Disposable molecular diagnostics: Microfluidic laboratories for the field Catherine Klapperich, Ph.D. Biomedical Microdevices and Microenvironments Laboratory www.klapperichlab.org Biomedical and Manufacturing Engineering Departments Boston University 3 October 2006

Microfluidics Applications Diagnostics/Management Point of Care Disease Surveillance Detection Homeland Security Fighting Force Protection High Throughput Screening Drug Discovery/Development Cell Based Assays Research Bench Applications Micro-Reactions Combinatorial Methods Living Tissue Arrays Drug Development

Global Impact Case finding Case management Surveillance 24% of the current burden of disease could be averted if 80% of the population of low income countries received the following: prenatal/delivery care, family planning, treatment of TB, management of sick children and case management of STDs. Implementation would cost $8/person per year. Nature Reviews Microbiology 2, 231-240 (2004) DIAGNOSTICS FOR THE DEVELOPING WORLD

State of the Art Microscopy Cell Culture EIA Parasitic and mycobacterial infections Requires well trained technician Cell Culture EIA Nucleic acid amplification All require specialized equipment and/or technicians.

MicroTAS for Diagnostics Sample introduction Cell sorting/separation Mixing Lysis Separation/concentration Detection Waste stream capture Sample Preparation Input Fluidics Detection Output Control and Signal Processing

System Schematic Detection Surfaces/Channels Antibodies Oligos PCR

Device Design Constraints Inexpensive materials Rapid prototyping Scale up/mass production Shelf life of 1 year or more Ease of use On-board reagents Disposable Little sample preparation off chip Low power or no power

Materials Requirements Optical properties UV transparent (for quantifying proteins, DNA and RNA) Transparent to excitation and detection wavelengths (488 nm, FITC) Thermal properties For PCR (95 degrees Celsius) For dimensional stability Surface chemistry Hydrophilic/hydrophobic Non-binding Binds specific molecules Shelf life issues

Engineering Polymers for Microfluidic Diagnostic Devices PMMA ZEONEX and ZEONOR by ZEON Tg= 85-105C Zeonor 750R, Tg 70C Zeonex 690R Tg 136C Ring Opening Polymerization Polycarbonate Tg= 140-150C ZEON Polymers are obtained by ring-opening metathesis polymerization (ROMP) of norbornene derivatives monomers followed by complete hydrogenation of double bonds. Polystyrene TOPAS by TICONA and APEL by Mitsui Tg= 90-110C Addition Polymerization R1,R2,R3; H

Advantages of Thermoplastic Chips Feature size is identical to PDMS but with long term dimensional stability. Surface treatments are robust and do not “age” as on PDMS devices. Permeability is low. Thermoplastics can be purchased in grades that are certified non-pyrogenic (do not contain DNA or RNA destructive enzymes). The per device material cost is low. The plastic chips can be easily manufactured in-house using rapid prototyping techniques in production materials to test and optimize new chip layouts and chemistries quickly. Internal structures (filters, valves, detection patches) can be fabricated in situ by light-directed processes. Acrylics and cyclic polyolefins have low autofluorescence for high detection signal to noise ratios. Acrylics and cyclic polyolefins are transparent to UV, which enables light directed processing of internal structures and UV detection of nucleic acid concentrations and integrity through the chip.

Rapid Prototyping A cyclic polyolefin (Zeonex 690R) was used as chip material Microchip fabricated by hot-embossing with a silicon master Photoresist Pressure and Heat Applied Si Wafer Polymer pellets UV light Mask Embossed substrate DRIE Thermally bonded channels

Scale-up Fabrication Completed fluidic card Repeat Glass or Si wafer Photoresist coat Mask, expose, develop photoresist Etch glass Remove photoresist Etched glass plate Electroplate Cover and seal Plastic cover layer Separate Metal electroform Repeat 1000’s of times Separate Molded plastic card Mold or emboss

Light-directed Processing in Formed Channels

In Situ Filter and Column Formation

Nucleic Acid Extraction

Moving Fluid Pressure Vacuum Electroosmotic Flow Surface Chemistry of Channels Simultaneous Assay and Device Development

Immobilized Surface Chemistries for Detection

Jessica Kaufman Arpita Bhattacharyya Justyn Jaworski Nathan Spencer Dominika Kulinski Dave Altman Amy VanHove Dr. Cassandra Noack Coulter Foundation Whitaker Foundation CIMIT NSF MUE Pria Diagnostics, Inc.