized, the chitosan layer can be retained on the electrode surface without the need for an applied voltage. Infrared (FT-IR) and electrospray mass spectrometry confirmed that the deposited material was chitosan. These results demonstrate that chitosan can be deposited and retained on electrode surfaces, and the potential advantages for applications in microfabricated devices are discussed.
Gelatin is one of the most commonly used biomaterials for creating cellular scaffolds due to its innocuous nature. In order to create stable gelatin hydrogels at physiological temperatures (37 degrees C), chemical crosslinking agents such as glutaraldehyde are typically used. To circumvent potential problems with residual amounts of these crosslinkers in vivo and create scaffolds that are both physiologically robust and biocompatible, a microbial transglutaminase (mTG) was used in this study to enzymatically crosslink gelatin solutions. HEK293 cells encapsulated in mTG-crosslinked gelatin proliferated at a rate of 0.03 day(-1). When released via proteolytic degradation with trypsin, the cells were able to recolonize tissue culture flasks, suggesting that cells for therapeutic purposes could be delivered in vivo using an mTG-crosslinked gelatin construct. Upon submersion in a saline solution at 37 degrees C, the mTG-crosslinked gelatin exhibited no mass loss, within experimental error, indicating that the material is thermally stable. The proteolytic degradation rate of mTG-crosslinked gelatin at RT was slightly faster than that of thermally-cooled (physically-crosslinked) gelatin. Thermally-cooled gelatin that was subsequently crosslinked with mTG resulted in hydrogels that were more resistant to proteolysis. Degradation rates were found to be tunable with gelatin content, an attribute that may be useful for either long-time cell encapsulation or time-released regenerative cell delivery. Further investigation showed that proteolytic degradation was controlled by surface erosion.
The amine-containing polysaccharide chitosan was selectively deposited onto patterned gold surfaces in response to an applied voltage. Standard microfabrication techniques were used to pattern gold onto silicon wafers, and these gold patterns served as templates for the electric field directed deposition of chitosan. Experiments conducted with a fluorescently labeled chitosan derivative demonstrated the spatially selective deposition of chitosan onto gold surfaces that were polarized to serve as negative electrodes. Studies with unlabeled chitosan demonstrated that a “templated” chitosan, deposited by voltage programming of electrodes, can subsequently react with standard amine-selective functional groups. This indicates that common coupling chemistries can be exploited to assemble a variety of compounds onto the deposited chitosan pattern. Thus, chitosan appears to be a unique interface material that can be “templated” onto patterned inorganic surfaces and is reactive for the subsequent assembly of organic and biological molecules.
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 hydrogel. 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.
MicroRNA (miRNA) and long non-coding RNA (lncRNA) have been demonstrated to participate in the progression of many cancers. Hepatocellular carcinoma (HCC) is one of the most common and aggressive malignant tumors worldwide, while the molecular mechanisms underlying HCC tumorigenesis are not completely clear. In this study, we showed that miR-92b was significantly upregulated in tumor tissue and plasma of HCC patients, and its expression level was highly correlated with gender and microvascular invasion. Functionally, miR-92b could promote cell proliferation and metastasis of HCC in vitro and in vivo. Mechanistic investigations suggested that Smad7, which exhibited an inverse relationship with miR-92b expression in HCC, was a direct target of miR-92b and could reverse its effects on HCC tumorigenesis. Furthermore, long non-coding RNA (lncRNA) X-inactive specific transcript (XIST) and miR-92b could directly interact with and repress each other, and XIST could inhibit HCC cell proliferation and metastasis by targeting miR-92b. Taken together, our study not only revealed for the first time the importance of XIST/miR-92b/Smad7 signaling axis in HCC progression but also suggested the potential value of miR-92b as a biomarker in the clinical diagnosis and treatment of HCC.
The enzyme tyrosinase was used for the in vitro conjugation of the protein gelatin to the polysaccharide chitosan. Tyrosinases are oxidative enzymes that convert accessible tyrosine residues of proteins into reactive o-quinone moieties. Spectrophotometric and dissolved oxygen studies indicate that tyrosinase can oxidize gelatin and we estimate that 1 in 5 gelatin chains undergo reaction. Oxidized tyrosyl residues (i.e., quinone residues) can undergo nonenzymatic reactions with available nucleophiles such as the nucleophilic amino groups of chitosan. Ultraviolet/visible, (1)H-NMR, and ir provided chemical evidence for the conjugation of oxidized gelatin with chitosan. Physical evidence for conjugation was provided by dynamic viscometry, which indicated that tyrosinase catalyzes the sol-to-gel conversion of gelatin/chitosan mixtures. The gels formed from tyrosinase-catalyzed reactions were observed to differ from gels formed by cooling gelatin. In contrast to gelatin gels, tyrosinase-generated gels had different thermal behavior and were broken by the chitosan-hydrolyzing enzyme chitosanase. These results demonstrate that tyrosinase can be exploited for the in vitro formation of protein-polysaccharide conjugates that offer interesting mechanical properties.
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.
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