Robert Y. Ofoli

An electrochemical interface for integrated biosensors

This paper presents an integrated, protein-based, biosensor that can be scaled to form high-density, multi-analyte sensor arrays physically integrated on a signal conditioning circuit die. A fully scalable, post-CMOS-compatible, three-electrode interface to biochemical sensors has been developed. A silicon substrate electrode system, consisting of Ti/Au working and auxiliary electrodes and a Ti/Au/Ag/AgCl reference electrode has been adapted to biomimetic sensors. The functional Ag/AgCl reference electrode is isolated from the environment using a Nafion cation-exchange membrane to extend operation lifetime. To complete the sensor structure, lipid bilayers have been deposited in passivation layer openings formed over individual working electrodes using a special tethering molecule. Total internal reflection microscopy (TIRFM) studies were done to confirm that a wide range of proteins, such as dehydrogenase enzymes and ion channels, can then be embedded into the lipid bilayers. These results verify the potential to form highly selective recognition elements with direct physical connection to readout electronics on the supporting silicon substrate.

In situ immobilization of palladium nanoparticles in microfluidic reactors and assessment of their catalytic activity.

We report on the synthesis and characterization of catalytic palladium nanoparticles (Pd NPs) and their immobilization in microfluidic reactors fabricated from polydimethylsiloxane (PDMS). The Pd NPs were stabilized with D-biotin or 3-aminopropyltrimethoxysilane (APTMS) to promote immobilization inside the microfluidic reactors. The NPs were homogeneous with narrow size distributions between 2 and 4 nm, and were characterized by transmission electron microscopy (TEM), selected-area electron diffraction (SAED), and x-ray diffraction (XRD). Biotinylated Pd NPs were immobilized on APTMS-modified PDMS and glass surfaces through the formation of covalent amide bonds between activated biotin and surface amino groups. By contrast, APTMS-stabilized Pd NPs were immobilized directly onto PDMS and glass surfaces rich in hydroxyl groups. Fourier transform infrared spectroscopy (FT-IR) and x-ray photoelectron spectroscopy (XPS) results showed successful attachment of both types of Pd NPs on glass and PDMS surfaces. Both types of Pd NPs were then immobilized in situ in sealed PDMS microfluidic reactors after similar surface modification. The effectiveness of immobilization in the microfluidic reactors was evaluated by hydrogenation of 6-bromo-1-hexene at room temperature and one atmosphere of hydrogen pressure. An average first-run conversion of 85% and selectivity of 100% were achieved in approximately 18 min of reaction time. Control experiments showed that no hydrogenation occurred in the absence of the nanocatalysts. This system has the potential to provide a reliable tool for efficient and high throughput evaluation of catalytic NPs, along with assessment of intrinsic kinetics.

Influence of lysophospholipid hydrolysis by the catalytic domain of neuropathy target esterase on the fluidity of bilayer lipid membranes

Neuropathy target esterase (NTE) is an integral membrane protein localized in the endoplasmic reticulum in neurons. Irreversible inhibition of NTE by certain organophosphorus compounds produces a paralysis known as organophosphorus compound-induced delayed neuropathy. In vitro, NTE has phospholipase/lysophospholipase activity that hydrolyses exogenously added single-chain lysophospholipids in preference to dual-chain phospholipids, and NTE mutations have been associated with motor neuron disease. NTEs physiological role is not well understood, although recent studies suggest that it may control the cytotoxic accumulation of lysophospholipids in membranes. We used the NTE catalytic domain (NEST) to hydrolyze palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (p-lysoPC) to palmitic acid in bilayer membranes comprising 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and the fluorophore 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (NBD-PC). Translational diffusion coefficients (D(L)) in supported bilayer membranes were measured by fluorescence recovery after pattern photobleaching (FRAPP). The average D(L) for DOPC/p-lysoPC membranes without NEST was 2.44 microm(2)s(-1)+/-0.09; the D(L) for DOPC/p-lysoPC membranes containing NEST and diisopropylphosphorofluoridate, an inhibitor, was nearly identical at 2.45+/-0.08. By contrast, the D(L) for membranes comprising NEST, DOPC, and p-lysoPC was 2.28+/-0.07, significantly different from the system with inhibited NEST, due to NEST hydrolysis. Likewise, a system without NEST containing the amount of palmitic acid that would have been produced by NEST hydrolysis of p-lysoPC was identical at 2.26+/-0.06. These results indicate that NTEs catalytic activity can alter membrane fluidity.

Biomimetic interfaces for a multifunctional biosensor array microsystem

Bioelectronic interfaces that allow dehydrogenase enzymes to electrically communicate with electrodes have potential applications in the development of biosensors and biocatalytic reactors. A fully scalable, post-CMOS-compatible, three-electrode interface to biochemical sensors, consisting of Ti/Au working and auxiliary electrodes and a Ti/Au/Ag/AgCl reference electrode, has been developed. Also described is a tri-functional linking molecule that binds the mediator and cofactor to the electrode in a unique spatial arrangement in which the dehydrogenase enzyme can bind to cofactor and multistep electron transfer between the electrode and enzyme is achieved. This approach provides greater flexibility in assembling complex bioelectronic interfaces than is possible with previously reported, linear linking molecules. A cysteine molecule was self-assembled on a gold electrode via a thiol bond. The electron mediator toluidine blue O (TBO) and the cofactor, /spl beta/-nicotinamide adenine dinucleotide phosphate (NADP/sup +/) were chemically attached to cysteine via the formation of amide bonds. Cyclic voltammetry, was used to demonstrate the electrical activity, and enzymatic activity of the resulting bioelectronic interface.

Effect of hydrogen bonding on the rotational and translational dynamics of a headgroup-bound chromophore in bilayer lipid membranes.

We have studied the interactions of the chromophore 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-7-nitro-2-1,3-benzoxadiazol-4-yl (18:1 NBD-PE) imbedded in the headgroup region of bilayer lipid membranes consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG). We have examined the molecular and mesoscale dynamics of the chromophore using time-correlated single photon counting (TCSPC) to measure rotational diffusion dynamics in lipid vesicles and fluorescence recovery after pattern photobleaching (FRAPP) to determine translational diffusion coefficients and mobile fractions in supported lipid bilayers. TCSPC data reveal that chromophore rotational diffusion rates in DOPG vesicles are statistically the same as in DOPC and mixed DOPC/DOPG vesicles, suggesting that the NBD-PE chromophore does not interact strongly with the headgroup region of these bilayers; however, FRAPP experiments show that lateral diffusion is statistically lower in mixed DOPC/DOPG-supported bilayers than in DOPC-supported bilayers. These results suggest that bilayers containing DOPG likely undergo interlipid headgroup hydrogen bonding interactions that suppress translational diffusion.

Prediction of temperature profiles in twin screw extruders

Abstract A model incorporating viscous dissipation effects and a heat transfer coefficient based on the Brinkman and Graetz numbers is presented for predicting the temperature profiles of non-Newtonian food doughs in a twin-screw extruder, assuming uniform product temperatures in the direction normal to the screw shafts. Experimental measurements were obtained to evaluate the model for three screw configurations: 30° forwarding paddles (30F), feed (two-start or double-flighted) screws and single-start (single-flighted or single-lead) screws. Model predictions were well within engineering accuracy for 30F paddles and feed screws under all experimental conditions. Predictions for single-start screws were inaccurate, with deviations of up to 50%. In general, results indicate that the one-dimensional energy equation is sufficient for heat transfer analysis of extruder sections configured with mixing paddles and feed screws, particularly at high flow rates, high RPM, or combinations of the two variables. However, the level of mixing provided by single-start screws over the RPM and flow rates used in this study does not justify the assumption of uniform temperatures in the transverse direction. For these screws, at least a two-dimensional formulation of the energy equation must be used.

Post-CMOS Compatible Microfabrication of a Multi-Analyte Bioelectrochemical Sensor Array Microsystem

A multi-analyte electrochemical sensor array platform has been developed for protein-based biosensors utilizing post-CMOS compatible fabrication procedures that enables formation of single-chip biosensor array microsystems. The process was developed on a glass substrate to emulate the surface of a CMOS chip. A three-electrode system, including an array of gold working electrodes (WE) and an on-chip Ag/AgCl reference electrode (RE), was formed to facilitate electrochemical analysis protein-based interfaces. SU-8 epoxy resin was subsequently applied and patterned as an insulation and planerization layer before application of microfluidic channels used to self-assemble novel bio-interfaces on individual working electrodes. The protein-based bio-interfaces provide selectivity, and the electrode floorplan maximizes sensitivity and minimizes solution resistance errors. The electrodes on the prototype platform can be easily scaled to form ~100 electrodes on the surface of a 10 mm2 CMOS chip.

Characterization of the swelling of starch doughs during extrusion

Abstract A simple but practical model has been presented for assessing the die swell of extruded food doughs for a given process history and in the absence of puffing, as a function of die geometry, shear rate and dough rheology. Since there are no simple yet effective models which account for these different phenomena, a phenomenological approach was adopted by combining parts of several models to achieve the objective of the study. The method offers two important advantages to die swell analysis. First, all predictions are made solely on the basis of die geometry, die temperature, pressure drop across the die, and dough rheological properties, thereby correlating die swell pressure to key operating conditions. Secondly, it provides a basis for developing die swell criteria that may be useful for incorporation in process control algorithms.

Effects of surface activation on the structural and catalytic properties of ruthenium nanoparticles supported on mesoporous silica

Using colloid-based methods to prepare supported catalytic metallic nanoparticles (NPs) often faces the challenge of removing the stabilizer used during synthesis and activating the catalyst without modifying the particles or the support. We explored three surface activation protocols (thermal oxidation at 150 °C, thermal reduction at 350 °C, and argon-protected calcination at 650 °C) to activate ruthenium NPs supported on mesoporous silica (MSU-F), and assessed their effects on the structural and catalytic properties of the catalysts, and their activity by the aqueous phase hydrogenation of pyruvic acid. The NPs were synthesized by polyol reduction using poly-N-vinyl-2-pyrrolidone (PVP) as a stabilizer, and supported on MSU-F by sonication-assisted deposition. The NPs maintained their original morphology on the support during activation. Ar-protected calcination was the most efficient of the three for completely removing PVP from particle surfaces, and provided the highest degree of particle crystallinity and a metal dispersion comparable to commercial Ru/SiO2. Its catalytic performance was significantly higher than the other two protocols, although all three thermally activated catalysts achieved higher activity than the commercial catalyst at the same Ru loading. Post-reaction analysis also showed that the supported catalyst activated at 650 °C retained its morphology during the reaction, which is an important requirement for recyclability.

Statistical method to evaluate the critical Reynolds number for pseudoplastic fluids in tubes

Abstract An experimental, non-intrusive tube system with appropriate instrumentation for measuring pressure drop and mass flow rate was developed to investigate the departure from laminar flow of pseudoplastic fluids (aqueous solutions of sodium carboxymethylcellulose). A critical Reynolds number was selected to correspond to the intersection of the two lines joining the laminar and turbulent flow data on the pressure drop versus Reynolds number plots. The standard deviation of pressure drop increased at these critical points. Results also indicated large spikes at these points in the plots of variance ratio of pressure drop versus the critical Reynolds number. This phenomenon could form the statistical basis for establishing the departure from laminar flow. In the laminar region, standard deviations were low, suggesting calm, stable flow of the fluids, a well known characteristic of laminar flow.