# Design of Antibody-Functionalized Polymeric Membranes for the Immunoisolation of Pancreatic Islets
## 2 Materials and Methods
### Site-Directed Functionalization of the Antibody and Characterization Procedures  
Preparation of the substrate for the immobilization of the protein antigenThe substrates were prepared by depositing a film of gold with a thickness of 250 nm (\(\pm\) 50 nm) on the top of an adhesive chromium layer (2.5 nm) deposited on a SiO\({}_{2}\)/Si (100 nm) wafer; afterwards, squared chips (area of about 1 cm\({}^{2}\)) were cleaved using a diamond-tipped scriber. The samples were rinsed with acetone, isopropyl alcohol, and ethanol (at least three times for each solvent) and blown dry with pure nitrogen. The gold surface was modified using a three-step procedure (Figure 1), before the immobilization of His-tagged protein. Gold-coated chips were immersed in a 10 mM solution of 11-mercapoundecanoic acid in ethanol (EtOH) overnight (ON) at room temperature (RT). Then, the substrates were rinsed with EtOH in order to remove MUA molecules not bound to the surface and dried with nitrogen (Figure 1a). The activation and amidation of the carboxylate groups on Au-MUA substrates were carried out in a double step process: firstly, the samples were exposed to 100 mM \(N\)-(3-Dimethylaminopropyl)-\(N^{\prime}\)-ethylcarbodiimide hydrochloride (EDC) and 20 mM \(N\)-Hydroxysulfosuccinimide (NHS) aqueous solution for 1.5 h at RT. Then, they were immersed in 40 mM \(N\alpha\),\(N\alpha\)-Bis(carboxymethyl)-L-lysine hydrate (NTA-lysine) aqueous solution (2 h at RT). Finally, the substrates were thoroughly rinsed with 150 mM NaCl and 10 mM HEPES aqueous solution (Figure 1b). The amino-nitrilotriacetic-Ni(II) complex (NTA-Ni\({}^{2+}\)) was formed by reaction of Au-MUA-NTA-lysine with nickel(II) chloride (1 mM) in 150 mM NaCl and 10 mM HEPES buffer pH 7.4 (2 h at RT) (Figure 1c). Atomic Force Microscope (AFM) characterizationNon-Contact mode Atomic Force Microscope (NC-AFM, XE-100 PSIA, Suwon, Korea) analysis was conducted to monitor the formation of MUA film onto gold surfaces. The Root Mean Square Roughness of AFM images is calculated on the basis of three independent measurements. Contact angle measurementsThe water contact angle (WCA) of the self-assembled monolayer (SAM) on gold surface was measured with a KSVCAM200, Kruss, Hamburg, Germany, contact angle goniometer, using distilled water droplets of 2 \(\upmu\)L. The average contact angle values with the corresponding standard deviations are reported for each sample based on five measurements. Measurements were carried out on the gold-coated substrates before MUA incubation, after 12 h incubation (MUA1), after 60 h incubation (MUA2), and after the double step of MUA2 and NTA-lysine incubation. Specific Immobilization of Histidine-Tagged antigen on Au-MUA-NTA-lysine-Ni(II), anchoring of target antibody and functionalization with Acryl-PEG-NHS. To promote the attachment of the histidine tags bearing proteins to the transition metal complex, the modified substrates were incubated with either GFP or B7-1 (400 nM in 150 mM NaCl and 10 mM HEPES buffer) overnight (ON) at 4 \({}^{\circ}\)C and then rinsed abundantly with phosphate buffer saline (PBS) 1X. Successively, the samples were exposed to a solution of the corresponding antibody (either anti-GFP or CTLA-4-Ig, 200 nM in PBS ON at 4 \({}^{\circ}\)C). Once the Ag-Ab interaction occurred, the antibody exhibited the constant fraction (Fc) on the top of the self-assembled monolayer (SAM).