Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N...

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


• 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

− ⋅∆

= ⋅


DNA Sequence Detection

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

Carolina Tropini, Andre Marziali, Biophys J. 2007


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


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)


Synthetic-organic hybrid nanosensor array

A25 : 75 – 115 mV


Label-free genotyping assay

• Sandwich Assay


Solid-State Nanopore Fabrication

TEM 1.5 nm

3.5 nm

10 nm

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


Noise reduction

0 1 2 3 4 5

-100

0

100

200

300

400

500

600

700

Cu

rren

t (p

A)


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


A25 : 75 – 115 mV


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


3 nm

Synthetic Nanopore Force Spectroscopy


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


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


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


Nanopore Force Spectroscopy





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:


Nanopore Force Spectroscopy





Prion Protein Structure Dynamics

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

1. Structure of misfolded PrP

2. Mechanism of conversion


Nanopore Analysis of Prion Proteins

� Measured signal: ionic current in the pore

� Key Advantages:

• Resolution of ~Å

• Observe repeated structural transitions on 1 molecule


Evidence of Structural Transitions


Long-Term Observation of a Single Molecule

dG ~10kcal/mol


Prion Protein Heterogeneity


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

Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N...

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