In this paper we report a label-free detection method of unmodified DNA using polypyrrole as an immobilization matrix by impedance measurement. A probe and a target complementary DNA sequence specific for the bacterial pathogen, <i>Bacillus cereus</i> are used. Impedance measurements are performed without using additional redox probes. The effects of hybridization and non-specific binding are compared when the Probe DNA molecules were immobilized by two different methods: electrochemical adsorption and entrapment. The probe DNA immobilized using electrochemical adsorption yielded better hybridization signals compared to that immobilized using the entrapment method. Control experiments were also performed to prove the specificity of the biosensor in the presence of non complementary DNA. Negligible unspecific binding with the immobilized probe was observed with the electrochemically adsorbed probe, whereas the entrapped probe responded to the non complementary target. The performance of the DNA sensor was characterized using both cyclic voltammetry and impedance spectroscopy techniques and proved to be effective in terms of specificity of hybridization events.
KeywordsImmobilizationLabel Free DNAPolypyrrole
M. V. Murugendrappa and M. V. N. Ambika Prasad, “Dielectric Spectroscopy of Polypyrrole-[Gamma]-Fe2O3 Composites,” Materials Research Bulletin, Vol. 41, No. 7, 2006, pp. 1364-1369. doi:10.1016/j.materresbull.2005.12.011
B. D. Malhotra, A. Chaubey and S. P. Singh, “Prospects of Conducting Polymers in Biosensors,” Analytica Chimica Acta, Vol. 578, No. 1, 2006, pp. 59-74. doi:10.1016/j.aca.2006.04.055
R. J. Geise, J. M. Adams, N. J. Barone and A. M. Yacynych, “Electropolymerized Films to Prevent Interferences and Electrode Fouling in Biosensors,” Biosensors & Bioelectronics, Vol. 6, No. 2, 1991, pp. 151-160. doi:10.1016/0956-5663(91)87039-E
N. J. Trujillo, M. C. Barr, S. G. Im and K. K. Gleason, “Oxidative Chemical Vapor Deposition (oCVD) of Patterned and Functional Grafted Conducting Polymer Nanostructures,” Journal of Materials Chemistry, Vol. 20, No. 19, pp. 3968-3972. doi:10.1039/b925736e
S. Liu, J. Q. Wang, D. Zhang, P. L. Zhang, J. F. Ou, B. Liu and S. R. Yang, “Investigation on Cell Biocompatible Behaviors of Polyaniline Film Fabricated via Electroless Surface Polymerization,” Applied Surface Science, Vol. 256, No. 11, pp. 3427-3431. doi:10.1016/j.apsusc.2009.12.046
F. Wei, W. Liao, Z. Xu, Y. Yang, D. T. Wong and C. M. Ho, “Bio/Abiotic Interface Constructed from Nanoscale DNA Dendrimer and Conducting Polymer for Ultrasensitive Biomolecular Diagnosis”, Small, Vol. 5, No. 15, 2009, pp. 1784-1790. doi:10.1002/smll.200900369
H. S. Kim, H. L. Hobbs, L. Wang, M. J. Rutten and C. C. Wamser, “Biocompatible Composites of Polyaniline Nanofibers and Collagen,” Synthetic Metals, Vol. 159, No. 13, 2009, pp. 1313-1318. doi:10.1016/j.synthmet.2009.02.036
N. K. E. Guimard, J. L.Sessler and C. E. Schmidt, “Toward a Biocompatible and Biodegradable Copolymer Incorporating Electroactive Oligothiophene Units,” Macromolecules, Vol. 42, No. 2, 2009, pp. 502-511. doi:10.1021/ma8019859
A. Ramanaviciene and A. Ramanavicius, “Application of Polypyrrole for the Creation of Immunosensors,” Critical Reviews in Analytical Chemistry, Vol. 32, No. 3, 2002, pp. 245-252. doi:10.1080/10408340290765542
A. Ramanavicius, A. Ramanaviciene and A. Malinauskas, “Electrochemical Sensors Based on Conducting Polymer-Polypyrrole,” Electrochimica Acta, Vol. 51, No. 27, 2006, pp. 6025-6037. doi:10.1016/j.electacta.2005.11.052
M. V. Murugendrappa and M. Prasad, “Chemical Synthesis, Characterization, and Direct-Current Conductivity Studies of Polypyrrole/Gamma-Fe2O3 Composites,” Journal of Applied Polymer Science, Vol. 103, No. 5, 2007, pp. 2797-2801. doi:10.1002/app.23868
S. Uzun and M. Can, “Oxidizing Effect on Chemical Polymerization of Pyrrole Monomer in Anhydrous Media,” Asian Journal of Chemistry, Vol. 22, No. 2, 2010, pp. 1321-1330.
S. Cavallaro, A. Colligiani and G. Cum, “Oxidative Chemical Polymerization of Pyrrole-Calorimetric and Kinetic Measurements,”Journal of Thermal Analysis and Calorimetry, Vol. 38, No. 12, 1992, pp. 2649-2655. doi:10.1007/bf01979741
J. Duchet, R. Legras and S. Demoustier-Champagne, “Chemical Synthesis of Polypyrrole: Structure-Properties Relationship,” Synthetic Metals, Vol. 98, No. 2, 1998, pp. 113-122. doi:10.1016/S0379-6779(98)00180-5
P. Dutta and S. K Mandal, “Charge Transport in Chemically Synthesized, DNA-Doped Polypyrrole,” Journal of Physics D-Applied Physics, Vol. 37, No. 20, 2004, pp. 2908-2913. doi:10.1088/0022-3727/37/20/019
A. K. Sharma, J. H. Kim and Y. S. Lee, “An Efficient Synthesis of Polypyrrole/Carbon Fiber Composite Nano-thin Films,” International Journal of Electrochemical Science, Vol. 4, No. 11, 2009, pp. 1560-1567.
S. Stankovic, R. Stankovic, M. Ristic, O. Pavlovic and M. Vojnovic, “Some Aspects of the Electrochemical Synthesis of Polypyrrole,” Reactive & Functional Polymers, Vol. 35, No. 3, 1997, pp. 145-151. doi:10.1016/S1381-5148(97)00090-4
K. Ghanbari, S. Z. Bathaie and M.F. Mousavi, “ElectroChemically Fabricated Polypyrrole Nanofiber-Modified Electrode as a New Electrochemical DNA Biosensor,” Biosensors & Bioelectronics, Vol. 23, No. 12, 2008, pp. 1825-1831. doi:10.1016/j.bios.2008.02.029
O. Fichet, F. Tran-Van, D. Teyssie and C. Chevrot, “Interfacial Polymerization of a 3, 4-Ethylenedioxythiophene Derivative Using Langmuir-Blodgett Technique. Spectroscopic and Electrochemical Characterizations,” Thin Solid Films, Vol. 411, No. 2, 2002, pp. 280-288. doi:10.1016/S0040-6090(02)00271-7
M. P. Srinivasan and F. J. Jing, “Composite Langmuir- Blodgett Films Containing Polypyrrole and Polyimide,” Thin Solid Films, Vol. 327-329, 1998, pp. 127-130. doi:10.1016/S0040-6090(98)00613-0
S. M. Shang, X. M. Yang, X. M. Tao and S. S. Lam, “Vapor-Phase Polymerization of Pyrrole on Flexible Substrate at Low Temperature and its Application in Heat Generation,” Polymer International, Vol. 59, No. 2, 2010, pp. 204-211.
H. Goktas, F. G. Ince, A. Iscan, I. Yildiz, M. Kurt and I. Kaya, “The Molecular Structure of Plasma Polymerized Thiophene and Pyrrole Thin Films Produced by Double Discharge Technique,” Synthetic Metals, Vol. 159, No. 19-20, 2009, pp. 2001-2008. doi:10.1016/j.synthmet.2009.07.003
Z. Zhang, P. Liang, X. Zheng, D. Peng, F. Yan, R. Zhao and C.-L. Feng, “DNA Immobilization/Hybridization on Plasma-Polymerized Pyrrole,” Biomacromolecules, Vol. 9, No. 6, 2008, pp. 1613-1617. doi:10.1021/bm800111a
Z. B. Bahsi, A. Buyukaksoy, S. M. Olmezcan, F. Simsek, M. H. Aslan and A. Y.Oral, “A Novel Label-Free Optical Biosensor Using Synthetic Oligonucleotides from E. coli O157:H7: Elementary Sensitivity Tests,” Sensors, Vol. 9, No. 6, 2009, pp. 4890-4900. doi:10.3390/s90604890
J. Liu, and Y. Lu, “Colorimetric and Fluorescent Biosensors Based on Directed Assembly of Nanomaterials with Functional DNA,” Integrated Analytical Systems, Springer, New York, 2009, pp. 155-178. doi:10.1007/978-0-387-73711-9_6
A. K. Singh, D. Senapati, S. G. Wang, J. Griffin, A. Neely, P. Candice, K. M. Naylor, B. Varisli, J. R. Kalluri and P. C. Ray, “Gold Nanorod Based Selective Identification of Escherichia coli Bacteria Using Two-Photon Rayleigh Scattering Spectroscopy,” Acs Nano, Vol. 3, No. 7, 2009, pp. 1906-1912. doi:10.1021/nn9005494
P. S. Petrou, S. E. Kakabakos and K. Misiakos, “Silicon Optocouplers for Biosensing,” International Journal of Nanotechnology, Vol. 6, No. 1-2, 2009, pp. 4-17.
S. J. P. Canete, W. W. Yang and R. Y. Lai, “Folding-Based Electrochemical DNA Sensor Fabricated by ‘Click’ Chemistry,” Chemical Communications, No. 32, 2009, pp. 4835-4837.
J. Jiang, D. Jiang, Y. Li, J. Wang and K. L. P. Sung, “Polypyrrole-Based Genolelectronics: Electrochemical DNA Biosensors,” Science & Technology Review, No. 11, 2009, pp. 93-101.
V. Velusamy, K. Arshak, O. Korostynska, K. Oliwa and C. Adley, “Design of a Real Time Biorecognition System to Detect Foodborne Pathogens-DNA Biosensor,” in SAS 2009. New Orleans, USA, 2009, pp. 38-41.
P. I. Reyes, Z. Zhang, H. H. Chen, Z. Q. Duan, J. Zhong, G. Saraf, Y. C. Lu, O. Taratula, E. Galoppini and N. N. Boustany, “A ZnO Nanostructure-Based Quartz Crystal Microbalance Device for Biochemical Sensing,” IEEE Sensors Journal, Vol. 9, No. 10, 2009, pp. 1302-1307. doi:10.1109/JSEN.2009.2030250
A. Tsortos, G. Papadakis and E. Gizeli, “Shear Acoustic Wave Biosensor for Detecting DNA Intrinsic Viscosity and Conformation: A study with QCM-D,” Biosensors & Bioelectronics, Vol. 24, No. 4, 2008, pp. 836-841. doi:10.1016/j.bios.2008.07.006
J. Wang, G. Rivas, C. Parrado, X. H. Cai and M. N. Flair, “Electrochemical Biosensor for Detecting DNA Sequences from the Pathogenic Protozoan Cryptosporidium parvum,” Talanta, Vol. 44, No. 11, 1997, pp. 2003-2010. doi:10.1016/S0039-9140(96)02191-1
D. Zhang and E. C. Alocilja, “Characterization of Nanoporous Silicon-Based DNA Biosensor for the Detection of Salmonella Enteritidis,” IEEE Sensors Journal, Vol. 8, No. 5-6, 2008, pp. 775-780. doi:10.1109/JSEN.2008.923037
V. Vyskocil, J. Labuda and J. Barek, “Voltammetric Detection of Damage to DNA Caused by Nitro Derivatives of Fluorene Using an Electrochemical DNA Biosensor,” Analytical and Bioanalytical Chemistry, Vol. 397, No. 1, pp. 233-241. doi:10.1007/s00216-010-3517-y
A. M. Nowicka, A. Kowalczyk, Z. Stojek and M. Hepel, “Nanogravimetric and Voltammetric DNA-Hybridization Biosensors for Studies of DNA Damage by Common Toxicants and Pollutants,” Biophysical Chemistry, Vol. 146, No. 1, pp. 42-53. doi:10.1016/j.bpc.2009.10.003
S. L. Yang, B. Y. Xia, X. D. Zeng, S. L. Luo, W. Z. Wei and X. Y. Liu, “Fabrication of DNA functionalized carbon nanotubes/Cu2+ complex by one-step electrodeposition and its sensitive determination of nitrite,” Analytica Chimica Acta, Vol. 667, No. 1-2, pp. 57-62. doi:10.1016/j.aca.2010.03.063
H. J. Qiu, Y. L. Sun, X. R. Huang and Y. B. Qu, “A Sensitive Nanoporous Gold-Based Electrochemical Aptasensor for Thrombin Detection,” Colloids and Surfaces B-Biointerfaces, Vol. 79, No. 1, pp. 304-308. doi:10.1016/j.colsurfb.2010.04.017
A. M. Tencaliec, S. Laschi, V. Magearu and M. Mascini, “A Comparison Study between a Disposable Electrochemical DNA Biosensor and a Vibrio Fischeri-Based Luminescent Sensor for the Detection of Toxicants in Water Samples,” Talanta, Vol. 69, No. 2, 2006, pp. 365-369. doi:10.1016/j.talanta.2005.09.042
G. Bagni, T. Baussant, G. Jonsson, J. Barsiene and M. Mascini, “Electrochemical Device for the Rapid Detection of Genotoxic Compounds in Fish Bile Samples,” Analytical Letters, Vol. 38, No. 15, 2005, pp. 2639-2652. doi:10.1080/00032710500371105
J. Y. Park and S. M.Park, “DNA Hybridization Sensors Based on Electrochemical Impedance Spectroscopy as a Detection Tool,” Sensors, Vol. 9, No. 12, 2009, pp. 9513-9532. doi:10.3390/s91209513
V. Velusamy, K. Arshak, O. Korostynska, K. Oliwa and C. Adley, “An Overview of Foodborne Pathogen Detection: In the Perspective of Biosensors,” Biotechnology Advances, Vol. 28, No. 2, 2010, pp. 232-254. doi:10.1016/j.biotechadv.2009.12.004
A. Bogomolova, E. Komarova, K. Reber, T. Gerasimov, O. Yavuz, S. Bhatt and M. Aldissi, “Challenges of Electrochemical Impedance Spectroscopy in Protein Biosensing,” Analytical Chemistry, Vol. 81, No. 10, 2009, pp. 3944-3949. doi:10.1021/ac9002358
C. Gautier, C. Esnault, C. Cougnon, J.-F. Pilard, N. Casse, and B. Chénais, “Hybridization-Induced interfacial changes detected by non-Faradaic impedimetric measurements compared to Faradaic approach,” Journal of Electroanalytical Chemistry, Vol. 610, No. 2，2007, pp. 227-233. doi:10.1016/j.jelechem.2007.07.013
C. Adley, K. Arshak, C. Molnar, K. Oliwa and V. Velusamy, “Design of Specific DNA Primers to Detect the Bacillus Cereus Group Species,” Sensors Applications Symposium, New Orleans, USA, 2009, pp. 236-239. doi: 10.1109/SAS.2009.4801807
F. Devreux, F. Genoud, M. Nechtschein and B. Villeret, “Electron-Spin-Resonance Investigation of Polarons and Bipolarons in Conducting Polymers-the Case of Polypyrrole,” Synthetic Metals, Vol. 18, No. 1-3, 1987, pp. 89-94. doi:10.1016/0379-6779(87)90859-9
A. O. Patil, A. J. Heeger and F. Wudl, “Optical-Properties of Conducting Polymers,” Chemical Reviews, Vol. 88, No. 1, 1988, pp. 183-200. doi:10.1021/cr00083a009
K. Arshak, V. Velusamy, O. Korostynska, K. Oliwa-Stasiak and C. Adley, “Conducting Polymers and Their Applications to Biosensors: Emphasizing on Foodborne Pathogen Detection,” IEEE Sensors Journal, Vol. 9, No. 12, 2009, pp. 1942-1951. doi:10.1109/JSEN.2009.2032052
D. S. Minehan, K. A. Marx and S. K. Tripathy, “DNA Binding to Electropolymerized Polypyrrole: The Dependence on Film Characteristics,” Journal of macromolecular science. Pure and applied chemistry, Vol. 38 2001, pp. 1245-1258. doi:10.1081/MA-100108381
K. L.Hanson, l. Filipponi and D. V. Nicolau, “Biomolecules and cells on surfaces: Fundamental concepts,” Uwe R. Müller and Dan V. Nicolau, Editors, Microarray Technology: Fundamentals, Fabrication and Applications, Springer Verlag, 2004, pp. 23-44.
L. A. Thompson, J. Kowalik, M. Josowicz and J. Janata, “Label-Free DNA Hybridization Probe Based on a Conducting Polymer,” Journal of the American Chemical Society, Vol. 125, No. 2, 2002, pp. 324-325. doi:10.1021/ja027929z
V. Chan, D. J. Graves, P. Fortina and S. E. McKenzie, “Adsorption and Surface Diffusion of DNA Oligonucleotides at Liquid/Solid Interfaces,” Langmuir, Vol. 13, No. 2, 1997, pp. 320-329. doi:10.1021/la960670b
V. Chan, S. E. McKenzie, S. Surrey, P. Fortina and D. J. Graves, “Effect of Hydrophobicity and Electrostatics on Adsorption and Surface Diffusion of DNA Oligonucleotides at Liquid/Solid Interfaces,” Journal of Colloid and Interface Science, Vol. 203, No. 1, 1998, pp. 197-207. doi:10.1006/jcis.1998.5495
A. M. Oliveira Brett, and A.-M. Chiorcea, “Atomic Force Microscopy of DNA Immobilized onto a Highly Oriented Pyrolytic Graphite Electrode Surface,” Langmuir, Vol. 19, No. 9, 2003, pp. 3830-3839. doi:10.1021/la027047d