|
TRACK 1
|
MICRO/NANOTECHNOLOGIES FOR
BIODEFENSE
|
|
T2 |
T3 |
Home |
Symposium Chairperson
Dr. Mauro Ferrari, The Ohio State University
2:00 The Force Discrimination Biosensor: An
Automated Immunoassay System for Rapid, Multiplexed Detection of Biological
Agents
Ms. Christina Cole, Nova Research, Inc. and the Naval Research
Laboratory
A portable immunoassay system has been developed for rapid, multiplexed
detection of biological agents with a response time of <15 minutes and
sensitivity 3 orders of magnitude higher than conventional ELISAs. The sensor
uses a magnetic field gradient to differentiate between specific and
nonspecific ligand-receptor/ligand-surface interactions. Based on a standard
sandwich assay, the Force Discrimination Biosensor (FDB) uses generically
functionalized paramagnetic beads (~0.8µm dia.) as optically detected labels
in lieu of conventional chromophores. After labeling, a magnetic force is
applied to remove weakly bound beads; only specifically bound beads remain to
be counted, reducing the background and the frequency of false positives.
Originally developed as a microtitre plate-based assay,1 a
second-generation system has been developed using an alumina ultrafiltration
membrane. Key advantages of the membrane-based system include enhancements in
sensitivity of ~2 orders of magnitude, reduction in assay time by a factor of
10, and an increase in portability. Additionally, a microfluidics cassette has
been designed for the membrane sensor, allowing the system to be automated.
Specific surface chemistries based on polymers
known to resist nonspecific protein adsorption have been developed to
introduce desired functionalities onto commercially available membranes
without compromising porosity. Semi-quantitative bench-top diagnostic methods
have also been developed to specifically correlate poly(ethyleneimine) and
poly(ethylene glycol) (PEG) densities to assay performance and to assure
lot-to-lot reproducibility of the functionalized filters: over 2500 membranes
have been functionalized with an assay-determined variance of +/–1%. Pulsed
laser transfer technologies developed at NRL are being adapted to pattern
antibody conjugates onto PEGylated filters. Assuming an average element
dimension of 100 x 100 µm2 and a spacing of 200 µm between
elements, a 10 x 10 array can be written in 3 mm2, permitting the
high-density arraying of the FDB filter for any variety of
clinical/environmental monitoring or diagnostic applications. To date, we have
demonstrated detection of protein toxins, viruses, and bacteria with high
sensitivity (<10 pg/ml, <102 cfu/ml, and <102
pfu/ml, respectively) and high specificity (> 99 %).
Connections for all sample and reagent handling
are made via hypodermic needles through a disposable PDMS cassette. Together
with compact optical components, the entire system is contained in a portable
28x25x36 cm3 box and can be easily connected to a computer for data
analysis. Work has already begun on a version of the immunoassay using
fluorescent paramagnetic labels. Converting the FDB system from a bead
counting system to one that counts photons should significantly simplify the
optics requirements, enabling a more compact instrument that maintains the
automation and high sensitivity of the original system.
2:20 Bacterial Blocking Agents for Design of
Protein Biochips
Dr. Michael R. Ladisch, Purdue University
Proteins and antibodies immobilized on microfabricated surfaces of
computer chips with channels through which m L quantities of fluid are
conveyed, are called protein biochips. The wells contain electrodes that can
detect living organisms by measurement of impedance, an accepted method of the
Association of Official Analytical Chemists (AOAC) for detecting bacteria in
clinical, food, and industrial samples. A necessary characteristic of a
protein biochip is that adsorption of microorganisms, other than the target
organism, be blocked. Since impedance only detects the presence of organisms,
but is not capable of distinguishing between species, capture of a target
organism will be obscured if other (non-target) organisms are retained on
surfaces to which the bioreceptor is attached. The oxide surfaces of the chip
will adsorb a wide range of bacterial species, and hence the surfaces must be
modified to avoid this. This paper discusses the serum protein, bovine serum
albumin (BSA) or biotinylated BSA as the blocking agent. While BSA is a
traditional blocking agent for immunoassays, this work presents BSA-based
blocking for a different application, i.e. for the fabrication of protein
biochips. Successful layering of the protein on the oxide surfaces requires
wet cleaning followed by dervitization with a hydrophobic coating that in
turns enables BSA to be adsorbed. The use of biotinylated BSA as an anchor for
antibodies on the surface of the chips combines the benefits of a blocking
agent and facile method for attaching bioreceptors. The combination of steps
resulting in surfaces that minimize bacterial adsorption are described, and
applications of this technique for the design of protein biochips is
presented.
2:40 Electronic Taste Chips Customized for
Biodefense Applications
Dr. John T. McDevitt, University of Texas, Austin
Recent work from The University of Texas at Austin has led to the
development of a powerful new "electronic taste chip" technology. By
mimicking the chemical features of the human taste bud, the chip has the
capacity to analyze rapidly the chemical and biochemical content of complex
fluids such as human blood, environmental samples, and bioaerosol specimens.
This technology is extremely versatile, making it suitable for the measurement
of electrolytes, protein antigens, antibodies, whole bacteria and DNA/RNA.
While these chips exhibit impressive analytical and diagnostic capabilities as
compared with gold standards such as pH meters for acidity and ELISA for
protein analysis, their compact design and low cost also allows for their use
in numerous military and civilian applications which require autonomous
operation. Moreover, because molecular detection is confined to a miniaturized
chamber etched into a silicon chip, multiple tests can be performed
simultaneously. The technology has the capacity to be mass-produced in
commercial quantities at minimal cost. Testing requires a single drop of fluid
and disposable cartridges, customized for specific applications can be created
using highly parallel chip fabrication and solid-state bead synthetic
procedures. This electronic taste chip technology can be used to identify and
quantify analytes in the solution-phase via colorimetric and fluorescence
changes to receptor and indicator molecules that are covalently attached to
the polymer microspheres. Sensor arrays are created by placing individual
microspheres into micromachined cavities in small silicon chips. The optical
response of each microsphere is monitored in real-time using a charged coupled
device (CCD), allowing for near-real-time analysis of complex fluids. Most
recently, microbead arrays have been fashioned specifically for the detection
of chemical weapons precursors and degradation products as well as for the
identification of bacterial spores from the bacillus family.
3:00 Speaker to be Announced
3:20-4:30 POSTER SESSION II
Refreshment Break with Exhibit and Poster Viewing
Saturday,
September 7
|
TRACK 2
|
BIOMEMS MATERIALS AND
FABRICATION METHODS
|
|
T1 |
T3 |
Home |
Symposium Chairperson
Dr. Goretty Alonso Amigo, Arlanzon Technologies, Inc.
2:00 Fabrication of Modular Microfluidic
Systems Containing High-Aspect-Ratio Microstructures in Polymers for the
Detection of Point Mutations in K-Ras Oncogenes Associated with Colorectal
Cancers
Dr. Steven A. Soper, Louisiana State University
We are currently developing modular microsystems for various biological
assays, such as single cell analysis of their intracellular contents and DNA
diagnostics and DNA sequencing. The modular format allows assembling devices
with discrete functions to carry out complex assays. One project that we are
currently involved with is the design and development of microfluidic devices
to carry out ligase detection reactions (LDR) for identifying low abundant
mutations in K-ras genes associated with colorectal cancers. The assay
involves processing tissue biopsy samples using a primary PCR reaction, LDR
reaction, and interrogation of the LDR products using either
micro-electrophoresis or DNA microarrays. The microfluidic devices are
fabricated using LIGA to manufacture Ni molding dies (metal electroforms)
consisting of high aspect ratio microstructures (>10:1). Our devices are
made from polymers, such as PMMA or PC that are embossed or molded from these
dies. In this presentation, we will discuss several functional platforms that
have been developed and tested in our laboratories to carry out PCR/LDR assays
for low abundant mutations, such as PCR thermal cyclers, micro-electrophoresis
and microfluidic chips for performing hybridization-based assays. The PCR
devices consist of isothermal zones with fluid packets (~1 nL total volume)
shuttled through a spiral channel of 1.5 m in total length (total number of
thermal cycles = 20) with a width of 50 m m and depth of 150 m m. Fluid
modeling of both pressure induced pumping (hydrostatic) and electrically
driven flows (electroosmotic) will be discussed. Experimental results using a
m -PIV system will be presented as well to allow direct visualization of these
fluid packets traversing the spiral channel configuration. We will also
discuss the integration (assembly) of the thermal cyclers to
micro-electrophoresis and DNA microarray modules for analyzing the products
generated from these units. The micro-electrophoresis was accomplished in
chips embossed into PMMA and contained a 9.5 cm length channel that could sort
the LDR products (mutated DNA) from the unligated (normal) DNA using an
entangled polymer solution. For the microarray assays, modification
chemistries were developed so as to allow immobilization of short
oligonucleotide probes to the surface of an embossed- PMMA microchannel.
Following spotting of the probes, the device could be assembled with a cover
plate and then used to analyze LDR products generated with fluorescently-labeled
primers and read with a confocal microscope.
2:20 Generalized MicroIntegration Method of
Chemical and Biosystems: Methodology and Examples
Dr. Takehiko Kitamori, University of Tokyo and Kanagawa Academy of
Science and Technology
The micro chemical systems integrated on microchips are attracting much
attention because of their high throughput processing, volume reduction, and
other merits. However, the application range of the present technology based
on the electrophoresis and electroosmosis is not so wide.
We have developed the design, fabrication,
control, and detection methods for micro chemical processes on microchips for
general purposes. They are the micro unit operation (MUO) and continuous flow
chemical processing (CFCP) methods for design, the 2D and 3D multi-phase
layered flow network for fluidic and process control, and the thermal lens
microscope for ultrasensitive detection of non-fluorescent molecules. These
methods have enabled to integrate any kinds of chemical systems on micro chips
almost freely. A total bio screening system is introduced for one of the
example of this integration method.
Microchips for cell incubation and stimulation,
enzymatic reaction, diazo coupling reaction were developed individually
applying MUO and CFCP methods. And the functions of these microchips were
confirmed and the optimized. Then, the each functions of these micro chemical
systems were assembled on a single microchip. Consequently, the total
functions of this microchip realized the high throughput screening system of
chemicals. Incubated macrophages in the chip were stimulated by a certain
chemical, and the cellar release by this stimulation was introduced to the
enzymatic reaction area. NO in the cellar release was oxidized to nitrous
acid, and the nitrous acid was detected by the thermal lens microscope via
Saltzman reaction. The total function was proved and the screening time was
reduced from several days to several ours.
This integration method is similar to the
design and integration method of LSI of electronics. In the electronics
technology, functions of several ICs are integrated on custom LSI for
realizing the large scale and more complicated functional devices. The
integration methods introduced here is expected to develop more large,
complicated and high throughput micro chemical systems on microchips.
2:40 Design and Fabrciation of Polymer-Based
Microfluidic Devices
Dr. Ralf-Peter Peters, STEAG microParts GmbH
Microfluidic devices are currently more and more established in laboratory
equipment for biomedical research and will soon penetrate the diagnostic
market for point-of-care and lab automation applications. This development is
driven by the capability of microfluidic devices to perform complete assay
protocols with very low sample and reagent consumption as well as minimum
external actuation or other interference. Microfluidic reaction platforms in
microplate or microscopic slide format can be designed to integrate typical
assay steps like flow propulsion, dosing, mixing or washing. By the use of
these devices the instrumentation for sample handling, incubation and
detection can be simplified considerably. However, the acceptance of such
BioMEMS devices strongly correlates with important features like low cost,
easy handling, high reliability and a consistent and mature fabrication
technology.
STEAG microParts uses micro-replication
techniques such as micro injection molding for the mass fabrication of polymer
based microfluidic devices. Recent developments in molding and assembly allow
the fabrication of polymer devices with superior flatness and extremely low
fluorescent background. Providing well suited surface properties STEAG
microParts has developed novel polymer based devices for integrated DNA arrays
and assays. Results on the performance of these BioMEMS will be discussed. By
example of such integrated assays the use of proven design elements and rapid
prototyping methods will be presented.
For the manufacture of integrated microfluidic devices a precise plastic
bonding technology is of equal importance as micro-replication and surface
engineering. Laser welding will be presented as a possible method to weld
plastic microstructures with micron precision. Different laser welding
concepts will be compared.
3:00 Label Free Detection
Dr. Jacob Thaysen , Cantion A/S
Micro cantilevers with piezoresistive readout used as a sensitive
biochemical sensors is a highly interesting technology, since it offers a
label detection of molecules. Basically, a biochemical reaction at the
cantilever surface can be monitored as a bending of the cantilever due to a
change in the surface stress. The change in surface stress is then transformed
into a change in the integrated piezoresistor, which is easily monitored by
simple instrumentation. Since a very small bending of a cantilever can be
measured, this detection method has proven to be very sensitive. Due to the
simple readout technique, this technology is ideal for de-central analysis
where limited sample preparation and instrumentation is necessary.
An array of cantilevers is placed in a
microliquid handling system, and the cantilevers are coated with a 'detector
film' that reacts with the biomolecules of interest in a test sample. By
coating each cantilever in the array with different 'detector films', a
multiple of different biomolecules can be detected simultaneously.
The change in surface stress on the cantilever
surface is related to the change of Gibbs' free energy during the molecular
interaction between the biomolecules of interest and the 'detector film', and
the micro cantilevers can therefore be used for detection of a wide variety of
molecules like DNA, proteins, antibody, etc.
Micro cantilever based sensors offer a platform
for highly sensitive, label free molecular recognition on small sample
volumes, which could be interesting for point of care diagnostic.
3:20-4:30 POSTER SESSION II
Refreshment Break with Exhibit and Poster Viewing
Saturday,
September 7
|
TRACK 3
|
POINT-OF-CARE DIAGNOSTIC APPLICATIONS |
|
T1 |
T2 |
Home |
Symposium Chairperson
Dr. Carlo S. Effenhauser, Roche Diagnostics GmbH
2:00 Microfluidic Technologies for PCR-Based
Diagnostics
Dr. Sundaresh Brahmasandra, HandyLab Inc.
Despite considerable progress reported towards the development of the
so-called "Lab-on-a-Chip" systems, commercialization of these
systems has proved to be a daunting challenge. There have been several reasons
for this discrepancy, the prime one being the lack of a microfluidic
technology capable of integrating all the operations necessary for true
"lab-on-a-chip" operation. In our view, such a system should be
capable of accepting, as input, crude "real world" samples and
perform all the operations necessary to produce a meaningful/desired result
without any external equipment or human intervention. While there has been
significant progress made in developing microfluidic reaction and separation
systems, the challenge of integrating a sample concentration and preparation
component are only beginning to be tackled. In addition, many of the developed
components have proved to be incompatible and hence, difficult to integrate
into a complete system. Consequently, the realization of a truly integrated,
hands-free PCR-based diagnostic system poses several significant challenges to
the microfluidics community.
HandyLab has developed several proprietary
microfluidic technologies that will alleviate a number of the problems
associated with current miniaturized components. The HandyLab proprietary
digital microfluidic technology allows for all the operations associated with
"real world" analyses to be performed in disposable cartridge
mounted on a base-station. Internally generated pressure gradients are used to
create and propel nanoliter size liquid plugs in a microchannel network. All
the fluid handling operations such as target concentration, nanoliter liquid
metering, mixing, heating/cooling, and fluorescence detection are performed in
a closed microchannel network without any human intervention. In addition,
since discrete drop based systems can handle crude environmental samples and
require very low power, such systems are optimized for field/portable
operation. Results to date showing significant promise for the development of
a fully automated, completely integrated PCR-based diagnostic system for
portable or clinical lab setting will be presented in the talk.
2:20 Silicon Microneedle Disposables for
Glucose Monitoring
Dr. Wilson Smart, Kumetrix Inc.
A unique minimally invasive system for alternative site testing has been
developed for painless one-step diabetic blood glucose measurement. The novel
component of this system, a disposable microsampling and assay chip, consists
of a tough, flexible silicon microneedle comparable in cross-section to a
human hair integrated with a silicon microcuvette. The microneedles retain the
single crystal silicon structure of the starting wafer to preserve strength in
the finished device, employ surface treatments to retard the formation of
microcracks to maximize needle strength and flexibility, and can easily
penetrate skin with a large safety factor relative to brittle fracture. This
microneedle is capable of reliably taking a very small sample of whole blood
completely painlessly, unlike sticks with the much larger metal lancet that
must be used in all other current systems. The disposable chip is fabricated
by silicon MEMS technology and can be produced in high volume at low unit
cost.
The one-step blood sampling and assay procedure
avoids the need to transfer blood from a skin puncture to a test strip, draws
only the blood volume required for the assay, eliminates mess and cleanup, and
does not require good eyesight or manual dexterity. The small hand-held
instrument, which contains a cartridge of disposable chips, is touched to the
skin of the arm or any other part of the body, not necessarily the finger, and
held there for one second. During this time, the microneedle is automatically
advanced and then withdrawn by a precision electromechanical actuator under
microprocessor control, puncturing the skin and drawing 60 nanoliters of blood
into the microcuvette. The assay is performed in five seconds by an
electrochemical glucose biosensor integrated into the microcuvette. The
instrument calculates blood glucose concentration, displays the result, and
stores it in memory.
The biosensor provides a linear output signal
over the diabetic range of glucose concentration (0 to 400 milligrams per
deciliter), and is insensitive to acetaminophen, ascorbate, and uric acid.
Sensors are integrated into the devices at the wafer level of processing. The
fabrication process combines conventional MEMS techniques (photolithographic
patterning, etching, inorganic thin film deposition) with precise deposition
of the organic biosensor constituents in a smooth series of steps. Pick and
place methods are required only for the packaging step after dicing.
This product will provide diabetics with a
painless, one-step, easy to use method for performing blood glucose
self-testing without the complexity, excess blood, and pain associated with
the lancet and test strip procedure. These significant advantages are expected
to encourage diabetics to follow the recommendations of the NIH Diabetes
Control and Complications Trial (DCCT) for much more frequent testing, leading
to a significant reduction in diabetic complications (blindness, amputation,
kidney failure).
This novel technique for painless blood testing
is an enabling technology applicable to many other analytes. It will be of
particular benefit to patient populations such as: diabetics, where very
frequent testing is now recommended by endocrinologists; older people, where
skin fragility, susceptibility to tissue damage, and bleeding problems are
common; and infants, where the painful heel stick is now used. Work is
proceeding in our laboratory to extend this technology to continuous
monitoring.
2:40 POC for Molecular
Diagnostics
Dr. Kurt Petersen, Cepheid
Molecular diagnostics continues to experience rapid grow and continues to
be applied for new, innovative purposes. NAT (nucleic acid testing) is now in
general use for the entire US blood supply, testing for HIV and HCV. The
"home-brew" market, in which numerous clinical laboratories use PCR
to test for infectious diseases, antibiotic resistance, and genomic
characteristics, is large and thriving. Molecular diagnostics was employed
extensively during last year's anthrax crisis to determine the presence of
spores in nearly 100K samples. Molecular techniques are now being used to
detect and identify infectious organisms in plants and animals. The use of
molecular or genomic analysis for cancer detection is also a rapidly
developing new technology. In fact, new applications and targets for molecular
diagnostics are published on a continuous basis.
Most of this sophisticated genomic analysis is
performed in well-equipped clinical or research laboratories. Not only are the
test procedures manually intensive, but they also must be performed by skilled
technicians or scientists. Molecular diagnostics will never move out of the
scientific laboratory, into general use, until these complex, tedious, manual
processes are fully integrated into small, easy-to-use disposable cartridges.
Achieving this ambitious goal requires the
development and commercialization of four complex technologies. First, a
versatile methodology for "sample prep" is required. Sample prep
involves the fluidic processing of a complex biological sample such as blood,
urine, emulsified tissue or food, or swab eluants in order to separate,
concentrate and purify the nucleic acid of interest. PCR, for example, is
extremely sensitive to "inhibiting" chemicals such as heme (common
in blood) and humic acid (common in simple dust) which prevent the reaction
from performing properly. Even today's automated robotic equipment takes
several hours to perform this step.
Second, a methodology for rapidly performing
the PCR amplification and detection is required. Real-time PCR is the
technology by which the PCR amplification process can be quantitatively
monitored while the reaction is actually proceeding. Several companies have
introduced thermal cycling instruments which can heat and cool a PCR reaction
tube rapidly to complete 40 PCR temperature cycles in around 20 minutes.
Third, the real-time PCR technology and
instrument must be capable of monitoring multiple nucleic acid
"targets" simultaneously in the same reaction tube. This capability
allows multiple tests to be performed in the same reaction tube and also, very
significantly, allows a reaction (and, therefore, a result) to be
diagnostically validated in a single reaction tube.
Fourth, PCR is a very fickle chemistry. PCR
reagents left on a bench for a day are significantly degraded. Technicians
often make mistakes mixing the complex PCR mastermix, which may require 10
different chemicals, all at accurate concentrations. A methodology is required
to stabilize these reagents and provide easy-to-use, user-friendly versions of
these reaction materials.
This presentation will describe how all these
technologies can be accomplished and can be integrated together into a unique,
simple system capable of being deployed in a POC context. The system is called
the GeneXpert and is now being commercialized by Cepheid.
3:00 Near Bedside Testing Technology Market
and Stat's Lessons Learned
Mr. Steve Cozzette, i-Stat
The i-STAT near bedside testing system was launched nearly 10 years ago.
Being the first of its kind to offer a complete solution to fast TAT of the
most commonly ordered blood tests and with connectivity to the hospital
information system, i-STAT has created a new market. The core technology of
the system is the multi-analyte microfabricated biosensor chips that are
assembled into disposable cartridges. Each cartridge is self-calibrating and
requires less than 100 uL of whole blood. The STAT-1 Analyzer is the current
handheld instrument and has many added features over the original analyzer.
Some of these features include barcode patient ID input, QA/QC features, etc.
The Central Data Station (CDS) enables the consolidation of several handheld
units and provides the link to the hospital information system.
The Market described in Enterprise Analysis
Corporation’s 2001 U.S. Hospital Point-of-Care Survey is growing in both
numbers of hospitals implementing as well as depth in the hospitals that have
implemented POC. The CVOR, ICU and NICU have lead the adoption of this system
and technology. i-STAT is the clear leader with 54% of the POC market of blood
gas and electrolytes. Expansion of the diagnostic menu to include coagulation
and immunoassays is key to our strategy.
Some of the lessons learned in growing a
company with new technology and a new medical market included the following;
