caa a22 4500 605479828 CHVBK 20210128100412.0 cr unu---uuuuu 210128e20150601xx s 000 0 eng 10.1007/s10544-015-9951-z doi (NATIONALLICENCE)springer-10.1007/s10544-015-9951-z Engineered self-assembling monolayers for label free detection of influenza nucleoprotein [Elektronische Daten] [Anton Le Brun, Andrei Soliakov, Deepan Shah, Stephen Holt, Alison McGill, Jeremy Lakey] Integrating nanotechnology into useable devices requires a combination of bottom up and top down methodology. Often the techniques to measure and control these different components are entirely different, so methods that can analyse the nanoscale component in situ are of increasing importance. Here we describe a strategy that employs a self-assembling monolayer of engineered protein chimeras to display an array of oriented antibodies (IgG) on a microelectronic device for the label free detection of influenza nucleoprotein. The structural and functional properties of the bio-interface were characterised by a range of physical techniques including surface plasmon resonance, quartz-crystal microbalance and neutron reflectometry. This combination of methods reveals a 13.5nm thick engineered-monolayer that (i) self-assembles on gold surfaces, (ii) captures IgG with high affinity in a defined orientation and (iii) specifically recognises the influenza A nucleoprotein. Furthermore we also show that this non-covalent self-assembled structure can render the dissociation of bound IgG irreversible by chemical crosslinking in situ without affecting the IgG function. The methods can thus describe in detail the transition from soluble engineered molecules with nanometre dimensions to an array that demonstrates the principles of a working influenza sensor. The Author(s), 2015 Protein engineering nationallicence Surface plasmon resonance nationallicence Quartz-crystal microbalance nationallicence Neutron reflectometry nationallicence Self-assembled monolayer nationallicence Influenza nationallicence BS(PEG)5 : Bis(succinimidyl) penta(ethylene glycol) nationallicence BSSS : Bis(sulfosuccinimidyl) suberate nationallicence ctOmpA : Circularly permuted transmembrane domain of E. coli outer membrane protein A nationallicence GGZctOmpA : N-terminal tandem pair of B domains from protein G with a C-terminal ctOmpA scaffold nationallicence IgG : Immunoglobulin G nationallicence mAb245 : Mouse monoclonal IgG raised against rNP nationallicence mAb108 : Mouse monoclonal IgG raised against rNP (different epitope to mAb245) nationallicence NP : Native nucleoprotein from influenza nationallicence NR : Neutron reflectometry nationallicence nSLD : Neutron scattering length density nationallicence PBS : Phosphate buffered saline nationallicence QCM-D : Quartz-crystal microbalance with dissipation monitoring nationallicence rNP : Recombinant nucleoprotein nationallicence SPR : Surface plasmon resonance nationallicence thioPEG : 1-mercaptoundec-11-yltriethylene glycol nationallicence Le Brun Anton Bragg Institute, Australian Nuclear Science and Technology Organisation, Locked Bag 2001, 2232, Kirrawee DC, NSW, Australia aut Soliakov Andrei Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Framlington Place, NE2 4HH, Newcastle upon Tyne, UK aut Shah Deepan Orla Protein Technologies Ltd, Biosciences Centre, International Centre for Life, Times Square, NE1 4EP, Newcastle upon Tyne, UK aut Holt Stephen Bragg Institute, Australian Nuclear Science and Technology Organisation, Locked Bag 2001, 2232, Kirrawee DC, NSW, Australia aut McGill Alison Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Framlington Place, NE2 4HH, Newcastle upon Tyne, UK aut Lakey Jeremy Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Framlington Place, NE2 4HH, Newcastle upon Tyne, UK aut Biomedical Microdevices Springer US; http://www.springer-ny.com 17/3(2015-06-01), 1-10 1387-2176 17:3<1 2015 17 10544 https://doi.org/10.1007/s10544-015-9951-z text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s10544-015-9951-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Le Brun Anton Bragg Institute, Australian Nuclear Science and Technology Organisation, Locked Bag 2001, 2232, Kirrawee DC, NSW, Australia aut NATIONALLICENCE 700 1- Soliakov Andrei Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Framlington Place, NE2 4HH, Newcastle upon Tyne, UK aut NATIONALLICENCE 700 1- Shah Deepan Orla Protein Technologies Ltd, Biosciences Centre, International Centre for Life, Times Square, NE1 4EP, Newcastle upon Tyne, UK aut NATIONALLICENCE 700 1- Holt Stephen Bragg Institute, Australian Nuclear Science and Technology Organisation, Locked Bag 2001, 2232, Kirrawee DC, NSW, Australia aut NATIONALLICENCE 700 1- McGill Alison Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Framlington Place, NE2 4HH, Newcastle upon Tyne, UK aut NATIONALLICENCE 700 1- Lakey Jeremy Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Framlington Place, NE2 4HH, Newcastle upon Tyne, UK aut NATIONALLICENCE 773 0- Biomedical Microdevices Springer US; http://www.springer-ny.com 17/3(2015-06-01), 1-10 1387-2176 17:3<1 2015 17 10544