•  To in situ fabricate ethylene dimethacrylate porous polymer monoliths within the confines of a commercial 20 μL polypropylene pipette tips.

•  To enhance the monolith surface area by immobilising AuNPs and then functionalise the AuNPs with ECL lectin for selective extraction of galactosylated proteins from complex media

Alwael, Hassan

Connolly, Damian

O'Connor, Brendan

Paull, Brett

Thompson, Roisin

Twamley, Brendan

English

DCU Online Research Access Service

AuNP-AGGLOMERATED MONOLITHS IN PIPETTE TIPS FOR LECTIN AFFINITY EXTRACTION OF GLYCOPROTEINS Hassan Alwael1, Damian Connolly1, Paul Clarke1, Roisin Thompson1, Brendan Twamley1, Brendan O’Connor1 and Brett Paull1,2	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  1. Irish Separation Science Cluster, NCSR and School of Chemical Sciences, DCU, Dublin, Ireland 2. Australian Centre for Research on Separation Science (ACROSS), School of chemistry, University of Tasmania, Australia. AIMS:   •  To in situ fabricate ethylene dimethacrylate porous polymer monoliths within the confines of a commercial 20 µL polypropylene pipette tips.  •   To enhance the monolith surface area by immobilising AuNPs and then functionalise the AuNPs with ECL lectin for selective extraction of galactosylated proteins from complex media. Nano-materials for Separation and Sensing  (WP 1.4) Bio-ligands & Purification  (WP 1.1) Advanced Polymer Substrates   (WP 1.3) Materials Technology Micro-fluidics  (WP 2.1) Advanced Platforms Column Technology   (WP 2.2) Instrumentation & Detection  (WP 2.3) Methods & Advanced Applications  (WP 2.4) Advanced Silica Support  & Bonded Phase Materials   (WP 1.2) Poster presentations •  35th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Boston, MA, USA. June 2010. •  Analytical Research Forum 2010, Loughborough University, UK, July 2010. •  3rd Annual Meeting of GlycoScience Ireland, UCD, Ireland, Aug-Sep 2010. •  28th International Symposium of Chromatography, Valencia, Spain, Sept 2010.    Oral Presentations: •  Conference on Analytical Science Ireland (CASi), DCU, Ireland, Feb 2011. •  22nd International Ion Chromatography Symposium, Cincinatti, USA, Sept 2010. 3. Project outputs   Publications: •  Pipette-tip selective extraction of glycoproteins with lectin modified gold nano-particles on a polymer monolithic phase, The Analyst, Accepted, 2011.  Authors: H. Alwael, D. Connolly, P. Clarke, R. Thompson, B. Twamley, B. O’Conner and B. Paull. 1. Porous monolith fabrication in a pipette-tip Fig. 1: SEM images of a monolith formed within a modified tip (a,c) and unmodified tip (b,d) prior to monolith polymerisation. •  Photo-grafted chains of poly(EDMA) were covalently attached to the polypropylene surface via hydrogen abstraction to facilitate subsequent covalent attachment of methacrylate monolith. (EDMA monolith formed in-situ via photo-initiated polymerisation. 2. Monolith surface modification with AuNPs Fig. 2: (left): Image of PP tips incorporating AuNP-agglomerated monolith at various stages of preparation. (right): Fe-SEM image of AuNP-agglomerated polymer monolith (60,000x) 3. Immobilisation of Erythrina cristagalli lectin (ECL) on Au-modified monoliths •  ECL is a lectin with specificity for glycans with terminal galactose. • Monolith was flushed with the amine-reactive bi-functional linker DTSP (3,3′-dithiodipropionic acid di(N-hydroxysuccinimide ester). • The covalent attachment of the lectin on the Au surface was achieved due to native lysine residues on ECL reacting with pendant succinimidyl groups. Fig. 3: Schematic diagram of the modification of the gold-immobilised monolith with ECL. 4. Bind-release and selectivity studies for selected glycoproteins •  Citrate-stabilised gold nanoparticles were immobilised upon the monolith after extensive amination of photo-grafted vinyl azlactone (amine reactive). •  Non-specific binding of protein was evaluated with a test mixture comprising selected non-glycosylated proteins as well as glycosylated proteins with and without terminal Gal residues. • Only glycoproteins bearing terminal galactose were extracted due to comprehensive blocking of any remaining unreacted bare gold nanoparticles or unreacted succinimidyl groups (Step 3, above) with 1 M Tris. • Fig 4 illustrates that ~35 % of a 20 µg/mL standard of transferrin was extracted, with recovery (via galactose wash) exceeding 86 %. • The %RSD for transferrin extraction (31 % average) was 14 % for six individually prepared extraction devices. OOOSO NOOOSO N+ OHOON+ OONOS OOS NH ECL OONOS OAu H2N Au Au Au ECL Fig. 4 (left): Test mix of proteins before (a) and (b) after passage through the affinity monolith. Chromatogram (c) is after a galactose wash. (Chromatographic conditions: Column: 100 cm x 100 µm LaMA monolith, 1 µL/min, TFA/ACN gradient. Peaks: (1): transferrin, (2): thyroglobulin. Fig. 5: Extraction of transferrin from a spiked E.coli cell lysate sample. Chromatograms are before ext ract ion (a) and af ter ex t rac t ion (b) . Chromatogram (c) is after a galactose wash. Chromatographic conditions as in Fig 4. (a) (b) (c) (1) (2) (a) (b) (c) (1) 5. Glycoprotein extraction from complex samples. • Transferrin was spiked into an E.coli cell lysate sample at a level of 20 ug/mL. • Significant amounts of transferrin were extracted from this complex sample, with no detectable levels of interfering matrix proteins being co-extracted. 

AuNP-Agglomerated monoliths in pipette tips for lectin affinity extraction of glycoproteins

•  To in situ fabricate ethylene dimethacrylate porous polymer monoliths within the confines of a commercial 20 μL polypropylene pipette tips.
•  To enhance the monolith surface area by immobilising AuNPs and then functionalise the AuNPs with ECL lectin for selective extraction of galactosylated proteins from complex media

O\u27Connor, Brendan

Irish Universities

•  To in situ fabricate ethylene dimethacrylate porous polymer monoliths within the confines of a commercial 20 μL polypropylene pipette tips.\ud
•  To enhance the monolith surface area by immobilising AuNPs and then functionalise the AuNPs with ECL lectin for selective extraction of galactosylated proteins from complex media

Alwael, Hassan Omar Salem

Name not available

http://doras.dcu.ie/17763/1/Monolith_Lectin_Poster_June_2011.pdf

AuNP-Agglomerated monoliths in pipette tips for lectin affinity extraction of glycoproteins

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