The goal of this project was to develop an accurate method for determination of ligand density on the surface of para-aminobenzamidine (pABA) linked regenerated cellulose membranes and other chromatography matrices. These membranes have been shown to capture the target serine hydrolase protein selectively from biological mixtures. However, the in-depth analysis of membrane’s protein binding efficiency requires an accurate estimation of the ligand density on membrane surface.  Quantification of amidine group in solid phase is challenging since the only known methods for this purpose are either colorimetric or require a disruption of the membrane structure. In this study, we were able to develop a fluorescence based assay for the estimation of ligand density (pABA) on cellulose membrane surface by reacting the immobilized benzamidine moiety with glyoxal and benzaldehyde in alkaline aqueous solution, leading to the formation of the fluorescent probe 2-aryl-4-arylidene-2-imidazolin-5–one (Ex 385 nm, Em 455 nm). The method was found to be linear up to 5 mM amidine concentration. While the pABA fluorescent tagging allowed us to perform a fast and reliable in situ analysis at varying ligand density without disrupting the membrane; imaging the fluorescently labeled membrane using Confocal Laser Scanning Microscopy (CLSM) facilitated the z-stack analysis of the membrane volume. An algorithm for pixel counting on membrane surface was developed to perform the analysis of CLSM images. The z-stack analysis confirmed that the ligand was immobilized uniformly across the membrane volume. <br/>

In situ analysis of ligand density on the surface of regenerated cellulose membranes

255th National Meeting (2018)

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