The in situ preparation of ethylene dimethacrylate porous polymer monoliths within 20 mu L polypropylene pipette tips, bound via surface grafted methacrylate anchor sites, is reported. Gold nano-particles (AuNPs) were immobilised onto the monolith pore surface utilising azlactone chemistry and coverage verified using field emission scanning electron microscopy. Erythrina cristagalli lectin (ECL) was immobilised upon the attached AuNPs via a bio-functional linker. The ECL-modified tip was successfully applied for the enrichment of galactosylated protein (desialylated transferrin) versus a non-galactosylated protein (ribonuclease B) due to the specificity of ECL. Reversed-phase capillary HPLC was used to validate the efficiency and selectivity of the developed micro-extraction phase which resulted in an increase in extraction recovery of similar to 95% due to the AuNP enhanced surface area. Further specificity of the ECL-modified tip was demonstrated with a complex mixture of non-glycosylated and glycosylated proteins with differing terminal sugar structures. Finally, the lectin affinity phase was applied to a galactosylated glycoproteins spiked Escherichia coli cell lysate to successfully demonstrate matrix tolerance.
History
Publication title
Analyst
Volume
136
Issue
12
Pagination
2619-2628
ISSN
0003-2654
Department/School
School of Natural Sciences
Publisher
Royal Society of Chemistry
Place of publication
Thomas Graham House, Science Park, Milton Rd, Cambridge, England, Cambs, Cb4 0Wf