Aptamer-modified monolithic capillary chromatography for protein separation and detection.

A capillary chromatography technique was developed for the separation and detection of proteins, taking advantage of the specific affinity of aptamers and the porous property of the monolith. A biotinylated DNA aptamer targeting cytochrome c was successfully immobilized on a streptavidin-modified polymer monolithic capillary column. The aptamer, having a G-quartet structure, could bind to both cytochrome c and thrombin, enabling the separation of these proteins from each other and from the unretained proteins. Elution of strongly bound proteins was achieved by increasing the ionic strength of the mobile phase. The following proteins were tested using the aptamer affinity monolithic columns: human immunoglobulin G (IgG), hemoglobin, transferrin, human serum albumin, cytochrome c, and thrombin. Determination of cytochrome c and thrombin spiked into dilute serum samples showed no interference from the serum matrix. The benefit of porous properties of the affinity monolithic column was demonstrated by selective capture and preconcentration of thrombin at low ionic strength and subsequent rapid elution at high ionic strength. The combination of the polymer monolithic column and the aptamer affinities makes the aptamer-modified monolithic columns useful for protein detection and separation.