Sequential affinity chromatography miniaturized within a “lab-on-valve” system

Abstract

An automatically renewable microcolumn, subjected to operation by programmable flow, is presented and for the first time used for separation and quantification of biomolecules on Sepharose Protein A beads, with absorbance measurement at 280 nm and a detection limit of 6 ng mouse IgG microL(-1).

Application of Moveable Suspensions of Solid Particles in Flow Analysis

“…Sepharose agarose beads were employed for affinity capture-release of biotincontaining conjugates with mass spectrometry (MS) detection [30]. Numerous biochemical applications have also been reported for the use of Protein A and Protein G Sepharose 4B conjugates [31][32][33]. Collagen-modified Cytodex 3 beads were used for non-covalent attachment of a phosphorescent oxygen probe (Pt-porphyrin complex) for detection of the oxygen consumption of adherent cell cultures [34].…”

“…After measurement, the beads are removed by retracking the capillary or reversing the fluid flow. This design was used in SIA determinations of the oxygen consumption of cultured cells with fluorescence detection [33]. Two versions of the jet ring cell with radial and axial probing light paths have been developed and used in FIA measurements for bioligand interaction assays [29,42].…”

Flow Injection Analysis in Industrial Biotechnology

“…The microaffinity chromatography in the LOV format has been demonstrated for separation of immunoglobulins or nucleic acids, as based on molecular recognition between a site fixed on the stationary phase and the target species that is being captured, while unwanted matrix components, such as salts and proteins, are washed out by the mobile phase. In contrast to conventional immunoaffinity chromatography it is readily feasible to control the operational pH, which is a very critical parameter, and compared to on-column spectroscopy, LOV-chromatography on a renewable bead column is more robust, since the volume of the beads captured within the microcolumn is far less critical, and also because the target analyte is eluted and monitored in solution and not on bead surfaces, thus precluding light scattering effects (67,76). Furthermore, by using a short pulse of eluant, the front elution contains almost all target molecules, yielding excellent limits of detection.…”

“…7B). While the first approach can be viewed as "on-column spectroscopy" (15,74,75), the latter one can be considered as miniaturized bioaffinity chromatography (59,60,67). Both methods employ in-valve bead injection analysis with renewable microcolumns and identical analytical instrumentation, whereby they can be exploited in a complementary fashion to get further knowledge on biomolecular association and dissociation reactions (76,88).…”

“…Combining LOV and bead injection (BI), Ruzicka and coworkers have in particular focused on cellular analyses and immunoassays (15,67,(74)(75)(76). The former are being used for screening of potential drugs by in-vitro evaluation of biological responses.…”