Ni-NTA-Gold Nanoparticles

Synthesis of Ni-NTA-5 nm Gold Nanoparticles.

http://www.google.com/patents/US20120244075

The Ni-NTA-5 nm gold nanoparticles were prepared by the synthesis of 5 nm nitrilotriacetic acid (NTA) gold nanoparticles followed by chelating to Ni2+. In addition to Ni2+, NTA gold nanoparticles can also chelate to other metal ions such as by way of example only, Cu2+ and Zn2+.

The 5 nm NTA gold nanoparticles were synthesized by addingsodium borohydride, NaBH4 (114 mg, 3 mmol) in 5 mL of deionized water to a mixture of Gold(III) chloride trihydrate, HAuCl4·xH2O (118 mg, 0.3 mmol, G4022-1G, $105.5), 2-(2′-(2″-methoxyethoxy)ethoxy)ethane thiol (15 mg, 0.08 mmol) and 5-(6′-mercaptohexanoylamino)-1-carboxypentylimino diacetate trisodium salt (32 mg, 0.08 mmol) in 55 mL of 6:1:1 methanol/acetic acid/H2O with rapid stirring. After two hours of stirring, the 5 nm NTA gold nanoparticles were pelleted down by centrifugation. The crude gold nanoparticles were redissolved in deionized water, and purified by filtration through a membrane with MW cut-off of 30,000. The purified NTA gold nanoparticles were diluted to 1 L with 50 mM MOPs, pH7.9. 600 μL of 0.2 N NiSO4 solution was added to the NTA gold solution with stirring. After two hours stirring, the solution was concentrated with membrane filtration (MWCO=30,000) and chromatographed over a desalting column, e.g., GH-25. The gold fractions were collected as Ni-NTA-5 nm gold. The prepared Ni-NTA-5 nm gold nanoparticles were nearly monodispersed, and its electron micrograph is shown in FIG. 2.

Sensitive Detection of a His-Tagged Protein Using Blots.

The 6x-His-tagged protein, ATF-1, was spotted onto nitrocellulose, depositing (from left to right) 100 ng, 50 ng, 10 ng, 5 ng, 1 ng, and 0.5 ng per spot. A second row of control proteins (all spots identical), E. Coli extract (1 μL of 2.03 mg/ml total protein, 2.03 μg per spot), was spotted directly below the test target ATF-1 protein spots. The blot was blocked with 5% non-fat dry milk in 20 mM Tris, 0.15 M NaCl, pH7.6 containing 0.1% (w/v) Tween® 20 (TBST), and incubated 30 min. with the Ni-NTA-5 nm gold nanoparticles in 50 mM MOPs pH7.9 (OD280 nm=1.5). The blot was then washed with 10 mM imidazole in TB ST for 2 min. After washing the membrane three times with water, the spots were catalytically enhanced with Gold Enhance EM (Nanoprobes) for 9 min. The membrane was then washed with water and dried. All target spots could be detected (FIG. 3), whereas all control spots were negative, indicating a sensitivity of 0.5 ng.

Detecting Polyhistidine Tagged Proteins on Western Blots.

As shown in FIG. 6, the indicated amounts of purified 6x-His-tagged ATF-1 (34 kDa), 6x-His-tagged YY1 (68 kDa), 6x-His-tagged Src (61.7 kDa) mixed with crude extract from E. coli cells, and the crude extract from E. coli cells (1.25 μg total protein per lane) were applied to a 4-15% SDS-polyacrylamide gel. The 6xHis Protein Ladder (6xHPL) consists of five 6x-His-tagged proteins ranging from 15 to 100 kDa. It was loaded as a molecular weight standard, and as a positive control for western blotting. After electrophoresis and Western transfer, 6x-His-tagged proteins were detected by Ni-NTA-5 nm gold nanoparticles followed by GoldEnhance EM (Nanoprobes Inc., catalog #2113). The Ni-NTA-5 nm gold nanoparticles selectively detected all poly His-tagged proteins including 6xHis Protein Ladder, 6x-His-tagged ATF-1, 6x-His-tagged YY1, and 6x-His-tagged Src, but have no detectable binding to the crude extract from E. coli cells.