This work describes a novel, fast and accurate fluorescence-based assay for amidine estimation in solid phase. The assay involves the reaction of benzamidine with glyoxal and an aromatic aldehyde. The so formed fluorophore can be analyzed and quantified by fluorescence spectroscopy and confocal microscopy. This reaction has been reported for aromatic amidine estimation in liquid phase previously; here we describe its application for the determination of para-amino benzamidine (pABA) in chromatography matrices, particularly benzamidine Sepharose and benzamidine-linked cellulose membranes which find important application in purification of trypsin type serine proteases. A sensitive and reproducible method for amidine quantitation in solid phase allows the tuning of the ligands density and therefore optimize the efficacy of a protein separation device. The glyoxal reaction was tested on microbead-sized Sepharose gel and cellulose membrane chemically modified with pABA. Calibration curves established linearity of 0-0.45 µmol per mL amidine for free amidine in solution, 0-0.45 µmol amidine per mL Sepharose gel, and 0-0.48 µmol per mL cellulose membrane. The assay showed high accuracy (~3.4 % error), precision (RSD &lt;2%) and reproducibility. Switching the aromatic aldehyde used in the glyoxal reaction from benzaldehyde to fluorene-2-carboxaldehyde led to the formation of a new fluorescent probe with different excitation and emission maxima, which expands the potential applications of this method and can provide a tool for imaging membranes and ligand distribution through laser scanning confocal microscopy.

In situ analysis and imaging of aromatic amidine at varying ligand density on cellulose membranes

2019 Spring National Meeting

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