Biomolecular Separation Multicomponent Magnetic Nanorods for Biomolecular Separations**

Nanomaterials have been used extensively in the development of high sensitivity and high selectivity biodetection schemes. Designer particles, including noble-metal polyhedral structures, quantum dots, nanopatterns, and nanorods have found application in many forms of biological tagging schemes, including DNA and protein detection, cell sorting, and histochemical staining. Significant advantages over conventional molecule-based fluorophore strategies have been identified for several of these structures. Other applications for nanomaterials in biology, beyond diagnostics, include therapeutics and separations. A key area for researchers working with proteins involves separation and purification. Traditionally, nickel columns have been used in conjunction with histidine tagged proteins to separate such structures from a matrix of other undesirable biological elements. We and others have been working with nanorod structures prepared by the porous template synthesis approach pioneered by Martin and the group of Moskovits. 17] This synthetic procedure allows one to prepare rods electrochemically with uniform diameters and with predefined block lengths of inorganic and organic materials with excellent control. We hypothesized that if nickel were introduced as one of the blocks in a single or multicomponent nanorod structure, it could be used as a magnetic nanosized affinity template for histidine tagged proteins and, therefore, an appropriately applied magnetic field could be used to effect separation of the protein–rod complex from a multicomponent solution. Herein, we demonstrate how one can use two-component triblock 330 nm diameter rod structures with gold end blocks and a nickel interior block as materials that can very efficiently separate His-tagged proteins from non-his tagged structures. This work builds on our work with the transport of His-tagged structures by dippen nanolithography (DPN) to bulk solid-state nickel oxide substrates and the work of others involving the generation of microscopic arrays of His-tagged proteins on bulk nickel oxide substrates. 21] Magnetic multisegment nanorods composed of nickel and gold blocks were synthesized by using the method of electrodeposition into a porous alumina membrane (experimental conditions are described in the Supporting Information). The gold portions of the three-component structure were used to prevent nickel domain etching during the silver dissolving procedure. Prior to use, the rods were repeatedly rinsed with distilled water until the pH of the solution was 7. The multicomponent nanorods were washed with methanol and ethanol to remove contaminants from their surfaces. This was done by using a magnetic field (BioMag, Polysciences, Inc.) to pull the rods to the sidewalls of a plastic vial while rinsing them with the appropriate solvent. The gold portions of the nanorods were passivated with 11-mercaptoundecyl-tri(ethylene glycol)(PEG–SH) by incubating the rod samples in 1 mL, 10 mm ethanolic solution of the surfactant for 2 h followed by copious rinsing with ethanol and then nanopure water (Barnstead International, Dubuque, IA, USA). Others have shown that alkylthiols preferentially modify the gold surface in such two component structures. The gold surface was modified with PEG–SH for two reasons. First, the PEG–SH minimizes nonspecific binding of proteins to the nanorod structures. 23] Second, it stabilizes the rods by minimizing bare gold surface–surface interactions. The specific interaction of polyhistidine (His > 6) with bulk oxidized nickel surface is well known. 21] Similarly, fluorescein-tagged poly-His (His > 6) binds specifically to the Ni portions of the substrate as evidenced by confocal fluorescence microscopy. In a typical experiment, Au-Ni-Au nanorods (10–10) were incubated in a 63 mm fluorescein labeled poly-His solution (1 mL, 0.1m PBS(phosphate buffered saline), pH 7.4) for 12 h at room temperature (22 8C). Then the nanorods were vigorously rinsed with phosphate buffered saline (PBS) solution followed by nanopure water. During each rinsing step, the rods were separated from the supernatant by using magnetic force. Fluorescence imaging shows that the fluorescein-tagged polyhistidines efficiently bind to the nanorod structures (Figure 1b). This reaction between the poly-His and Au-Ni-Au rods can be monitored with the naked eye simply by watching the color of the solution decrease in intensity as a function of reaction time (Figure 1c). A quantitative analysis of the efficiency of polyHis adsorption was performed by preparing a standard calibration curve from the fluorophore-labeled poly-His over a range of concentrations starting with the experimental poly-His initial concentration of 63 mm and going to 0.16 mm (inset Figure 2). The fluorescence emission intensity of supernatant solution isolated from the reacted nickel nanorods shows that 90% of the poly-His was captured by the rods from the starting solution (Figure 2). As a control experiment, pure Au nanorods (no Ni), passivated with PEG–SH, were incubated in the poly-His solution (63 mm in 0.1m PBS, pH 7.4) under nearly identical conditions, and little interaction between the poly-His molecules and PEG–SH modified Au particles was observed (i.e., no detectable change in the emission of the fluoroscein as measured by fluorescence spectroscopy, data not shown). The Au-Ni-Au rods can be used in a novel scheme for separating His-tagged proteins from structures without His[*] K.-B. Lee, S. Park, Prof. C. A. Mirkin Department of Chemistry and Institute for Nanotechnology Northwestern University 2145 Sheridan Road, Evanston, IL 60208-3113 (USA) Fax: (+1)847-467-5123 E-mail: [email protected] [] These authors contributed equally to this work.