Stable Immobilization of DNA to Silica Surfaces by Sequential Michael Addition Reactions Developed with Insights from Confocal Raman Microscopy.

The immobilization of DNA to surfaces is required for numerous biosensing applications related to the capture of target DNA sequences, proteins, or small-molecule analytes from solution. For these applications to be successful, the chemistry of DNA immobilization should be efficient, reproducible, and stable and should allow the immobilized DNA to adopt a secondary structure required for association with its respective target molecule. To develop and characterize surface immobilization chemistry to meet this challenge, it is invaluable to have a quantitative, surface-sensitive method that can report the interfacial chemistry at each step, while also being capable of determining the structure, stability, and activity of the tethered DNA product. In this work, we develop a method to immobilize DNA to silica, glass, or other oxide surfaces by carrying out the reactions in porous silica particles. Due to the high specific surface area of porous silica, the local concentrations of surface-immobilized molecules within the particle are sufficiently high that interfacial chemistry can be monitored at each step of the process with confocal Raman microscopy, providing a unique capability to assess the molecular composition, structure, yield, and surface coverage of these reactions. We employ this methodology to investigate the steps for immobilizing thiolated-DNA to thiol-modified silica surfaces through sequential Michael addition reactions with the cross-linker 1,4-phenylene-bismaleimide. A key advantage of employing a phenyl-bismaleimide over a comparable alkyl coupling reagent is the efficient conversion of the initial phenyl-thiosuccinimide to a more stable succinamic acid thioether linkage. This transformation was confirmed by in situ Raman spectroscopy measurements, and the resulting succinamic acid thioether product exhibited greater than 95% retention of surface-immobilized DNA after 12 days at room temperature in aqueous buffer. Confocal Raman microscopy was also used to assess the conformational freedom of surface-immobilized DNA by comparing the structure of a 23-mer DNA hairpin sequence under duplex-forming and unfolding conditions. We find that the immobilized DNA hairpin can undergo reversible intramolecular duplex formation based on the changes in frequencies and intensities of the phosphate backbone and base-specific vibrational modes that are informative of the hybridization state of DNA.