Shell Nanoparticles for

Jun 24, 2009 - Fang Bao, Jian-Lin Yao,* and Ren-Ao Gu*. Department of Chemistry, Chemical Engineering and Materials Science, Soochow University, ...
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Synthesis of Magnetic Fe2O3/Au Core/Shell Nanoparticles for Bioseparation and Immunoassay Based on Surface-Enhanced Raman Spectroscopy Fang Bao, Jian-Lin Yao,* and Ren-Ao Gu* Department of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, China Received April 15, 2009. Revised Manuscript Received May 12, 2009 Magnetic Fe2O3/Au core/shell nanoparticles can be particularly used in biological separation, but the development of an appropriate technique including a production process for higher efficient separation and the subsequent immunoassay for lower level still represent a great challenge. In this article, Fe2O3/Au core/shell nanoparticles with different Au ratios were prepared by reducing HAuCl4 on the surface of γ-Fe2O3 nanoparticles. Scanning electron microscopy (SEM) images and surface-enhanced Raman spectroscopy (SERS) spectra clearly show that the surfaces of Fe2O3 nanoparticles were covered by Au. SERS signals of pyridine (Py) have been obtained on the Fe2O3/Au nanoparticles, and it has been found that the SERS intensity enhanced with the increase of iterative additions of HAuCl4. The antigens in test solution have been effectively separated by the magnetic Fe2O3/Au core/shell nanoparticles, and subsequent rapid detection was examined by immunoassay analysis based on SERS. The result demonstrates that the magnetic bioseparation program used by this magnetic Fe2O3/Au core/shell nanoparticles could separate almost all of the antigens in test solution. The ease of operation and good separation efficiency of this effective method has shown a potential application for magnetic Fe2O3/Au core/shell nanoparticles in bioseparation.

Introduction The development and implementation of innovative processes in the field of bioseparation have been extensively studied in recent years because of high costs in biotechnological processing.1-3 Among them, the application of the magnetic nanomaterials in bioseparation processing is of particular interest.4,5 The combination of magnetic nanoparticles with bioactive molecules for the separation of proteins has been proposed.6 However, the development of an appropriate technique including a production process for higher efficient separation and the subsequent immunoassay for lower level still represent a great challenge. Many efforts have been made to design appropriate magnetic separation nanoparticles and nanocomposites,7-9 but one of the major obstacles is the lack of surface tunability for biocompatible applications. Coating the magnetic particles with a gold shell provides an intriguing class of biomaterials to overcome such an obstacle because the well-established surface chemistry and biological reactivity of gold can impart the magnetic particles with the desired chemical or biomedical properties.10-12 *Corresponding authors. Telephone: +86-512-65880399. E-mail: ragu@ suda.edu.cn (R.-A.G.); [email protected] (J.-L.Y.). (1) Hickstein, B.; Peuker, U. A. Biotechnol. Prog. 2008, 24, 409. (2) Desaia, T. A.; Hansford, D.; Ferrari, M. J. Membr. Sci. 1999, 159, 221. (3) Keller, K.; Friedmann, T.; Boxman, A. Trends Biotechnol. 2001, 19, 11. (4) Son, S. J.; Reichel, J.; He, B.; Schuchman, M.; Lee, S. B. J. Am. Chem. Soc. 2005, 127, 7316. (5) Bao, J.; Chen, W.; Liu, T.; Zhu, Y.; Jin, P.; Wang, L.; Liu, J.; Wei, Y.; Li, Y. ACS Nano 2007, 1(4), 293. (6) Gu, H.; Xu, K.; Xu, C.; Xu, B. Chem. Commun. 2006, 941. (7) Hyeon, T. Chem. Commun. 2003, 927. (8) Lu, A. H.; Salabas, E. L.; Schiith, F. Angew. Chem. 2007, 46, 1222. (9) Jeong, U.; Teng, X.; Wang, Y.; Yang, H.; Xia, Y. Adv. Mater. 2007, 19, 33. (10) Cui, Y.; Wang, Y.; Hui, W.; Zhang, Z.; Xin, X.; Chen, C. Biomed. Microdevices 2005, 7, 153. (11) Yu, H.; Chen, M.; Rice, P. M.; Wang, S. X.; White, R. L.; Sun, S. Nano Lett. 2005, 5, 2. (12) Xu, Z.; Hou, Y.; Sun, S.; Xu, Z.; Hou, Y.; Sun, S. J. Am. Chem. Soc. 2007, 129, 8698.

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Considering the amount of applications and publications on Fe oxide/Au composite nanoparticles, Lyon et al. provided a rapid and effective route to magnetic core/shell particles that are soluble in aqueous media.13 Park et al. recently reported findings of the fabrication and characterization of gold-coated iron oxide (Fe2O3 and Fe3O4) core@shell nanoparticles (Fe oxide@Au) toward novel functional biomaterials.14 This method has been applied to separate the protein capped with BSA-capped nanoparticles and bioassay of gold-coated iron oxide core@shell nanoparticles. The further application of Fe2O3/Au core/shell magnetic nanoparticles used in bioseparation would be exploited. Immunoassay is a common and useful means of biochemical analysis.15,16 The strong, specific binding of an antibody to its antigen has been widely exploited in biochemical studies, clinical diagnostics, sensor design, and environmental monitoring. In the past years, many different approaches such as fluorescence,17 chemiluminescence,18 electrochemical detection,19 enzymatic,20 and surface plasmon resonance (SPR)21 have been developed for a direct measurement of antigen-antibody binding. Of these techniques, the application of surface-enhanced Raman spectroscopy (SERS) in immunoassay is an important aspect. SERS has (13) Lyon, J. L.; Fleming, D. A.; Stone, M. B.; Schiffer, P.; Willians, M. E. Nano Lett. 2004, 4, 719. (14) Park, H. Y.; Schadt, M. J.; Wang, L.; Lim, I-Im. S.; Njoki, P. N.; Kim, S. H.; Jang, M. Y.; Luo, J.; Zhong, C. J. Langmuir 2007, 23, 9050. (15) Kanda, V.; Kariuki, J. K.; Harrison, D. J.; McDermott, M. T. Anal. Chem. 2004, 76, 7257. (16) Cui, Y.; Ren, B.; Yao, J. L.; Gu, R. A.; Tian, Z. Q. J. Phys. Chem. B 2006, 110, 4002. (17) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013. (18) Yakovleva, J.; Davidsson, R.; Lobanova, A.; Bengtsson, M.; Eremin, S.; Laurell, T.; Emneus, J. Anal. Chem. 2002, 74, 2994. (19) Duan, C.; Meyerhoff, M. E. Anal. Chem. 1994, 66, 1369. (20) Gosling, J. P. Clin. Chem. 1990, 36, 1408. (21) Oh, B. K.; Kim, Y. K.; Lee, W.; Bae, Y. M.; Lee, W. H.; Choi, J. W. Biosens. Bioelectron. 2003, 18, 605.

Published on Web 06/24/2009

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Article Scheme 1. Schematic Illustration of SERS Immunoassay

demonstrated its ability to characterize the process occurring on metal surfaces on the molecular level.22,23 Among the metallic substrates used for surface-enhanced Raman scattering are metal nanoparticles (e.g., gold and silver colloids). Detection by SERS has several potentially valuable attributes with respect to the noted signal transduction methods.24 First, excitation in the red spectral region is used for Au nanoparticles, which can minimize possible interference from native fluorescence. Second, Raman processes are less susceptible to photobleaching, enabling the use of signal averaging to increase sensitivity and lower detection levels. Finally, the widths of Raman spectral bands are 10-100 times narrower than those of fluorescence bands, which reduces the potential for spectral overlap from multiple labels and thus facilitates multiplexed applications. As illustrated in Scheme 1, SERS immunoassay generally has three steps.25 It involves the immobilization of capture antibodies on a substrate, the use of the immobilized antibodies to capture antigens from solution, and indirect Raman detection via gold nanoparticles labeled with both antibodies and intrinsically strong Raman scatterers (i.e., Raman reporter molecules). The antigen is therefore selectively sandwiched between a substrate and gold nanoparticles by the capture and labeling antibodies. This technique has been applied in immunoassay with limited success due to the low level of detection sensitivity.26,27 In this Article, Fe2O3/Au core/shell nanoparticles with different Au shell thicknesses were prepared by reducing HAuCl4 on the surface of γ-Fe2O3 nanoparticles. The size and morphology properties of these nanoparticles were characterized by scanning electron microscopy (SEM). The SERS activities of these nanoparticles have been tested by using pyridine (Py) as a probe molecule. The antigens in test solution were separated by Fe2O3/ Au magnetic nanoparticles capped with antibody expediently, and the separation efficiency was detected by immunoassay analysis based on SERS.

Experimental Section Chemicals. Ferrous chloride (FeCl2 3 4H2O), iron(III) chloride hexahydrate (FeCl3 3 6H2O), sodium hydroxide (NaOH), hydrochloric acid (HCl), hydroxylamine hydrochloride (NH2OH 3 HCl), trisodium citrate (Na3C6H5O7 3 2H2O), chloroauric acid (HAuCl4 3 4H2O), Tween 80, tris(hydroxymethyl)aminomethane (Tris), 4-mercaptobenzoic acid (MBA), and pyridine (Py) were purchased from Sinopharm Chemical Reagent Co., Ltd. Goat anti-mouse IgG, goat anti-human IgG, human IgG, and mouse IgG were obtained from Sino-American Biotechnology Co., Ltd. Bovine serum albumin (BSA) was acquired from Sigma. Ultrapure water with a conductivity of 18 MΩ cm-1 was used in all experiments. (22) Cao, Y. C.; Jin, R.; Mirkin, C. A. Science 2002, 297, 1536. (23) Nie, S.; Steven, R. E. Science 1997, 275, 1102. (24) Porter, M. D.; Lipert, R. J.; Siperko, L. M.; Wang, G.; Narayanana, R. Chem. Soc. Rev. 2008, 37, 1001. (25) Ni, J.; Lipert, R. J.; Dawson, G. B.; Porter, M. D. Anal. Chem. 1999, 71, 4903. (26) Grubisha, D. S.; Lipert, R. J.; Park, Y.; Driskell, J.; Porter, M. D. Anal. Chem. 2003, 75, 5936. (27) Narayanan, R.; Lipert, R. J.; Porter, M. D. Anal. Chem. 2008, 80, 2265.

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The following buffer solutions were used: borate buffer (BB, 2 mM, pH = 9), PBS buffer (KH2PO4/K2HPO4, 150 mM NaCl, pH = 7.6), TBS buffer (10 mM Tris, 150 mM NaCl, pH = 7-8), and TBS/0.1% Tween buffer (10 M Tris, 150 mM NaCl, 0.1% Tween, pH = 7-8). Synthesis and Preparation. Fe2O3 (γ-Fe2O3) was prepared by oxidizing Fe3O4 nanoparticles in the aqueous phase.28 A total of 2.6 g of FeCl3 and 1.0 g of FeCl2 was successively dissolved in a solution of 0.43 mL of concentrated HCl (12 M) in 12.5 mL of H2O. The solution was added dropwise into 125 mL of a 1.5 M NaOH solution with vigorous stirring. The generated black precipitate was collected on a magnet, and the supernatant was removed from the precipitate by decantation. After being washed three times with water, 250 mL of a 0.01 M HCl solution was added to the precipitate to neutralize the anionic charges on the nanoparticles. The resulting colloidal was again isolated by the magnet and washed twice by water. The fresh Fe3O4 nanoparticles were dissolved in 0.01 M HNO3 and heated with stirring at 90-100 °C for 1 h to completely oxidize the particles to γ-Fe2O3. The solution was cooled to room temperature and washed twice with water. The oxidized γ-Fe2O3 remain stable for several months. Fe2O3/Au core/shell nanoparticles were synthesized by deposition of Au on the preformed Fe2O3 nanoparticles using a modification of Lyon’s iterative hydroxylamine seeding procedure.13 First, The Fe2O3 colloidal was diluted to 1.1 mM in water, and 1 mL was stirred with 1 mL of 0.1 M sodium citrate for 10 min. Next, the solution was diluted with H2O to 20 mL, and 0.1 mL of NH2OH 3 HCl solution of 80 mmol/L was added. Then, 1% HAuCl4 was incrementally added dropwise upon stirring each for 2 mL. Three additions (each for NH2OH 3 HCl and HAuCl4) were totally performed during the reaction, and the stirring continued for at least 50 min after each addition. The clear yellow solution became blue at first addition and changed to garnet after three iterative additions.

Preparation of Immuno-Fe2O3/Au Nanoparticles for Bioseparation. The resultant Fe2O3/Au nanoparticles were separated from the solution by centrifugation and resuspended with 2.0 mL of borate buffer. Next, 10 μL of 3.34 mg/mL goat antihuman IgG was added to the above nanoparticles and incubated at room temperature for 1 h. After being purified by centrifugation and resuspended with 2.0 mL of borate buffer, 20 μL of BSA (5%) was added to the immuno-Fe2O3/Au nanoparticles to block active sites between antibodies. The solution was incubated for 1 h at room temperature and then again centrifugated and resuspended in 2.0 mL of borate buffer. The resulting immuno-Fe2O3/ Au nanoparticles were stored at 4 °C.

Preparation of Raman Reporter-Labeled Immunogold Nanoparticles. The Au nanoparticles were prepared according

to Frens’ method.29 In a typical process, 100 mL of 0.01% HAuCl4 aqueous solution was heated to boiling with vigorous stirring, to which 1 mL of a 1% trisodium citrate solution was added. The mixture was then kept boiling for 30 min. Afterward, the solution was allowed to cool to room temperature with continuous stirring. The resulting red Au colloidal was about 30 nm in diameter. (28) Kang, Y. S.; Risbud, S.; Rabolt, J. F.; Stroeve, P. Chem. Mater. 1996, 8, 2209. (29) Frens, G. Nat. Phys. Sci. 1973, 241, 20.

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Article The Raman reporter-labeled immunoassay Au nanoparticles were prepared by following a procedure reported by Ni et al. with a slight modification.25 A total of 2.5 μL of 1 mM probe molecule (MBA) in ethanol was added to 1.0 mL of Au nanoparticles, and the resultant mixture was allowed to shake for 1 h. The amount of MBA, based on an estimation of the colloidal surface area, was chosen to coat only a portion of the colloidal surface. The reporter-labeled nanoparticles were then separated from the solution by centrifugation at 5000g for 10 min and resuspended with 1.0 mL of borate buffer. Next, 5 μL of 3.34 mg/mL antibody (goat anti-human or goat anti-mouse IgG) was added to 1.0 mL of MBA-labeled Au nanoparticles with gentle agitation. This amount of antibody was ∼50% more than the minimum amount for coating the unmodified portion of the colloidal gold surface. After incubation at room temperature for 1 h, the MBA-labeled immunogold nanoparticles were purified by centrifugation and resuspended with 1.0 mL of borate buffer. Then 10 μL of BSA (5%) was added to the above MBA-labeled immunogold nanoparticles to make sure that no bare sites on Au nanoparticles were left. The mixture was incubated for 1 h at room temperature and then centrifugated and resuspended in 1.0 mL of borate buffer. Preparetion of Capture Antibody Substrates. The substrates were microscopic glass slides coated with multiple layers of materials as described below and were donated by Full Moon BioSystems. The slide surface was first coated with a buffer layer of Ni-Cr using a vacuum deposition process and then coated with a thin layer of silver. After being activated, the surface was covered with a polymer layer, which contains specifically designed functional groups (-COOH) that can bind to the -NH2 groups of antibodies. This particular binding arrangement allows antibodies to be erected on the surface without compromising their biological activities. Next, 50 μL of 50 μg/mL antibody (goat anti-human or goat anti-mouse IgG) was dropped onto the 1 cm2 substrate. After being placed in a chamber with a relative humidity of 65-75% for over 12 h, the substrates were allowed to dry at room temperature for 30 min. The substrates were then incubated in 5% BSA for 1 h to block active sites between antibodies, rinsed with water, and dried under nitrogen. Immunoassay Protocol. The immunoassays were conducted following the typical procedure for a sandwich-type assay. Mouse IgG and human IgG were used as the test antigens. In each case, the capture antibody-coated substrate was immersed in the solution to be tested. After the tube had been gently shaken at room temperature for about 2-4 h on a shaker, the substrates were taken out and washed three times with TBS/0.1% Tween at room temperature and then washed two times with TBS. After being rinsed with copious amounts of water, the substrates were placed in a tube containing reporter-labeled immunogold nanoparticles. The tube was gently shaken at room temperature for 2 h, and then all of the samples were rinsed with TBS/0.1% Tween, TBS, and ultrapure water successively and dried under nitrogen. Measurements and Instrumentation. The study of the binding between test antigen solution and Fe2O3/Au nanoparticles fixed with goat anti-human IgG was carried out by mixing them. The test antigen solution of 1 mL was prepared by mixed 1 μg/mL of different IgG (mouse IgG and human IgG) with same volume. The immuno-Fe2O3/Au nanoparticles were then mixed with the test antigen solution at room temperature for 2 h. Each time, about 0.2 mL of upper solution was taken out for immunoassay after using a NdFeB magnet to collect the reaction product for different times. Raman spectra were obtained using a confocal microprobe Raman system (HR800, Jobin Yvon). It is a single spectrograph instrument equipped with a holographic notch filter and a CCD detector. The size of the silt and pinhole were 100 and 400 μm, respectively. A long working distance 50 objective was used to collect the Raman scattering signal. The excitation wavelength was 632.8 nm from a He-Ne laser, and the greatest laser power 10784 DOI: 10.1021/la901337r

Bao et al. was 10 mW. Scanning electron microscopy (SEM) was conducted with a field-emission microscope (Leo1530) operated at an accelerating voltage of 20 kV.

Results and Discussion Morphological Characterization of Fe2O3/Au Core/Shell Nanoparticles. SEM images of γ-Fe2O3 nanoparticles and Fe2O3/Au core/shell nanoparticles prepared from the first, second, and third iterative additions of HAuCl4 are shown in Figure 1, and the insets are the corresponding magnified SEM images. Figure 1A gives a SEM image of the Fe2O3 nanoparticles with a mean diameter of 10 ( 2 nm. Because the partially oxidized Fe3O4 nanoparticles are more resistant to Au deposition than γ-Fe2O3 nanoparticles, the surfaces of the Fe2O3/Au nanoparticles are jagged after the initial addition of HAuCl4 and NH2OH 3 HCl to the γ-Fe2O3 solution and become more spherical after subsequent iterations.13 The measured average diameters of Fe2O3/Au nanoparticles for Figure 1B-D are about 148 ( 16 nm, 154 ( 19 nm, and 163 ( 11 nm, respectively. The Fe2O3/Au nanoparticles prepared for the first iteration show a very jagged appearance originally from the Au3+ initially reduced onto specific sites of the Fe oxide core surface (Figure 1B). Then, the second iterative addition of HAuCl4 filled the empty surface sites with Au, so the prepared Fe2O3/Au nanoparticles become more spherical as shown in Figure 1C. When the iterative additions of HAuCl4 increased to three times, the prepared Fe2O3/Au nanoparticles showed nearly spherical particles from the SEM image in Figure 1D; the Fe2O3 core has been covered by Au. From Figure 1, we can also see that no small particles were observed in these SEM images, indicating that there are no Au nanoparticles in the resulting solution to influence the next step. SERS Activity of Magnetic Fe2O3/Au Core/Shell Nanoparticles. Because the Fe2O3/Au core/shell nanoparticles have the core Fe2O3 with magnetic properties and the shell Au is a wellknown SERS active material, the Fe2O3/Au nanoparticles would be collected by magnet and also have SERS activity. To analyze both of these characteristics, we put the nanoparticles in a vial, a magnet was placed under the vial to collect the particles, and then in situ SERS activity of the solution at the position of onto the magnet was detected (Scheme 2). In this experiment, Py was used as the probe molecule, because of its well-established Raman spectral data and large

Figure 1. SEM images γ-Fe2O3 nanoparticles (A) and Fe2O3/Au core/shell nanoparticles prepared from the first (B), second (C), and third (D) iterative additions of HAuCl4. Langmuir 2009, 25(18), 10782–10787

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Figure 2. In situ SERS spectra of Py adsorbed on Fe2O3/Au core/ shell nanoparticles prepared from one incremental addition of Au3+ depending on collecting time: (a) 0 min, (b) 10 min, (c) 20 min, and (d) 30 min.

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Figure 3. SERS spectra of Py absorbed on pure γ-Fe2O3 (a) and Fe2O3/Au core/shell nanoparticles for Au iterative addition numbers 1-3 (b-d).

Scheme 2. Schematic Illustration of In Situ SERS Detection of Py Absorbed on Magnetic Fe2O3/Au Core/Shell Nanoparticles

Scheme 3. Illustration of the Separation of Two Different Antigens with Fe2O3/Au Magnetic Nanoparticles (A) and the Detection of the Separation Efficiency with SERS (B)

Figure 4. SERS spectra of immunoassay for human IgG of various concentrations.

Raman scattering cross section.30 Figure 2 displays the in situ SERS spectra for 1  10-5 M Py adsorbed on Fe2O3/Au core/ shell nanoparticles prepared from one incremental addition of Au3+ depending on collecting time. From Figure 2, we can find that the SERS signal intensity is dependent on the collecting time by the magnet: When the magnet was not placed, there were no obvious peaks on the SERS spectra (a); as the external magnetic field was put on for 10 min, the SERS spectra (b) showed clearly a peak at 1012 cm-1 corresponding to ring breathing (v1) vibration modes of Py adsorbed on Au, which is characteristic of the expected signature.31 The SERS signal became stronger with the increase of the collecting time (c), and the intensity stabilized at about 30 min of collection time (d). The above observation demonstrates that Fe2O3 core nanoparticles were covered by Au and the label molecule can be immobilized on the Au shell of the Fe2O3/Au nanoparticles. To further study the influence of Au (30) Huang, Q. J.; Lin, X. F.; Yang, Z. L.; Hu, J. W.; Tian, Z. Q. J. Electroanal. Chem. 2004, 563, 121. (31) Wu, D. Y.; Ren, B.; Jiang, Y. X.; Xu, X.; Tian, Z. Q. J. Phys. Chem. A 2002, 106, 9042.

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Figure 5. SERS immunoassay for products separated by Fe2O3/ Au nanoparticles capped with goat anti-human IgG with different times: (a) 0 h, (b) 6 h, (c) 12 h, and (d) 24 h. A magnet was used to collect the particles. DOI: 10.1021/la901337r

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Figure 6. SERS spectra of products separated by magnet of different times to the test solution which contains two components of antigen for the Fe2O3/Au nanoparticles and detected by MBA-labeled immunogold nanoparticles: (A) human IgG and (B) mouse IgG.

ratio on the SERS signal intensity of Py adsorbed on Fe2O3/Au nanoparticles, SERS spectra of as-prepared Fe2O3/Au nanoparticles collected with 30 min for each iterative Au deposition are shown in Figure 3. As can been seen in Figure 3, the SERS spectra of pure γ-Fe2O3 did not have any characteristic peak of Py. And the stronger SERS signal can be obtained as the ratio of Au to Fe oxide increased; this would be attributed to that Au as a SERS active metal becomes thicker with increasing iterative addition numbers. Bioseparation Using the Fe2O3/Au Core/Shell Magnetic Nanoparticles. For the Fe2O3/Au core/shell nanoparticles used for bioseparation, both the magnetic properties of the core and the IgG binding properties of the gold shell in the Fe2O3/Au nanoparticles are important. The test result of the preceding paragraph demonstrated clearly that the Fe2O3/Au nanoparticles are magnetically active, which is desired for the bioseparation application. We chose the Fe2O3/Au nanoparticles prepared from one incremental addition of Au3+ in the bioseparation experiment for two reasons: one is that the thicker Au shell would weaken the magnetic of Fe2O3/Au nanoparticles, and the other is that the nanoparticles prepared from one incremental addition of Au3+ already have high SERS intensity demonstrating that Fe2O3 core nanoparticles are covered by Au and the antibodies can be immobilized the Fe2O3/Au nanoparticles. Scheme 3 illustrates the process of separating two different antigens with Fe2O3/ Au magnetic nanoparticles and detecting the separation efficiency with SERS. The preparation of immuno-Fe2O3/Au nanoparticles fixed with antibody was introduced in the Experimental Section. After the immuno-Fe2O3/Au nanoparticles (e.g., fixed with goat antihuman IgG) were immersed in the test antigen solution, the corresponding specific antigen (human IgG) would be immobilized on them owing to that antibody molecules interact highly specifically with their corresponding antigens. This provided an effective means for the separation of the test antigen solution via application of a magnetic field. And the next stage is a detection step for the specific antigen (human IgG) in the product after the separation to demonstrate the efficiency of this separation method. The resulting solution was reacted with Au nanoparticles capped with both antibody (goat anti-human IgG) and a Raman 10786 DOI: 10.1021/la901337r

label (e.g., MBA (Experimental Section)). If there is any remaining antigen (human IgG) in the solution, the antigen would be therefore selectively sandwiched between gold nanoparticles and substrate by the capture and labeling antibody. This sandwich assay format (a) as an immunoassay readout method based on SERS has been demonstrated to detect antigen at very low concentration.25 The detection limit of this approach would be affected by many factors (capture substrates, extrinsic Raman labels, size and shape of nanoparticles, etc.),24 and our system about the immunoassay has been introduced in detail in the Experimental Section. The results of our SERS-based detections for human IgG are shown in Figure 4. Test solutions were made by serial dilution of a 1 μg/mL human IgG standard to cover the range from 1 fg/mL to 10 pg/mL. The spectra in Figure 4 were obtained after completion of the immunoassay protocol outlined above. As is evident, the features diagnostic of the MBAlabeled nanoparticles exhibit a strong increase as the IgG level increases. The detection limit of antigen was down to 1 fg/mL, thus encompassing the concentration critical to antigen in the test solution. Figure 5 shows a set of SERS spectra of products separated by applying a magnetic field of different times to the test solution for the Fe2O3/Au nanoparticles capped with goat anti-human IgG and detected by MBA-labeled immunogold nanoparticles. The test solution contains 1 μg/mL human IgG diluted with PBS buffer solution. The SERS spectrum of the original test solution detected by MBA-labeled immunogold nanoparticles showed mainly two peaks at about 1081 and 1594 cm-1, which correspond to v8a and v12 aromatic ring vibrations of MBA, respectively.32 After the test solution reacted with immuno-Fe2O3/Au nanoparticles and was collected by using a magnet for 6 h, the test antigen in the solution significantly reduced, which can be obtained from the decrease of SERS signal intensity in Figure 5b. After 12 h separation, there is only a little test antigen in solution as seen from the weak SERS signal detected by labeled immunogold nanoparticles. And there was no obvious peak in the SERS spectra after 24 h separation. According to the detection limit of antigen mentioned above, the amount of test antigen that (32) Michota, A.; Bukowska, J. J. Raman Spectrosc. 2003, 34, 21.

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remained in solution after 24 h bioseparation is less than 1 fg/mL. Therefore, in our experiment, 24 h bioseparation for the test solution can almost completely separate the test antigen from the solution. This demonstrates the viability of exploiting Fe2O3/Au nanoparticles for magnetic bioseparation of a single type of antigen from a test solution. However, there are generally multiple antigens in solution to be tested such as blood or other proteins. In order to see whether other components will interfere with this bioseparation method, the solution containing two components of antigen would be separated by Fe2O3/Au magnetic nanoparticles. Figure 6 displays the SERS spectra of products separated by magnet of different times to the test solution for the Fe2O3/Au nanoparticles and detected by MBA-labeled immunogold nanoparticles. The immuno-Fe2O3/Au nanoparticles were capped with goat antihuman IgG, and the test solution was made by mixing 1 μg/mL human IgG and 1 μg/mL mouse IgG. As can be seen in Figure 6A, the SERS signal decreased with the increase of separation time, indicating that human IgG remaining in solution gradually reduced. The similar good separation efficiency of human IgG in the two component antigen solution suggested that the other components did not interfere with the bioseparation course. The other antigen (mouse IgG) in the remaining solution, for which the corresponding capture antibody did not assemble on the Fe2O3/Au nanoparticles, was also detected by MBA-labeled immunogold nanoparticles (B). Comparison of the SERS signal detected before and after the bioseparation showed only a slight decline of mouse IgG in the remaining solution, indicating the scarce influence of immunoFe2O3/Au nanoparticles on other antigens in solution. And the slight decline may be due to the nonspecific adsorption of immuno-Fe2O3/Au nanoparticles.25 The results above clearly demonstrate the effective separation of a multiple antigen solution by Fe2O3/Au magnetic nanoparticles.

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Conclusions An effective bioseparation method based on Fe2O3/Au magnetic nanoparticles with ease of operation, good separation efficiency, and rapid detection has been introduced in this paper. Fe2O3/Au core/shell nanoparticles with different Au ratios were prepared by reducing HAuCl4 on the surface of γ-Fe2O3 nanoparticles. We have confirmed that the surfaces of Fe2O3 nanoparticles have been covered by Au with SEM images and SERS; the SERS intensity of probe molecules as Py absorbed on Fe2O3/ Au nanoparticles enhanced with the increase of iterative additions of HAuCl4. A single type of antigen and multiple types of antigens in test solutions both have been separated by magnetic Fe2O3/Au core/shell nanoparticles. The remaining antigens in solution have been assayed by the reaction between MBA-labeled immunogold nanoparticles and substrates captured by corresponding antibodies. The result of immunoassay detected by SERS demonstrated that the magnetic bioseparation program used by this Fe2O3/Au core/shell nanoparticles could separate almost all of the antigens in the test solution. The high separation efficiency of this effective method has shown a potential application for magnetic Fe2O3/Au core/shell nanoparticles in bioseparation. And trying to quantify the separation efficiency is part of our ongoing work. Acknowledgment. We gratefully acknowledge the support from the Natural Science Foundation of China (NSFC) (20573076, 20503019, 20773091), the NSF of Jiangsu Province (BK2005032), and the Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP) (20050285019). J.-L.Y. is thankful for the support from the Program of Innovative Research Team of Suzhou University. We thank Full Moon BioSystems Inc. (www.Fullmoonbiosystems.com, Sunnyvale, CA 94085) for the kind donation of the substrates for our experiment. We also thank Dr. P. G. Cao for amendment of the article.

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