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Scanning Electrochemical Microscopy (SECM) Based Detection of Oligonucleotide Hybridization and Simultaneous Determination of the Surface Concentration of Immobilized Oligonucleotides on Gold
Author(s) -
Nunes Kirchner Carolina,
Szunerits Sabine,
Wittstock Gunther
Publication year - 2007
Publication title -
electroanalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.574
H-Index - 128
eISSN - 1521-4109
pISSN - 1040-0397
DOI - 10.1002/elan.200703862
Subject(s) - ultramicroelectrode , scanning electrochemical microscopy , oligonucleotide , streptavidin , chemistry , polymer , substrate (aquarium) , pyrrole , biotin , analytical chemistry (journal) , chemical engineering , nanotechnology , electrochemistry , materials science , chromatography , dna , organic chemistry , biochemistry , electrode , cyclic voltammetry , oceanography , engineering , geology
The direct mode of scanning electrochemical microscopy (SECM) was used for the local deposition of oligonucleotide (ODN) patterns on thin gold films and the generation‐collection (GC) mode was applied for the determining the amount of surface‐accessible oligonucleotides. The local deposition was achieved through the micrometer‐sized formation of a conducting polymer bearing 15 mer single‐stranded oligonucleotide strands. After the interaction of the oligonucleotide with its biotin‐labeled complimentary strand, streptavidin was bound. The molecular assembly was completed by linking biotin‐labeled β ‐galactosidase from Escherichia coli to the streptavidin. The activity of the linked β ‐galactosidase was mapped with SECM in the GC mode by monitoring the oxidation of p ‐aminophenol (PAP) formed in the enzyme‐catalyzed hydrolysis of p ‐aminophenyl‐ β ‐ D ‐galactopyranoside. The feedback effect due to recycling of the reaction product at the gold surface was analyzed. It was shown experimentally that this effect becomes insignificant at ultramicroelectrode (UME)‐substrate distances larger than 3 UME radii. The flux of formed PAP allowed the determination the surface density of accessible oligonucleotide strands in the functionalized polymer. It was shown that that thicker pyrrole/ODN–Pyrrole polymer films do not lead to a significantly increased accessible ODN surface concentration.