process-making-three-dimensional-microstructures-using-vaporization

Process of Making Three-dimensional Microstructures using Vaporization of a Sacrificial Component

Vascular structures in natural systems are able to provide high mass transport through high surface areas and optimized structure. Few synthetic material ...

University of California, Irvine

N-terminal protein modification through a biomimetic transamination reaction.

A new bioconjugation reaction has been developed based on the chemoselective modification of anilines through an oxidative coupling pathway. Aryl amines were installed on the surface of protein substrates through lysine acylation reactions or through the use of native chemical ligation techniques. Upon exposure to NaIO4 in aqueous buffer, the anilines coupled rapidly to the aromatic rings of N,N-dialkyl-N'-acyl-p-phenylenediamines. The identities of the reaction products were confirmed using ESI-MS and through comparison to small molecule analogs. Control experiments indicated that none of the native amino acids participated in the reaction. The resulting bioconjugates were found to be stable toward hydrolysis from pH 4 to pH 11 and in the presence of many commonly used oxidants, reductants, and nucleophiles. A fluorescent phenylenediamine reagent was synthesized for the selective detection of aniline labeled proteins in mixtures, and the reaction was used to append the C-terminus of the green fluorescent protein with a single PEG chain. When combined with techniques for the incorporation of unnatural amino acids into proteins, this bioorthogonal coupling method should prove useful for a number of applications requiring a high degree of labeling specificity.

Modification of aniline containing proteins using an oxidative coupling strategy.

Protein-cross-linked polymeric materials through site-selective bioconjugation.

The diverse functional repertoire of proteins promises to yield new materials with unprecedented capabilities, so long as versatile chemical methods are available to integrate biomolecules with synthetic components. As a demonstration of this potential, we have used site-selective strategies to cross-link polymer chains using the N- and C-termini of a metallothionein derived from a pea plant. This arrangement directly relates the swelling volume of the polymer to the folded state of the protein. The material retains the protein's ability to remove heavy metal ions from contaminated water samples, and can be regenerated through the subsequent addition of inexpensive chelators. The change in hydrogel volume that occurs as metal ions are bound allows the detection of contaminants through simple visual inspection. The utility of this bulk property change is demonstrated in the construction of a low-cost device that can report heavy metal contamination with no external power requirements. Most importantly, the generality of the protein modification chemistry allows the immediate generation of new hybrid materials from a wide range of protein sequences.

Metallothionein-cross-linked hydrogels for the selective removal of heavy metals from water.

For the autonomous repair of damaged materials, microcapsules are needed that release their contents in response to a variety of physical and chemical phenomena, not just by direct mechanical rupture. Herein we report a general route to programmable microcapsules. This method creates core-shell microcapsules with polymeric shell walls composed of self-immolative polymer networks. The polymers in these networks undergo a head-to-tail depolymerization upon removal of the triggering end group, leading to breakdown of the shell wall and subsequent release of the capsule's liquid interior. We report microcapsules with shell walls bearing both Boc and Fmoc triggering groups. The capsules release their contents only under conditions known to remove these triggering groups; otherwise, they retain their contents under a variety of conditions. In support of the proposed release mechanism, the capsule shell walls were observed to undergo physical cracking upon exposure to the triggering conditions.

Programmable microcapsules from self-immolative polymers.

The diverse functional repertoire of proteins promises to yield new materials with unprecedented capabilities, so long as versatile chemical methods are available to introduce synthetic components at specific sites on biomolecule surfaces. As a demonstration of this potential, we have used site-selective strategies to attach antifreeze proteins found in Arctic fish and insects to polymer chains. This multivalent arrangement increases the thermal hysteresis activity of the proteins and leads to materials that can be cast into thin films. The polymer-protein conjugates retain the ability of the proteins to slow ice growth in subzero water and can inhibit ice formation after attachment to glass surfaces. These inexpensive materials may prove useful as coatings for device components that must function at low temperature without ice buildup. The polymer attachment also allows higher thermal hysteresis values to be achieved while using less protein, thus lowering the cost of these additives for biomedical applications.

Incorporation of antifreeze proteins into polymer coatings using site-selective bioconjugation.

Chemical reactions that facilitate the attachment of synthetic groups to proteins are useful tools for the field of chemical biology and enable the incorporation of proteins into new materials. We have previously reported a pyridoxal 5'-phosphate (PLP)-mediated reaction that site-specifically oxidizes the N-terminal amine of a protein to afford a ketone. This unique functional group can then be used to attach a reagent of choice through oxime formation. Since its initial report, we have found that the N-terminal sequence of the protein can significantly influence the overall success of this strategy. To obtain short sequences that lead to optimal conversion levels, an efficient method for the evaluation of all possible N-terminal amino acid combinations was needed. This was achieved by developing a generalizable combinatorial peptide library screening platform suitable for the identification of sequences that display high levels of reactivity toward a desired bioconjugation reaction. In the context of N-terminal transamination, a highly reactive alanine-lysine motif emerged, which was confirmed to promote the modification of peptide substrates with PLP. This sequence was also tested on two protein substrates, leading to substantial increases in reactivity relative to their wild-type termini. This readily encodable tripeptide thus appears to provide a significant improvement in the reliability with which the PLP-mediated bioconjugation reaction can be used. This study also provides an important first example of how synthetic peptide libraries can accelerate the discovery and optimization of protein bioconjugation strategies.

Identification of highly reactive sequences for PLP-mediated bioconjugation using a combinatorial peptide library.

Three-dimensional microvascular fiber-reinforced composites.

When heated, poly(lactic acid) (PLA) fibers depolymerize in a controlled manner, making them potentially useful as sacrificial fibers for microchannel fabrication. Catalysts that increase PLA depolymerization rates are explored and methods to incorporate them into commercially available PLA fibers by a solvent mixture impregnating technique are tested. In the present study, the most active catalysts are identified that are capable of lowering the depolymerization temperature of modified PLA fibers by ca. 100 °C as compared to unmodified ones. Lower depolymerization temperatures allow PLA fibers to be removed from a fully cured epoxy thermoset resin without causing significant thermal damage to the epoxy. For 500 μm diameter PLA fibers, the optimized treatment involves soaking the fibers for 24 h in a solvent mixture containing 60% trifluoroethanol (TFE) and 40% H(2)O dispersed with 10 wt % tin(II) oxalate and subsequent air-drying of the fibers. PLA fibers treated with this procedure are completely removed when heated to 180 °C in vacuo for 20 h. The time evolution of catalytic depolymerization of PLA fiber is investigated by gel permeation chromatography (GPC). Channels fabricated by vaporization of sacrificial components (VaSC) are subsequently characterized by scanning electron microscopy (SEM) and X-ray microtomography (Micro CT) to show the presence of residual catalysts.

Chemical treatment of poly(lactic acid) fibers to enhance the rate of thermal depolymerization.

For the capture of CO(2) from mixed gas streams, materials for increased gas exchange are necessary. Efficient gas exchange systems already exist in the form of vascularized lung-tissue. Herein we report a fabrication technique for the synthesis of three-dimensional microvascular gas exchange units capable of removing CO(2) from flowing gas created using the recently reported Vaporization of a Sacrificial Component (VaSC) technique. We demonstrate the spatiotemporal pattern of CO(2) reactivity in the microvascular gas exchange unit using colorimetric, pH sensitive dyes. Control over three-dimensional placement of channels is shown to increase capture efficiencies. A computational finite element model validates and explains the experimental observations.

A three-dimensional microvascular gas exchange unit for carbon dioxide capture.

We present a primary example of a cell surface modified with a synergistic combination of agonists to tune immune stimulation. A model cell line, Lewis Lung Carcinoma, was covalently modified with CpG-oligonucleotides and lipoteichoic acid, both Toll-like receptor (TLR) agonists. The immune-stimulating constructs provided greater stimulation of NF-κB in a model cell line and bone marrow-derived dendritic cells than the components unconjugated in solution.

Aaron P. Esser-Kahn

Department of Chemistry

University of California, Irvine

Process of Making Three-dimensional Microstructures using Vaporization of a Sacrificial Component

Aaron P. Esser-Kahn

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Chemistry

University of California, Irvine

Process of Making Three-dimensional Microstructures using Vaporization of a Sacrificial Component

Department of Chemistry