Elsevier

Biosensors and Bioelectronics

Volume 47, 15 September 2013, Pages 133-140
Biosensors and Bioelectronics

Polypyrrole–poly(3,4-ethylenedioxythiophene)–Ag (PPy–PEDOT–Ag) nanocomposite films for label-free electrochemical DNA sensing

https://doi.org/10.1016/j.bios.2013.02.049Get rights and content

Highlights

  • PPy–PEDOT–AgNP nanocomposite has been synthesized using a simple chemical method.

  • DNA hybridization sensor based on thiolated ssDNA immobilized on this nanocomposite has been fabricated.

  • Highly sensitive, stable and reproducible sensor has been achieved.

  • The developed DNA sensor effectively discriminates between single and double base mismatch target DNA.

Abstract

The electrochemical DNA hybridization sensing of bipolymer polypyrrole and poly(3,4-ethylenedioxythiophene) (PPy–PEDOT) nanotubes functionalized with Ag nanoparticles has been investigated. The bipolymer nanotubes are prepared by simple chemical route and silver nanoparticles (Ag) further deposited over the PPy–PEDOT nanotubes to form PPy–PEDOT–Ag nanocomposite films. DNA labeled at 5′end using 6-mercapto-1-hexhane (HS-ssDNA) is immobilized on the PPy–PEDOT–Ag surface to form PPy–PEDOT–Ag–S-ssDNA and hybridization sensing is done in phosphate buffer. The presence of Ag nanoparticles (~28±5 nm) well dispersed in the polymer composite with high surface area, high electrical conductivity and catalytic activity provides desirable microenvironment for the immobilization of probe DNA with controlled orientation leading to increased hybridization efficiency with target DNA. The morphological and structural characterizations by a scanning electron microscope (SEM) and X-ray diffraction (XRD) confirm the nanotube structure of composite polymer while Raman measurements indicate the efficient interactions between the PPy, PEDOT, Ag and HS-ssDNA. The sensor effectively discriminates different target DNA sequences with PPy–PEDOT–Ag–S-ssDNA substrate. The observed dynamic detection range is found between 1×10−11 M and 1×10−14 M with the lowest detection limit (3 σ/b) of 5.4×10−15 M. This observed value is of higher sensitivity than that for MWCNT–Ag, PANi–Au, MWCNT–PPy–Au and PPy–PANi–Au composites reported previously.

Introduction

The sequence-specific detection of DNA targets (for genetic or pathogenic diseases) are receiving considerable attention in molecular diagnostics (Stobiecka et al., 2007, Karimi et al., 2012, Stobiecka et al., 2012, Carlsson et al., 2011, Dar et al., 2011, Prabhakar et al., 2012, Hepel and Stobiecka, 2011, Jin et al., 2012, Mascolo et al., 2011, Shankar and Deka, 2011). The detection of DNA sequence requires the development of easy to-use, fast, inexpensive, miniaturized analytical devices. So, there is an urgent need for a faster, cheaper, and simpler DNA assay that would tackle the demands of modern medical diagnostics and biomedical research. In the past decade, a variety of techniques have been developed for the detection of DNA hybridization such as electrochemical (Wang, 2002, Katz et al., 2005), optical (Liu et al., 2005, Peter et al., 2001) and acoustic (Zhou et al., 2001). Among them, electrochemical sensing has attracted paramount interest in the development of DNA sensors due to its simplicity in experimental design and mass fabrication of sensors element. In the direction of fabrication of highly sensitive and selective transducers, nanogold (Castaneda et al., 2007, Ensafi et al., 2011, Liu et al., 2011a), quantum dots (Zhang et al., 2005, Peng et al., 2007a), carbon nanotubes (Martinez et al., 2009, Tang et al., 2006), graphene (Mohanty and Berry, 2008, Kuila et al., 2011) and conducting polymers (Peng et al., 2009, Kannan et al., 2011) are being investigated for DNA sensing. Among them, conducting polymers are well-known functional materials for biosensing applications due to their unique properties. Particularly, the nanostructured conducting polymers such as nanowire, nanotubes, and micro-structured films have been currently demonstrated to improve the sensitivity of the sensors. The nanostructured conducting polymers have many additional advantages over conventional conducting polymers including high effective surface area, low density, along with special chemical and physical properties. Generally, immobilization of biomolecules on the conducting polymer surfaces falls into two classes: (i) non-covalent doping of DNA by co-polymerization of both monomer and DNAs (Cai et al., 2003). However, the capture probes are less accessible for hybridization due to their imperfect orientation. (ii) Covalent attachment of the 5′ end functionalized DNA on the –COOH functionalized monomers was carried out by the 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide/N-hydroxy succinimide (EDC/NHS) coupling (Peng et al., 2007b). Unfortunately, it involves complex chemistry for the functionalization of the native monomer. Currently, the conducting polymer and its composites with metal nanoparticles and nanocomposites are attractive materials for the development of electrochemical DNA sensors. The major advantages of this biosensor are (i) we can avoid functionalization steps on the native monomer, (ii) the noble silver metal nanoparticles are able to provide a stable immobilization of biomolecules that retain their bioactivity and (iii) simple and novel bipolymer nanocomposite preparation with excellent porosity which eliminates the conventional templates and surfactants. Recently, conducting polymer metal nanocomposites such as PEDOT–AuNP, PANi–AuNP, MWCNT–PPy–AuNP and PPy–PANi–AuNP have been used for DNA sensing applications (Spain et al., 2012, Feng et al., 2008, Liu et al., 2011b, Wilson et al., 2012). However, in these work, they have followed complex nanomaterials preparation and DNA immobilization. For example, Spain et al. (2012) recently developed a DNA sensor based PEDOT–AuNP composite prepared by the vapor phase polymerization method. In addition, in this work horseradish peroxidase (HRP) enzyme tagged probe DNA was used to improve the sensitivity and selectivity. This enzyme tagged method has shortcomings arising from limited tagging efficiency and complex multistep analysis. In recent years, Ag nanoparticles are extensively used in DNA hybridization sensing for amplification of hybridization event (Wang, 2003, Fu et al., 2005, Niu et al., 2009, Cai et al., 2002) owing to their catalytic properties and also exhibit the highest electrical and thermal conductivities among all the metals. Also, the Ag nanoparticles demonstrate many advantages when compared to Au nanoparticles, such as higher extinction coefficients, sharper extinction bands and higher field enhancement (Caro et al., 2010). Many techniques including chemical reduction in aqueous solution with or without stabilizing agent, thermal decomposition in organic solvents, chemical, photochemical and microwave irradiation have been employed in the synthesis of Ag nanoparticles. However, these methods are obviously involving complicated procedure with high energy consuming and sophisticated techniques.

So, in the present work, Ag nanoparticles functionalized PPy–PEDOT nanotubes are prepared by a simple method without using any expensive capping or reducing agent and the time required for the synthesis is around 2 h only. Further, the Ag nanoparticles have been uniformly deposited along the walls of the nanotubes. Subsequently, the HS-ssDNA is immobilized onto the Ag nanoparticles (Fu et al., 2005, Niu et al., 2009) and used for the label-free DNA hybridization sensing in phosphate buffer for the first time. The sensor behavior is characterized by cyclic voltammetry and impedance. The morphological changes of the modified electrode surfaces are examined by SEM, Raman and FT-IR techniques.

Section snippets

Materials

Pyrrole monomer and silver nitrate (AgNO3) were purchased from SRL (Mumbai, India) and used as received. 3,4-ethylenedioxythiophene (EDOT), sodium-saline citrate (SSC) buffer, 6-mercaptohexanol (MCH) and ferric chloride (FeCl3) were used as received from Sigma-Aldrich (Bangalore, India) and sodium dodecyl sulfate (SDS), methyl orange (MO) were purchased from Himedia (Mumbai, India). All other reagents were of analytical grade and used without further purification.

Thiolated short chain 27 mer

FT-IR, Raman, XRD and UV–vis characterizations of PPy–PEDOT–Ag composites

The FT-IR spectra of PPy, PEDOT, PPy–PEDOT and PPy–PEDOT–Ag composites are presented in Fig. S1. The main characteristic peaks of PPy were assigned as follows: 3429 cm−1 (N–H stretching), 1524 cm−1 (C=C and C–H stretching), 1440 cm−1 (N–H stretching) and 1280 cm−1 (C–H and N–H deformation) in the pyrrole ring. In the FT-IR spectra of PEDOT, peaks seen at 1543 and 1311 cm−1 (C=C and C–C in the thiophene ring), 905 cm−1 (C–S bond), 668 cm−1 (C–S–C stretching) in the thiophene ring and 905 cm−1

Conclusion

We have demonstrated a simple, convenient electrochemical approach to fabricate PPy–PEDOT–Ag nanocomposite films modified GC electrode for DNA sensor. The microdispersed Ag nanoparticles in the PPy–PEDOT nanotubes provide a good platform for anchoring the thiolated HS-ssDNA in a controlled manner. The hybridization efficiency is enhanced by 2.7 times due to the fact that this platform accommodates the optimum probe DNA density (27.198×10−13 mol/cm2) and the microdispersed Ag nanoparticles in

Acknowledgments

S.R. thanks the DST, India and MEST-NRF, Korea for the award of Indo-Korean Research Internship (IKRI). J.W. acknowledges the UGC for funding (UGC. F. no. 36-2/2008). Ahmad Umar would like to acknowledge the Ministry of Higher Education, Kingdom of Saudi Arabia (PCSED-001-11) under the Promising Center on Sensor and Electronic Devices at Najran University, Kingdom of Saudi Arabia.

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