A Multistep Chemical Modification Procedure To Create DNA Arrays on Gold Surfaces for the Study of Protein−DNA Interactions with Surface Plasmon Resonance Imaging

• Published in: Journal of the American Chemical Society 1999-09, Vol.121 (35), p.8044-8051
• Main Authors: Brockman, Jennifer M, Frutos, Anthony G, Corn, Robert M
• Format: Article
• Language: English
• Subjects: 11-mercaptoundecylamine; DNA micrarrays; single-stranded DNA-binding protein; surface plasmon resonance
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja991608e

A multistep surface modification procedure for the creation of DNA arrays on chemically modified gold surfaces that can be used in surface plasmon resonance (SPR) imaging studies of protein−DNA interactions is demonstrated. The multistep procedure is required to create an array of spots that are surrounded first by a hydrophobic background which allows for the pinning of aqueous DNA solutions onto individual array elements and then to replace that hydrophobic background with one that resists the nonspecific adsorption of proteins during in situ SPR imaging measurements. An amine-terminated alkanethiol monolayer is employed as the base layer, and Fmoc and PEG modifiers are used to create the sequentially hydrophobic and protein adsorption-resistant surfaces, respectively. Specifically, the chemical modification steps are the following:  (1) the adsorption and self-assembly of an 11-mercaptoundecylamine (MUAM) monolayer on an evaporated gold thin film, (2) the reaction of the MUAM monolayer with an Fmoc protecting group to create a hydrophobic surface, (3) the photopatterned removal of the alkanethiol followed by (4) the readsorption of MUAM to create an array of MUAM squares (750 × 750 μm) surrounded by a hydrophobic MUAM-Fmoc background that can pin drops of aqueous solution, (5) the attachment of oligonucleotide sequences onto the MUAM squares by the reaction of the amine-terminated surface with the heterobifunctional cross linker SSMCC followed by a coupling reaction to a small volume (0.1 μL) of thiol-modified DNA, and (6) the removal of the Fmoc protecting group followed by (7) a pegylation reaction of the MUAM with PEG-NHS to create a protein adsorption-resistant background. A combination of polarization-modulation FTIR spectroscopy, contact angle, and scanning angle SPR measurements is used to characterize the surface modification procedure. An SPR imaging measurement of the adsorption of single-stranded DNA binding protein (SSB) onto an oligonucleotide array created by this procedure is used to demonstrate the utility of these surfaces.