In situ analysis and imaging of aromatic amidine at varying ligand densities in solid phase

Abstract

We report the development of a fast and accurate fluorescence-based assay for amidine linked to cellulose membranes and Sepharose gel. The assay is founded on the glyoxal reaction, which involves reaction of amidine group with glyoxal and an aromatic aldehyde, leading to the formation of a fluorophore that can be analyzed and quantified by fluorescence spectroscopy and imaging. While the assay has been reported previously for aromatic amidine estimation in solution phase, here we describe its adaptation and application to amidine linked to diverse forms of solid matrices, particularly benzamidine Sepharose and benzamidine-linked cellulose membranes. These functionalized porous matrices find important application in purification of serine proteases. The efficacy of a protein separation device is determined by, among other factors, the ligand (amidine) density. Hence, a sensitive and reproducible method for amidine quantitation in solid phase is needed. The glyoxal reaction was carried out on microbead-sized Sepharose gel and cellulose membranes. Calibration curves were developed for each phase, which established linearity in the range 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 <2%) and reproducibility. Finally, we show how this fluorescent labeling (glyoxal) method can provide a tool for imaging membranes and ligand distribution through confocal laser scanning microscopy.

Cellulose acetate microwell plates for high-throughput colorimetric assays

“…In the past, we have modified CA discs chemically and demonstrated their feasibility as a template for fluorometric estimations. 49 The CA microwell plates will similarly form excellent platforms for fluorescence-based assays too. CA is highly amenable to chemical modification.…”

“…26 In previous studies reported by our group, we chemically functionalized commercially available cellulose acetate (CA) discs and demonstrated their efficacy in uorescence-based detection of analytes. 27,28 The growing interest in PADs, coupled to our previous experience in developing chemically derivatized CA discs for analyte detection, has inspired the work being reported in this manuscript. Amidst growing concerns over the staggering amount of plastic waste being generated in scientic laboratories worldwide, 6 and an immediate need to curb the use of single use plasticware, 3 here we report the fabrication of 96-well-format cellulose acetate (CA) plates as viable substitutes for plastic multiwell plates.…”