Papers

AVS1996 Session BI+MM-MoM: Biosensor-Biology Interface

Monday, October 14, 1996 8:20 AM in Room 203A

Monday Morning

Start	Invited?	Item
8:20 AM		BI+MM-MoM-1 What's Chemical Sensing Have to Do with Interfaces? F. Bright, C. Ingersoll, J. Lundgren, J. Jordon (State University of New York, Buffalo) As a nation we spend billions of dollars on diagnostic assays annually. Unfortunately, most of these assays are performed in well-outfitted laboratories and require skilled personnel, large amounts of costly reagents, and often demand long analysis times to quantify clinically important analytes. In order to overcome the disadvantages inherent with this approach, one must develop advanced sensing/quantification schemes that are reliable, inexpensive, fast, simple to construct and operate, redundant and self-checking, accurate and precise, with analyte detection limits in the range of interest. In a generic biosensor, a surface-immobilized biomolecule or fragment thereof serves to selectively recognize the target analyte and the binding or conversion (if the analyte is a substrate) event leads eventually to an optical, mass, thermal, or electrochemical response (signal) that is associated to the equilibrium concentration of the analyte in the sample. Of course, there are many steps associated with the actual construction and development of any real world biosensor. For example, one must choose an appropriate biorecognition element to selectively "recognize" the analyte, one needs to investigate, develop, and/or decide upon available detection scheme, and one must 'immobilize' the biomolecule such that it retains its activity or affinity, it remains stable over time, and it can be reset/reused. The decision on a particular analyte-biorecognition element pair is seemingly straightforward. One choice depends on the analyte of interest, its concentration in the sample, and the availability of suitable, stable biorecognition elements and their activities or affinities. The choice of detection method depends on issues like dynamic range and the requisite detection limits needed for quantifying a particular analyte. Biorecognition element immobilization is NOT nearly so straightforward and to complicate matters further this step )often a series of steps) controls ultimately all analytical figures of merit associated with a given biosensor. This presentation will summarize our work on the behavior and performance of biorecognition elements immobilized at or in interfaces and discuss new immobilization schemes as such apply to the development of discrete analytical biosensors and biosensor arrays
9:00 AM		BI+MM-MoM-3 "Smart" Polymer-Protein Conjugates P. Stayton, A. Hoffman, Z. Ding, C. Long (University of Washington) "Stimuli-responsive" polymers exhibit reversible phase changes in water in response to small changes in temperature, pH or other physical, chemical or biochemical stimuli. Such responsive polymers have been conjugated to antibodies, Protein A and enzymes for phase-separation applications in affinity separations, immunoassays, and enzyme recovery and recycle. We report here a new concept based on conjugation of a responsive polymer to a specific site near the ligand-binding pocket of a genetically-engineered protein, which provides sensitive environmental control of the ligand-protein recognition process. For example, a temperature-sensitive polymer has been conjugated to a cysteine thiol in the biotin binding pocket of streptavidin, and the reversible, thermally-induced collapse of the polymer has been used as a "molecular gate" to control the association of biotin with streptavidin, which is bound on the surface of a microporous membrane. The environmentally-triggered "gating" and "switching" capabilities of these "intelligent" polymer-protein conjugates have many potential uses, including eluate-free recovery of affinity ligands, regeneration of "fouled" affinity biosensor surfaces, triggered release of therapeutics, and storage/retrieval of electro-optical information from spatial arrays of immobilized conjugates.
9:20 AM		BI+MM-MoM-4 An Electrochemical Method Towards Assembling Microscopic Arrays of Biological Receptors L. Tender, R. Worley, H. Fan (University of New Mexico); R. Harris (Commonwealth Biotechnologies, Inc.); G. Lopez (University of New Mexico) An Electrochemical method enabling patterning of many different biological receptors into prototype microscopic biosensor arrays will be described. This method involves the manipulation of self-assembled monolayers on gold substrates onto which receptors are either covalenty attached or adsorbed, is non-destructive to the activity of the receptors, and does not require micro-manipulation to deliver receptors and/or monolayer forming comounds to their intended array elements. The basis of this method is the selective desorption of a monolayer film or a monolayer receptor/film from one of many individually electrically addressable monolayer modified microscopic gold array elements and the subsequent sole re-modification of that specific element by immersion of the entire array into solution of a different self-assembling compound and/or receptor. It will be shown that, by sequentially re-modifying new array elements with different monolayer/receptor films in this manner, a multi receptor microscopic array cn be quiclky built up.
9:40 AM		BI+MM-MoM-5 Cell-Based Sensors D. Stenger (Naval Research Laboratory); J. Hickman (Science Applications International Corporation); G. Kovacs (Stanford University) We are building a self-contained system to allow automatous operation of a biosensor utilizing a living neuron as the sensing element. We are using neuroblastoma cells which act as sensing elements as well as transducers for toxin detection. The receptors on the cell act as the sensor which responds to toxins by stopping the electronic signal (action potentials) generated by the cell. The electrical signal (or lack thereof) is sensed by a microelectrode placed close to the neuronal-derived cell and then is amplified by the accompanying microelectronics. We have developed strategies to manipulate the cell to increase desired operational properties. We have also engineered systems for signal amplification, conditions for long-term longevity (>3 months), and automatous microfluids operation. These systems will be described in detail as well as the results generated with biological and chemical toxins.
10:00 AM		BI+MM-MoM-6 Combinatorial Chemistry H. Geysen (Glaxo Wellcome, Inc.) Combinatorial approaches in chemistry and biology have in the last 15 years revolutionized the problem of locating protein epitopes responsible for eliciting biological function, and for finding small molecule inhibitors of those functions.The initial focus of the parallel synthetic procedures producing libraries of peptides was to identify epitopes (both sequential and discontinuous) of proteins inducing an antibody response. These techniques were rapidly extended to include epitopes important in activities of T-cells, and there after the identification of short peptides which inhibited receptor ligand binding.Once the principles of combinatorial chemistry were well understood, their application to the synthesis of small organic molecule libraries suitable for use in the drug discovery process has and is revolutionizing all aspects of the drug discovery process.However, even with the explosion of chemistries, automated synthesis and assay machines and techniques now available, many challenges still face this rapidly developing field.These include: - Encoding and decoding strategies - More efficient interface between chemical libraries and the ever increasing variety of biological assays - Parallelization of routine chemical procedures - Library design - Data handling and analysis
10:40 AM		BI+MM-MoM-8 Advantages of Miniaturizing DNA-based Diagnostic Instrumentation M. Northrup, B. Beeman, D. Hadley, P. Landre, S. Lehew (Lawrence Livermore National Laboratory) Significant advantages can be attained by miniaturizing components of diagnostic instruments. Theory predicts huge gains can be made in efficiency and speed of analysis for chemical separation systems such as those used in chromatography and electrophoresis, and several research groups are taking advantage of these favorable scaling laws. Similar advantages are afforded by the miniaturization of chemical reactors allowing for new levels of performance and efficacy. We will show how these advantages are being used to build a miniature, low-cost, low-power, and high efficiency PCR instrument. In this report we detail the design and development of a miniature thermal cycling instrument for performing and detecting the polymerase chain reaction (PCR) that uses microfabricated, silicon-based reaction chambers. Several different reaction chamber designs have been modeled, built, and tested. Each design incorporates an integrated thin film heater, passive silicon cooling surfaces, and optical windows for detection of the reaction. A highly efficient, battery-operated controller has been implemented that shows significant improvements over commercial thermal cycling instrumentation. We have named the instrument the Miniature Analytical Thermal Cycling Instrument (MATCI) for convenience. The following technical demonstrations have been accomplished with the MATCI: 1) low power operation (average 1.2 W per reaction chamber), 2) high cycling speed (10x commercial instruments) with high product specificity, 3) multiplex (8 simultaneous amplicons) PCR, 4) real-time detection of DNA production with an LED and photodiode/CCD in the miniature system, 5) specific probe, energy-transfer-based detection of PCR product production (commercial system for the beta actin gene on human genomic DNA) with the diode detection system, 6) immobilized probe, reverse-dot-plot detection of products, 7) rapid amplification of viral, human genomic, and pathogenic bacteria targets, 8) the functionality of disposable plastic liners and 9) the combination of microPCR and microelectrophoresis (20 minute amplification and 1 minute separation/detection of the product). The results from the MATCI indicate a new ability to perform detailed studies of the reaction kinetics and improve the efficiency of this important diagnostic technique. Due to the use of microelectromechanical systems (MEMS) technology, we have shown that low- cost, portable, DNA-based, biotechnological and clinical diagnostic instrumentation is a reality. Work performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory, contract number W-7405-ENG-48. The authors acknowledge the support of the MEMS program of the Advanced Research Projects Agency. We would also like to acknowledge the collaboration of Roche Molecular Systems of Alameda, Ca.
11:20 AM		BI+MM-MoM-10 Influence of Surface Morphology in Surface-based DNA Computing W. Cai, L. Smith, R. Corn, A. Condon, A. Thiel, Q. Liu, Z. Guo, A. Frutos, J. Gray, Z. Fei, M. Lagally (University of Wisconsin, Madison) The possibility of using DNA combinatorial chemistry to build a highly parallel computer has recently been raised (1). Computations may be conducted by chemically searching through all possible solutions of a problem, each of which is represented by a DNA molecule with a unique base sequence. We have begun a project to assess the viability of this novel concept, using a surface-based approach, which is a promising paradigm in many areas of chemical synthesis and analysis, rather than the initially proposed test tube approach (1). At this stage, in order to search through 32 binary solutions, we have immobilized different DNA molecules with a unique 5-base sequence in 32 spots on a surface. The DNA molecules are searched in 5 cycles for the solutions, using chemical manipulations that correspond to operations in an algorithm. We have achieved the selection of the solution by single-base mismatch hybridization for 2 different molecules, and we can eliminate the remaining DNA molecule by exonuclease degradation. The answer would be read through polymerase chain reaction. A number of issues arise in immobilization of DNA on surfaces. For example, to investigate non-specific binding between the DNA and the surface, we show using atomic force microscopy that attachment efficiency is related to surface roughness for a range of substrates, including glass, gold films, and silicon wafers with a native oxide. We are also characterizing the evolution of the surface morphology with surface chemical modification, generally the first step in immobilizing DNA on the surface, demonstrating a general roughening of the surface by this step.
11:40 AM		BI+MM-MoM-11 Physical Characterization and Hybridization Reactions of Surface-Bound DNA Probes T. Herne (National Institute of Standards & Technology); M. Tarlov (National Institute of Standards &Technology); K. McKenney (National Institute of Standards & Technology) We have investigated the surface conformations and hybridization efficiency of single-stranded DNA on gold surfaces. A better understanding of surface immobilized DNA is critical for the development of array based genetic screening and sequencing devices. The thiol-derivatized DNA probes, abbreviated HS-DNA, are 25-mers with a thiol group and a 6-methylene group spacer at the 5-prime end of the DNA. The HS-DNA monolayer surfaces are analyzed using x-ray photoelectron spectroscopy (XPS), ellipsometry, Fourier-transform infrared spectroscopy, and \super 32\P radiolabeling. We have found that the adsorption of DNA depends strongly on ionic strength. No HS-DNA adsorbs on gold from a pure water solution. As the ionic strength is increased from 2.7 x 10\sub -4\ to 1.O M KH\sub 2\PO\sub 4\, the amount of adsorbed DNA increases dramatically (five-fold). We have determined that essentially all of the HS-DNA is adsorbed on gold specifically through the thiol functionality. The role of surface coverage in maximizing hybridization efficiency was also explored. We found that hybridization of the surface-bound probe is sterically and electrostatically hindered on the tightly-packed, pure HS-DNA monolayer. Surface coverage of the probe was varied by producing mixed monolayers of HS-DNA and another thiol molecule, mercaptohexanol (MCH). MCH served another important role, in that it displaces non-specifically bound probe. Using \super 32\P radiolabeled complement, the hybridization efficiency of the surface bound probes was determined as a function of surface coverage. The robustness of the surface-bound probe as a function of temperature was examined. Preliminary results on the melting behavior of surface bound probes will also be presented.