Immobilization of DNA onto Poly(dimethylsiloxane) Surfaces and Application to a Microelectrochemical Enzyme-Amplified DNA Hybridization Assay

• Published in: Langmuir Vol. 20; no. 14; pp. 5905 - 5910
• Main Authors: Liu, Daojun, Perdue, Robbyn K, Sun, Li, Crooks, Richard M
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 06.07.2004
• Subjects: Alkaline Phosphatase - chemistry; Dimethylpolysiloxanes - chemistry; DNA - chemistry; Electrochemistry; Enzymes, Immobilized - chemistry; Microelectrodes; Nucleic Acid Hybridization - methods; Particle Size; Silicones - chemistry; Surface Properties
• ISSN: 0743-7463; 1520-5827
• DOI: 10.1021/la049605p

This paper describes immobilization of DNA onto the interior walls of poly(dimethylsiloxane) (PDMS) microsystems and its application to an enzyme-amplified electrochemical DNA assay. DNA immobilization was carried out by silanization of the PDMS surface with 3-mercaptopropyltrimethoxysilane to yield a thiol-terminated surface. 5‘-acrylamide-modified DNA reacts with the pendant thiol groups to yield DNA-modified PDMS. Surface-immobilized DNA oligos serve as capture probes for target DNA. Biotin-labeled target DNA hybridizes to the PDMS-immobilized capture DNA, and subsequent introduction of alkaline phosphatase (AP) conjugated to streptavidin results in attachment of the enzyme to hybridized DNA. Electrochemical detection of DNA hybridization benefits from enzyme amplification. Specifically, AP converts electroinactive p-aminophenyl phosphate to electroactive p-aminophenol, which is detected using an indium tin oxide interdigitated array (IDA) electrode. The IDA electrode eliminates the need for a reference electrode and provides a steady-state current that is related to the concentration of hybridized DNA. At present, the limit of detection of the DNA target is 1 nM in a volume of 20 nL, which corresponds to 20 attomoles of DNA.