Isoelectric Point Determination of Proteins Using Track-Etched Single-Nanopore Membrane

Abstract

In this study, we have investigated a new method to determine the pI values of proteins compared to the conventional methods. We developed a new sensing platform and paradigm to succeed it. The track-etched poly ethylene terephthalate PET membranes were used to fabricate both single and multipore membranes. Two-step chemical etching method was prefered to fabricate single nanopore in a reproducible manner. EDC-coupling was used to modify and attach the proteins inside the nanopore surface. By the aid of ion current rectification, the surface charge characteristics of nanopores were identified with and without protein. A close approximation was successfully made to the literature values for the pI values of proteins using nanopore membranes respect.

Keywords

• Track-etched polymer membrane,ion-current rectification,nanopore sensor,electrochemical sensor

İz-Aşındırılmış Tekli Nano Membran Kullanarak Proteinlerin İzoelektrik Noktalarının Belirlenmesi

Abstract

B u çalışmada geleneksel yöntemlere kıyasla proteinlerin pI değerlerinin belirlenmesi için yeni bir yöntem geliştirilmesini araştırdık. Tekli ve çoklu nanogözeneklerin üretilmesi için iz-aşındırılmış polietilen tereftalat membranlar kullanılmıştır. Tekli nanogözeneklerin tekrarlanabilir şekilde üretilmesi için iki basamaklı kimyasal aşındırma yöntemi tercih edilmiştir. Nanogözeneklerin iç yüzeyine proteinlerin tutturulması için EDC bağlanması kullanılmıştır. İyon-akım rektifikasyonun yardımıyla protein varlığında ve yokluğunda yüzey yük karakteristiği belirlenmiştir. Nanogözenek membranlar kullanarak elde edilen veriler ile literatürde ki pI değerlerine başarı ile yaklaşılmıştır

Keywords

• İz-aşındırılmış polimer membran,iyon-akım rektifikasyonu,nanogözenek sensörü,elektrokimyasal sensör

References

1. Ai, Y., Liu, J., Zhang, B.K. & Qian, S.Z. Ionic current rectification in a conical nanofluidic field effect transistor, Sensor Actuat B-Chem, 157 (2011) 742-751 .
2. Wang, C., Xu, J.J., Chen, H.Y. & Xia, X.H. Mass transport in nanofluidic devices, Sci China Chem, 55 (2012) 453-468 . Diameter and Debye Length on Ion Current Rectification in a Fluidic Bipolar Diode, J Phys Chem C, 115 (2011) 22917-22924.
3. Plecis, A., Pallandre, A. & Haghiri-Gosnet, A.M. Ionic and mass transport in micro-nanofluidic devices: a matter of volumic surface charge, Lab Chip, 11 (2011) 795-804.
4. 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 (2008) 16351-16357.
5. Siwy, Z. et al. Protein biosensors based on biofunctionalized conical gold nanotubes, J Am Chem Soc, 127 (2005) 5000-5001.
6. Xie, R. et al. Characterization of microstructure of poly (N-isopropylacrylamide)-grafted polycarbonate track-etched membranes prepared by plasma-graft pore-filling polymerization, J Membrane Sci, 258 (2005) 157-166.
7. Wang, J. & Martin, C.R. A new drug-sensing paradigm based on ion-current rectification in a conically shaped nanopore, Nanomedicine-Uk, 3 (2008) 13-20.
8. Kececi, K., Sexton, L.T., Buyukserin, F. & Martin, C.R. Resistive-pulse detection of short dsDNAs using a chemically functionalized conical nanopore sensor, Nanomedicine-Uk, 3 (2008) 787-796.

9. Sexton, L. T. et al. Resistive-pulse studies of proteins and protein/antibody complexes using a conical nanotube sensor, J Am Chem Soc, 129 (2007) 13144- 13152.
10. Wanunu, M. & Meller, A. Chemically modified solid- state nanopores, Nano Lett, 7 (2007) 1580-1585.
11. Hu, K.K. et al. Open Carbon Nanopipettes as Resistive-Pulse Sensors, Rectification Sensors, and Electrochemical Nanoprobes, Anal Chem, 86 (2014) 8897-8901.
12. Chen, P. et al. Atomic layer deposition to fine-tune the surface properties and diameters of fabricated nanopores, Nano Lett, 4 (2004) 1333-1337.
13. Nguyen, Q.H., Ali, M., Neumann, R. & Ensinger, W. Saccharide/glycoprotein recognition inside synthetic ion channels modified with boronic acid, Sensor Actuat B-Chem, 162 (2012) 216-222.
14. Wharton, J.E. et al. A method for reproducibly preparing synthetic nanopores for resistive-pulse biosensors, Small, 3 (2007) 1424-1430.
15. Dhopeshwarkar, R., Crooks, R.M., Hlushkou, D. & Tallarek, U. Transient effects on microchannel electrokinetic filtering with an ion-permselective membrane, Anal Chem, 80 (2008) 1039-1048.
16. Kovarik, M.L., Zhou, K.M. & Jacobson, S.C. Effect of Conical Nanopore Diameter on Ion Current Rectification, J Phys Chem B, 113 (2009) 15960- 15966.
17. Righetti, P.G., Tudor, G. & Ek, K. Isoelectric Points and Molecular-Weights of Proteins - a New Table, J Chromatogr, 220 (1981) 115-194.
18. McCafferty, E. & Wightman, J. P. Determination of the surface isoelectric point of oxide films on metals by contact angle titration, J Colloid Interf Sci, 194 (1997) 344-355.