Novel Microfluidic Valving and Packaging Designs for Protein Containing Biochips

Transcription

1 Novel Microfluidic Valving and Packaging Designs for Protein Containing Biochips Chunmeng Lu and L. James Lee Department of Chemical and Biomolecular Engineering The Ohio State University

2 Lab-on on-a-chip O.R.C.A microfluids (Micronics, Inc.) LabCard TM (ACLARA BioSciences, Inc.) Microtiterplate Lilliput (STEAG microparts GmbH) NanoChip TM (Nanogen, Inc.) LabChip (Caliper, Inc.) Point of care -- fast response Small amount of sample < μl Parallel detection Information storage

3 Lab-on on-a-chip 10~100μm 10~10,000nm Application DNA/Gene chips PCR chips Enzyme assays Immunoassays Cell chips (cell reactors) Drug delivery systems Biological functions Micro/nano fluidic Platforms Micro/nano fluidics Fabrication Packaging Pumping Valving (Switching) Mixing/Diluting Metering (Splitting) Separation Master Making - CNC Machining - Lithography - DRIE - Electroplating Replication - Casting - Embossing - Injection molding Surface Modification Platform Bonding Reagent Loading Integration Control Circuit Sensors/Detectors

4 ELISA Enzyme-linked Immuno-Sorbent Assay Bioassays in life sciences research Food Safety detection of foodborne pathogens and toxins Biodiagnostics detection of cancer and immuno diseases Environmental pollutants National security detection of bio- and chemi-warfare agents $5-10B/yr. ELISA market for cancer, HIV, food and water detection!

5 Schematic of ELISA Incubation Washing Incubation Washing Incubation Washing Incubation Washing Blocking protein Antigen Conjugate Substrate 1 st antibody adsorption Blocking 1 st antibody Blocking protein Antigen Conjugate (2 nd antibody) Antibody - antigen reaction Enzyme Substrate Conjugate binding Detectable product Enzymatic reaction High Selectivity!! High Sensitivity!!

6 Conventional 96-well ELISA Time consuming (hours to 2~3 days) Immunoreaction is diffusion controlled Long incubation time (several hours) Relatively large reagent consumption (several hundred μl) Labor intensive Inconsistent results

7 CD Microfluidic Platform System Point of care -- fast response Small amount of sample -- < μl Parallel detection Information storage

8 Pumping and Valving Driving Force Centrifugal force Capillary Valve Balance of centrifugal force and capillary force Center of CD F C R 2 R 1 F S Chamber θ ω f b = π 2 γ sin θ ρ R ΔR d H

9 M. J. Madou, L.J. Lee, S. Daunert, K. W. Koelling, S. Lai, C-H Shih, Biomedical Microdevices, 2001 Pumping and Valving - Flow Sequencing Flow Sequencing 1400 Burst frequencies of different chambers based on DI water Burst Frequency (rpm) Design First CD Second CD Displacement in Optode Chamber # Chamber index: 1- Calibration 1; 2- Wash 1; 3- Calibration 2; 4- Wash 2; 5- Sample

10 CD-ELISA Chip Design Automation Parallel substance conjugate primary antibody washing solution blocking protein antigen waste measurement

11 Issues Design: dimension control; aspect ratio; multiple depth; many reservoirs Protein blocking: Valving Protein preloading: Bonding

12 CD Manufacturing Mask fabrication (metal mask) multi-layer photolithography (SU-8 mold) Assembly CAD design Electroplating (Nickel mold)

13 Design Issues Higher aspect ratio desirable Multiple depth is needed for a larger design window for 9-reservoir CD and bubble free sample loading Mold fabrication is more difficult Mold release First layer Second layer Electroplating Nickel mold

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15 Issues Flow sequence: dimension; aspect ratio; multiple depth, more reservoirs Protein blocking: Valving Protein preloading: Bonding

16 Protein Issues in Capillary Valving Surface property changes after protein blocking The valving capacity lost or reduced w/o protein blocking with protein blocking

17 Principle of Super-hydrophobicity Wensel s theory cos θ = ( γ γ ) / γ r sv sl lv (1) r=real surface area/projected surface area (roughness factor) Fig.1 Cassie s theory cosθ* = f cosθ (1 f ) (2) f is the solid fraction at the interface Fig.2

18 Plasma Treated and Surface Microstructured PMMA Typical PMMA θ~ 73 º Fluorine plasma (CHF3) treated PMMA surface θ ~ 108º Fluorine plasma treated micro-patterned PMMA surface θ > 160º, f=0.1

19 Effect of Protein Blocking on Contact Angle Treat method and substrate materials 0 min 5 min No plasma without protein treatment (PMMA) Picture of Teflon-PLA Edited image Young-Laplace Equation: γ sv γ sl cosθ = γ Lv Calculated result γlv γsl θ Reference Plane γsv No plasma with protein treatment (PMMA) No plasma with protein treatment (PLGA) No plasma with protein treatment (Tape) Controlled environment CCD camera Drop profile analysis Treatment BSA solution=1.0% Testing BSA solution=0.2% No plasma with protein treatment(coc) With plasma with protein treatment (PMMA) With plasma without protein treatment (PMMA) With plasma with protein (PMMA with microfeaturel)

20 Fishbone Design Based on Superhydrophobicity valve l Hydrophilic surface Protein solution d w Fishbone design Advantages: Protein-proof with superhydrophobic property Easy fabrication (embossing or injection molding) Easy alignment of bonding Protein solution With food color

21 Blocking Process Simulation (Flow-3D ) and Visualization Channel: 200µm) Flow speed: 2mm/s Hydrophobic Hydrophilic PMMA surface w/o plasma treatment m Hydrophobic surface with plasma treatment

22 calculated burst frequency Top view φ Top Hc φ Bottom F Bottom Side view PMMA F Right Wc θ F Left θ Tape f = ω / 2π = 2γ sinθ γ (cosθtop + cosθ Wc H c 2 4π ρδrr bottom ) 1/ 2 Side view Top and bottom view

23 Holding pressure test Micrometer Optical microscope Water tank Microchip Static pressure Centrifugal force

24 Experimental vs. theoretical Burst frequency Single channel -Syringe injection plus vacuum removal CD device -Centrifugal force Parameter Protein Treatment Type R1 (mm) R2 (mm) R_delta Width Valve wt% BSA soak Valve wt% BSA soak Valve3 0.1 wt% BSA soak Valve4 0.1 wt% BSA soak Valve5 0.1 wt% BSA soak Why discrepant? -bonding defect -local defect Depth(mm) (mn/m) Density Burst Freq loading defect Exp. Burst Freq Match? YES NO YES NO YES

25 Issues Flow sequence: dimension; aspect ratio; multiple depth; more reservoirs Protein issues: Valving Protein preloading: Bonding

26 Platform Bonding Methods Silicon/Glass/Metal Materials: Anodic bonding Fusion bonding Eutectic bonding Adhesive bonding Polymeric Materials: Welding (hot plate, laser, ultrasound) Lamination ( adhesive tape, film thermal bonding) Chemical (solvent) bonding Well developed in IC industry Usually high temperature, high voltage, or high pressure most not applicable to polymers Well developed in polymer industry Applicable mainly to relatively large features (several hundred microns) Typical dimensions in BioNEMS/MEMS applications: 10 nm ~ 100 μm

27 Surface T g of PS under CO 2 Particle Penetration Depth Surface Layer Thickness Surface T g Y. Yang, L.J. Lee

28 CO 2 Bonding Experimental Setup CO 2 gas Heating blocks N 2 gas Chip assembly Stopper CO2 bonding (200psi, 75psi, 1 hour)

29 CO 2 Bonding and Testing Results 140 Normalized Signal Intensity CO2 bonding (200psi, 75psi, 1 hour, PLGA interlayer) 0 Thermal CO2 Control CO2 bonded: 120umx150um Bonding Method (injection molded) Thermal lamination: 140ºC, 10sec

30 Collaboration with Ritek and Tecan BioLOC/OSU Microfluidics design Surface modification Reagent loading Packaging RITEK CD manufacturing Tecan Reader (detection/equipment) Software development