title: Chemical and biological characterisation of a sensor surface for bioprocess monitoring.
abstract: This paper describes the step-wise fabrication and characterisation of a multi-layer dual polarization interferometry (DPI) based biosensor utilising Protein G (ProG) as the bio-recognition layer for the detection of a fragment antibody (Fab'). The biosensor is capable of monitoring the concentration of Fab' product within the extracellular medium of a fed-batch fermentation after leakage from Escherichia coli (E.coli). The activity, stability and functionality of each sensor layer were analysed in situ using DPI, whilst the chemical identity and homogeneity of the chemical layers were assessed ex situ using X-ray photoelectron spectroscopy (XPS) and secondary ion mass spectrometry (SIMS). Two different biotin linkers were found to produce hugely differing surfaces after the capture of NeutrAvidin™ (NA) and biotinylated Protein G (b-ProG). The hydrophilic (PEG)(4)-biotin linker resulted in a surface where the b-ProG layer was deposited and organised above the NA layer producing an active and stable surface, whilst the hydrophobic LC-biotin linker generated a surface where the b-ProG layer was buried within the NA layer leading to variable surfaces and poor binding of the Fab' target. The biosensor has a detection limit of 1.7 μg/ml with a dynamic range covering two orders of magnitude. The sensor can detect the onset of Fab' leakage as early as 2h following product induction, with high signal-to-noise ratios and little interference from extracellular components. Leakage of Fab' followed a biphasic profile, switching to a more rapid rate 20 h after induction, indicating accelerated product loss and the need for cultivation harvest.
authors.affiliation: National Physical Laboratory, Hampton Road, Teddington, Middlesex TW11 0LW, United Kingdom. jonathan.moore@npl.co.uk
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Moore
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Perez-Pardo
authors.forename: Jonathan F
authors.initials: JF
authors.lastname: Popplewell
authors.forename: Steve J
authors.initials: SJ
authors.lastname: Spencer
authors.forename: Santanu
authors.initials: S
authors.lastname: Ray
authors.forename: Marcus J
authors.initials: MJ
authors.lastname: Swann
authors.forename: Alex G
authors.initials: AG
authors.lastname: Shard
authors.forename: Walis
authors.initials: W
authors.lastname: Jones
authors.forename: Anna
authors.initials: A
authors.lastname: Hills
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Bracewell
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D001710:Biotin; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D055767:Immobilized Proteins; D007140:Immunoglobulin Fab Fragments / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D007368:Interferometry; D009419:Nerve Tissue Proteins; D056951:Photoelectron Spectroscopy; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D018629:Spectrometry, Mass, Secondary Ion; D013499:Surface Properties
chemical_list: C029152:G-substrate; D055767:Immobilized Proteins; D007140:Immunoglobulin Fab Fragments; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D001710:Biotin