1. Nanofluidic Microsystems for Advanced Biosample Preparation Ying-Chih Wang (ycwang@mit.edu) 1, Jianping Fu, Yong-Ak Song and Jongyoon Han 2,3 1 Department of Mechanical Engineering, 2 Biological Engineering Division, 3 Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02139 October 3 rd, 2006 Microfluidics Tech Fair 2006

2. Separation The need and the market for sample preparation  Market of Proteomics, $1.3 billion and growing (13% annually)  Greatest challenge in proteomics  Sample complexity (>20,000 different proteins)  Purification required  2D gel electrophoresis, $800 M in 2004 ($1.8B 2011)  Time and labor consuming  Poor recovery for low abundance sample after multiple steps  Consumers: Biologist, Pharmaceutical R&D, clinical diagnostics Fraction (Mass Spectrometry) Detection (2D gel analysis) IN

3. Our Approach (gel free) Complex peptide/protein mixture Sensing/Detection Droplet/Electrospray Size-based separation Charge-based separation Preconcentration + - - + - - + + Microchip Sample preparation in microfluidic chip

4. Our Core (Patented) Techniques  Nanofluidic molecular sieving Continuous biomolecule size separation  Microfluidic charge-based sorting Continuous biomolecule charge separation  Electrokinetic nanofluidic preconcentrator Rapid molecular trapping Enable rapid and economical low-abundant sample identification

5. Fabrication Method of Nanofluidic Devices Fabrication DO NOT need nanolithography Thin channel instead of Narrow channel Uniform, flat nanofluidic channel confirmed down to 20nm Pan Mao and Jongyoon Han, 2005, EECS / BE / MIT Making nanofluidic (20 nm) device using standard microfabrication techniques!

6. Core Technology I: Nanofluidic filter array for size-based separation

7. Molecular Separation in 2-D Nanofilter Arrays  Physically hinders protein migration  Continuous two- dimensional separation

8. Continuous flow separation video (All 20  Speed)  PCR marker.  E x =70 V/cm, E y =100 V/cm Ogston sieving, DNA (50 bp – 766 bp, 5 bands)  DNA - Hind III digest.  E x =380 V/cm, E y =400 V/cm Entropic trapping, DNA (2 kbp – 23 kbp, 6 bands) Large Small Small Large Size: 1960 µm 4080 µm Large Small  SDS-Protein complex.  E x =150 V/cm, E y =200 V/cm. Ogston sieving, protein complex (11 kDa vs. 116 kDa)  A general but unique size-based separation tool. Even larger DNA (~Mbp) possible with this method. J. Fu, A. Stevens, S. R. Tannenbaum & J. Han. subminted

9. Core Technology II: Charge separation driven by diffusion potential (no external power) c=200mM c=1mM Different diffusivities of the buffer ions generate a diffusion potential across the liquid junction Potential gradient (electric field) utilized for binary sorting

11. Ampholyte-free pI-based separation Continuous-flow operation Song, Y.-A., Hsu, S., Stevens, A.and Han, J. Anal. Chem. (2006).

12. Core Technology III: Preconcentration by Ion Selective Nanofluidic Channels Fabrication: Mao et al., Lab Chip, 2005, (8),837-844 Wang et al, Anal. Chem., 77, 4293 Electrical double layer overlapping: Device layout: 20 mm Allen, Bard “Electrochemical Methods” micro channel: cross section 1x10  m ~50  mx50  m length 1 -2 cm nano channel: cross section 40nm x20  m ~40  mx200  m length 100 -200  m

13. Preconcentration Mechanism t = 0 t = 40 min E T ( )

14. 10 5 10 7 Million-fold Protein Concentration Enhancement Regular, stable pore size contributes its long term stability Wang, Y.-C., Stevens, A. L.and Han, J. Anal. Chem. 77, 4293-4299 (2005).

15. Vision: The Integrated sample preparation device Size separation Charge Separation Pre-concentration pI-based Sorter size-based Sorter Protein Concentrator Silicon-based technologies make integration easier

16. Developing Timeline Our advantages:  Rapid biomolecule separation (<30 mins)  Minimum sample consumption (<1  l)  Automated solution for biomarker discovery/ tracking  Better recovery compares to 2D gel (no post processing)  Direct coupling to mass spectroscopy or immunoassay Principal Investigator: Jongyoon (Jay) Han, jyhan@mit.edu