protein translocation through artificial nanopores€¦ · protein translocation through artificial...

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