Surface immobilization of nucleic acids for genosensor development.

摘要

Research to discover new treatments for genetic diseases and drug discovery programs require the development of novel methods for investigating the interactions of genes with regulatory proteins. Current methodologies are indirect, often difficult to reproduce and time-consuming. The long turnover time and cost of each assay severely limits applicability to the large-scale screening of drug candidates. The biosensor format uses immobilized nucleic acid probes coupled with an optical, electrochemical or piezoelectric surface which can be used in a flow-injection analysis system to provide fast, non-labor intensive nucleic acid assays. Immobilization of nucleic acids on biosensor surfaces is a critical endeavor, as it is desired to immobilize nucleic acid receptor as possible onto the surface in a controlled fashion while retaining an essentially monolayer-type structure. Silanization is a useful method of functionalizing sensor surfaces, however, the mechanism is poorly understood. Novel methods of evaluating the role of surface moisture were invented, including the extraction of D2O-laden silica surfaces with toluene followed by reaction with phenyllithium to create deuterobenzene which was subsequently quantitated by GC-MS. The substrate involved in silanization should be hydrated, while the solvent should be moisture-free to create the best films. Hydration of the substrates in a controlled environment is the key to creating highly reproducible films. Bifunctional alkyltrichlorosilanes were synthesized and used to create ate monolayer films with high loading potential. 1-(Bromoacetato)-11-(trichlorosilyi)-undecane (BATU) and 1-(thiotrifluoroacetato)-11-(uichlorosilyl)-undecane (TM were synthesized and characterized. Three types of probe molecules were synthesized to probe various aspects of surface thiol chemistry. The probes consisted of a pentafluorophenyl ring attached to various sulfur-reactive groups such as iodoacetyl, thiol, and pyridyldisulfide, were reacted with TTU and BATU surfaces and analyzed by X-ray photoelectron spectroscopy (XPS). The results showed that 33% of the BATU surface and up to 88% of the thiol groups on the TTU surface are available for immobilization. The thiol surface is sensitive to atmospheric oxygen, which causes adjacent thiol groups on the surface to form disulfide linkages, which can also be formed by the action of alkylpyridyl disulfides. The TTU surface was compared with the well known silane, 3-mercaptopropytrimethoxysilane (MPS), and was found to load a greater quantity of perfluorinated probe than do multilayer films of MPS, while retaining a monolayer structure. New methods of chemically modifying synthetic DNA were developed and assessed by a novel ]HPLC protocol involving tagging unmodified DNA with fluorescein-based reagents, which subsequently cause noticeable retention time shifts. The assay indicated that modification of thiol-DNA by 2,2 '-dipyridyldisulfide to produce a DNA-alkylpyridyldisulfide species was successful. Attempts to modify amine-DNA with various NHS-ester based bifunctional reagents were largely unsuccessful due to the pH-sensitive nature of the amine group. These results show that immobilization of nucleic acids under mild conditions is now possible.