4.3.1. Functionalisation of the Sensor Surface

The sensor surface was derivatised by several routes to form reactive functional groups for covalent immobilisation of the protein immunoreagents (ZON-specific antibodies or ZON-BSA conjugate) (Figure 1). Proteins were immobilised to the derivatised sensor chip in a flow-through system using a 42 mM Tris running buffer (pH 7.4). Reactive epoxy groups were formed on the sensor surface by heating the sensor chips to 60 ◦C in 10% GOPS in toluene for 20 hrs, followed by washing the chips with toluene and further heating to 100 ◦C for 1 hr. Epoxy functionalised surfaces allowed for direct anchoring of biomolecules carrying amino or hydroxy moieties by nucleophilic addition to them in alkaline medium (pH > 8.5) [81]. Thus, protein immunoreagents were injected onto the epoxylated sensor chips at 1–20 µg/mL concentrations in 0.2 M carbonate buffer at pH 9.5, followed by buffer exchange to 42 mM Tris buffer at pH 7.4, and removal of unbound proteins from the surface by injecting 0.1 M aqueous hydrochloric acid. Amino groups were formed on the surface of the sensor by treating the chips at 75 ◦C with 10% APTS at pH 3.0 for 4 hrs, followed by washing with distilled water and heat treatment at 95 ◦C for 6 hrs [81]. The amino functionality was activated with 2.5% aqueous glutaraldehyde (GA), allowing direct additive anchoring biomolecules carrying amino groups. The immobilisation reaction was carried out within the flow-through cuvette, by injecting GA into the flowing distilled water medium, followed by medium exchange to Tris buffer (42 mM, pH 7.4), subsequent injection of the proteins at 1–20 µg/mL concentration, and elution of the unbound reagent fraction by the injection of 0.1 M aqueous hydrochloric acid. Alternatively, amino groups were modified to carboxyl groups by derivatisation with succinic anhydride (SA), and were utilised for covalent attachment of biomolecules by the activated ester method using NHS with a dehydrating agent EDC. Carboxylation was carried out in separate vessels with 1% SA in dry ethanol at 25 ◦C for 1 hr, followed by drying the chips at 90 ◦C for 15 min. Active ester formation on the chip surface was carried out in a stopped flow mode by injecting a 1:1 solution of 0.2 M EDC and 0.05 M NHS, incubating at room temperature for 10 min, rinsing with Tris buffer (42 mM, pH 7.4), adding the protein-ZON conjugate at 1–20 µg/mL concentration in 10 mM sodium acetate buffer at pH 4.0, and incubating again at room temperature for 10 min. Alternatively, active ester formation could be carried out outside the OWLS 210 instrument, on a Petri dish, under similar reaction conditions. Residual active ester functional groups were finally deactivated by the injection of 1 M ethanolamine at pH 8.5 for 10 min.
