New Approach to Oligonucleotide Microarrays Using Zirconium Phosphonate-Modified Surfaces

Abstract

A new approach to oligonucleotide arrays is demonstrated that utilizes zirconium phosphonate-derivatized glass slides. The active slides are prepared by binding Zr⁴⁺ to surfaces terminated with organophosphonate groups previously deposited using either Langmuir−Blodgett or self-assembled monolayer methods. Oligonucleotide probes modified with a terminal phosphate bind strongly to the active zirconium phosphonate monolayer, and arrays for detecting fluorescent targets have been prepared using commercial spotting and scanning instruments. Preferred binding to the surface of the terminal phosphate of the modified probes instead of the internal phosphate diester groups is demonstrated and shown to yield increased fluorescence intensity after hybridization with labeled targets. A significant decrease in background signal is achieved by treating the slides with bovine serum albumin after spotting and before hybridization. A further increase in fluorescence after hybridization is observed when using a poly-guanine spacer between the probe oligomer and the terminal phosphate. Combining these modifications, an intensity ratio of nearly 1000 is achieved when comparing 5‘-phosphate-modified 33-mer probes with unmodified probes upon hybridization with fluorescent targets.