Jason C. Harper

Supramolecular self-assembled chaos: polyphenolic lignin's barrier to cost-effective lignocellulosic biofuels

Phenylpropanoid metabolism yields a mixture of monolignols that undergo chaotic, non-enzymatic reactions such as free radical polymerization and spontaneous self-assembly in order to form the polyphenolic lignin which is a barrier to cost-effective lignocellulosic biofuels. Post-synthesis lignin integration into the plant cell wall is unclear, including how the hydrophobic lignin incorporates into the wall in an initially hydrophilic milieu. Self-assembly, self-organization and aggregation give rise to a complex, 3D network of lignin that displays randomly branched topology and fractal properties. Attempts at isolating lignin, analogous to archaeology, are instantly destructive and non-representative of in planta. Lack of plant ligninases or enzymes that hydrolyze specific bonds in lignin-carbohydrate complexes (LCCs) also frustrate a better grasp of lignin. Supramolecular self-assembly, nano-mechanical properties of lignin-lignin, lignin-polysaccharide interactions and association-dissociation kinetics affect biomass deconstruction and thereby cost-effective biofuels production.

Maleimide-Activated Aryl Diazonium Salts for Electrode Surface Functionalization with Biological and Redox-Active Molecules

A versatile and simple method is introduced for formation of maleimide-functionalized surfaces using maleimide-activated aryl diazonium salts. We show for the first time electrodeposition of N-(4-diazophenyl)maleimide tetrafluoroborate on gold and carbon electrodes which was characterized via voltammetry, grazing angle FTIR, and ellipsometry. Electrodeposition conditions were used to control film thickness and yielded submonolayer-to-multilayer grafting. The resulting phenylmaleimide surfaces served as effective coupling agents for electrode functionalization with ferrocene and the redox-active protein cytochrome c. The utility of phenylmaleimide diazonium toward formation of a diazonium-activated conjugate, followed by direct electrodeposition of the diazonium-modified DNA onto the electrode surface, was also demonstrated. Effective electron transfer was obtained between immobilized molecules and the electrodes. This novel application of N-phenylmaleimide diazonium may facilitate the development of bioelectronic devices including biofuel cells, biosensors, and DNA and protein microarrays.

A Multifunctional Thin Film Au Electrode Surface Formed by Consecutive Electrochemical Reduction of Aryl Diazonium Salts

A multifunctional thin film surface capable of immobilizing two diverse molecules on a single gold electrode was prepared by consecutive electrodeposition of nitrophenyl and phenylboronic acid pinacol ester (PBA-PE) diazonium salts. Activation of the stacked film toward binding platinum nanoparticles (PtNPs) and yeast cells occurred via chemical deprotection of the pinacol ester followed by electroreduction of nitro to amino groups. FTIR spectral analysis was used to study and verify film composition at each stage of preparation. The affect of electrodeposition protocol over the thickness of the nitrophenyl and PBA-PE layers was explored and had a profound impact on the film properties. Thicker nitrophenyl films led to diminished PBA-PE diazonium reduction currents during assembly and decreased phenylboronic acid (PBA) layer thickness while allowing for higher PtNP loading and catalytic currents from PtNP-mediated peroxide reduction. Multilayer PBA films could be formed over the nitrophenyl film; however, only submonlayer PBA films permitted access to the underlying layer. The sequence of functional group activation toward binding was also shown to be significant, as perchlorate used to remove pinacol ester also converted aminophenyl groups accessible to the solution to nitrophenyl groups, preventing electrostatic PtNP binding. Finally, SEM images show PtNPs immobilized in close proximity (nanometers) to captured yeast cells on the PBA-aminophenyl-Au film. Such multibinding functionality films that maintain conductivity for subsequent electrochemical measurements hold promise for the development of electrochemical and/or optical platforms for fundamental cell studies, genomic and proteomic analysis, and biosensing.

Multiphoton Lithography of Nanocrystalline Platinum and Palladium for Site-Specific Catalysis in 3D Microenvironments.

Integration of catalytic nanostructured platinum and palladium within 3D microscale structures or fluidic environments is important for systems ranging from micropumps to microfluidic chemical reactors and energy converters. We report a straightforward procedure to fabricate microscale patterns of nanocrystalline platinum and palladium using multiphoton lithography. These materials display excellent catalytic, electrical, and electrochemical properties, and we demonstrate high-resolution integration of catalysts within 3D defined microenvironments to generate directed autonomous particle and fluid transport.

Rational Redesign of Glucose Oxidase for Improved Catalytic Function and Stability

Glucose oxidase (GOx) is an enzymatic workhorse used in the food and wine industries to combat microbial contamination, to produce wines with lowered alcohol content, as the recognition element in amperometric glucose sensors, and as an anodic catalyst in biofuel cells. It is naturally produced by several species of fungi, and genetic variants are known to differ considerably in both stability and activity. Two of the more widely studied glucose oxidases come from the species Aspergillus niger (A. niger) and Penicillium amagasakiense (P. amag.), which have both had their respective genes isolated and sequenced. GOx from A. niger is known to be more stable than GOx from P. amag., while GOx from P. amag. has a six-fold superior substrate affinity (K M) and nearly four-fold greater catalytic rate (k cat). Here we sought to combine genetic elements from these two varieties to produce an enzyme displaying both superior catalytic capacity and stability. A comparison of the genes from the two organisms revealed 17 residues that differ between their active sites and cofactor binding regions. Fifteen of these residues in a parental A. niger GOx were altered to either mirror the corresponding residues in P. amag. GOx, or mutated into all possible amino acids via saturation mutagenesis. Ultimately, four mutants were identified with significantly improved catalytic activity. A single point mutation from threonine to serine at amino acid 132 (mutant T132S, numbering includes leader peptide) led to a three-fold improvement in k cat at the expense of a 3% loss of substrate affinity (increase in apparent K M for glucose) resulting in a specify constant (k cat/K M) of 23.8 (mM−1 · s−1) compared to 8.39 for the parental (A. niger) GOx and 170 for the P. amag. GOx. Three other mutant enzymes were also identified that had improvements in overall catalysis: V42Y, and the double mutants T132S/T56V and T132S/V42Y, with specificity constants of 31.5, 32.2, and 31.8 mM−1 · s−1, respectively. The thermal stability of these mutants was also measured and showed moderate improvement over the parental strain.

Biocompatible Microfabrication of 3D Isolation Chambers for Targeted Confinement of Individual Cells and Their Progeny

We describe a technique to physically isolate single/individual cells from their surrounding environment by fabricating three-dimensional microchambers around selected cells under biocompatible conditions. Isolation of targeted cells is achieved via rapid fabrication of protein hydrogels from a biocompatible precursor solution using multiphoton lithography, an intrinsically 3D laser direct write microfabrication technique. Cells remain chemically accessible to environmental cues enabling their propagation into well-defined, high density populations. We demonstrate this methodology on gram negative (E. coli), gram positive (S. aureus), and eukaryotic (S. cerevisiae) cells. The opportunities to confine viable, single/individual-cells and small populations within user-defined microenvironments afforded by this approach should facilitate the study of cell behaviors across multiple generations.

Cell-Directed Localization and Orientation of a Functional Foreign Transmembrane Protein within a Silica Nanostructure

A simple procedure for introducing functional exogenous membrane-bound proteins to viable cells encapsulated within a lipid templated silica nanostructure is described. In one method, bacteriorhodopsin (bR) was added directly to a Saccharomyces cerevisiae solution along with short zwitterionic diacylphosphatidylcholines (diC(6) PC) and mixed with equal volumes of a sol precursor solution. Alternatively, bR was first incorporated into liposomes (bR-proteoliposomes) and then added to an S. cerevisiae solution with diC(6) PC, and this was followed by mixing with sol precursor solution. Films prepared from bR added directly to diC(6) PC resulted in bR localization near S. cerevisiae cells in a disordered and diffuse fashion, while films prepared from bR-proteoliposomes added to the diC(6) PC/yeast solution resulted in preferential localization of bR near yeast cell surfaces, forming bR-containing multilayer vesicles. Importantly, bR introduced via proteoliposomes was observed to modulate pH gradients developed at the cell surface, demonstrating both retained functionality and preferential orientation. Localization of liposome lipid or bR did not occur around neutrally charged latex beads acting as cell surrogates, demonstrating that living cells actively organize the multilayered lipid during evaporation-induced self-assembly. We expect this simple procedure for introducing functional and oriented membrane-bound proteins to the surface of cells to be general and adaptable to other membrane-bound proteins. This advance may prove useful in fundamental studies of membrane protein function and cell-cell signaling and in imparting non-native characteristics to arbitrary cells.

Fabrication and Testing of a Microneedles Sensor Array for p-Cresol Detection with Potential Biofuel Applications

We present a miniaturized high-throughput sensor array that will augment biofuel technology by facilitating in situ biochemical measurements upon micrometer-scale surfaces of leaves, stems, or petals. We used semiconductor processing to photopattern Foturan glass wafers and fabricated gold-plated microscopic electrode needles (ElectroNeedles) that pierced 125-mum-thick surfaces without deformation. The 5 x 5 or 10 x 10 arrays of ElectroNeedles can analyze 25 or 100 samples simultaneously, increasing throughput. Each microneedle in the array can also be individually addressed and selectively functionalized using diazonium electrodeposition, conferring multiplexing capability. Our microfabrication is a simple, inexpensive, and rapid alternative to the time-, cost-, and protocol-intense, deep-reactive-ion-etching Bosch process. We validated the system performance by electrochemically detecting p-cresol, a phenolic substrate for laccase, an enzyme that is implicated in lignin degradation and therefore important to biofuels. Our limits of detection (LOD) and quantization (LOQ) for p-cresol were 1.8 and 16microM, respectively, rivaling fluorescence detection (LOD and LOQ = 0.4 and 3microM, respectively). ElectroNeedles are multiplexed, high-throughput, chip-based sensor arrays designed for minimally invasive penetration of plant surfaces, enabling in situ and point-of-test analyses of biofuel-related biochemicals.

Orthogonal Cell-Based Biosensing: Fluorescent, Electrochemical, and Colorimetric Detection with Silica-Immobilized Cellular Communities Integrated with an ITO–Glass/Plastic Laminate Cartridge

This is the first report of a living cell-based environmental sensing device capable of generating orthogonal fluorescent, electrochemical, and colorimetric signals in response to a single target analyte in complex media. Orthogonality is enabled by use of cellular communities that are engineered to provide distinct signals in response to the model analyte. Coupling these three signal transduction methods provides additional and/or complementary data regarding the sample which may reduce the impact of interferants and increase confidence in the sensors output. Long-term stability of the cells was addressed via 3D entrapment within a nanostructured matrix derived from glycerated silicate, which allows the device to be sealed and stored under dry, ambient conditions for months with significant retention in cellular activity and viability (40% viability after 60 days). Furthermore, the first co-entrapment of eukaryotic and bacterial cells in a silica matrix is reported, demonstrating multianalyte biodetection by mixing disparate cell lines at intimate proximities which remain viable and responsive. These advances in cell-based biosensing open intriguing opportunities for integrating living cells with nanomaterials and macroscale systems.

A parallel microfluidic channel fixture fabricated using laser ablated plastic laminates for electrochemical and chemiluminescent biodetection of DNA

Herein is described the fabrication and use of a plastic multilayer 3-channel microfluidic fixture. Multilayer devices were produced by laser machining of plastic polymethylmethacrylate and polyethyleneterapthalate laminates by ablation. The fixture consisted of an array of nine individually addressable gold or gold/ITO working electrodes, and a resistive platinum heating element. Laser machining of both the fluidic pathways in the plastic laminates, and the stencil masks used for thermal evaporation to form electrode regions on the plastic laminates, enabled rapid and inexpensive implementation of design changes. Electrochemiluminescence reactions in the fixture were achieved and monitored through ITO electrodes. Electroaddressable aryl diazonium chemistry was employed to selectively pattern gold electrodes for electrochemical multianalyte DNA detection from double stranded DNA (dsDNA) samples. Electrochemical detection of dsDNA was achieved by melting of dsDNA molecules in solution with the integrated heater, allowing detection of DNA sequences specific to breast and colorectal cancers with a non-specific binding control. Following detection, the array surface could be renewed via high temperature (95 °C) stripping using the integrated heating element. This versatile and simple method for prototyping devices shows potential for further development of highly integrated, multi-functional bioanalytical devices.