Conjugation of nanoparticles with antibody

The GNP, SNP and GNR were conjugated with anti-mitochondria antibodies (MAB 1273, Millipore, Schwalbach, Germany) as follows. Exactly 10 mM MUA and 30 mM MCH dissolved in ethanol were added to a nanoparticle solution suspended in water, followed by sonication for 10 min and incubation for 2 h 30 min to form a self-assembled monolayer (SAM) of MUA-MCH on the NPs surface. The monolayer was then washed with ultra-pure water by centrifugation (12 000 rpm, 90 min, 4 °C). The NPs-MUA-MCH was re-suspended in 50 mM MES and 0.1 M NaCl (pH 6.0). These procedures allowed for the formation of a carboxylic acid-terminated alkanethiol monolayer on the surface of the NPs. Conjugation of NPs with antibody was carried out using EDC and Sulfo-NHS. EDC (40 μg, 2 mg/mL in 50 mM MES pH 6.0) and Sulfo-NHS (196 μg, 2 mg/mL in 1 × PBS, pH 7.4) were added to the NPs-MUA-MCH solution. After stirring at room temperature for 40 min, NPs-MUA-MCH-NHSS was washed using centrifugation (17 000 rpm, 30 min, 4 °C) and then re-dissolved in PBS. The anti-mitochondria antibody solution was diluted 400 times in PBS (pH 7.4), added to the activated GNP, SNP, and GNR, and allowed to react using a rotary shaker for 4 h at room temperature. The mixture was then stored for 12 h at 4 °C. Tris (10 mM, pH 7.5) and 1 M glycine were added and the solution was incubated for 15 min to quench the excess hydroxylamine. The final nanoparticle-antibody suspension was centrifuged (17 000 rpm, 90 min, 4 °C) to remove any unbound antibody and re-suspended in PBS buffer and stored at 4 °C.