DNA Capture Into Solid-State Nanopores€¦ · Using osmotic pressure to enhance DNA capture Rate...
Transcript of DNA Capture Into Solid-State Nanopores€¦ · Using osmotic pressure to enhance DNA capture Rate...
DNA Capture Into Solid‐State Nanopores
Meni WanunuMeni Wanunu
ERBM4, Liege, 2008
Solid‐state nanopores for single‐molecule (SM) analysis
• Chemically robust (pH, solvents, ionic strengths, oxidizers, etc.)
• Physically robust (vibrations, pressure changes)
• Tunable size and interface
• Fixed coordinates (pore position always the same)
Capture rate determines sensitivity
h d f d f “ l l l ” lThousands of events are required for “single‐molecule” analysis
α‐hemolysin solid‐state pores
Song, 1996 Kim, 2006
Meller, 2003Chen, 2004,
DNA capture displays non‐obvious behavior3 5 nm SiN nanopore
Voltage DNA size
3.5 nm SiN nanopore
Temperature
1 nM 400 bp DNA
400 bp1,200 bp6,000 bp
D 1~η∝ RD 1
∝
DdcRc π2≠
Tη
gR
Q: How can we improve capture efficiency?
A: Pressure!
Using osmotic pressure to enhance DNA captureRate enhanced with sucrose or PEG in trans chamber
PEG 12,000water
30% sucrose
ΔP ≈ (RT vw ) ln(χ cis χ trans)wv = molar volume of water
χ = water molar fraction
See also: Gu et al, PNAS, 2003
Osmotic gradient causes fluid “bathtub” effect
320=IcR v
c ICapture rate in drain with flow
For a hemispherical point absorber (no bias):
321 −− ec vICapture rate in drain with flow
DIr vc π43= Defined capture radius
F l i (i β 0)
PLREPLI d
pv Δ≈+Δ=η
σπβκσ8
4
For purely osmotic case (i.e., β=0):
σ: osmolyte filtration coefficient
water
σ: osmolyte filtration coefficient,30% sucrose β: electro‐osmotic flow coefficient,
κ: pore conductivity
But: Sucrose ≤1 nmPore: 3.5 nm
There is always a compromise…
Increasing the Voltage:
• Increases capture rate, decreases signal duration
Decreasing temperature:
• Decreases ion mobility degrading the signal• Decreases ion mobility, degrading the signal
Addition of osmolytes:
• Increases viscosity, which decreases ion mobility
• Results in frequent pore clogging
• Limited examples of osmolytes, particularly for larger pores
Q2: How else can we improve capture efficiency?
A2: Salt gradient (in the right direction)
Salt gradient (in the right direction) enhances DNA capture
1M/1M 0.2M/1M 1M/0.2M
⎥⎦
⎤⎢⎣
⎡−= −
−
trans
ciso Cl
ClF
RTV log⎦⎣
Capture rate still scales with DNA concentration
Experiments reveal Poissonian δt distributions, linearity of Rc with [DNA]:
Enhancement not due to multiple blockades from same molecule
Picomolar/attomole detection of 8,000 bp
38 pM 8,000 bp
1 3 l ll
b
1‐3 μl cell
3.8 pM 8,000 bp
Q3: What about the translocation dynamics?
Translocations are slower with lower salt in cis chamber!
It’s not the gradient, it’s the ionic strength
20.1ms
8,000 bp
8.1ms
7.9ms
Going from 1M to 0 2M:Going from 1M to 0.2M:• 40% change for 400 bp• 280% change for 2,000 bp!
Summary
Capture in SiN pores:
• V, T dependence suggest diffusion isn’t the rate‐limiting step, but the time near
the pore!the pore!
Osmotic pressure:
• enhances DNA capture rate
• can be explained by osmotically‐driven convection
Salt gradient
d l d h d h• dynamics suggest electrostatic interactions dominate with reducing ionic strength
• capture rate is enhanced dramatically
This enables
• attomole detection
• working at physiological conditions
Acknowledgements
BU Harvard
Organizers
Amit Meller
Jason Sutin
Gautam Soni
David Nelson
Greg Lakatos
Center for Nanoscale Systems (CNS)Gautam Soni
Ben McNally
Alon Singer
Allison Squires
Center for Nanoscale Systems (CNS)
The rest of the Meller group
ERBM4 Boncelles BE, 2008