DO ANTIBODIES LIE OR ARE THEY UPSTANDING? A neutron reflectometry study of biosensor surfaces Peter W. Akers, Nam Cao Hoai Le, Andrew R. J. Nelson, Vladimir Gubala, David E. Williams and Duncan McGillivray

DO ANTIBODIES LIE OR ARE THEY UPSTANDING? Biosensors Antibodies and ELISAs Neutron Reflectometry Our experiment Summary and future

DO ANTIBODIES LIE OR ARE THEY UPSTANDING? Biosensors Antibodies and ELISAs Neutron Reflectometry Our experiment Summary and future

DO ANTIBODIES LIE OR ARE THEY UPSTANDING? Biosensors Antibodies and ELISAs Neutron Reflectometry Our experiment Summary and future

DO ANTIBODIES LIE OR ARE THEY UPSTANDING? Biosensors Antibodies and ELISAs Neutron Reflectometry Our experiment Summary and future

DO ANTIBODIES LIE OR ARE THEY UPSTANDING? Biosensors Antibodies and ELISAs Neutron Reflectometry Our experiment Summary and future

BIOSENSORS In situ detection of analytes Clinical, environmental, agricultural applications Big business: global sales worth US$ 9.9 billion in 2011

ANTIBODIES AND ELISAs Enzyme-linked immunosorbent assays (ELISAs) use surface- immobilised antibodies to detect presence of antigen Commonly used in point-of- care diagnostic tests

NEUTRON REFLECTOMETRY Detector Used to measure structure of thin films Ideally suited for probing the structure of polymer films and biological samples

OUR EXPERIMENT PMMA films spin- coated and linkers attached Anti-human IgG attached using common protein coupling chemistry Antibody surfaces exposed to human IgG Do antibodies on “soft” surfaces capture more antigen than those on “hard” surfaces?

LINKERS COOH and NH 2 - terminated tetraethylene glycol ‘CA-PEG-4’ ‘SOFT’ SURFACE Aminotriester ‘HARD’ SURFACE

LINKERS Generation 4.5 PAMAM dendrimer – SOFT SURFACE

DATA AND MODELLING Samples measured under D 2 O and H 2 O contrasts and corefined Data modelled using motofit Successive layers treated as a series of slabs

PMMA AND LINKERS PMMA films 50 – 150 Å Dendrimer structure not as expected

ANTIBODY ATTACHMENT Anti-IgG tethered to all surfaces in ‘flat’ orientation

ANTIGEN CAPTURE CA-PEG-4 and dendrimer appear to capture more antigen

SUMMARY Aminotriester not hydrated, CA-PEG-4 hydrated Dendrimer flatter, denser layer than expected Antibodies in flat orientation. They Lie! Modelling indicates that dendrimer and PEG linker capture more antigen than aminotriester and bare PMMA in accordance with ellipsometry results

SUMMARY AND FUTURE Antigen-capture performance of linkers as expected – but our hypothesis was not completely correct Surface structure of dendrimers – rescheduled! More modelling – continuous distribution method? More experiments! Quartz Crystal Microbalance More linkers! We need to test a wider range of linkers to know more about why some perform better than others

Thank you! Rayomand Shahlori Amy Xu Anton Le Brun Joseph Nichols Bryon Wright AINSE Post Graduate Research Award The University of Auckland – Faculty of Science