Symposium III
Concurrent Tracks
Saturday, September 7








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

Refreshment Break with Exhibit and Poster Viewing



Saturday, September 7







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.

Refreshment Break with Exhibit and Poster Viewing


Saturday, September 7







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;

    • New technology requires significant energy, money and commitment to become established,

    • People with passion for the technology are key to its success,

    • Big partners may not have the answers or be able to provide better solutions to technology or marketing issues,

    • Regulatory compliance for new technologies can be tricky and therefore planned for early on in the development and company growth cycle.

Refreshment Break with Exhibit and Poster Viewing




Gain visibility for your research by participating in the poster session.
Posters will be judged by a Scientific Advisory Board; over 60 posters are expected to be submitted. Cash prizes will be awarded.
Please fill out the registration form, giving the poster title and the poster's primary author. All submissions will be reviewed for possible inclusion for poster presentation.
Click here for poster instructions
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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