synthetic nanopore force- spectroscopyvincent tabard-cossa, dhruti trivedi, matthew wiggin, nahid n...
TRANSCRIPT
![Page 1: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/1.jpg)
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
![Page 2: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/2.jpg)
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
− ⋅∆
= ⋅
![Page 3: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/3.jpg)
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
![Page 4: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/4.jpg)
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
![Page 5: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/5.jpg)
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)
![Page 6: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/6.jpg)
UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory
Synthetic-organic hybrid nanosensor array
A25 : 75 – 115 mV
![Page 7: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/7.jpg)
UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory
Label-free genotyping assay
• Sandwich Assay
![Page 8: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/8.jpg)
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
![Page 9: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/9.jpg)
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)
![Page 10: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/10.jpg)
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
![Page 11: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/11.jpg)
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
![Page 12: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/12.jpg)
UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory
3 nm
Synthetic Nanopore Force Spectroscopy
![Page 13: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/13.jpg)
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
![Page 14: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/14.jpg)
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
![Page 15: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/15.jpg)
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
![Page 16: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/16.jpg)
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
![Page 17: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/17.jpg)
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:
![Page 18: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/18.jpg)
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
![Page 19: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/19.jpg)
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
![Page 20: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/20.jpg)
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
![Page 21: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/21.jpg)
UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory
Evidence of Structural Transitions
![Page 22: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/22.jpg)
UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory
Long-Term Observation of a Single Molecule
dG ~10kcal/mol
![Page 23: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/23.jpg)
UBC Department of Physics and Astronomy / UBC Engineering Physics Applied Biophysics Laboratory
Prion Protein Heterogeneity
![Page 24: Synthetic Nanopore Force- SpectroscopyVincent Tabard-Cossa, Dhruti Trivedi, Matthew Wiggin, Nahid N Jetha, and Andre Marziali, Nanotechnology, 2007 Noise reduction . UBC Department](https://reader035.vdocuments.us/reader035/viewer/2022071008/5fc5bc236fa4fd1caa61b95e/html5/thumbnails/24.jpg)
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