Hellmanex

The surface of SPGE (Wara Microcircuit, Samut Prakan, Thailand) was subjected to 1% v/v Hellmanex (Hellma, Müllheim, Germany) in water. The cleaned SPGE was immersed in 3 M HCl for 5 min, and dried with argon gas (LabGas, Pathumthani, Thailand). Membrane solution (2.5 µL) was applied on the working electrode of SPGE, and incubated at room temperature for 3 h. The membrane solution was prepared using THF as a solvent (200 µL) for dissolving co-PVC (14.7 mg), plasticizer (44 mg), and cadmium ionophore I (4 mg, 6% w/w). Then, the membrane-modified SPGE was immersed into a supporting electrolyte. Electropolymerization was performed by using five scans of 10th cycle of cyclic voltammetry with an applied voltage of 1.0 to −1.0 V with Ag/AgCl (3M KCl) at a scan rate of 0.05 V/s.

Monolayers of polystyrene particles were prepared according to the procedure of Retsch et al.42 Cationically functionalized glass slides were spin cast with a 3 wt% particle dispersion at a speed of 4000 rpm. Subsequently, the coated glass substrates were immersed in a 0.1 mM SDS solution in MilliQ. The aqueous phase was adjusted to pH 12 by adding a few drops of NH₃. A monolayer was formed at the liquid/air interface by self-assembly of the detaching particles. The monolayer was transferred to a 1 × 1 inch glass substrate and dried in air. The monolayers were etched in a plasma reactor MiniFlecto (Plasma Technology GmbH, Herrenberg, Germany) with 75% argon and 25% oxygen at 80 W at a pressure of 0.14 mbar to obtain non-close packed monolayers. A 3 nm chromium layer and 50 nm gold were deposited using a Balzers BA360 thermal evaporation chamber. The layer thickness was monitored via a SQM 160 microbalance (Sigma Instruments, Schaefer Technologie GmbH). Afterwards, the particles were removed using Scotch® tape (3M) giving the nanohole arrays. The Au substrates were cleaned for 10 min in an ultrasonic bath with a 2% aqueous Hellmanex (Hellma GmbH, Mühlheim, Germany) solution in MilliQ water. The surfactant was extensively rinsed off with MilliQ water, and the substrates were placed in the ultrasonic bath in ethanol for 10 minutes and dried with compressed air.

Absolute ethanol 99.8%, bovine serum albumine (BSA), dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium hydroxide, dithioerythritol (DTE), dithiothreitol (DTT), 3-glycidyloxypropyl trimethoxysilane (GOPTS), guanidinium hydrochloride (GuHCl), hydrogen chloride (37%), methanol, Pluronic^® F-127, poly(ethylene glycol) diglycidyl ether (diepoxy-PEG, M_N = 500), potassium dihydrogen phosphate, sodium azide, sodium carbonate, sodium chloride, sodium dodecyl sulfate (SDS), sodium hydrogen carbonate, fuming sulfuric acid, D-(+)-trehalose dihydrate, tris(hydroxymethyl)aminomethane Sigma 7–9^® (TRIS), Tween^®-20 and urea were obtained from Sigma-Aldrich (Taufkirchen, Germany). 3-(N-morpholino)-propanesulfonic acid (MOPS PUFFERAN^®) and Menzel glass slides (76 mm × 26 mm × 1 mm) were obtained from Carl Roth (Karlsruhe, Germany). Hellmanex was obtained from Hellma GmbH (Mannheim, Germany). Jeffamine^® ED-2003 polyetheramine (DAPEG) was obtained from Huntsman Belgium BVBA (Everberg, Belgium) as a gift. WESTAR supernova ELISA luminol and hydrogen peroxide were purchased from Cyanagen (Bologna, Italy). The ARcare 90106 adhesive film was obtained from Adhesive Research Ireland (Limerick, Ireland). The poly(methyl methacrylate) support for the chip was produced in our workshop. 384-well PP flat bottom microtiter plates (MTPs) were obtained from Greiner GmbH (Frickenhausen, Germany).

SPR experiments used BIAcore Au sensor chips in a BIAcore X100. The Au chip was cleaned by using piranha solution (3:1 H₂SO₄/H₂O₂ by volume) on the gold surface for 15 min followed by copious washing with 2 % Hellmanex (Hellma, UK) and ethanol with >18 MΩ water washes between each step. The sensor was immersed in a 1 % (v/v) β-mercaptoethanol (BME) in ethanol solution to passivate the gold surface (Terrettaz et al. 2002 (link)), washed with 1 % (w/v) SDS followed by >18 MΩ water. The running buffer for all experiments was phosphate buffered saline (PBS, 20 mM sodium phosphate pH 7.6, 137 mM NaCl and 2.7 mM KCl). The volumes and flow rates used for the assembly of the GGZctOmpA array; binding and crosslinking of antibody; and binding of antigen and secondary antibody are outlined in Supplementary Tables S2, S3 and S4 respectively with experiments repeated in triplicate.