Protein Translocation Through Artificial Nanopores
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1 Protein Translocation Through Artificial Nanopores Marc Creus University of Basel ERBM4 Liège A WHOLE nano-world to be explored! Institute of Microtechnology (Neuchâtel) Dr Urs Staufer Dr Anpan Han Prof Nico de Rooij Institute of Chemistry (Neuchâtel) Dr Marc Creus Prof Thomas Ward
2 First patent application for Coulter Counter: 1949 You cannot patent a hole! US Patent granted: 1953, USPT Wallace H. Coulter ( ) Engineer, Inventor, Entrepreneur, Visionary
3 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 CBCs are performed on instruments using the Coulter Principle.
4 Micro vs Nano 10µm 1nm (e.g. diametre of a small protein, which is x smaller than this small cell)
5 Since the nano-scale corresponds to the size of biological macromolecules, nanopores could be useful in biochemical analyses of proteins. ~5nm 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
6 Genetic data Primary structure Polymer of A, T, C, G Secondary structure - B helix Diameter Structure of DNA 2 nm Tertiary structure Alberts et al.
7 Primary structure Structure of Proteins 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
8 Properties of macromolecules: Surface charge: positive, neutral or negative -DNA (an acid) is usually negatively charged Acid (low) ph Basic (high) ph -Proteins can be basic or acidic and have different charges depending on the ph Alberts et al.
9 More properties of macromolecules: Specific interactions Proteins are designed for recognition: antibodies, hormones, enzymes, structural proteins, toxins, etc
10 How can a biochemist make use of synthetic nanopores? Measure size, charge, structural properties and interactions of proteins, in real-time and in solution?
11 Process flow chart nanopore fabrication 500 nm SiO 2 20 nm Si 3 N 4 AZ 1518 both sides KOH etching rinsing spin PMMA 20nm backside alignment optical lithography oxidation e-beam litho. RIE, stripping chip level PDMS bonding RIE, stripping silicon PMMA Si 3 N 4 PDMS SiO 2 AZ 1518
12 Wafer-level nanopore fabrication process PDMS lp= 20nm Si 3 N 4 25 nm SiO 2 Si SiO 2 Si 3 N 4
13 Experiment setup
14 Count the numbers of spikes per minute Number of spikes proportional to concentration Measured parameters Individual spikes Duration: t Current change: I I t
15 Four different proteins, differing in size and charge properties Streptavidin Mut. S27a Human Serum Albumin Ovalbumin Avidin comp. Biotin. Le Trong et al PDB: 1N9Y S.Sugio, et al., 1998 PDB: 1BM0 Stein, Leslie, 1990 PDB: 1OVA Livnah et al 1993, PDB: 2AVI Notes Mass (kda) r stoke (nm) pi isof pi ef Streptavidin (SAV) Recombinant BSA >99% electrophoresis Ovalbumin (OA) Grade VII (>98% elph.) Avidin (AV) Heterogeneous 72/
16 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 first approximation we can ignore protein-pore interactions
17 Protein charge explored by nanopores Valleys (50mV) Peaks (-50mV)
18 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:
19 Spikes: ph-dependence of shape and duration Duration of blockage-events varies with ph: longer (and more complex) signals closer to pi BSA 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:
20 Variety of spikes with complex fine-structure BSA ph3 BSA ph6 AV ph6 SAV ph5 Han, Creus, Schürmann,Lindner, Ward, Staufer, Analytical Chemistry (2008) 89:
21 Time resolution Our calculations suggest that at ph8 BSA translocates the 20nm porelength 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 BSA BSA Han, Creus, Schürmann,Lindner, Ward, Staufer, Analytical Chemistry (2008) 89:
22 Slowing down by ph E r I ph close to pi ph far from pi t
23 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:
24 Protein diameter measured by nanopores Pore Protein I (na) d p (nm) ΔI (na) d m (nm) A OA ph 6, 100mV OA, BSA, SAV B C OA OA D BSA E BSA F BSA d OA = 7.3 nm ± 0.2 nm d BSA = 8.7 nm ± 0.1 nm 1 nm = 10 hydrogen atoms (10 Å)
25 Quantifying molecules by exploiting specific interactions of proteins IgG (hcg)= 4µg/ml Time (s) Time (s) Time (s)
26 Interpretation E r I t
27 Nanopore bioassays 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 Titrations can be employed for quantification (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)
28 New methods bring surprising outcomes SAV (calculated pi= 6.5) is apparently very heterogeneous, with both positively and negatively-charged tetramers at any given ph SAV SDS-PAGE gel
29 SAV apparently pure? Mass Reconstruction of Streptavidin Wildtype. Applied Biosystems/ Sciex QTrap Mass Spectrometer: Electrospray Low Resolution, Positive Ion Mode Acetonitrile/Water (1:1) + 1%HFo Avi Sav Sav (theory)= Sav (found) = Da Da Sav + Ca2+= Da Sav + 2x Ca 2+= Da Sav + 3x Ca2+ = ~16552 Da Isoelectric Focusing Lutter et al. Electrophoresis 2001, 22:
30 New methods bring surprising outcomes SAV (calculated pi= 6.5) is apparently very heterogeneous, with both positively and negatively-charged tetramers at any given ph SAV Charge heterogeneity? Binding to counterions?
31 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
32 Questions and outlook 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 heterogeneity Biomolecular interactions & affinities Paradigm shift (beyond DNA): Since nanopores are easy to use and informative, they may become a useful analytical tool for the biochemist
33 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)
34 Wafer-level nanopore fabrication process Si 3 N 4 SiO 2 Si SiO 2 Si 3 N 4
35 Wafer-level nanopore fabrication process Resist Si 3 N 4 SiO 2 Si SiO 2 Si 3 N 4
36 Wafer-level nanopore fabrication process e-beam exposure Resist Si 3 N 4 SiO 2 Si SiO 2 Si 3 N 4 Resist optical lithography
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