UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

Andre MarzialiApplied Biophysics Laboratory

Department of Physics and AstronomyUniversity of British Columbia

Synthetic Nanopore Force-

Spectroscopy

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

• Receptor-Ligand Interactions- Avidin-biotin (model system)- Protein detection

• DNA-DNA Interactions- Genotyping (SNP)

• Protein Structural Transition- β-sheet melting/annealing

Nanopore force spectroscopy

b barrier

B

E f x

k T

D eτ τ

− ⋅∆

= ⋅

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

DNA Sequence Detection

Jonathan Nakane, Matthew Wiggin, Andre Marziali, Biophys J. 2004

Carolina Tropini, Andre Marziali, Biophys J. 2007

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

0.0001

0.001

0.01

0.1

1

10

100

1000

-100-90-80-70-60-50-40-30-20-100

mV

seconds .

3G12G 7C

7T 10C

PC rev PC

Dissociation time for the match is 100 times the nearest same base mismatch.

5 measurements per data point

Single Nucleotide Resolution

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

Nanopore-based genotyping

500 nM of target DNA

rs7242-G rs7242-T rs7242-GT --0.0

0.2

0.4

0.6

0.8

1.0

% c

alle

d

Haplotype

Demonstration on organic pores:

• SNP from gene SERPINE1 (rs7242) is associated with increase risk in SEPSIS patient

% called for SNP_G% called for SNP_T

1E-4 1E-3 0.01 0.1 1 10

1E-3

0.01

0.1

1

PM+7C Data

PM Data

7C Data

PM+7C Fit

Psu

rviv

al

Time (s)

Comparison of Force Spectroscopy Results

for Mixed vs. Pure Analytes (-50mV)

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

Synthetic-organic hybrid nanosensor array

A25 : 75 – 115 mV

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

Label-free genotyping assay

• Sandwich Assay

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

Solid-State Nanopore Fabrication

TEM 1.5 nm

3.5 nm

10 nm

• Individual Nanopores fabricated with single nanometre precision in thin SiNx membranes

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

Noise reduction

0 1 2 3 4 5

-100

0

100

200

300

400

500

600

700

Cu

rren

t (p

A)

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

200 mV Applied Potential

1/f Noise

2 2

1 2 3/S a a f a f pA Hz= + +

Thermal Noise

Dielectric Noise

Vincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007

Noise reduction

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

A25 : 75 – 115 mV

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

λ-DNA (48.5 kbp dsDNA) at +150 mV in 1M KCl, ~ 7nm pore

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

3 nm

Synthetic Nanopore Force Spectroscopy

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

0.1 1 10 1000.01

0.1

1

τττταααα

PM= 6.74

MM

Surv

ival P

rob

abili

ty

Time (s)

PM

τττταααα

MM= 0.24

Force applied -150mV

1E-4 1E-3 0.01 0.1 1 100.01

0.1

1

Force applied -50mV

Surv

ival P

robab

ility

Time (s)

PM

τττταααα

PM= 0.77

τττταααα

MM= 0.003

MM

� DNA duplex dissociation under force

α-HL data SiNx data

Perfect Match: 5’-GGTTTGGTTGGTGG-3’

Mismatch: 5’-GGTTTGCCTTGGTGG-3’

Single Base Pair Resolution

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

Single Base Pair Resolution

-225 -200 -175 -150 -125 -100 -75 -50

0.1

1

10

PM pore1

MM pore1MM pore2

(2)

(4)

(4)

(2)

(1)

(2)

Ch

ara

cte

ristics T

ime

scale

, τα

Applied Force (mV)

(1)

Comparison of PM & MM Analytes:Dissociation Timescales During Force Spectroscopy

-27.4 kBTMM

-37.7 kBTPM 5’-GGTTTGGTTGGTGG-3’

5’-GGTTTGCCTTGGTGG-3’

DNA Sequences used :

PM = Perfect Match, MM = MisMatch

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

DNA-pore interactions

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

-100

0

100

200

300

400

500

600

700

0

20

40

60

80

100

Cu

rre

nt

(pA

)

Time (s)

Ap

plie

d P

ote

ntia

l (mV

)

0.01 0.1 1 10 100 10000.0

0.1

0.2

0.3

0.4

0.5

Co

eff

icie

nt

Timescale (s)

Fit:

/ it

i

i

Aeτ

∑

ln( / ) 2 3B i j BE k T k Tτ τ∆ = ⋅ ≈ −

Eb

Energ

yx

Eb+∆∆∆∆E

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

Nanopore Force Spectroscopy

• Receptor-Ligand Interactions- Avidin-biotin (model system)- Protein detection

• DNA-DNA Interactions- Genotyping (SNP)

• Protein Structural Transition- β-sheet melting/annealing

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

Strength of Molecular Bonds

1E-3 0.01 0.1 1 10

0.01

0.1

1

400 mV

500 mV

600 mV

700 mV

800 mV

Su

rviv

al P

rob

ab

ility

Time (sec)

(Neutr)Avidinbiotin

40 50 60 70 80 90 100 110

1E-3

0.01

0.1

1

10

400 500 600 700 800 900

Applied Voltage (mV)

<τ>

Force (pN)

Merkel et al. (1999) Nature. 397:50–53. & Izrailev, et al. (1997) Biophys. J. 72:1568–1581. & Pincet et al. (2005) Biophys. J. 89:4374–4381.

∆xb

Effective charge z ~ 0.3

b barrier

B

E f x

k T

D eτ τ

− ⋅∆

= ⋅

Kramer’s rate theory:

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

Nanopore Force Spectroscopy

• Receptor-Ligand Interactions- Avidin-biotin (model system)- Protein detection

• DNA-DNA Interactions- Genotyping (SNP)

• Protein Structural Transition- β-sheet melting/annealing

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

Prion Protein Structure Dynamics

� Progress on diagnostics & cures is limited by not knowing:

1. Structure of misfolded PrP

2. Mechanism of conversion

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

Nanopore Analysis of Prion Proteins

� Measured signal: ionic current in the pore

� Key Advantages:

• Resolution of ~Å

• Observe repeated structural transitions on 1 molecule

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

Evidence of Structural Transitions

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

Long-Term Observation of a Single Molecule

dG ~10kcal/mol

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

Prion Protein Heterogeneity

UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory

Vincent Tabard-Cossa

Jason Dwyer

Matthew Wiggin

Nahid Jetha

Dhruti Trivedi

Carolina Tropini

Chris Feehan

Thanks

St. Paul’s Hospital – iCAPTURE center

Scott Tebbutt

Keith Walley

Prion Collaboration

Dr. Neil Cashman (UBC)

Will Guest

Dr. David Wishart (U of A)

Bow Suriyamongkol