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TRACK 1
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MICROARRAYS AND BIOCHIPS
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T2 |
T3 |
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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 |
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1:00-2:00 Session II Poster Setup
Saturday,
September 7
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
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12:20-2:00 Luncheon Co-Sponsored by Kluwer
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1:00-2:00 Session II Poster Setup
Saturday,
September 7
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TRACK 3
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THERAPEUTIC
MICRO/NANOTECHNOLOGY
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T1 |
T2 |
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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
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12:20-2:00 Luncheon Co-Sponsored by Kluwer
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1:00-2:00 Session II Poster Setup
