JoVE Journal > Bioengineering
Summary
Abstract
Protocol
Discussion
Divulgazioni
Acknowledgements
Materials
Riferimenti
Traduzione automatica
Please note that all translations are automatically generated. Click here for the English version.
Bioingegneria

监测与固态纳米孔蛋白质吸附

Published: December 02, 2011
doi:
10.3791/3560
,
1Department of Physics,Syracuse University

Summary

一个方法是使用固态纳米孔监测到无机表面蛋白质的非特异性吸附描述。该方法采用电阻脉冲的原则，允许实时和单分子水平探讨吸附。因为单一的蛋白质吸附过程是远离平衡，我们建议平行合成纳米孔阵列的就业，使表观一级反应速率常数蛋白质吸附定量测定以及和Langmuir吸附常数。

Abstract

固态纳米孔已被用来执行在单分子水平测量，以考察当地的结构和灵活性 1-6核酸， 蛋白质 7开展，8种不同的配体结合的亲和力。通过这些孔洞耦合9-12电阻脉冲技术，这样的测量，可以做各种各样的条件下，没有标签 3 。电阻脉冲技术，离子的盐溶液中引入纳米孔的两侧。因此，离子驱动从一个会议厅一侧的其他应用的跨膜电位，造成在一个稳定的电流。分析物的纳米孔的分割，导致在当前的定义良好的偏转，它可以分析提取单分子信息。使用这种技术，纳米孔壁的单个蛋白质的吸附作用，可以广泛的监视下条件13。蛋白质吸附的重要性越来越大，因为微流体器件尺寸的缩小，这些系统与单一蛋白质的相互作用成​​为一个令人关注的问题。这一协议描述为氮化物薄膜，它可以很容易地扩展到其他薄膜进行纳米孔钻，或功能化氮化物表面蛋白结合的快速检测。下一个广泛的解决方案和变性条件下，可以探索多种蛋白质。此外，该协议可用于探索使用纳米孔光谱的基本问题。

Protocol

1。氮化硅膜固态纳米孔的制造把费Tecnai F20的S /透射电镜一个200千伏的加速电压。如果使用不同的S /透射电子显微镜，加速电压应大于或等于200千伏9 TEM样品架装入一个20纳米厚的SPI氮化硅窗口电网和清洁除去持有​​人的任何污染物与氧气30秒的等离子体。 装入的S / TEM样品，并允许为真空抽空。一旦S /透射电镜抽空真空，发现在明场TEM模式通过寻找光明广场上的Ronc…

Discussion

自发到固态表面27-29上的蛋白质吸附在一些领域，如生物芯片的应用和设计一个新的功能混合生物材料类，是从根本上重要的。以往的研究表明，吸附到固体表面的蛋白质不显示横向调动或显著解吸率，因此蛋白质的吸附通常被认为是一个不可逆转的和非特异性的过程30-32。到固态表面的蛋白质吸附被认为是由于多种因素， 包括 13在固-液界面的蛋白质和活性基团的侧链之?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

作者想约翰Grazul（康奈尔大学），安德烈Marziali（在温哥华不列颠哥伦比亚大学）和外袍，科萨文森特（渥太华大学），以感谢他们的意见。这项工作是由美国国家科学基金会（DMR – 0706517和DMR – 1006332）和国立卫生研究院（R01 GM088403）资助部分经费。纳米孔钻在康奈尔大学材料研究中心（CCMR）从国家科学基金会支持的电子显微镜机制 – 材料研究科学和工程中心（MRSEC）程序（DMR 0520404）。氮化硅膜的制备是在康奈尔大学的纳米基金，国家纳米技术基础设施网络的成员，这是由国家科学基金会（格兰特ECS – 0335765）的支持。

Materials

Name of the reagent Company Catalogue number Comments
Tecnai F20 S/TEM FEI   S/TEM requires acceleration voltage ≥200kV and field-emission source.
20 nm thick silicon nitride membrane window for TEM SPI 4163SN-BA  
Axon Axopatch 200B patch-clamp amplifier Molecular Devices    
Axon Digidata 1440A Molecular Devices    
pCLAMP 10 software Molecular Devices   Electrophysiology Data Acquisition and Analysis Software
Sulfuric Acid Fisher Scientific A300  
hydrogen peroxide Fisher Scientific H325  
silicone O-rings McMaster-Carr 003 S70 Alternatively use PDMS
silver wire Sigma-Aldrich 348759 For electrodes
SPC Technology, D sub contact, pin Newark 9K4978 For electrodes
potassium chloride Sigma P9541  
potassium phosphate dibasic Sigma P2222  
potassium phosphate monobasic Sigma P5379  
PDMS Dow Corning   Sylgar 184 Elastomer set. For making chamber.
Kwik-Cast Sealant World Precision Instruments KWIK-CAST Fast acting silicone sealant
hot plate Fisher Scientific    
Faraday cage      
DOWNLOAD MATERIALS LIST

Riferimenti

  1. Li, J. Ion-beam sculpting at nanometre length scales. Nature. 412, 166-169 (2001).
  2. Li, J., Gershow, M., Stein, D., Brandin, E., Golovchenko, J. A. DNA molecules and configurations in a solid-state nanopore microscope. Nat. Mater. 2, 611-615 (2003).
  3. Dekker, C. Solid-state nanopores. Nature. Nanotechnology. 2, 209-215 (2007).
  4. Branton, D. The potential and challenges of nanopore sequencing. Nat. Biotechnol. 26, 1146-1153 (2008).
  5. Wanunu, M., Morrison, W., Rabin, Y., Grosberg, A. Y., Meller, A. Electrostatic focusing of unlabelled DNA into nanoscale pores using a salt gradient. Nat. Nanotechnol. 5, 160-165 (2010).
  6. Peng, H., Ling, X. S. Reverse DNA translocation through a solid-state nanopore by magnetic tweezers. Nanotechnology. 20, 185101-185101 (2009).
  7. Talaga, D. S., Li, J. Single-molecule protein unfolding in solid state nanopores. J. Am. Chem. Soc. 131, 9287-9297 (2009).
  8. Zhao, Q. Detecting SNPs using a synthetic nanopore. Nano. Lett. 7, 1680-1685 (2007).
  9. Storm, A. J., Chen, J. H., Ling, X. S., Zandbergen, H. W., Dekker, C. Fabrication of solid-state nanopores with single-nanometre precision. Nat. Mater. 2, 537-540 (2003).
  10. Sexton, L. T. Resistive-pulse studies of proteins and protein/antibody complexes using a conical nanotube sensor. J. Am. Chem. Soc. 129, 13144-13152 (2007).
  11. Martin, C. R., Siwy, Z. S. Chemistry. Learning nature’s way: biosensing with synthetic nanopores. Science. 317, 331-332 (2007).
  12. Sexton, L. T. An adsorption-based model for pulse duration in resistive-pulse protein sensing. J. Am. Chem. Soc. 132, 6755-6763 (2010).
  13. Niedzwiecki, D. J., Grazul, J., Movileanu, L. Single-molecule observation of protein adsoption onto an inorganic surface. J. Am. Chem. Soc. 132, 10816-10822 (2010).
  14. Wanunu, M., Meller, A., Selvin, P. R., Ha, T. . Single-molecule techniques: a laboratory manual. , 395-420 (2008).
  15. Han, A. Label-free detection of single protein molecules and protein-protein interactions using synthetic nanopores. Anal. Chem. 80, 4651-4658 (2008).
  16. Han, A. Sensing protein molecules using nanofabricated pores. Appl. Phys. Lett. 88, (2006).
  17. Fologea, D., Ledden, B., McNabb, D. S., Li, J. Electrical characterization of protein molecules by a solid-state nanopore. Appl. Phys. Lett. 91, (2007).
  18. 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. , (2010).
  19. Pedone, D., Firnkes, M., Rant, U. Data analysis of translocation events in nanopore experiments. Anal. Chem. 81, 9689-9694 (2009).
  20. Oukhaled, A. Dynamics of Completely Unfolded and Native Proteins through Solid-State Nanopores as a Function of Electric Driving Force. ACS. Nano.. , (2011).
  21. Yusko, E. C. Controlling protein translocation through nanopores with bio-inspired fluid walls. Nat. Nanotechnol. 6, 253-260 (2011).
  22. Arafat, A., Schroen, K., de Smet, L. C., Sudholter, E. J., Zuilhof, H. Tailor-made functionalization of silicon nitride surfaces. J. Am. Chem. Soc. 126, 8600-8601 (2004).
  23. Wanunu, M., Meller, A. Chemically modified solid-state nanopores. Nano. Lett. 7, 1580-1585 (2007).
  24. Movileanu, L., Cheley, S., Bayley, H. Partitioning of individual flexible polymers into a nanoscopic protein pore. Biophys. J. 85, 897-910 (2003).
  25. Aksimentiev, A. Deciphering ionic current signatures of DNA transport through a nanopore. Nanoscale. 2, 468-483 (2010).
  26. Timp, W. Nanopore Sequencing: Electrical Measurements of the Code of Life. IEEE Trans. Nanotechnol. 9, 281-294 (2010).
  27. Roach, P., Farrar, D., Perry, C. C. Surface tailoring for controlled protein adsorption: effect of topography at the nanometer scale and chemistry. J. Am. Chem. Soc. 128, 3939-3945 (2006).
  28. Roach, P., Farrar, D., Perry, C. C. Interpretation of protein adsorption: surface-induced conformational changes. J. Am. Chem. Soc. 127, 8168-8173 (2005).
  29. Brewer, S. H., Glomm, W. R., Johnson, M. C., Knag, M. K., Franzen, S. Probing BSA binding to citrate-coated gold nanoparticles and surfaces. Langmuir. 21, 9303-9307 (2005).
  30. Schon, P., Gorlich, M., Coenen, M. J., Heus, H. A., Speller, S. Nonspecific protein adsorption at the single molecule level studied by atomic force microscopy. Langmuir. 23, 9921-9923 (2007).
  31. Rabe, M., Verdes, D., Zimmermann, J., Seeger, S. Surface organization and cooperativity during nonspecific protein adsorption events. J. Phys. Chem. B. 112, 13971-13980 (2008).
  32. Rabe, M., Verdes, D., Rankl, M., Artus, G. R., Seeger, S. A comprehensive study of concepts and phenomena of the nonspecific adsorption of beta-lactoglobulin. Chemphyschem. 8, 862-872 (2007).
  33. Micic, M., Chen, A., Leblanc, R. M., Moy, V. T. Scanning Electron Microscopy Studies of Protein-Functionalized Atomic Force Microscopy Cantilever Tips. Scanning. 21, 394-397 (1999).
  34. Grant, A. W., Hu, Q. H., Kasemo, B. Transmission electron microscopy ‘windows’ for nanofabricated structures. Nanotechnology. 15, 1175-1181 (2004).
  35. Giannoulis, C. S., Desai, T. A. Characterization of proteins and fibroblasts on thin inorganic films. J. Mater. Sci: Mater. Med. 13, 75-80 (2002).
  36. Gustavsson, J. Surface modifications of silicon nitride for cellular biosensors applications. J. Mater. Sci: Mater. Med. 19, 1839-1850 (2008).
  37. Shirshov, Y. M. Analysis of the response of planar polarization interferometer to molecular layer formation: fibrinogen adsorption on silicon nitride surface. Biosens. Bioelectron. 16, 381-390 (2001).
  38. Chen, P. Atomic layer deposition to fine-tune the surface properties and diamters of fabricated nanopores. Nano. Lett. 4, 1333-1337 (2004).
  39. Siwy, Z. Protein biosensors based on biofunctionalized conical gold nanotubes. J. Am. Chem. Soc. 127, 5000-5001 (2005).
  40. Ding, S., Gao, C., Gu, L. Q. Capturing Single Molecules of Immunoglobulin and Ricin with an Aptamer-Encoded Glass Nanopore. Anal. Chem. , (2009).
  41. Kim, M. -. J., Wanunu, M., Bell, C. D., Meller, A. Rapid Fabrication of Uniform Size Nanopores and Nanopore Arrays for Parallel DNA Analysis. Adv. Mater. 18, 3149-3153 (2006).
  42. Bezrukov, S. M. Ion channels as molecular Coulter counters to probe metabolite transport. J. Membr. Biol. 174, 1-13 (2000).

Tags

Solid-state NanoporesProtein AdsorptionSingle-molecule MeasurementsNucleic AcidsProtein UnfoldingBinding AffinityResistive-pulse TechniqueLabeling-free MeasurementsTransmembrane PotentialAnalyte PartitioningNanopore WallsMicrofluidic DevicesProtein Binding AssayNitride FilmsThin FilmsFunctionalized SurfacesDenaturing Conditions
check_url/it/3560?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citazione di questo articolo
Niedzwiecki, D. J., Movileanu, L. Monitoring Protein Adsorption with Solid-state Nanopores. J. Vis. Exp. (58), e3560, doi:10.3791/3560 (2011).
Scarica citazione
Ristampe e autorizzazioni

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image