Imaging of DNA hybridization on microscopic polypyrrole patterns using scanning electrochemical microscopy (SECM): the HRP bio-catalyzed oxidation of 4-chloro-1-naphthol.

We illustrate in this paper the successful combination of the direct and feedback mode of scanning electrochemical microscopy (SECM) for the writing of oligonucleotide patterns on thin gold films alongside the imaging of DNA hybridization. The patterning process was achieved using the direct mode of SECM, where the electrical field established between the SECM tip and the gold interface was used to drive the local deposition of micrometre sized polypyrrole spots to which a 15(mer) oligonucleotide (ODN) strand was linked covalently. Imaging of the deposited polypyrrole-ODNs was achieved by means of the feedback mode of SECM using Ru(NH(3))(6)(3+) as the mediator. The detection of the hybridization reaction of the ODN probes with their biotinylated complementary strands using SECM was possible after subsequent reactions with streptavidin and biotinylated horseradish peroxidase (HRP). The HRP-biocatalyzed oxidation of 4-chloro-1-naphthol (1) in the presence of H(2)O(2), and the precipitation of the insoluble product 4-chloro-1-naphthon (2) on the hybridized areas on the gold film caused a local alteration of conductivity. Such a change in conductivity was sensitively detected by the SECM tip and allowed imaging of DNA arrays in a fast and straightforward way.

[1]  Thierry Livache,et al.  Micro‐Imprinting of Oligonucleotides and Oligonucleotide Gradients on Gold Surfaces: A New Approach Based on the Combination of Scanning Electrochemical Microscopy and Surface Plasmon Resonance Imaging (SECM/ SPR‐i) , 2005 .

[2]  T. Livache,et al.  Reversible oligonucleotide immobilisation based on biotinylated polypyrrole film , 2001 .

[3]  Jun Wang,et al.  Scanning electrochemical microscopic imaging of surface-confined DNA probes and their hybridization via guanine oxidation , 2002 .

[4]  J. Heinze,et al.  Applications of scanning ultra micro electrodes for studies on surface conductivity , 1995 .

[5]  M. Mirkin,et al.  Electroanalytical measurements using the scanning electrochemical microscope , 2000 .

[6]  G. Wittstock,et al.  Patterns of functional proteins formed by local electrochemical desorption of self-assembled monolayers , 2001 .

[7]  E. Paleček,et al.  Past, present and future of nucleic acids electrochemistry. , 2002, Talanta.

[8]  T. Livache,et al.  Biosensing effects in functionalized electroconducting conjugated polymer layers: addressable DNA matrix for the detection of gene mutations , 1995 .

[9]  D. Wipf,et al.  Localized avidin/biotin derivatization of glassy carbon electrodes using SECM. , 1998, Analytical chemistry.

[10]  T. Livache,et al.  Polypyrrole DNA chip on a silicon device: example of hepatitis C virus genotyping. , 1998, Analytical biochemistry.

[11]  Heinz Schmid,et al.  Microfluidic Networks for Chemical Patterning of Substrates: Design and Application to Bioassays , 1998 .

[12]  W. Schuhmann,et al.  Formation and imaging of microscopic enzymatically active spots on an alkanethiolate-covered gold electrode by scanning electrochemical microscopy , 1997 .

[13]  T. Livache,et al.  Polypyrrole electrospotting for the construction of oligonucleotide arrays compatible with a surface plasmon resonance hybridization detection , 2001 .

[14]  W. Schuhmann,et al.  Visualization of micropatterned complex biosensor sensing chemistries by means of scanning electrochemical microscopy. , 2004, Biosensors & bioelectronics.

[15]  S. Cosnier.  Biomolecule immobilization on electrode surfaces by entrapment or attachment to electrochemically polymerized films. A review. , 1999, Biosensors & bioelectronics.

[16]  Jan Greve,et al.  Vibrating mirror surface plasmon resonance immunosensor , 1991 .

[17]  W. Heineman,et al.  Spatially addressed deposition and imaging of biochemically active bead microstructures by scanning electrochemical microscopy. , 2000, Analytical chemistry.

[18]  C. Kranz,et al.  Lateral deposition of polypyrrole lines over insulating gaps. Towards the development of polymer-based electronic devices† , 1995 .

[19]  Anthony G. Frutos,et al.  Peer Reviewed: SPR of Ultrathin Organic Films , 1998 .

[20]  H. Kondo,et al.  Visualization of DNA microarrays by scanning electrochemical microscopy (SECM). , 2001, The Analyst.

[21]  Thierry Livache,et al.  Electronically conductive polymer grafted with oligonucleotides as electrosensors of DNA: Preliminary study of real time monitoring by in situ techniques , 2001 .

[22]  A. Bard,et al.  Scanning electrochemical microscopy. Theory of the feedback mode , 1989 .

[23]  M. Billon,et al.  Scanning electrochemical microscopy (SECM): localized glucose oxidase immobilization via the direct electrochemical microspotting of polypyrrole–biotin films , 2005 .

[24]  Gordon G. Wallace,et al.  Coupling conducting polymers and mediated electrochemical responses for the detection of Listeria , 2003 .

[25]  Joseph Wang.  Electrochemical nucleic acid biosensors , 2002 .

[26]  G. Wittstock,et al.  Scanning electrochemical microscopy for detection of biosensor and biochip surfaces with immobilized pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase as enzyme label. , 2005, Biosensors & bioelectronics.

[27]  W. Schuhmann,et al.  Lateral deposition of polypyrrole lines by means of the scanning electrochemical microscope , 1995 .

[28]  I. Willner,et al.  Liposomes labeled with biotin and horseradish peroxidase: a probe for the enhanced amplification of antigen--antibody or oligonucleotide--DNA sensing processes by the precipitation of an insoluble product on electrodes. , 2001, Analytical chemistry.

[29]  A. Peterson,et al.  Surface Plasmon Resonance Spectroscopy as a Probe of In-Plane Polymerization in Monolayer Organic Conducting Films , 2000 .

[30]  G. Wittstock,et al.  Generation of Periodic Enzyme Patterns by Soft Lithography and Activity Imaging by Scanning Electrochemical Microscopy , 2002 .

[31]  J. Kauffmann,et al.  Hydrogen peroxide sensitive amperometric biosensor based on horseradish peroxidase entrapped in a polypyrrole electrode. , 2002, Biosensors & bioelectronics.

[32]  Jun Wang,et al.  Silver-Enhanced Imaging of DNA Hybridization at DNA Microarrays with Scanning Electrochemical Microscopy , 2002 .

[33]  W. Schuhmann,et al.  Imaging of microstructured biochemically active surfaces by means of scanning electrochemical microscopy , 1997 .

[34]  Compton,et al.  Laser-activated voltammetry: measurement of the diffusion coefficients of electropassivating species. Application to pyrrole and phenol in aqueous solution , 2000, Analytical chemistry.

[35]  Wolfgang Schuhmann,et al.  Imaging immobilised ssDNA and detecting DNA hybridisation by means of the repelling mode of scanning electrochemical microscopy (SECM). , 2004, Biosensors & bioelectronics.

[36]  T. Wink,et al.  Liposome-mediated enhancement of the sensitivity in immunoassays of proteins and peptides in surface plasmon resonance spectrometry. , 1998, Analytical chemistry.

[37]  T. Livache,et al.  New approach to writing and simultaneous reading of micropatterns: combining surface plasmon resonance imaging with scanning electrochemical microscopy (SECM). , 2004, Langmuir : the ACS journal of surfaces and colloids.

[38]  T. Matsue,et al.  Patterning and characterization of surfaces with organic and biological molecules by the scanning electrochemical microscope. , 2000, Analytical chemistry.

[39]  R. E. Gyurcsányi,et al.  Chemical imaging of biological systems with the scanning electrochemical microscope. , 2004, Bioelectrochemistry.

[40]  S. Rotenberg,et al.  Scanning electrochemical microscopy of living cells: different redox activities of nonmetastatic and metastatic human breast cells. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[41]  F Lesbre,et al.  Characterization and optimization of a real-time, parallel, label-free, polypyrrole-based DNA sensor by surface plasmon resonance imaging. , 2000, Analytical chemistry.

[42]  P. Caillat,et al.  Electroconducting polymers for the construction of DNA or peptide arrays on silicon chips. , 1998, Biosensors & bioelectronics.

[43]  Minhee Yun,et al.  Glucose oxidase entrapped in polypyrrole on high-surface-area Pt electrodes: a model platform for sensitive electroenzymatic biosensors , 2005 .

[44]  T. Livache,et al.  Preparation of a DNA matrix via an electrochemically directed copolymerization of pyrrole and oligonucleotides bearing a pyrrole group. , 1994, Nucleic acids research.

[45]  E. García-Ruiz,et al.  Amperometric cholesterol biosensors based on the electropolymerization of pyrrole and the electrocatalytic effect of Prussian-Blue layers helped with self-assembled monolayers. , 2004, Talanta.

[46]  A. Heller,et al.  Scanning electrochemical microscopy. 44. Imaging of horseradish peroxidase immobilized on insulating substrates. , 2002, Analytical chemistry.