Detection and Isolation of Salivary Biomarkers Using a Novel Hybrid Size Exclusion - Immobilized Metal Affinity Chromatography Matrix

Abstract

Biomarkers are a vital tool used in the diagnosis of multiple different diseases, from the presence of creatine kinase in blood indicating muscle damage, to the presence of glucose in urine signaling high blood sugar and possibly diabetes. One major limitation for biomarkers comes in the difficulty of isolating them from biological fluids (such as blood, urine, saliva, etc.). Another big problem is that biomarkers are usually found in low concentrations together with large amounts of proteins that makes the isolation of target biomarkers quite difficult. Isolating a desired biomarker from a patient sample requires multiple steps and these steps can vary depending on the bodily fluid being sampled.This work deals with novel separations of small molecular size compounds from large molecules. This technology combines, in the same separation medium, the characteristics and advantages of size exclusion and affinity adsorptive protein separations methods. The novel features involve the covalent attachment of permeation polymers to a matrix so that after modification preferentially "rejects" biomolecules of a certain size (large proteins, for example), and allows only compounds of appropriate size (small biomarkers, for example) to penetrate the "rejection" zone, thus allowing them to interact with affinity groups previously attached on the surface of the matrix. The main objective of this work was to develop a method to isolate specific biomarkers directly from saliva, a quite relevant biological fluid. The hybrid chromatographic matrix in this case consisted of size exclusion chromatographic matrices functionalized with specific affinity ligands (Iminodiacetic acid (IDA) and Dipicolylamine (DPA) as chelators) and polymers (polyethylene glycol, PEG). In this approach, PEG acts as a blocking polymer that acts as a permeating barrier that allows only small proteins to permeate and interact with the chelator on the surface, larger proteins cannot penetrate permeating barrier. The method was used to isolate the small biomarkers, creatinine, spermine, choline and isethionate and the small protein myoglobin from synthetic saliva preparations. Myoglobin has been linked as a salivary biomarker for myocardial necrosis, creatinine has been linked to chronic kidney disease and spermine, choline and isethionate have been shown to be promising salivary biomarkers for breast cancer. The novel approach was effective at isolating and almost completely recovering most of the target biomarkers from the other components of the synthetic saliva used in the research.