12 mercaptododecanoic acid

SAMs were prepared using a protocol modified from our previous study (15 (link)). Briefly, the Au surfaces (40 nm) were deposited on glass coverslips following a Ti layer (40 nm), using an ANELVAL-400EK electron beam evaporator (Canon Anelva Corp., Kanagawa, Japan). The silicon wafers coated with Au (Zhongding Ltd., Yangzhou, China) were washed with triple-distilled water in an ultrasonic bath for 10 min, and immersed in 1 Mm 1-undecanethiol (-CH₃; Sigma, St. Louis, MO, USA), 11-amino-1-undecanethiol (-NH₂; Sigma), 11-hydroxy-1-undecanethiol (-OH; Sigma), 12-mercaptododecanoic acid (-COOH; Sigma) for 12 h (16 (link)). Following the self-assembly process, the substrates were washed with triple-distilled water and dried with nitrogen.

The linker MDDA (12-Mercaptododecanoic acid, Sigma-Aldrich) conjugates between the GNP and the barbiturate. One side of the MDDA chain (thiol) connects to the gold via semi-covalent bonding, while the other side of the MDDA chain, carboxylic acid, binds to the negatively charged oxygen of the barbiturate. To each solution of the various GNP sizes, MDDA was added in excessive amounts, and the mixture was stirred for another four hours. Following this step, the solutions were centrifuged in order to reach higher concentrations. Next, the activating agents EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl, Thermo Scientific) and NHS (N-Hydroxysulfosuccinimide sodium salt, Chem-Impex International) were added to the mixture together with the barbiturate, and stirred overnight (Figure 8). The solutions were centrifuged again in order to increase concentration.Figure 8

Synthesis of GNPs. Schematic diagram of the synthesis of GNPs and functionalization with barbiturate. In order to conjugate the glucosamine to the GNP, the linker 12-Mercaptododecanoic acid (MDDA) was utilized. EDC and NHS were added in order to activate the carboxylic acids of the linker. One side of the MDDA chain (thiol) connects to the gold via semi-covalent bonding, while the other side, carboxylic acid, binds to the negatively charged oxygen of the barbiturate.

MDCK (Madin-Darby Canine Kidney Cells) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) with 1% Penicillin/Streptomycin and 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA) in the humidified atmosphere in 5% CO₂ at 37 °C.
H1N1 (A/Netherlands/2009) was a kind gift from Prof. M Schmolke (University of Geneva) and it was reproduced in MDCK cells and titrated by immunocytochemistry assay.
Heptakis-(6-deoxy-6-iodo)-β-Cyclodextrin was purchased from AraChem (Tilburg, Netherlands) and dried under high vacuum for 48 h prior to use. 12-mercaptododecanoic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dried in the same way. Neu5Acα2-6Galβ1-4GlcNAc-β-ethylamine was purchased from Asparia Glycomics (Donostia, Spain). Maleimide-PEG8-CH₂CH₂COOH was purchased from PurePEG (San Diego, CA, USA). All the other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) as reagent grade and used without further purification.