Jaime L. McClain

High-efficiency magnetic particle focusing using dielectrophoresis and magnetophoresis in a microfluidic device

We describe a novel technique that utilizes simultaneous implementation of dielectrophoresis (DEP) and magnetophoresis (MAP) to focus magnetic particles into streams for optical analysis of biological samples. This technique does not require sheath flow and utilizes a novel interdigitated electrode array chip that yields multiple streams of flowing magnetic particles in single-file columns. The MAP force placed particles in close proximity to the microelectrodes where they were subjected to a strong DEP force that generated the particle focusing effect. Particle focusing efficiency was improved using this combination DEP–MAP technique compared to DEP alone: particle stream widths were reduced ~47% and stream width variability was reduced 80% for focused streams of 8.5 µm diameter magnetic particles. 3 µm diameter magnetic particles were strongly focused with DEP–MAP (~4 µm wide streams with sub-µm variability in stream width) while DEP alone provided minimal focusing. Additional components of a prototype detection system were also demonstrated including an integrated magnetic pelleting component, a hand-held MHz frequency signal generator and a bench-top near-confocal microscope for optical analysis of flowing particles. Preliminary testing of a sandwich assay performed on the surface of magnetic particles showed 50 ppb detection levels of a surrogate biotoxin (ovalbumin) in a raw milk sample.

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.

Active MEMS Valves for Flow Control in a High-Pressure Micro-Gas-Analyzer

We present active electrostatic MEMS gas valves for Micro-Gas-Analyzer (MGA) flow control. These unique valves enable extremely low dead volume, highly integrated flow control chips for the MGA application, and potentially others (e.g., propulsion, pneumatic, and thermodynamic microsystems). We have demonstrated low leak rates ( <; 0.025 sccm, <; 0.0025 sccm on a similar passive valve design), high operating pressures 6.9×105 N/m2 (100 psig), a high-pressure record for valves of this size and type, and high flow rates (>; 25 sccm) using control voltages on the order of 100 V. The valve designs presented eliminate charge build-up issues associated with insulating materials and are closely tied to a base-lined microfabrication process (SUMMiT), allowing mass production. Using this process, which incorporates only CMOS compatible materials, eliminates outgassing and absorption problems inherent to microvalve designs that incorporate elastomers or organic bonding layers, and reduces contamination when the valve is part of the chemical analysis flowpath. The results obtained indicate that even higher performance level valves (>; 1.4 × 106 N/m2 or 200 psig operating pressure, at similar control voltage, flow rates, and leak rates) are possible.

Passive MEMS Valves With Preset Operating Pressures for Microgas Analyzer

In this paper, we present integrated disk-in-cage poppet valves with tuned spring stiffness for gas flow control of a microgas analyzer. The valves require zero power and close at preset offset pressures (0-35 psig) to switch from gas sample loading onto a preconcentrator to concentrated constituent sample injection into a microgas chromatograph. Air flow rates of 4.5 mL/min at pressures of - 2.5--5 psig (vacuum sample loading) were measured. Hydrogen leak rates of 0.1 muL/s (0.006 mL/min) were measured with valves closed at 15 psig. Analytical and numerical modeling was used to guide design of valve spring constants (ranging from 10 to 1500 N/m) that control the valve open position, flow rate, and closing pressure. The parameter design space is limited to a range of seat overlap, valve size, and spring stiffness that will allow adequate flow rate, sealing, and closing at predictable pressures. A linear curve defining closing pressure as a function of spring constant, valve gap, valve size, and seat overlap fit measured closing pressure data and can be used to predict closing pressure for future designs.

Low leak rate mems valves for micro-gas-analyzer flow control

We present MEMS polysilicon microvalves for flow control of a rapid analytical microsystem (Micro-Gas-Analyzer, MGA). All valve components (boss, seat, springs, electrodes, and stops) are surface micromachined in the SUMMiTTM microfabrication process. The valves have been characterized at high flow rate when open (60 ml/min air), low leak rate when closed (≪0.0025 ml/min Hydrogen, H2), and tunable closing pressures (1 to 35 psig). Active electrostatic valves have been shown to hold closed (voltage on) against a high pressure (≫40 psig) for sample loading, open for gas chromatograph (GC) loading (voltage off), and reclose against low pressure 2–5 psig.

Orthogonal, Spectroscopic High Throughput Screening of Laccase-Catalyzed p-Cresol Oxidation

There is considerable interest in the oxidative fate of phenols such as p-cresol as environmental pollutants and uremic toxins. We supply a menu of spectroscopic options for the high throughput screening of laccase oxidation of p-cresol through multiple modes of detection. Laccase activity was monitored kinetically at pH 4.5 by absorption changes at 250 nm, 274 nm or 297 nm, and in endpoint mode by the bathochromic shift in absorption to 326 nm in 50 mM NaOH. Laccase oxidation of p-cresol was also detected by product fluorescence at 425 nm after excitation at 262 nm or 322 nm in 50 mM NaOH. We optimized the kinetic parameters for p-cresol oxidation (pH optimum 4.5-5.1; 37 degrees C; Km = 2.2 mM) resulting in laccase limits of detection and quantitation of 25 pg/microL and 75 pg/microL, respectively (approximately 360 pM; 25 ppb). The sensitivity for p-cresol was similar to previously reported values. The small (approximately 20%) decrease in signal strength after six cycles of excitation over a 3 h period was attributed to photobleaching or photodegradation of the emitter and not due to fluorescence decay (photoinstability). Halide inhibition was characteristic of laccases (IC(50) = 25 mM NaCl). A unique advantage of our assay is that laccase catalysis could be interrogated using multi-mode absorption or fluorescence under acidic or basic conditions, in real time or endpoint modes. Orthogonal interrogation facilitates ratiometric analysis enabling high specificity while minimizing interferences during compound library screening. The phenolic alcohol p-cresol may be a model for monolignol oxidation. Our studies might find applications in biofuels, to triage dialysis patients, or for the environmental bioremediation of phenols.

Medically relevant ElectroNeedle technology development.

ElectroNeedles technology was developed as part of an earlier Grand Challenge effort on Bio-Micro Fuel Cell project. During this earlier work, the fabrication of the ElectroNeedles was accomplished along with proof-of-concept work on several electrochemically active analytes such as glucose, quinone and ferricyanide. Additionally, earlier work demonstrated technology potential in the field of immunosensors by specifically detecting Troponin, a cardiac biomarker. The current work focused upon fabrication process reproducibility of the ElectroNeedles and then using the devices to sensitively detect p-cresol, a biomarker for kidney failure or nephrotoxicity. Valuable lessons were learned regarding fabrication assurance and quality. The detection of p-cresol was accomplished by electrochemistry as well as using fluorescence to benchmark ElectroNeedles performance. Results from these studies will serve as a guide for the future fabrication processes involving ElectroNeedles as well as provide the groundwork necessary to expand technology applications. One paper has been accepted for publication acknowledging LDRD funding (K. E. Achyuthan et al, Comb. Chem. & HTS, 2008). We are exploring the scope for a second paper describing the applications potential of this technology.

Quantification of false positive reduction in nucleic acid purification on hemorrhagic fever DNA.

Columbia University has developed a sensitive highly multiplexed system for genetic identification of nucleic acid targets. The primary obstacle to implementing this technology is the high rate of false positives due to high levels of unbound reporters that remain within the system after hybridization. The ability to distinguish between free reporters and reporters bound to targets limits the use of this technology. We previously demonstrated a new electrokinetic method for binary separation of kb pair long DNA molecules and oligonucleotides. The purpose of this project 99864 is to take these previous demonstrations and further develop the technique and hardware for field use. Specifically, our objective was to implement separation in a heterogeneous sample (containing target DNA and background oligo), to perform the separation in a flow-based device, and to develop all of the components necessary for field testing a breadboard prototype system.