Nanofluidic Microsystems for Advanced Biosample Preparation

Ying-Chih Wang (ycwang@mit.edu)1, Jianping Fu, Yong-Ak Song and Jongyoon Han 2,3

1Department of Mechanical Engineering, 2Biological Engineering Division, 3Department of Electrical Engineering and Computer Science,

Massachusetts Institute of Technology, Cambridge, MA 02139 October 3rd, 2006

Microfluidics Tech Fair 2006

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

Our Approach (gel free)

Complex peptide/protein mixture

Sensing/Detection

Droplet/Electrospray

Size-based separation

Charge-based separation

Preconcentration

+ -

- +

- -+

+

Microchip

Sample preparationin microfluidic chip

Our Core (Patented) Techniques

Nanofluidic molecular sieving Continuous biomolecule size separation

Microfluidic charge-based sortingContinuous biomolecule charge separation

Electrokinetic nanofluidic preconcentratorRapid molecular trapping

Enable rapid and economical low-abundant sample identification

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!

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

microfluidic buffer channels reservoir

1mm

samplereservoir

10µm

10µm 2µm

10µm

Molecular Separation in 2-D Nanofilter Arrays

Physically hinders protein migration

Continuous two- dimensional separation

Continuous flow separation video (All 20 Speed)

PCR marker. Ex=70 V/cm, Ey=100 V/cm

Ogston sieving, DNA (50 bp – 766 bp, 5 bands)

DNA - Hind III digest. Ex=380 V/cm, Ey=400 V/cm

Entropic trapping, DNA (2 kbp – 23 kbp, 6 bands)

Large Small Small LargeSize:

19

60

µm

19

60

µm

40

80

µm

Large Small

SDS-Protein complex. Ex=150 V/cm, Ey=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

Core Technology II:Charge separation driven by diffusion potential (no external power)

c=200mMc=1mM

• Different diffusivities of the buffer ions generate a diffusion potential across the liquid junction• Potential gradient (electric field) utilized for binary sorting

Ampholyte-free pI-based separation Continuous-flow operation

Song, Y.-A., Hsu, S., Stevens, A.and Han, J. Anal. Chem. (2006).

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 1x10m ~50mx50mlength 1 -2 cmnano channel:cross section 40nm x20m ~40mx200mlength 100 -200m

Preconcentration Mechanism

t = 0 t = 40 min

ET ( )

105 107

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).

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

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