Stoichiometric Control of Bismaleimide Conjugation of DNA to Silica Surfaces Through Quantitative Fluorescence Analysis of Thiolated DNA

Surface immobilization of DNA for biosensing or separations applications requires covalent attachment chemistry that is efficient, reproducible, and stable. In this work, an approach to link thiol-functionalized DNA to thiol-modified silica surfaces using N,N'-1,4-phenylene-bismaleimide is optimized by developing an efficient, one-pot synthesis of the maleimide-conjugated DNA followed by its immediate reaction with thiolated porous silica particles. The methodology takes advantage of a Michael addition reaction that couples a phenyl-bismaleimide cross-linking reagent and thiol-modified DNA to form a monomeric DNA-maleimide conjugate. The 1:1 stoichiometry of this reaction must be carefully controlled to avoid excess thiol-DNA, which generates unreactive bismaleimide-linked DNA dimers, or excess bismaleimide, which competes with the DNA-maleimide conjugate for reaction with the thiolated silica surface. To achieve control over the reaction forming the DNA conjugate, we adapt a fluorescence assay for free-thiols using 7-diethylamino-3-(4-maleimidophenyl)-4-methyl-coumarin (CPM) to determine the concentration of thiol-modified DNA that emerges from its synthesis, disulfide labeling, reduction to a thiol, and purification. The fluorescence response of the CPM reagent was calibrated using reduced glutathione as a standard, which allowed determination of the concentrations of thiolated-DNA and control over the stoichiometry of its reaction with a bismaleimide linker. The maleimide-conjugated DNA product thus formed was then reacted with thiolated-silica in order to bind the DNA to the internal surfaces of porous silica, whose surface populations were determined in individual particles by confocal Raman microscopy. Self-modeling curve resolution of the Raman spectra of surface-bound molecules validated the efficiency of the bismaleimide:thiolated DNA reaction, which provided stoichiometric control over formation of the monomeric DNA-maleimide conjugate and its optimized reaction with thiolated-silica surfaces.