In situ analysis and imaging of aromatic amidine at varying ligand densities in solid phase
| Autor: | Sean B. Yeldell, Adriana N. Santiago-Ruiz, Vibha Bansal, Adaliz J Torres-Rosado, Jose Sotero-Esteva, Ezio Fasoli, Rolando Oyola, Jomarie Jiménez-Gonzalez, Christian J Ortiz-Hernandez, Ivan J. Dmochowski |
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| Rok vydání: | 2018 |
| Předmět: |
Chromatography
Fluorophore 010401 analytical chemistry 02 engineering and technology 021001 nanoscience & nanotechnology 01 natural sciences Biochemistry Benzamidine Fluorescence spectroscopy Article 0104 chemical sciences Analytical Chemistry Sepharose Amidine chemistry.chemical_compound Membrane chemistry Glyoxal Cellulose 0210 nano-technology |
| Zdroj: | Analytical and bioanalytical chemistry. 411(8) |
| ISSN: | 1618-2650 |
| Popis: | 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 an 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. Graphical abstract. |
| Databáze: | OpenAIRE |
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