A high throughput assay for the detection of cancer biomarkers via the novel redox-initiated radical chain polymerization signal amplification method.

A29

The significance of molecular diagnostics includes the potential to extract genetic information, thereby revealing sub-cellular alterations before extensive and abnormal cellular proliferation. However, challenges arise when designing an effective assay capable of screening large asymptomatic populations. An ideal biomarker screening assay would meet the demands of requiring a non-invasive biological sample, providing facile and robust experimental steps, utilizing inexpensive reagents and offering high amplification factors able to detect low concentrations of biomarkers. A proposed solution to these demands involves radical chain polymerization wherein the formation of a radical species rapidly initiates the assembly of a high molecular weight polymer chain from monomer units. Since the radical group exclusively originates from an initiator molecule, the specific allocation of this initiator at surface locations undergoing specific bio-recognition events permits a prompt signal amplification of relevant biological molecules. The specific coupling of this initiator to the bio-recognition event occurs using a dual-functional molecule, containing a Streptavidin linked initiator, on a biotinylated surface. This polymerization reaction reliant upon specific biological interactions provides a novel signal amplification technique for the detection of cancer biomarkers without reliance on costly reagents, prolonged assays or complicated enrichment schemes. Moreover, the high throughput nature of this amplification method derives from the ability to utilize the same initiator and monomer detection reagents for all screened biomarkers.
 Preliminary studies demonstrate the potential of this novel and facile polymerization assay for the detection of genetic biomarkers present in cancer, specifically single base pair mutations in the K-ras gene. First, studies revealed the ascorbic acid and hydrogen peroxide redox pair would generate radicals capable of polymerizing fluorescent water-soluble monomers. A rapid and specific polymerization occurred only from locations on a microarray surface containing ascorbic acid after introducing the entire surface with a mixture of hydrogen peroxide and fluorescent monomer. These polymeric spots on the microarray surface were both visible with the unaided eye and evident from fluorescent images obtained with a microarray scanner. Further efforts demonstrate the potential to synthesize a dual-functional macromolecule containing both ascorbic acid, capable of generating a radical species in the presence of hydrogen peroxide, and a Streptavidin linker group that joins the initiator to locations on the microarray containing biotin. The application of this polymerization assay in the detection of single base pair mutations in codon 12 of the K-ras gene is currently being investigated. The proper design of capture probes permitted the utilization of a primer extension assay with the Klenow fragment to both biotinylate the capture probe and discriminate between single base pair mutations.