Fundamentals and Applications of Chromatographic Adsorption



Journal Title

Journal ISSN

Volume Title



The affinity and capacity of ion-exchange adsorbents of a given total-charge density are improved by immobilization of the charges in pre-ordered clusters, rather than individually in random locations. Here, we demonstrate that polyamine spermine can serve as an alternative to our previously described expensive peptide clustered ligands. Spermine Sepharose exhibited superior α-lactalbumin binding capacity (Qmax > 1.6 and 1.3-fold higher than those for Qiagen DEAE and GE DEAE Sepharose adsorbents of much greater charge density, respectively) and higher initial binding affinity (Qmax/KD 2.4 and 2.1-fold higher, respectively). The new spermine-based matrix also exhibited a higher value of the salt-sensitivity Z parameter, suggesting an increased number of apparent interaction sites between the protein and the resin, and functioned well in column mode. In another study, we developed an ultra-sensitive immunochromatographic lateral flow assay (LFA) for the detection of MS2 bacteriophage. LFAs are a well-established point-of-care diagnostic method, but the sensitivities of the standard gold and latex particle formats are not sufficient for many potential applications. Here we present a novel reporter, horseradish peroxidase (HRP)-labeled immuno-phage particles, for sensitivity-enhanced LFAs. Two different types of approaches were employed to manufacture antibody- and peroxidase-doubly-modified M13 bacteriophage, and these novel reporters were successfully integrated into an LFA. The sensitivity of the assay for a model viral pathogen, MS2, was 1000-fold greater than the traditional gold nanoparticle LFA with the same antibodies and results could easily be visually assessed, even without instruments. In another study in collaboration with the group of C. Landes of Rice University, we employed a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT) to study complex mechanistic details involved in protein adsorption. We demonstrated that clusters of charges are necessary to create detectable adsorption sites and that even chemically-identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Simulated elution profiles predicted from the molecular-scale data suggest that if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five.



Spermine Sepharose, Chromatography, Lateral flow assay, Single-molecule super-resolution imaging, Motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT)


Portions of this document appear in: Dhamane, Sagar, Federico Ruiz-Ruiz, Wen-hsiang Chen, Katerina Kourentzi, Jorge Benavides, Marco Rito-Palomares, and Richard C. Willson. "Spermine Sepharose as a clustered-charge anion exchange adsorbent." Journal of Chromatography A 1324 (2014): 135-140. And in: Adhikari, Meena, Sagar Dhamane, Anna EV Hagström, Gavin Garvey, Wen-Hsiang Chen, Katerina Kourentzi, Ulrich Strych, and Richard C. Willson. "Functionalized viral nanoparticles as ultrasensitive reporters in lateral-flow assays." Analyst 138, no. 19 (2013): 5584-5587. And in: Kisley, Lydia, Jixin Chen, Andrea P. Mansur, Bo Shuang, Katerina Kourentzi, Mohan-Vivekanandan Poongavanam, Wen-Hsiang Chen, Sagar Dhamane, Richard C. Willson, and Christy F. Landes. "Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations." Proceedings of the National Academy of Sciences 111, no. 6 (2014): 2075-2080.