Symposium II
Concurrent Tracks
11:00am-2:00pm
Saturday, September 7

 

TRACK 1

MICROARRAYS AND BIOCHIPS

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Symposium Chairperson
Dr. Ernest Kawasaki, National Cancer Institute

11:00 Microarrays: Where Do We Go From Here?
Mr. Neil Winegarden, University Health Network in Toronto
Microarray technology has rapidly taken over as the de facto tool for monitoring gene expression. This technology has proven to be the most effective realisation of functional genomics approaches to date. With the advent of microarrays, scientists have switched from focusing on a handful of genes at a time to assessing the activity of large sets of genes, in some organisms all the genes, at one time. Concomitant with this change in focus is a paradigm shift that represents a change from purely hypothesis driven research to hypothesis generating research. Classical technologies require that researchers decide ahead of time which genes they believe are involved in a particular process. With microarrays, it is not necessary to make these decisions ahead of time.

Microarrays are attractive tools because they allow researchers to assay many thousands of genes at a time in an efficient and robust manner. Although this technology provides a powerful tool that is widely utilised for gene expression, it is just beginning to find applications outside of genomics. Now researchers are applying the lessons learned from DNA microarrays to proteins, cells, tissues and even small molecules. The advantages of microarray technology make adaptation to these other areas of research highly attractive.

Despite the relative maturity of the DNA microarray, there are still issues that need to be addressed, and these new adaptations of microarrays provide even greater challenges. New technologies and approaches that improve manufacturing efficiency, reduce variability, and increase sensitivity will be essential to provide microarray technology even greater potential.

Although the name microarray indicates that these tools are very small in nature, they still lack the density that may be required for some applications. Current technologies allow deposition of several thousand spots; in some cases 30 to 40 thousand spots may be placed on a single one by three inch microscope slide. This number, although seeming an impressive quantity, is far below what may be necessary for some technologies. The human genome is thought to contain 30 to 40 thousand genes. As such, a microarray could potentially be manufactured to contain a probe for every gene. However, it is believed that with alternative splicing, polymorphisms and other modifications, there are hundreds of thousands of different RNAs, which could potentially be involved, in the human transcriptome. This gains a further level of complexity when we look at proteins, which involve post-translational modifications. As such, there may be millions of protein in the human proteome. Current technologies would not allow a single microarray to contain all of these products. Integration of current microarray technologies with advances in microfluidics, and nanotechnology will allow this tool to mature to a level that will allow microarrays to find a roll in other aspects of genomics, proteomics and drug discovery.

11:20 The Use of Short Oligos, Long Oligos, and cDNA Clones In the Analysis of Gene Expression with Microarrays
Dr. Ernest Kawasaki, National Cancer Institute
One of the goals of NCI Advanced Technology Center (ATC) is to provide the NCI researcher with the latest technologies for the analysis of gene expression using microarrays. Several methodologies have evolved over the years for this purpose. Originally, arrays were constructed from PCR products synthesized from cDNA clones. Concurrently, very high density, short oligonucleotide arrays were made in-situ by photolithographic methods similar to the construction of computer chips. These two methods have been the mainstay for array research until now. With the completion of the sequence of the human genome and many others, it is now a "simple" task to design relatively long oligonucleotide sequences that can cover the entire gene complement of a species. These oligo sequences range in size from 40 to 80 bases in length, and may possess some advantages over the other array types. With modern equipment and software, the design and synthesis of the oligos are relatively straightforward, and custom arrays can be developed in short order. Arrays made from these oligos can be hybridized with about the same stringency as arrays made from cDNA clones, and may have better performance characteristics due their single stranded nature. However, all new methods must be proven to be as good or better than standard technologies before being accepted for widespread use. The NCI ATC labs have begun a study to qualify the long oligo protocol in microarray gene expression experiments. The experimental design will include a ~17,000 70mer human array from Operon; a ~17,000 25mer array from Affymetrix; and a ~10,000 PCR product array from Incyte. The Operon and Incyte arrays will be printed in house and the 25mer arrays will be obtained from Affymetrix. Large amounts of RNA for probe synthesis are being prepared from two breast cancer cell lines and two prostate cancer lines, so that the same RNA can be used throughout the entire experiment. Expression of genes that are contained in all three array types will be analyzed, and a number of their corresponding mRNAs will be quantitated by real time RT-PCR to determine how well each system reflects actual expression levels. Published results and preliminary internal studies show that the long oligo system works quite well, and we hope to confirm the efficacy of these types of arrays and provide a solid basis for researchers to choose among the three different methodologies.

11:40 Advance in Protein Microarray Platforms
Dr. Karen Woodward, PerkinElmer Life Sciences
Protein microarrays hold the promise of a faster and more complete understanding of proteomes. Advances in this field will enhance drug discovery by improving target validation and lead optimization. Although the Human Proteome project is still far away, practical applications of protein microarrays in research is possible today using commercially available microarray instrumentation and consumables. PerkinElmer Life Sciences has developed an integrated product line that addresses the full range of needs for proteomics research in a microarray platform. Microarrays produced with PerkinElmer’s non-contact PiezotipnologyÔ dispensing have spots with uniform morphology and highly reproducible quantities of probe. HydroGelÔ coated slides comprise a proprietary three-dimensional matrix for probe immobilization in a protein-friendly, hydrophilic environment. Proteomics applications performed with this substrate demonstrate that the substrate has unsurpassed protein loading capacity, retains probe functionality, and exhibits low inherent fluorescence. The ScanArrayÔ confocal laser scanner is a versatile instrument for data capture that offers the use of a wide variety of fluorescent dyes for signal detection. Coupled with QuantArrayâ microanalysis software, this system provides one-step, automatable scanning and quantitation of data for bioinformatics analysis. A variety of protein assays in microarray format will be presented and compared to traditional, non-microarray methodologies.

12:00 Microarrays on Polymer Platforms: Genotyping, Expression Profilng, and Proteomics
Dr. Søren Møller, Exiqon A/S
We have developed a microarray platform based on a variety of technologies facilitating complex microarray studies of the human genome. 
These technologies include:
- LNA (Locked Nucleic Acids) - a DNA derivative with unprecedented high binding affinity and selectivity in hybridization based assays
- A polymer platform for construction of microarray slides and chips. The polymer format has been adapted to a microchip with integrated microfluidics for analysis of low volume samples
- Software tools for easy parallel design of highly complex microarrays - including data analysis
- Anthraquinone based photo-immobilization for homogeneous and polarized attachment of oligonucleotides to the polymer surface
Using these technologies we have demonstrated robust SNP genotyping, sensitive expression profiling and protein microarray applications.

12:20-1:00 Session I Poster Breakdown

12:20-2:00 Luncheon Co-Sponsored by Kluwer


1:00-2:00 Session II Poster Setup

 

 

Saturday, September 7

TRACK 2

BIOSENSORS

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Symposium Chairperson
Dr. Nitish V. Thakor, Johns Hopkins School of Medicine

11:00 Biosensors and Biochips: From Research at the Single-Cell Level to the Clinical Potential
Dr. Tuan Vo-Dinh, Oak Ridge National Laboratory
This presentation will discuss the development and application of advanced biosensors and biochips for biomedical diagnostics. An important area in chemical and biological sensing is the sensitive detection and selective identification of biochemical compounds (carcinogens, metabolites, proteins, etc.) or living systems (bioaerosols, bacteria, viruses or related components) at ultra-trace levels in complex biological systems, tissues and organs. The development of biosensors is aimed at providing selective identification of biochemical compounds at ultra-trace levels biological systems (e.g., bacteria, viruses, cells, or tissue components).

We will discuss the development of biosensors and nanosensors with antibody probes, which have recently been developed for the detection of biological species or xenobiotic chemical compounds in a single cell. Combining the exquisite specificity of biological recognition probes and the excellent sensitivity of laser-based optical detection, these nanosensors are capable of detecting and differentiating biochemical constituents of complex systems in order to provide unambiguous identification and accurate quantitation in a single cell. The development of nanosensors opens new horizons to biomolecular research at the single-cell level, and permits the ability to probe the intact cellular architecture for biomedical applications.

We will discuss the development of an integrated Multi-functional Biochip (MFB), which allows simultaneous detection of several disease end-points using different bioreceptors such as DNA, antibodies, enzymes, cellular probes) on a single biochip system. An important element in the development of the MFB involves the design and development of an integrated circuit (IC) electro-optic system for the microchip detection elements using the complementary metal oxide silicon (CMOS) technology. With this technology, highly integrated biochips are made possible partly through the capability of fabricating multiple optical sensing elements and microelectronics on a single system. The MFB device is a self-contained system based on an integrated circuit including photodiode sensor arrays, electronics, amplifiers, discriminators and logic circuitry on board. The highly integrated biochip is produced using the capability of fabricating multiple optical sensing elements and microelectronics for up to 100 sensing channels on a single IC. Probe recognition is based on the nucleic acid hybridization process (DNA probes) as well as on the immunological binding process (antibody probes). Biologically active probes are directly immobilized on optical transducers, which allow detection of fluorescent probe labels. The usefulness and potential applications of biochips in clinical diagnostics at the point-of-care will be discussed.

11:20 Spore-Based Biosensor for Detecting and Identifying Bacteria in Real Time
Dr. Boris Rotman, BCR Corporation and Brown University
We have developed a real-time biosensing system using microbial spores as nanodetectors responding to proximal bacterial cells by emitting fluorescent light signals.1 As compared to other cells, spores have unique levels of functionality particularly suited for biosensing, i.e., they are extremely rugged cells without detectable metabolic activity (dormant), but still capable of responding to specific external stimuli (germinants) by rapidly reactivating normal-cell functions.

The biosensor’s operating system, termed LEXSAS™ (Label-free Exponential Signal-Amplification System), consists of spores suspended in buffer containing a "germinogenic" substrate and diacetate fluorescein (DAF). Germinogenic indicates an enzyme substrate producing a germinant upon catalysis. It is critical that germinogenic substrates do not stimulate per se spore germination. Inosine-5’-phosphate (IP), for example, is germinogenic because it produces inosine (a germinant) when hydrolyzed by phosphatases. IP is a general germinogenic substrate because phosphatases are ubiquitous in bacteria but are not expressed in spores. DAF is a universal fluorogenic substrate of esterases and functions as a fluorescent indicator.

The LEXSAS™ principle is illustrated using IP as an example. A bacterial analyte triggers a chain reaction among the spores as follows: (1) bacteria hydrolyze IP producing inosine which stimulates spores near the analyte to germinate and produce de novo phosphatase; (2) the de novo phosphatase activity generates more inosine (from IP), which in turn, produces more de novo phosphatase activity; (3) the chain reaction is monitored using fluorescence produced by DAF hydrolysis due to de novo acetyl esterase activity which parallels the de novo phosphatase activity. The fluorescence produced in the LEXSAS™ is digitally captured at time intervals and converted to kinetic data profiles that are used for detecting and identifying the bacteria in the sample.

A feature of the LEXSAS™ is that assay sensitivity can be simply increased by reducing the reaction volume. For example, real-time detection of a single bacterium becomes feasible by reducing the reaction volume to about 5-picoliter (5 x 10-12 L). Other properties of the biosensor include low cost, ability to test samples with little or no preparation, portability, applicability to automated high-throughput testing, and linear dynamic range extending from 1 to 10,000 bacterial cells per sample.

The LEXSAS™ is designed to operate in a chip (20 mm dia) consisting of an array of tens of thousands of microsieves of 5-picoliter volume each. The chip serves two important functions: (1) it eliminates soluble materials (or small particles) that may be present in the sample, and (2) the array configuration allows for conducting tens of thousands of parallel assays using a charge-coupled device (CCD) imaging system.

A real-time bacteriologic biosensor would have a critical impact in public health and other major industries now relying on culture-based testing that usually requires 16-48 hours for completion. Potential applications of the technology include: clinical diagnostics; bioterrorism defense, testing blood products intended for transfusion; screening food and beverages; environmental monitoring; and sterility testing.

11:40 Neurally Inspired Sensors and VLSI
Dr. Nitish V. Thakor
Neurons not only function through their electrical connectivity but also through their chemical connectivity. Neurotransmitters bind to receptors on neurons, produce electrical excitation, which eventually results in excitation and firing of the neuron. Neurotransmitters are quite sensitive to pathological response of the neurons as well as the environmental influences from toxins. Therefore, a neurally inspired sensor and circuit interface would be expected to have the sensitivity and selectivity of such environmental influences and could form the basis for a biomimetic sensor.

A variety of neurotransmitters and neuronal messengers, such as dopamine and Nitric Oxide, respectively, influence the electrical excitability of neurons. A neurally inspired sensor would transduce the association of the neurochemical and produce a small electrical current. The neurally inspired interface would transduce the electrical current and produce a suitable output in the form of a voltage, digital data stream or even a spike pattern. To meet this objective, we have proposed and developed a neurochemical sensor and a and a very large scale integrated (VLSI) circuit chip circuit interface.

The sensor comprises of a novel microfabricated carbon sensor coated with suitable polymers to impart sensitivity and selectivity. The sensor is fabricated using a novel screen printing process in which carbon ink is patterned over photolithographically placed electrodes. The sensors are made selective to the appropriate neurotransmitters by coating polymers such as Nafion, and O-phenyl-diamine. Further, application of appropriate redox potential in 0.6 to 0.9 results in current due to the selected neurotransmitter. Our sensor has been optimized for sensing NO, and in addition, to sense NO from multiple sources, an array of NO sensors has been fabricated.

The VLSI interface is a potentiostat chip that converts the small current produced at the sensor due to redox reaction, into a proportional voltage. The chip circuitry is designed using current mode circuit design principles to make it relatively noise immune and low power. In this first design, eight potentiostats are placed on a single chip, and the output of the potentiostats is then multiplexed and then digitized. Digitization is done using a sigma-delta analog-to-digial converter providing a very wide dynamic range and a high resolution. As a result, the output of this design is a serial pulse stream in digital fashion encoding the output of the electrochemical sensor. This design is quite amenable to further processing so that the output of the chip is presented in an encoded form, very much like that of a neuron.

Performance of the integrated sensor and VLSI chip system as well as the results of its in vitro and in vivo tests will be presented. Applications of this integrated sensor and VLSI system in mimicking and modeling neurons, in studying the response of brain to drugs and ischemic injury, as well as in cellular and neuronal toxicity research will be discussed.

Acknwoledgements: Research supported by grant MH62444 from the NIH and fellowship support from the Army Research Laboratory.

12:00 Design of a Subcutaneous Implantable Biochip
Dr. Anthony Guiseppi-Elie, Virginia Commonwealth University
The design and fabrication of an implantable amperometric biochip for the continuous in vivo monitoring of physiological analytes is described. The 2mm x 4mm x 0.5mm biochip (Figure 1) contains two platinum working enzyme electrodes that adopt the microbore design to minimize diffusional limitations associated with enzyme kinetics. This configuration permits either dual analyte sensing or a differential response analytical methodology during amperometric detection of a single analyte. The working enzyme electrodes are each complemented by a platinized platinum counter electrode with an effective area that is 125 times the working electrode area. A common silver reference electrode is patterned on the biochip to form the third electrode in a co-planar configuration. The biorecognition layer of the working electrodes was fabricated from a c. 1.0 m thick composite membrane of principally cross-linked poly(2-hydroxyethyl methacrylate) that also contains a derivatized polypyrrole component and a biomimetic methacrylate component with pendant phosphorylcholine groups. These two additional components were introduced to aid in interference screening and in vivo biocompatibility respectively. Preliminary studies revealed that similar composite hydrogel membranes were very effective in screening common physiologic interferents, ascorbic acid, uric acid and l-cysteine. The composite biorecognition membrane is rendered biospecific by the covalent immobilization of the appropriate oxidase enzyme during fabrication. Thus, by simply varying the nature of the immobilized enzyme in the composite membrane, biochips for continuous in vivo monitoring of various clinically important metabolites can be readily fabricated.

12:20-1:00 Session I Poster Breakdown

12:20-2:00 Luncheon Co-Sponsored by Kluwer

1:00-2:00 Session II Poster Setup

 

Saturday, September 7

TRACK 3

THERAPEUTIC MICRO/NANOTECHNOLOGY

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Symposium Chairperson
Dr. Tejal Desai, Boston University

11:00 Microsystems for Cellular Manipulation
Dr. Murat Okandan, Sandia National Laboratories
Micromachining technology has demonstrated the possibility and feasibility of intricate and complex mechanical structures. Ultimately, a true "system on a chip" will involve integration of microfluidic, mechanical, optical and electrical components, and our current technology development is aimed at providing the required core components for this integration. The microtransfection device is a demonstration tool for the feasibility and utility of such integration of mechanical, microfluidic and electrical components. Other prototype devices fabricated in this technology include separation sub-systems, valves, pumps and reaction chambers.

To deliver large molecules into cells, electroporation or chemical permeabilization methods are used to allow traversal of the cellular membrane. The microtransfection device is designed to mechanically disrupt the cell membrane to allow delivery or extraction of large molecules in a controllable, repeatable manner. An example application of this device would involve delivery of recombinant DNA, RNA, proteins, fluorescent molecules or other large molecules of interest into the cellular medium. This device can also be used to lyse cells with a known and repeatable physical mechanism to simplify following sample preparation steps.

We are continuing the development and characterization of devices for manipulation and characterization of cells and/or particles. This technology development effort and the resulting devices are expected to enable new and exciting research directions in biological and medical sciences.

11:20 Drug Delivery and Therapeutic Applications of the Photothermal Properties of Gold Nanoshells
Dr. Naomi Halas, Rice University

11:40 Implantable Wireless Microsystems
Dr. Babak Ziaie, University of Minnesota
The ability to use wireless techniques for measurement and control of various physiological parameters inside human body has been a long-term goal of physicians and biologists. From early on, it was recognized that this capability could provide effective diagnostic, therapeutic, and prosthetic tools in physiological research and pathological intervention. However, this goal eluded scientists prior to the discovery of transistor in 1947. During the ensuing decades a variety of custom-made implantable devices with telemetry capabilities for various physiological measurements (e.g., temperature, pressure, force, flow, etc.) were developed. These were mostly designed and fabricated using hybrid techniques, thus making them bulky and in most cases with limited reliability and functionality. Recent advances in microelectromechanical (MEMS) based transducer and packaging technology, new and compact power sources (high efficiency inductive powering and miniature batteries) and CMOS low-power wireless integrated circuits have provided a major impetus to the development of wireless implantable microsystems. These advances have created new opportunities for increased reliability and functionality, which has been hard to achieve with pervious technologies. In this presentation, we will discuss some of these recent advances in the context of implantable neuromuscular prosthetic devices (microstimulator) and passive microtransponders for various physiological measurements. Various design issues such as power transfer, transducer design, and packaging will be discussed to demonstrate numerous possibilities afforded by some of these emerging technologies.

12:00 Kinetics of in Vivo Drug Release from Implantable MEMS Array
Dr. Michael J.Cima, Massachusetts Institute of Technology
It is well known that the method by which a drug is delivered can have a significant effect on the drug’s therapeutic efficacy. Most drugs have a concentration range in which they have maximum efficacy. Conventional drug delivery regimens result in sharp changes in systemic drug levels that can be toxic. Controlled drug delivery can alleviate the problems associated with conventional therapy by providing stable drug bioavailability in a therapeutically meaningful range and in addition can be used to localize the therapy to the tissue site of interest. We have shown that it is possible to fabricate a solid-state MEMS device in which a number of chemicals or drugs can be stored in individual micro-reservoirs and released on demand by electrochemically dissolving the gold cap in saline solutions with an external trigger. One advantage of this novel controlled release system is that it allows for simultaneous release of multiple drugs in complex release profiles. One can potentially develop a device that can be pre-programmed to deliver combinations of drugs in a pre-determined fashion. We believe that this novel delivery technology has broad utility in the biomedical areas such as local delivery of anesthetics for pain management, subdermal delivery of vaccines, periodontal delivery of antibiotic and anti-inflammatory agents, localized delivery of anti-tumor and neoplastic agents, gene delivery, delivery of antiarrhythmic agents. This talk reports on MEMS arrays that have been designed for the release of drugs in vivo. The spatial and temporal kinetics of in vivo release in a subcutaneous rat model are discussed. Devices with fluorescent dye were activated and the spatial profile of the dye release was examined by sectioning the surrounding tissue. Other devices with radiolabeled carmustine were activated multiple times and the temporal release profile assayed by blood analysis.

12:20-1:00 Session I Poster Breakdown

12:20-2:00 Luncheon Co-Sponsored by Kluwer

1:00-2:00 Session II Poster Setup

 

 


 

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PUBLICATION OF ARCHIVAL PAPERS IN BIOMEDICAL MICRODEVICES
Full-length papers based on podium or poster presentation at the conference will be reviewed for fast-track publication in Biomedical Microdevices. Submitted typed manuscripts are due at the conference.
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