Stochastic sensing of proteins with receptor-modified solid-state nanopores


Solid-state nanopores are capable of the label-free analysis of single molecules. It is possible to add biochemical selectivity by anchoring a molecular receptor inside the nanopore, but it is difficult to maintain single-molecule sensitivity in these modified nanopores. Here, we show that metallized silicon nitride nanopores chemically modified with nitrilotriacetic acid receptors can be used for the stochastic sensing of proteins. The reversible binding and unbinding of the proteins to the receptors is observed in real time, and the interaction parameters are statistically analysed from single-molecule binding events. To demonstrate the versatile nature of this approach, we detect His-tagged proteins and discriminate between the subclasses of rodent IgG antibodies.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Chemical modification of metallized solid-state nanopores and stochastic sensing of proteins.
Figure 2: Single-molecule binding kinetics of His6-tagged protein A/G/L to a bisNTA modified nanopore.
Figure 3: Quantitation of dissociation rates and influence of receptor position.
Figure 4: Influence of receptor multivalence on binding stability.
Figure 5: Antibody-selective nanopore.


  1. 1

    Howorka, S. & Siwy, Z. Nanopore analytics: sensing of single molecules. Chem. Soc. Rev. 38, 2360–2384 (2009).

  2. 2

    Chen, P. et al. Atomic layer deposition to fine-tune the surface properties and diameters of fabricated nanopores. Nano Lett. 4, 1333–1337 (2004).

  3. 3

    Wanunu, M. et al. Rapid electronic detection of probe-specific microRNAs using thin nanopore sensors. Nature Nanotech. 5, 807–814 (2011).

  4. 4

    Robertson, J. W. F. et al. Single-molecule mass spectrometry in solution using a solitary nanopore. Proc. Natl Acad. Sci. USA 104, 8207–8211 (2007).

  5. 5

    Fologea, D., Ledden, B., McNabb, D. S. & Li, J. L. Electrical characterization of protein molecules by a solid-state nanopore. Appl. Phys. Lett. 91, 053901 (2007).

  6. 6

    Han, A. et al. Label-free detection of single protein molecules and protein–protein interactions using synthetic nanopores. Anal. Chem. 80, 4651–4658 (2008).

  7. 7

    Talaga, D. S. & Li, J. L. Single-molecule protein unfolding in solid state nanopores. J. Am. Chem. Soc. 131, 9287–9297 (2009).

  8. 8

    Uram, J. D., Ke, K., Hunt, A. J. & Mayer, M. Submicrometer pore-based characterization and quantification of antibody–virus interactions. Small 2, 967–972 (2006).

  9. 9

    Smeets, R. M. M., Kowalczyk, S. W., Hall, A. R., Dekker, N. H. & Dekker, C. Translocation of RecA-coated double-stranded DNA through solid-state nanopores. Nano Lett. 9, 3089–3095 (2009).

  10. 10

    Van Dorp, S., Keyser, U. F., Dekker, N. H., Dekker, C. & Lemay, S. G. Origin of the electrophoretic force on DNA in solid-state nanopores. Nature Phys. 5, 347–351 (2009).

  11. 11

    Firnkes, M., Pedone, D., Knezevic, J., Doblinger, M. & Rant, U. Electrically facilitated translocations of proteins through silicon nitride nanopores: conjoint and competitive action of diffusion, electrophoresis, and electroosmosis. Nano Lett. 10, 2162–2167 (2010).

  12. 12

    Movileanu, L. Interrogating single proteins through nanopores: challenges and opportunities. Trends Biotechnol. 27, 333–341 (2009).

  13. 13

    Keyser, U. F. Controlling molecular transport through nanopores. J. R. Soc. Interface 8, 1369–1378 (2011).

  14. 14

    Iqbal, S. M., Akin, D. & Bashir, R. Solid-state nanopore channels with DNA selectivity. Nature Nanotech. 2, 243–248 (2007).

  15. 15

    Bayley, H. & Cremer, P. S. Stochastic sensors inspired by biology. Nature 413, 226–230 (2001).

  16. 16

    Liu, A. H., Zhao, Q. T. & Guan, X. Y. Stochastic nanopore sensors for the detection of terrorist agents: current status and challenges. Anal. Chim. Acta 675, 106–115 (2010).

  17. 17

    Braha, O. et al. Simultaneous stochastic sensing of divalent metal ions. Nature Biotechnol. 18, 1005–1007 (2000).

  18. 18

    Gu, L. Q., Braha, O., Conlan, S., Cheley, S. & Bayley, H. Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter. Nature 398, 686–690 (1999).

  19. 19

    Howorka, S., Cheley, S. & Bayley, H. Sequence-specific detection of individual DNA strands using engineered nanopores. Nature Biotechnol. 19, 636–639 (2001).

  20. 20

    Movileanu, L., Howorka, S., Braha, O. & Bayley, H. Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore. Nature Biotechnol. 18, 1091–1095 (2000).

  21. 21

    Dekker, C. Solid-state nanopores. Nature Nanotech. 2, 209–215 (2007).

  22. 22

    Wanunu, M. & Meller, A. Chemically modified solid-state nanopores. Nano Lett. 7, 1580–1585 (2007).

  23. 23

    Sexton, L. T. et al. Resistive-pulse studies of proteins and protein/antibody complexes using a conical nanotube sensor. J. Am. Chem. Soc. 129, 13144–13152 (2007).

  24. 24

    Yusko, E. C. et al. Controlling protein translocation through nanopores with bio-inspired fluid walls. Nature Nanotech. 6, 253–260 (2011).

  25. 25

    Kowalczyk, S. W. et al. Single-molecule transport across an individual biomimetic nuclear pore complex. Nature Nanotech. 6, 433–438 (2011).

  26. 26

    Siwy, Z. et al. Protein biosensors based on biofunctionalized conical gold nanotubes. J. Am. Chem. Soc. 127, 5000–5001 (2005).

  27. 27

    Ali, M. et al. Biosensing and supramolecular bioconjugation in single conical polymer nanochannels. Facile incorporation of biorecognition elements into nanoconfined geometries. J. Am. Chem. Soc. 130, 16351–16357 (2008).

  28. 28

    Vlassiouk, I., Kozel, T. R. & Siwy, Z. S. Biosensing with nanofluidic diodes. J. Am. Chem. Soc. 131, 8211–8220 (2009).

  29. 29

    Ding, S., Gao, C. & Gu, L-Q. Capturing single molecules of immunoglobulin and ricin with an aptamer-encoded glass nanopore. Anal. Chem. 81, 6649–6655 (2009).

  30. 30

    Actis, P., Jejelowo, O. & Pourmand, N. Ultrasensitive mycotoxin detection by STING sensors. Biosens. Bioelectron. 26, 333–337 (2010).

  31. 31

    Han, C. P. et al. Enantioselective recognition in biomimetic single artificial nanochannels. J. Am. Chem. Soc. 133, 7644–7647 (2011).

  32. 32

    Wei, R. S., Pedone, D., Zurner, A., Doblinger, M. & Rant, U. Fabrication of metallized nanopores in silicon nitride membranes for single-molecule sensing. Small 6, 1406–1414 (2010).

  33. 33

    Sexton, L. T. et al. An adsorption-based model for pulse duration in resistive-pulse protein sensing. J. Am. Chem. Soc. 132, 6755–6763 (2010).

  34. 34

    Tinazli, A. et al. High-affinity chelator thiols for switchable and oriented immobilization of histidine-tagged proteins: a generic platform for protein chip technologies. Chem. Eur. J. 11, 5249–5259 (2005).

  35. 35

    Raiber, K., Terfort, A., Benndorf, C., Krings, N. & Strehblow, H-H. Removal of self-assembled monolayers of alkanethiolates on gold by plasma cleaning. Surf. Sci. 595, 56–63 (2005).

  36. 36

    Prime, K. L. & Whitesides, G. M. Adsorption of proteins onto surfaces containing end-attached oligo(ethylene oxide)—a model system using self-assembled monolayers. J. Am. Chem. Soc. 115, 10714–10721 (1993).

  37. 37

    Sigal, G. B., Bamdad, C., Barberis, A., Strominger, J. & Whitesides, G. M. A self-assembled monolayer for the binding and study of histidine tagged proteins by surface plasmon resonance. Anal. Chem. 68, 490–497 (1996).

  38. 38

    Zevenbergen, M. A. G., Singh, P. S., Goluch, E. D., Wolfrum, B. L. & Lemay, S. G. Stochastic sensing of single molecules in a nanofluidic electrochemical device. Nano Lett. 11, 2881–2886 (2011).

  39. 39

    Lata, S. & Piehler, J. Stable and functional immobilization of histidine-tagged proteins via multivalent chelator headgroups on a molecular poly(ethylene glycol) brush. Anal. Chem. 77, 1096–1105 (2005).

  40. 40

    Hill, A. V. The possible effects of the aggregation of the molecules of haemoglobin on its dissociation curves. J. Physiol. 40, iv–vii (1910).

  41. 41

    Colquhoun, D. Binding, gating, affinity and efficacy: the interpretation of structure–activity relationships for agonists and of the effects of mutating receptors. Br. J. Pharmacol. 125, 924–947 (1998).

  42. 42

    Woodhull, A. M. Ionic blockage of sodium channels in nerve. J. Gen. Physiol. 61, 687–708 (1973).

  43. 43

    Gu, L. Q. & Bayley, H. Interaction of the noncovalent molecular adapter, β-cyclodextrin, with the staphylococcal α-hemolysin pore. Biophys. J. 79, 1967–1975 (2000).

  44. 44

    Mathe, J., Visram, H., Viasnoff, V., Rabin, Y. & Meller, A. Nanopore unzipping of individual DNA hairpin molecules. Biophys. J. 87, 3205–3212 (2004).

  45. 45

    Strunz, T., Oroszlan, K., Schumakovitch, I., Guntherodt, H. J. & Hegner, M. Model energy landscapes and the force-induced dissociation of ligand–receptor bonds. Biophys. J. 79, 1206–1212 (2000).

  46. 46

    Valiokas, R. et al. Self-assembled monolayers containing terminal mono-, bis-, and tris-nitrilotriacetic acid groups: characterization and application. Langmuir 24, 4959–4967 (2008).

  47. 47

    Pedone, D., Firnkes, M. & Rant, U. Data analysis of translocation events in nanopore experiments. Anal. Chem. 81, 9689–9694 (2009).

  48. 48

    Palmqvist, N., Foster, T., Tarkowski, A. & Josefsson, E. Protein A is a virulence factor in Staphylococcus aureus arthritis and septic death. Microb. Pathog. 33, 239–249 (2002).

  49. 49

    Lindmark, R., Thorentolling, K. & Sjoquist, J. Binding of immunoglobulins to protein-A and immunoglobulin levels in mammalian sera. J. Immunol. Methods 62, 1–13 (1983).

  50. 50

    Rousseaux, J., Picque, M. T., Bazin, H. & Biserte, G. Rat IgG subclasses: differences in affinity to protein A–sepharose. Mol. Immunol. 18, 639–645 (1981).

  51. 51

    Escribano, M. J., Haddada, H. & de Vaux Saint Cyr, C. Isolation of two immunoglobulin G subclasses, IgG2 and IgG1, from hamster serum using protein A–sepharose. J. Immunol. Methods 52, 63–72 (1982).

  52. 52

    Coe, J. E. Humoral immunity and serum-proteins in syrian-hamster. Fed. Proc. 37, 2030–2031 (1978).

Download references


The authors thank G. Abstreiter for his support, and M. Firnkes, D. Pedone, A. Kleefen and M. Langecker for discussions. This work was supported by the German Research Foundation DFG (SFB 863 and SFB 807), the TUM Institute for Advanced Study, the Federal Ministry of Education and Research BMBF (0312031/0312034), and the Clusters of Excellence Nanosystems Initiative Munich and Macromolecular Complexes (at the Goethe-University Frankfurt).

Author information

U.R., R.We. and R.T. devised the research. R.We. prepared the pores and performed experiments. V.G. and R.Wi. synthesized the NTA compounds. R.We., U.R. and R.T. analysed the data. R.We. and U.R. wrote the paper.

Correspondence to Robert Tampé or Ulrich Rant.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 3073 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Wei, R., Gatterdam, V., Wieneke, R. et al. Stochastic sensing of proteins with receptor-modified solid-state nanopores. Nature Nanotech 7, 257–263 (2012).

Download citation

Further reading