Shell Nanoparticles Modified with Ni2+−Nitrilotriacetic

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Fe3O4/Au Core/Shell Nanoparticles Modified with Ni2+-Nitrilotriacetic Acid Specific to Histidine-Tagged Proteins Hai-Yan Xie,* Rui Zhen, Bo Wang, Yong-Jun Feng, Ping Chen, and Jian Hao School of Life Science, Beijing Institute of Technology, Beijing 100081, People’s Republic of China ReceiVed: NoVember 12, 2009; ReVised Manuscript ReceiVed: February 16, 2010

Well-defined Fe3O4/Au core/shell nanoparticles were successfully prepared with polyethyleneimine (PEI) as a linker, which are of good monodispersity and strong magnetism. The intact gold shell made these nanoparticles easily modified and biofunctionalized for different biodetection and biosensing purposes. Hereby, they were surface modified with mercaptopropionic acid, followed by conjugating nitrilotriacetic acid (NTA) and subsequently chelating Ni2+. The resulting biofunctionalized Fe3O4/Au-NTA-Ni2+ composite nanoparticles were used to enrich and separate the histidine-tagged (His-Tag) maltose-binding protein (MBP) directly from the mixture of lysed cells. It has been found that Fe3O4/Au-NTA-Ni2+ can be used for rapid, efficient, and specific enrichment and separation of His-Tag fusion proteins. The enrichment efficiency of the Fe3O4/ Au-NTA-Ni2+ nanoparticles is significantly higher than that of metal-chelate affinity chromatography (MCAC). The detection limit of the current method coupled with facile SDS-PAGE can be lower than 5.5 × 10-8M. Due to the ease of operation and good efficiency of separation, diverse bifunctional and even multifunctional nanomaterials can be further developed for biological applications. 1. Introduction The biotechnologies today enable proteins to express very easily, whereas the purification of protein is still very difficult. The development of methods for enrichment and purification of proteins, especially low-abundance proteins, is currently a hot topic. Many target proteins are usually expressed with a tag for affinity separation, while histidine-tagged (His-Tag) fusion proteins are preferably considered in protein preparation. The as-prepared products can be conveniently purified by metal-chelate affinity chromatography (MCAC) based on the formation and disassociation of the coordination bonds between the six consecutive histidine residues (6 × His) and Ni2+-nitrilotriacetate complex (NTA-Ni2+) under different conditions.1-3 The MCAC method is universal, simple in the chemical structure of coordination group, and mild in separation conditions; nevertheless, it also has shortcomings such as its tedious operation, low protein binding capacity, and less efficiency for low-abundance proteins. Magnetic nanoparticles (NPs) are of unique superiorities in separation and analysis due to their highly specific surface area, good solubility, and magnetic manipulability. With the development of magnetic NP-based bioseparation and analytical techniques,4-8 magnetic NPs have been used to enrich and separate histidine-tagged proteins. The protein-binding efficiency for MCAC with magnetic NPs is much higher than that with micrometer-scale packing, with a remarkable decrease in nonspecific adsorption of the surface for proteins.9-15 However, magnetic NPs are easy to aggregate, surface unstable, and even easy to lose magnetism when being used for complicated systems, which limits their applications. Therefore, it is worth exploiting higher enrichment and separation efficiency and the lower detection limit of magnetic nanoparticles used in bioseparation. Gold nanoparticles have been widely applied in biosensing and immunoassay16-18 since they have good bio* To whom correspondence should be addressed: phone, 0086-1068915940; fax, 0086-10-68915956; e-mail, [email protected].

compatibility, chemical stability, and special optical properties and are easy to biofunctionalize. Composite nanomaterials of gold-coated magnetic NPs are becoming more and more popular for their integration with magneto-manipulability, stability, and biocompatibility. Gold-coated magnetic nanoparticles facilitate to extend the applications of both nanogold and magnetic NPs in biology.19-31 Some Fe2O3/Au composite nanoparticles were used to separate proteins. Bao et al.19 synthesized Fe2O3/Au core/ shell nanoparticles and used them for antigen separation and immunoassay coupled with surface-enhanced Raman scattering (SERS). Park et al.23 prepared Fe oxide@Au core@shell nanoparticles by thermal processing, with the particle size and the shell thickness being controllable, and used the resulting nanoparticles for immunoseparation with SERS detection. Evidently, SERS detection needs a Raman spectrometer, and the pretreatment of materials is tedious. Additionally, commonly used organic phase synthesis of nanoparticles needs to be carried out at high temperatures and requires a large amount of organic solvents, and at the same time, resultant nanoparticle products need to be transferred into aqueous phase by ligand exchange prior to use for bioassay. Bao et al.20 fabricated Au-Fe3O4 NPs by chemical bonding, which were successfully used for protein separation. But the reported nanocomposite had no intact Au shell, thus the stability of Fe3O4 NPs was less improved and the homogeneity of their surface functionalization was still not so good. Evidently, it is worth developing and applying Fe3O4/ Au core/shell nanoparticles in biopurification and bioseparation. In the present work, intact Fe3O4/Au core/shell nanoparticles were prepared by aqueous phase synthesis according to the recent report by Goon et al.32 Subsequently, biofunctionlized magnetic Fe3O4/Au-NTA-Ni2+ nanoparticles were successfully fabricated by modifying Fe3O4/Au with mercaptopropionic acid (MPA) followed by conjugating NTA and chelating Ni2+. Furthermore, maltose-binding protein (MBP) was enriched and separated directly from the mixture of lysed cells with these nanoparticles. The present method is facile and rapid, and its enrichment efficiency is much higher than that of MCAC. With

10.1021/jp910753f  2010 American Chemical Society Published on Web 03/01/2010

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such a strategy, a variety of biospecific magnetic nanomaterials are expectedto be constructed for enrichment and separation of different types of proteins. 2. Materials and Methods 2.1. Chemicals. Polyethyleneimine (PEI, Mw ≈ 25000 g mol-1), N-(3-(dimethylamino)propyl)-N′-ethyl carbodiimide (EDC), N-hydroxysuccinimide (NHS), and nitrilotriacetic acid (NTA) were purchased from the Sigma-Aldrich. 3-Mercaptopropionic acid (MPA, 99.9%) was purchased from Alfa Aesar. All other chemicals were obtained from Shanghai Bio Life Science & Technology. Strains: E. coli BL21 (DE3), which has malE gene of recombinant plasmid pET28a+YS19, was transferred in our lab. 2.2. Synthesis and Characterization of Au-Coated Fe3O4 Nanoparticles. Au-coated Fe3O4 core/shell NPs (Fe3O4/Au) were prepared according to the reported method with some modifications.32 First, PEI-functionalized Fe3O4 nanoparticles (Fe3O4-PEI) and citrate-coated Au NPs were synthesized. Then the Fe3O4-PEI suspension was rinsed and sonicated. Subsequently, 1 mL of the above Fe3O4-PEI suspension was added to 90 mL of colloidal gold solution. After 2 h of stirring, Au NPs could be electrostatically attracted onto the surface of Fe3O4-PEI NPs, leading to formation of the Fe3O4-Au seed particles. The Fe3O4-Au seeds were magnetically isolated from excess Au colloid solution and suspended in 10 mL of Milli-Q water. The particles were then coated again with PEI by heating at 60 °C for 1 h in the presence of PEI (5 mg mL-1) followed by rinsing for 5 times. Finally, the obtained NPs were dispersed in water. In the coating process, 10 mL of the above solution was added to 55 mL of 0.01 M NaOH under mechanical stirring. The NH2OH · HCl (0.2 M) and HAuCl4 (1%) were added consecutively to the reaction flask containing Fe3O4-Au seeds. In the first iteration, 0.25 mL of HAuCl4 and 0.375 mL of NH2OH · HCl were injected into the reaction flask, waiting for 1 min between injections. After the mixture was stirred for 10 min, 0.25 mL of HAuCl4 and 0.125 mL of NH2OH · HCl were injected into the reaction flask in the second iteration, and this process was repeated nine times. After ten iterations, the Fe3O4/ Au core/shell NPs would be produced. And they were separated from the reaction mixture by a magnetic field and rinsed, followed by dispersing in 10 mL of water. The resultant products were characterized by transmission electron microscopy (TEM, JEM-2010, JEOL Company of Japan) operated at 200 kV, X-ray diffraction (XRD, D8 Advance, Bruker, Germany), vibrating sample magnetometry (VSM), and X-ray photoelectron spectroscopy (XPS, XSAM800, Kratos Ltd.U.K.). 2.3. Surface Modification of the Fe3O4/Au Core/Shell NPs. Ten milliliters of Fe3O4/Au NPs was washed with water and then washed with 0.5 M NaCl, 0.1 M sodium borate solution, and 0.1 M sodium acetate four times. Finally, they were washed with 0.5 M NaCl six times followed by dispersing in 10 mL of water. Two milliliters of these Fe3O4/Au NPs were added to a 25 mL flask and diluted with 8 mL of water. Purging with Ar flow, 0.1 mL of MPA was dropwised into the flask. After the mixture was stirred overnight, the MPA-capped Fe3O4/Au NPs were separated from the reaction mixture by a magnetic field and finally dispersed in 2 mL of water. XPS was used to characterize the modification. 2.4. Fabrication of Biofunctionalized Magnetic Fe3O4/ Au-NTA-Ni2+ Nanoparticles. A 0.5 mL portion of MPAcapped Fe3O4/Au NPs was magnetically separated from solution and dispersed in 0.5 mL of 0.01 M PBS (pH ) 6.5) with EDC/

Xie et al. NHS (50 mM/50 mM). The mixture was then shaken for 30 min at room temperature (RT) at a rotation speed of 120 revolutions min-1, followed by magnetic separation. After the supernatant was removed, 0.3 mL of 0.01 M PBS (pH ) 7.4) and 0.2 mL of 0.05 M NTA solution, which was dissolved in 0.01 M PBS (pH ) 7.4), were added to dissolve the precipitate. The mixture was then shaken for 4-6 h at room temperature. Subsequently, the Fe3O4/Au-NTA particles were magnetically separated from the solution and dispersed in 0.4 mL of water, and then 0.5 mL of 0.1 M NiCl2 was added to react for 1-2 h. After separation from the reaction mixture by a magnetic field, the precipitate was washed two times with water. The content of Ni2+ in the magnetic-NTA-Ni2+ nanoparticles was analyzed using an inductively coupled plasma optical emission spectrometer (ICP-AES, Intrepid XSP Radial, Thermo. USA). 2.5. Extraction and Purification of Maltose-Binding Protein (MBP). After being transfected, the recombinant strain of E. coli BL21 (DE3) was inoculated into LB culture medium containing Kanamycin and the contents was shaken for 12-16 h for activating culture. The activated cells were inoculated into LB culture medium and incubated for 3 h at 37 °C. When the cell turbidity OD reached to 0.3, 1 mM isopropyl β-Dthiogalactoside (IPTG) was added into culture media. Then the cells were inductively cultured for 10 h at 37 °C. After that, the cells were centrifuged at 12000g for 10 min, and the pellet was resuspended in 20 mM Tris-HCl (pH ) 8.0) and sonicated 60 times, 3 s every time at an interval time of 5 s. After that, the cell debris was centrifuged at 12000g for 10 min to obtain the cell lysate. The total protein concentration of the cell lysate was measured by the Coomassie brilliant blue method. 2.6. Protein Enrichment and Separation Using Biofunctionalized Magnetic Fe3O4/Au-NTA-Ni2+ Nanoparticles. First, Fe3O4/Au-NTA-Ni2+ nanoparticles were separated from the solution (500 µL, 0.4 mg mL-1). After being washed three times with deionized water, they were added directly into 500 µL of mixture of lysed cells and were shaken for 30 min at a rotation speed of 120 revolutions min-1. Second, Fe3O4/ Au-NTA-Ni2+ nanoparticles having captured MBP were isolated from the solution using a magnet and washed three times with deionized water in order to remove any residual uncaptured proteins. Then, the targeting nanoparticles were washed with 500 µL of imidazole solution to disassociate histidine-tagged proteins from their surface. SDS-PAGE was applied to detect the released proteins via silver staining imaging, while the preconcentration voltage was 60 V and the separation voltage was 90 V. The binding capacity of protein was determined by UV-visible absorption spectra (using UV-2550, Shimadzu Corporation of Japan) by combining with the Coomassie brilliant blue staining. The targeting materials can be recovered and reused by washing sequentially with EDTA (0.1 M) and NiCl2 solution (0.2 M). 3. Results and Discussion 3.1. Preparation and Characterization of Fe3O4/Au Core/ Shell Nanoparticles. The aqueous phase synthesis of Fe3O4/ Au core/shell nanoparticles includes three steps. First, PEIcoated Fe3O4 NPs and citrate-coated gold nanoparticles (Au NPs) were synthesized in aqueous phase. Second, Fe3O4 NPs were surface immobilized with a number of Au NPs by electrostatic interaction, denoted as Fe3O4-Au seeds. Third, Au shell-coated Fe3O4 nanoparticles, denoted as Fe3O4-Au coats, were fabricated by reducing HAuCl4 on Au NPs, attached to Fe3O4 surface, as crystal seeds to form an Au shell (Figure 1). In strong alkaline solution, ferrous ions were precipitated to produce Fe(OH)2, followed by adding PEI as a stabilizer, heated,

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Figure 1. Schematic representation of the preparation procedure of biofunctionalized magnetic Fe3O4/Au-NTA-Ni2+ nanoparticles and their enrichment and separation of the proteins.

Figure 2. TEM images for the same batch of Fe3O4 NPs, Fe3O4-Au seeds and Fe3O4-Au coats: (a and b) PEI-coated Fe3O4 NPs, interplane interval d ) 0.25 nm; (c and d) Fe3O4-Au seeds; (e and f) Fe3O4-Au coats NPs, interplane interval d ) 0.201 nm.

and oxidized for 2 h, with brown-black magnetic NPs obtained. The results from XRD and crystal phase purity analysis suggested that the resulting product was highly pure and crystalline Fe3O4 NPs. TEM results indicated that they had a uniform size distribution of ca. 20 nm and a well-defined PEI coating (Figure 2a). PEI is a kind of water-soluble, low toxicity polymer,33 whose chain contains a lot of imine groups positively charged under the synthesis conditions. At the same time, PEI has a big steric hindrance, thus greatly improving the antiag-

gregation capability of Fe3O4 NPs, which makes them capable of dispersing stably in aqueous solution for a long time. Au NPs were synthesized by reducing HAuCl4 with sodium borohydride in the presence of sodium citrate as a stabilizer, by which citrate-coated Au NPs could be obtained. TEM results indicated a uniform size distribution. And the size could be tuned by changing the amount of HAuCl4. Au NPs can be densely attached to Fe3O4 NPs by electrostatic interaction between citrate on Au NPs and positively charged PEI on the surface of Fe3O4 NPs since citrate has three negatively charged carboxyl groups, producing Fe3O4-Au seeds. The product exhibited pink in aqueous solution. From TEM images it can be clearly seen that a lot of small-sized Au NPs were attached to Fe3O4 NPs (Figure 2, panels c and d). Au NPs on the surface of Fe3O4-Au seeds can be used as crystal seeds to directly produce an intact Au shell. Using NH2OH · HCl to repeatedly reduce HAuCl4 makes Au NPs grow, gradually connect to each other, and finally form an intact Au shell to obtain Fe3O4-Au coats, the solution exhibiting dark blue. The product has good monodispersity. When 20 nm of Fe3O4 NPs and 4 ( 1 nm of Au NPs were used, the size of Fe3O4-Au coats was about 30 ( 5 nm (Figure 2e). From Figure 2b, it can be seen that the interplane interval of Fe3O4 NPs was 0.25 nm, in agreement with that of (311) facet of face-centered cubic Fe3O4 crystal. Additionally, the interplane interval of 0.201 nm in Figure 2f was consistent with that of (111) facet of facecentered cubic Au crystal. Therefore, these results could prove that the resultant Fe3O4-Au coats were Fe3O4/Au core/shell nanocomposite. In comparison with the procedure for preparing Fe3O4-Au coats reported by Goon et al.,32 we used more than five reduction cycles, namely 10 iterations to produce a complete Au shell, which is expected to be more beneficial to further functionalization of Fe3O4-Au coats. Relative to Fe3O4 NPs and Fe3O4-Au seeds, Fe3O4-Au coats decreased in magnetism, with a magnetization of about 10 emu g-1 (Figure 3). It was found that the Fe3O4-Au coats obtained could readily redisperse in solution after removing the external magnetic field, very similar to the performance of superparamagnetic particles. However, considering their slight magnetic remanence and coercivity (Figure 3), they should be ferromagnetic in a way. Fe3O4-Au coats dispersing in solution could be completely isolated within 5 min with a NdFeB magnet. The current method for preparing Fe3O4-Au coats is facile and safe, and the resulting product was very stable. The thickness of the Au shell

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Figure 3. Room temperature hysteresis curves for (a) Fe3O4 NPs, (b) Fe3O4-Au seeds, and (c) Fe3O4-Au coats.

Figure 4. XRD spectra for the Au NPs and the same batch of Fe3O4 NPs, Fe3O4-Au seeds and Fe3O4-Au coats.

and the size and magnetism of Fe3O4-Au coats can be efficiently tuned by controlling the amount of PEI and Au NPs. To our knowledge, the present method for preparation of Fe3O4/ Au core/shell nanoparticles is one of the most repeatable. XRD spectrum of Fe3O4-Au coats had a sharp peak, which was consistent just with that of Au nanocrystal, and no peak was observed for Fe3O4 NPs (Figure 4), which is consistent with the results for concluding “complete coverage” or “core/shell” reported by other groups,26,28 further indicating that Fe3O4 in Fe3O4-Au coats was completely coated by a thick Au shell. XPS results of the same batch of Fe3O4-Au seeds and Fe3O4-Au coats suggested that from Fe3O4-Au seeds to Fe3O4-Au coats the content of Au element on the nanoparticle surface dramatically increased (Figure 5B), while the peaks characteristic of the Fe2p of Fe3O4-Au coats disappeared (Figure 5A), indicating that little Fe element existed on the surface of Fe3O4-Au coats NPs. These confirmed that an intact Au shell on Fe3O4 NPs had formed with a thickness of more than 5 nm.34 Here, all the results from TEM, XRD and XPS support a conclusion that an intact Au shell can be obtained by increasing the iteration of HAuCl4 reduction. Importantly, an intact Au shell will make the nanoparticles quite stable and biocompatible and also enable us to rapidly, homogeneously, and efficiently biofunctionalize Fe3O4-Au coated NPs with multifarious biofunctional molecules; therefore, it would greatly facilitate their applications in biology and medicine. 3.2. Modification and Biofunctionalization of Fe3O4-Au Coated Nanoparticles. Modification of Au surface with mercapto compounds has been intensively studied. In this work, mercaptopropionic acid (MPA) was used to modify the surface of Fe3O4-Au coated nanoparticles. Subsequently, the carboxyl group was first activated using 1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide hydrochloride and N-hydroxyl suc-

Xie et al. cinimide (EDC/NHS) and then was coupled with the amino group of nitrilotriacetic acid (NTA), followed by chelating Ni2+, with magnetic Fe3O4/Au-NTA-Ni2+ nanocomposite obtained (Figure 1). The resultant product was characterized by XPS (Figure 6). In Figure 6A, XPS peaks fitted with a nonlinear least-squares fitting (NLSF) program (XPS peak software 4.1, Raymund W. M. Kwork) gave a value of 162.6 eV for S 2p peak, close to that for S 2p of Au-S bond in binding energy, implying that -SH of MPA had bonded via the Au-S bond to the surface of Fe3O4-Au coated NPs. No S 2p peak (164-165 eV) for free mercapto group was observed.35 Figure 6B is the high-resolution C 1s XPS spectrum of MPAmodified Fe3O4-Au coats. Evidently, a peak appeared at 284.9 eV corresponding to C 1s binding energy for -(CH2)-, while a dragging peak was observed around 289.1 eV corresponding to C 1s binding energy of -COOH. Fitting the spectrum with the NLSF program gave three peaks at 284.9, 287.1, and 289.1 eV, of which the peak at 287.1 eV could be assigned to the C 1s binding energy contributed from both the C in -CS- group and the β-C in -CH2COOH group. In conclusion, Fe3O4-Au coats had been successfully modified with MPA. ICP-AES was used to determine Ni2+ content in magnetic Fe3O4/Au-NTA-Ni2+ nanoparticles. The AES peak around 231.6 nm was consistent with that of Ni2+. For six repeated determinations an average content of 1.58 × 10-7 mol mg-1 for Ni2+ was obtained. 3.3. Enrichment and Purification of His-Tag Proteins in Cell Lysate with Magnetic Fe3O4/Au-NTA-Ni2+ Nanoparticles. Theoretically, all the proteins with a 6 × His tag should be able to be captured and preconcentrated by such a MCAC method. In the present work, the resulting magnetic Fe3O4/ Au-NTA-Ni2+ nanoparticles were used for direct preconcentration and purification of His-Tag fusion MBP in crude cell lysate. After washing with water to remove nonspecifically adsorbed MBP, the Fe3O4/Au-NTA-Ni2+-bound proteins were disassociated with imidazole. Separately collected MBP solutions were detected by SDS-PAGE. In Figure 7, the MW of MBP is 44 kD. As can be seen, the Fe3O4/Au-NTA-Ni2+ nanoparticles can be used to efficiently enrich target proteins from cell lysate. No impurity proteins were detected, indicating that the method is of high specificity. Over a concentration range of 100-500 mM the quantity of disassociated proteins increased with incremental concentration of imidazole, finally to a plateau (Figure 7a). Fe3O4/Au-NTA-Ni2+ nanoparticles can be repeatedly used in the experiment, if it was treated with EDTA and NiCl2 on demand. As shown in Figure 7b, the specificity and affinity of the Fe3O4/Au-NTA-Ni2+ nanoparticles remained unaffected after being recovered and reused. By incubating identical MBP solutions with Fe3O4/Au nanoparticles, Fe3O4/Au-NTA nanoparticles, and Fe3O4/ Au-NTA-Ni2+ nanoparticles, it has been found that the quantities of MBP bound to magnetic Fe3O4/Au and magnetic Fe3O4/Au-NTA by nonspecific adsorption were much less than those specifically captured by magnetic Fe3O4/Au-NTA-Ni2+ (Figure 7c, lanes 3-5), suggesting that the enrichment of MBP indeed resulted from 6 × His specific affinity for Ni2+ via an Ni2+-nitrilotriacetate complex (NTA-Ni2+). The capacity of magnetic Fe3O4/Au-NTA-Ni2+ nanoparticles capturing MBP at about 2 mg protein mg-1 from lane 5 in Figure 7c is much higher than that of commercial microbeads at only 10-12 µg of protein mg-1.15 Such a high efficiency comes from the highly specific surface of the nanoparticles and corresponding high content of Ni2+ on the surface of nanoparticles. As mentioned above, the content of Ni2+ on the magnetic nanoparticles was

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Figure 5. XPS spectra of Fe 2p and Au 4f of the same batch of (A) Fe3O4-Au seeds and (B) Fe3O4-Au coats.

Figure 6. XPS spectra of MPA-modified Fe3O4-Au coats (A: S 2p, black, raw data; blue, baseline; red, deconvolution peak; B: C 1s, black, raw data; green, baseline; red, blue, and pink, deconvolution peaks).

Figure 7. Analysis of the cell lysate by SDS-PAGE. (a) The fractions washed off from the Fe3O4/Au-NTA-Ni2+ NPs: lane 1, marker; lane 2, the cell lysate; lane 3, the fraction washed off from the commercial Ni2+-NTA column; lanes 4-7, the fractions washed off from the Fe3O4/ Au-NTA-Ni2+ NPs when different amount imidazole solution was used (lane 4, 100 mM; lane 5, 250 mM; lane 6, 500 mM; lane 7, 1 M). (b) The fractions washed off from the reused Fe3O4/Au-NTA-Ni2+ NPs using imidazole solution: lane 1, marker; lane 2, the cell lysate; lane 3, 100 mM; lane 4, 250 mM; lane 5, 500 mM. (c) Specific detection of the Fe3O4/Au-NTA-Ni2+ NPs: lane 1, marker; lane 2, the cell lysate; lane 3, the fraction washed off from the Fe3O4/Au nanoparticles; lane 4, the fraction washed off from the Fe3O4/Au-NTA NPs; lanes 5-9, the fraction washed off from the Fe3O4/Au-NTA-Ni2+ nanoparticles when different amounts of cell lysate were used (lane 5, 2.2 × 10-5 mol L-1; lane 6, 2.2 × 10-6 mol L-1; lane 7, 2.2 × 10-7 mol L-1; lane 8, 1.1 × 10-7 mol L-1; lane 9, 5.5 × 10-8 mol L-1).

1.58 × 10-7 mol mg-1. Thus, one of 3.5 Ni2+ ions bound a MBP molecule. Considering the steric hindrance on the nanoparticles, it can be seen that the availability of Ni2+ was very high, suggesting that magnetic Fe3O4/Au-NTA-Ni2+ nanoparticles can specifically bind target proteins with a great efficiency. The quantity of proteins captured by magnetic Fe3O4/ Au-NTA-Ni2+ depended directly on the concentration of proteins in cell lysate (Figure 7c lane 5-9). By coupling with facile SDS-PAGE, the magnetic nanoparticles can be used to detect target proteins at a detection limit lower than 5.5 × 10-8 M. When the concentration of MBP was very low, the

supernatant after preconcentration and magnetoseparation with magnetic Fe3O4/Au-NTA-Ni2+ scarcely contained remanent target protein; on the contrary, the target protein could be found in the eluate obtained by disassociating the magnetic nanoparticles, which have been used for enriching the protein, with imidazole. This indicates that the magnetic Fe3O4/ Au-NTA-Ni2+ nanoparticles can be applied to total enrichment and purification of low-abundance target proteins. Increasing the concentration of magnetic Fe3O4/Au-NTA-Ni2+ nanoparticles can further improve the detection limit for proteins, thus improving their capability to detect trace targets, which is highly

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significant for separation and analysis of biomacromolecules and trace target species in complex real samples. 4. Conclusions Well-defined Fe3O4/Au core/shell nanoparticles have been fabricated via a linker of polyethyleneimine. The Au shell can facilitate improving the biocompatibility of Fe3O4/Au nanoparticles, which makes their biofunctionalization facile through Au-S bonding. Magnetic Fe3O4/Au-NTA-Ni2+ nanoparticles have been successfully assembled using MPA to modify Fe3O4/ Au followed by conjugating NTA and then chelating Ni2+, producing biofunctionalized magnetic Fe3O4/Au-NTA-Ni2+ nanoparticles with strong affinity for His-Tag fusion proteins. These magnetic nanoparticles can efficiently capture, enrich, and purify the MBP directly from the cell lysate. The enrichment efficiency of the magnetic nanoparticles is much higher than that of MCAC. The present method is facile in operation, rapid, and low cost and it does not need sample pretreatment, which is suitable for preconcentrating, purifying, and detecting target His-Tag proteins in complex samples. The magnetic nanoparticles having been used can be reused after being treated with EDTA and NiCl2. On the basis of this work, more bioapplications of Fe3O4/Au nanoparticles can be realized by utilizing good biocompatibility and easily biofunctionalizable superiority of the Au shell. If some other biotargeting molecules are used to modify Fe3O4/Au nanoparticles, corresponding new nanomaterials for preconcentration and purification of other proteins or other molecules in complex samples will be produced. For example, conjugating glutathione to Fe3O4/Au surface, the glutathione S-tranferase tagged proteins will be able to be separated. The present strategy should be helpful for developing methods for bioseparation and analysis based on nanomaterials. Acknowledgment. This work was supported by the National Key Scientific Program (973)-Nanoscience and Nanotechnology (No. 2006CB933100), the Program for New Century Excellent Talents in University (No. NCET-08-0046), the National Natural Science Foundation of China (No. 20975013), the Excellent Young Scholars Research Fund of the Beijing Institute of Technology (No. 2007YS0603), and the Ministry of Public Health of China (No. 2009ZX10004-107). The authors thank Mr. Peng Jiang, Dr. Zhi-Quan Tian, and Dr. Min Xie for helpful discussions. References and Notes (1) Ahrends, R.; Pieper, S.; Ku¨hn, A.; Weisshoff, H.; Hamester, M.; Lindemann, T.; Scheler, C.; Lehmann, K.; Taubner, K.; Linscheid, M. W. Mol. Cell. Proteomics 2007, 6, 1907–1916. (2) Ahrends, R.; Pieper, S.; Neumann, B.; Scheler, C.; Linscheid, M. W. Anal. Chem. 2009, 81, 2176–2184. (3) Kim, J.; Park, H. Y.; Kim, J.; Ryu, J.; Kwon, D. Y.; Grailhe, R.; Song, R. Chem. Commun. 2008, 1910, 1912.

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