Designed affinity surfaces for biomolecule immobilisation and biosensor construction
|Paul A. Millner , Alexander Vakurov , Henry Hays , Sophie Weiss , Morsaline Billah , Tim Gibson|
University of Leeds (UOL), Woodhouse Lane, Leeds ls2-9jt, United Kingdom
Nanoscale design and functionalisation of transducer surfaces is vital for the effective and rapid assembly of biosensors devices. Substrates can be initially functionalised in a variety of ways; screen printed carbon electrodes have been aminated of via electrochemical reduction of nitrobenzene diazonium, whilst silica or platinum have been aminated via treatment with aminosilanes such as APTES; finally gold/silver are most easily modified but thiol-terminated reagents which chemisorb to the metal surface. An alternative strategy with conducting surfaces is the electropolymerisation of conducting polymers such as polyaniline and polypyrrole, which also provide surface amino functions for subsequent modification.
Once the base layer is in place then various affinity strategies exist for rapid and gentle attachment of the sensing biomolecule, usually an enzyme or antibody. Non- specific affinity strategies have been used to capture enzymes and antibodies. Acetylcholine esterase was immobilised via multipoint electrostatic interaction to multilayers of the polyanion PEI, to construct an organophosphate pesticide sensor. Antibodies to various analytes have been tethered in the same way to polypyrrole and polyaniline bases layers on Au and carbon using multiple alternate layers of the polyanion PDDM and polycation PSS.
Specific affinity interactions have also been used for biomolecule immobilisation. Attachment of a biotin group to surface amines on polyaniline subsequently permits tethering of biotinylated antibody via avidin as a multivalent crosslinker. The same strategy has also been used to attach biotinylated antibodies to transducer surfaces supporting a mixed self assembled monolayer, which comprises the alkanethiol MHDA and intercalated affinity lipid, DPPE-caproyl-biotin. A similar approach can be used to immobilise recombinant sensor molecules, e.g. Fab, or Scfv, which bear a terminal His6 tag, to the affinity lipid DPPE-Ni2+-NTA. Such mixed self assembled monolayer incidentally also function well as a 'non-specific' His6' tagged protein sensor.
Finally, approaches to characterising the assembly of such nanostructured surfaces and biosensors will be described. These include quartz crystal microbalance (QCM) to observe the kinetics of sensor assembly and analyte recognition, AFM to delineate surface topology, radiolabelling components for quantification, and amperometric and impedimetric interrogation of the sensor at various stages of assembly.
Presentation: Tutorial lecture at SMCBS'2005 Workshop, by Paul A. Millner
See On-line Journal of SMCBS'2005 Workshop
Submitted: 2005-07-27 08:52 Revised: 2009-06-07 00:44
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