A novel high throughput nucleic acid sandwich hybridization assay on a gold substrate

Abstract

Biosensors will become increasingly important for medical diagnostics. An essential part of biosensor development is assay optimization; to find the optimal conditions a large number of variables need to be tested and high throughput online measurements are time-consuming or sometimes impossible. In this study, an offline high throughput nucleic acid sandwich hybridization assay is developed for pre-screening assay conditions on a gold substrate. The assay is performed in a 48-well plate using alkanethiolated ssDNA molecules, which bind to the substrate surface through a specific thiol–gold interaction and function as the recognition site for target DNA. The hybridization reaction was analyzed using an enzymatic read-out system measuring DNA–DNA interactions in a highly specific and sensitive manner, since only fully complementary nucleotide sequences were detected with a lower limit of detection of 10 pmol l⁻¹. This high throughput low cost offline method has been demonstrated to be flexible and is expected to improve and accelerate biosensor development.