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Protein Translocation Through
Artificial Nanopores
Marc Creus
University of Basel
ERBM4 Liège
Institute of Microtechnology (Neuchâtel)Dr Urs StauferDr Anpan HanProf Nico de Rooij
Institute of Chemistry (Neuchâtel)Dr Marc CreusProf Thomas Ward
A WHOLE nano-world to be explored!
First patent application for Coulter Counter: 1949
US Patent granted: 1953, USPT 2656508“You cannot patent a hole!”
Wallace H. Coulter (1913-1998)Engineer, Inventor, Entrepreneur, Visionary
Application of Coulter Principle:Blood-Cell Counter
The complete blood count or “CBC” is one of the most commonly ordered diagnostic tests worldwide.
Today, ninety-eight percent of CBCsare performed on instruments using the Coulter Principle.
Micro vs Nano
10µm
1nm(e.g. diametre ofa small protein,which is10 000 x smallerthan this small cell)
“We have friends in other fields---in biology, for instance. We physicists often look at them and say, (…) ``You should use more mathematics, like we do.'' They could answer us (…) ``What you should do in order for us to make more rapid progress is to make the electron microscope 100 times better.''
Richard Feynman, December 29th 1959“There’s plenty of room at the bottom”http://www.zyvex.com/nanotech/feynman.html
Since the nano-scale corresponds to the size of
biological macromolecules, nanopores could be
useful in biochemical analyses of proteins.
~5nm
Structure of DNA
• Genetic data• Primary structure
– Polymer of A, T, C, G
• Secondary structure- B helix– Diameter
2 nm
• Tertiary structure Alberts et al.
Structure of Proteins
• Primary structure– Polymer of 20 amino acids
• Secondary structure– α-helix, β-strands, coils
• Tertiary structure
• Quaternary structure– Multi protein complex, filaments
• Typical diameter: 1 - 20 nm Ovalbumin Deposition: Stein , Leslie,
1990 PDB: 1OVA
• Surface charge: positive, neutral or negative
Properties of macromolecules:
Alberts et al.
-DNA (an acid) is usually negatively charged
Acid (low) pH Basic (high) pH
-Proteins can be basic or acidicand have different chargesdepending on the pH
More properties of macromolecules:Specific interactions
• Proteins are designed for recognition: antibodies, hormones, enzymes, structural proteins, toxins, etc…
• Measure size, charge, structural properties and interactions of proteins, in real-time and in solution?
How can a biochemist make use of synthetic nanopores?
Process flow chart nanopore fabrication
500 nm SiO220 nm Si3N4
spin PMMA
e-beam litho.
RIE, stripping
AZ 1518 both sides
backside alignmentoptical lithography
RIE, stripping
KOH etching rinsing
oxidation
chip level PDMS bonding
silicon
PMMA
Si3N4
PDMS
SiO2
AZ 1518
20nm
Wafer-level nanopore fabrication process
Si
SiO2
Si3N4
PDMS
SiO2
Si3N4
25 nm
lp= 20nm
Experiment setup
Measured parameters
• Count the numbers of spikes per minute– Number of spikes
proportional to concentration
• Individual spikes– Duration: ∆t– Current change: ∆I
∆t
∆I
Four different proteins, differing in size and charge properties
Human Serum Albumin
S.Sugio, et al., 1998 PDB: 1BM0
Ovalbumin
Stein , Leslie, 1990 PDB: 1OVA
Avidin comp. Biotin.
Livnah et al 1993, PDB: 2AVI
Streptavidin Mut. S27a
Le Trong et al. 2002 PDB: 1N9Y
10.5-72/62HeterogeneousAvidin (AV)
4.64.542.744Grade VII (>98% elph.)Ovalbumin (OA)
4.255.3 3.566>99% electrophoresisBSA
6.5-66RecombinantStreptavidin (SAV)
pIefpIisofrstoke (nm)Mass (kDa)Notes
Translocation by electrophoresis
Electrode bias set at 50 mV (or -50mV)pH 6, citrate, 1M KCl, 1 µg BSA/mL
Since pore is considerably larger than proteins, at a firstapproximation we can ignore protein-pore interactions
Protein charge explored by nanopores
Valleys (50mV) Peaks (-50mV)
Protein charge explored by nanopores
BSA
BSA is reported to have pI 4.2 in presence of KCl
(reports that pI is reduced from 5.3 due to binding of Cl-)
Suggests importance of counterions?
Han, Creus, Schürmann,Lindner, Ward, Staufer, Analytical Chemistry (2008) 89:4651-4658
Duration of blockage-events varies with pH: longer (and more complex) signals closer to pI
Fewer, sharp spikes when pH is distant from pI
Suggests time resolution is a critical issue
Han, Creus, Schürmann,Lindner, Ward, Staufer, Analytical Chemistry (2008) 89:4651-4658
Spikes: pH-dependence of shape and duration
BSA
Variety of spikes with complex fine-structure
Han, Creus, Schürmann,Lindner, Ward, Staufer, Analytical Chemistry (2008) 89:4651-4658
BSA pH3 BSA pH6 AV pH6 SAV pH5
Our calculations suggest that at pH8 BSA translocates the 20nm pore-length in about 2µs
Even with 100kHz bandwidth, practical time resolution is only 40µs
Very fast translocations will not be resolved
Can slow down by measuring with pH close to pI
Han, Creus, Schürmann,Lindner, Ward, Staufer, Analytical Chemistry (2008) 89:4651-4658
Time resolution
BSA
BSA
Slowing down by pH
E
r
t
I
pH close to pI
pH far from pI
Protein translocation explored by nanopores
BSA
BSA is reported to have pI 4.2 in presence of KCl
(reports that pI is reduced from 5.3 due to binding of Cl-)
Very few translocations of BSA at pH4
Han, Creus, Schürmann,Lindner, Ward, Staufer, Analytical Chemistry (2008) 89:4651-4658
Protein diameter measured by nanopores
8.80.3128.915.9BSAF
8.70.2726.514.5BSAE
8.60.3126.414.0BSAD
7.30.2326.314.0OAC
7.50.2522.010.8OAB
7.10.2121.910.7OAA
dm (nm)ΔΔΔΔI (nA)dp (nm)I (nA)ProteinPore
pH 6, 100mVOA, BSA, SAV
dBSA = 8.7 nm ± 0.1 nm
dOA = 7.3 nm ± 0.2 nm
1 nm = 10 hydrogen atoms (10 Å)
Quantifying molecules by exploitingspecific interactions of proteins
Time (s)
0 2 4 6 8 10
Time (s)
0 2 4 6 8 10
Time (s)
0 2 4 6 8 10
IgG (hCG)= 4µg/ml
Interpretation
E
r
t
I
• The principle of the assay is general & can be applied wherever
two molecules combined give a different signal from signals of either molecules alone
A + B = C
Nanopore bioassays
•
Titrations can be employed forquantification (e.g. measures of affinity)
Statistical calculations: 1000 counts (C.V. 3.2%)
Counting 1000 proteins in 1ml volumes is not “zeptoM sensitivity”, due to limitations:
- Time: 500 counts/min (25nM antibody)- Affinities (for biomolecular interactions)
New methods bring surprising outcomes…
SAV
• SAV (calculated pI= 6.5) isapparently very heterogeneous, with both positively and negatively-charged tetramers atany given pH
SDS-PAGE gel
SAV apparently pure?
16430.0
Mass Reconstruction of Streptavidin Wildtype.
Applied Biosystems/ Sciex QTrap Mass Spectrometer:Electrospray Low Resolution, Positive Ion ModeAcetonitrile/Water (1:1) + 1%HFo
Sav (theory)= 16423 DaSav (found) = 16430 Da
Lutter et al. Electrophoresis 2001, 22: 2888-2897
Sav + Ca2+= 16470 DaSav + 2x Ca 2+= 16510 DaSav + 3x Ca2+ = ~16552 Da
Isoelectric Focusing
Avi Sav
New methods bring surprising outcomes…
SAV
• SAV (calculated pI= 6.5) isapparently very heterogeneous, with both positively and negatively-charged tetramers atany given pH
• Charge heterogeneity?• Binding to counterions?
Summary
• Protein sensing using nanopores: label-free, in solution, in
real time
– Exquisitely sensitive: proteins analysed one-by-one
– Diameter precisely determined with 0.2nm reproducibility
– Charge-properties and interactions between proteins can be measured
– Label-free immunoassays
– Counting just 1000 molecules is required for accuracy, which
could be found in tiny volumes
What are the effects of counterions?
What is the significance of the fine-structure of spikes?
Structural/ biophysical properties:Explore orientation of translocation Sequence proteins: beyond genomics?Protein folding (time resolution)Domain movements (time resolution)
Nanopore Assays:Protein heterogeneityBiomolecular interactions & affinities
Questions and outlook
Paradigm shift (beyond DNA):Since nanopores are easy to use and informative, theymay become a useful analytical tool for the biochemist
Acknowledgements
• Canton de Neuchâtel
• Swiss National Science Fund
• Danish Research Agency
for financial support
• The staff of COMLab & the joint clean-room facility of IMT and
CSEM for their technological support
• Prof. Urs Staufer (now at Delft Technical University)
• Dr Anpan Han (now in Copenhagen)
Wafer-level nanopore fabrication process
Si
SiO2
Si3N4
SiO2
Si3N4
Wafer-level nanopore fabrication process
Si
SiO2
Si3N4
Resist
SiO2
Si3N4
Wafer-level nanopore fabrication process
Si
SiO2
Si3N4
Resist
e-beam exposure
Resist
SiO2
Si3N4
optical lithography