Publications

Sensors and Actuators, B: Chemical

Flemming, Jeb H.; Baca, Helen K.; Werner-Washburne, Margaret; Brozik, Susan M.; López, Gabriel P.

We present and evaluate a new approach to cell immobilization for use in cell-based biosensors. We have fabricated a microfluidic channel using poly(dimethylsiloxane) (PDMS) with cell entrapment posts for the gentle packing and immobilization of yeast cells. This method of immobilization allows for a density of metabolically active cells greater than 8.0 × 106 cells/mm3. The packed microcolumn approach addresses simple diffusional limitations inherent in traditional suspension and membrane entrapment techniques. By utilizing genetically engineered whole cells, rather then cellular components, the sensor is capable of detecting and responding to a wide range of biologically active compounds. In this study, Saccharomyces cerevisiae was genetically engineered to produce yellow fluorescent protein (YFP) when exposed to galactose. Fluorescence response of packed cells (G 1 phase) to galactose required 40% longer than the fluorescent response of cells grown in suspension. To address concerns of long-term viability (>60 days) and cell overgrowth, stationary phase cells were also tested in the microfluidic channel. Response time required approximately 50% longer than non-stationary phase cells packed inside the microfluidic channel. Additionally, cellular response as a function of the target analyte concentration was investigated and response time versus analyte concentration is reported. This report demonstrates proof-of-concept of using protein expression-based biosensors, based upon a packed, microcolumn architecture, as a dependable long-term storage platform. © 2005 Elsevier B.V. All rights reserved.

Apblett, Christopher A.; Novak, James L.; Hudgens, James J.; Podgorski, Jason R.; Brozik, Susan M.; Flemming, Jeb H.; Ingersoll, David I.; Eisenbies, Stephen E.; Shul, Randy J.; Cornelius, Christopher J.; Fujimoto, Cy F.; Schubert, William K.; Hickner, Michael A.; Volponi, Joanne V.; Kelley, Michael J.; Zavadil, Kevin R.; Staiger, Chad S.; Dolan, Patricia L.; Harper, Jason C.; Doughty, Daniel H.; Casalnuovo, Stephen A.; Kelley, John B.; Simmons, Blake S.; Borek, Theodore T.; Meserole, Stephen M.; Alam, Todd M.; Cherry, Brian B.; Roberts, Greg

Abstract not provided.

Proposed for publication in Science.

Brinker, C.J.; Fan, Hongyou F.; Flemming, Jeb H.; Dunphy, Darren R.; Singh, Seema S.; Brozik, Susan M.

Abstract not provided.

Brozik, Susan M.; Carles, Elizabeth L.; Flemming, Jeb H.; Bachand, George B.; Frink, Laura J.

The challenge of modeling the organization and function of biological membranes on a solid support has received considerable attention in recent years, primarily driven by potential applications in biosensor design. Affinity-based biosensors show great promise for extremely sensitive detection of BW agents and toxins. Receptor molecules have been successfully incorporated into phospholipid bilayers supported on sensing platforms. However, a collective body of data detailing a mechanistic understanding of membrane processes involved in receptor-substrate interactions and the competition between localized perturbations and delocalized responses resulting in reorganization of transmembrane protein structure, has yet to be produced. This report describes a systematic procedure to develop detailed correlation between (recognition-induced) protein restructuring and function of a ligand gated ion channel by combining single molecule fluorescence spectroscopy and single channel current recordings. This document is divided into three sections: (1) reported are the thermodynamics and diffusion properties of gramicidin using single molecule fluorescence imaging and (2) preliminary work on the 5HT{sub 3} serotonin receptor. Thirdly, we describe the design and fabrication of a miniaturized platform using the concepts of these two technologies (spectroscopic and single channel electrochemical techniques) for single molecule analysis, with a longer term goal of using the physical and electronic changes caused by a specific molecular recognition event as a transduction pathway in affinity based biosensors for biotoxin detection.

Brinker, C.J.; Sinclair, Michael B.; Timlin, Jerilyn A.; Cesarano, Joseph C.; Brinker, C.J.; Baca, Helen K.; Flemming, Jeb H.; Dunphy, Darren R.; Brozik, Susan M.; Werner-Washburne, Margaret

This report summarizes the development of new biocompatible self-assembly procedures enabling the immobilization of genetically engineered cells in a compact, self-sustaining, remotely addressable sensor platform. We used evaporation induced self-assembly (EISA) to immobilize cells within periodic silica nanostructures, characterized by unimodal pore sizes and pore connectivity, that can be patterned using ink-jet printing or photo patterning. We constructed cell lines for the expression of fluorescent proteins and induced reporter protein expression in immobilized cells. We investigated the role of the abiotic/biotic interface during cell-mediated self-assembly of synthetic materials.

Wong, Chungnin C.; Flemming, Jeb H.; Manginell, Ronald P.; Kottenstette, Richard K.; Frye-Mason, Gregory C.

Preconcentration is a critical analytical procedure when designing a microsystem for trace chemical detection, because it can purify a sample mixture and boost the small analyte concentration to a much higher level allowing a better analysis. This paper describes the development of a micro-fabricated planar preconcentrator for the {mu}ChemLab{trademark} at Sandia. To guide the design, an analytical model to predict the analyte transport, adsorption and resorption process in the preconcentrator has been developed. Experiments have also been conducted to analyze the adsorption and resorption process and to validate the model. This combined effort of modeling, simulation, and testing has led us to build a reliable, efficient preconcentrator with good performance.