Scanning electrochemical microscopy (SECM) based detection of oligonucleotide hybridization and simultaneous determination of the surface concentration of immobilized oligonucleotides on gold

作者：Kirchner Carolina Nunes; Szunerits Sabine^*; Wittstock Gunther

来源：Electroanalysis, 2007, 19(12): 1258-1267.

DOI：10.1002/elan.200703862

• scanning electrochemical microscopy
• beta-galactosidase
• polypyrrole
• DNA chip
• hybridization
• surface concentration

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(mcr) 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 P-galactosidase from Escherichia coli to the streptavidin. The activity of the linked beta-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-beta-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.

• 出版日期2007-6