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Magnetic assistance highly sensitive protein assay based on surface-enhanced resonance Raman scattering
Journal of Colloid and Interface Science 368 (2012) 282–286
Contents lists available at SciVerse ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Magnetic assistance highly sensitive protein assay based on surface-enhanced resonance Raman scattering Lei Chen a,b, Wonjin Hong b, Zhinan Guo a, Youngjo Sa b, Xu Wang a, Young Mee Jung b,⇑, Bing Zhao a,⇑ a b
State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun 130012, PR China Department of Chemistry, Institute for Molecular Science and Fusion Technology, Kangwon National University, Chunchon 200-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 28 September 2011 Accepted 28 October 2011 Available online 6 November 2011 Keywords: SERS AgMNPs Avidin Protein detection
a b s t r a c t A simple and effective surface-enhanced Raman scattering (SERS)-based protocol for the detection of protein–small molecule interactions has been developed. We employed silver-coated magnetic particles (AgMNPs), which can provide high SERS activity as a protein carrier to capture a small molecule. Combining magnetic separation and the SERS method for protein detection, highly reproducible SERS spectra of a protein–small molecule complex can be obtained with high sensitivity. This time-saving method employs an external magnetic field to induce the AgMNPs to aggregate to increase the amount of atto610-biotin/ avidin complex in a unit area with the SERS enhancement. Because of the contribution of the AgMNP aggregation to the SERS, this protocol has great potential for practical high-throughput detection of the protein–small molecule complex and the antigen–antibody immunocomplex. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Surface-enhanced Raman scattering (SERS) and surface-enhanced resonance Raman scattering (SERRS) have potential for the highly selective and sensitive detection of molecules, including single molecules. SERS and SERRS have recently attained considerable prominence as powerful analytical techniques for the detection of biomaterials such as proteins, DNA, and cells [1–5]. Applications of protein detection based on SERS and SERRS can be divided into two major detection modes: directly detected target protein signal (label-free detection protocol) and indirectly employed Raman-label compound detection (label detection protocol). Label detection protocols have been used widely because of the high Raman scattering cross-sections for dye molecules. Raman-label compounds provide much greater enhancement to the SERS spectra than direct detection methods. This enhancement is important for achieving simple, multiple, and sensitive signals in protein detection based on SERS spectra [6–9]. Many SERS-based label protocols have been previously proposed, such as Coomassie Brilliant Blue (CBB) as a SERS probe for protein concentration evaluations [10], SERS-based immunoassay with probe-labeling immunogold nanoparticles [11], and SERS markers embedded into biocompatible materials (e.g., polyelectrolytes and silicon dioxide) for multiplex immunoassay [6,12]. However, most of the previous
⇑ Corresponding authors. E-mail addresses: [email protected] (Y.M. Jung), [email protected] (B. Zhao). 0021-9797/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2011.10.069
methods have focused on the introduction of different kinds of labeling methods for ultrasensitive protein detection. Magnetic nanoparticles have been widely used in protein separation and purification, drug delivery, and medical imaging because of the potential benefits for biomedical applications [13–15]. Silica-coated, iron-oxide-based magnetic nanoparticles are of particular interest because silica-rich surfaces can easily react with various coupling agents to covalently attach specific ligands. Silica-coated magnetic particles combined with different nanoscale materials may lead to the development of multifunctional nano-assembled systems that simultaneously exhibit novel optical, electronic, and magnetic properties. Magnetic nanoparticles that exhibit superparamagnetism can provide a large surface area. More importantly, these magnetic nanoparticles have no magnetic memory and can be easily redispersed by removal of the external magnetic field [16–20]. Magnetic nanoparticles coated with Ag or Au have been applied to protein detection and analysis based on SERS [21–24]. In SERSbased immunoassays, antigen-capped gold nanoparticles were generally labeled with Raman-active molecules such as mercaptobenzoic acid, 4-aminothiophenol, 4-methylbenzenethiol, or benzenethiol. These labeled nanoparticles can be specifically recognized using antibodies conjugated to gold-coated magneticbead nanoparticles [22–24]. In this paper, we introduce silver-coated magnetic nanoparticles (AgMNPs) that exhibited strong magnetic properties as well as high SERS activity in protein assays. High-quality SERS (SERRS) spectra with both high sensitivity and reproducibility were obtained with these particles. Quality, sensitivity, and reproducibility
L. Chen et al. / Journal of Colloid and Interface Science 368 (2012) 282–286
are crucial for any analytical method. We demonstrate that with the use of the magnetic nanoparticles, the avidin–biotin complex separation and SERS assay can be finished in minutes. By decreasing the amount of magnetic nanoparticles and by increasing the incubation time for proteins and small molecules, the SERS intensity of atto610 was increased. Employing the optimal conditions, we could ultimately detect avidin at very low levels (5 pg/mL) using the proposed method.
283
(XRD) using a Siemens D5005 X-ray powder diffractometer with a Cu Ka radiation source at 40 kV and 30 mA was used. X-ray photoelectron spectroscopy (XPS) was performed on an ESCLAB MKII using Al as the excitation source. Magnetization measurements as a function of the applied field at room temperature were performed in a Quantum Design Magnetic Properties Measurement System. 2.6. SERS measurements
2. Experimental section 2.1. Materials Ferric chloride (FeCl36H2O), sodium acetate, ethylene glycol, tetrakis(hydroxymethyl)phosphonium chloride (THPC), tetraethylorthosilicate (TEOS), ammonium hydroxide, gold chloride hydrate, avidin, atto610-biotin (MW = 801.43 g/mol), bovine serum albumin (BSA), silver enhancer kit, solution A and B, (3-aminopropyl)trimethoxysilane (APTMS), and poly(diallyldimethylammonium chloride) (PDDA) were purchased from Sigma–Aldrich at the highest purity available and used as received without further purification. Ultrapure water (18 MX cm 1) was used throughout the present study. 2.2. Preparation of Fe3O4, core–shell Fe3O4@SiO2, and AgMNPs The Fe3O4 and Fe3O4@SiO2 nanoparticles were prepared according to a previously reported method [25]. The magnetic Fe3O4@SiO2 nanoparticles were dispersed in 1% APTMS solution and stirred for 24 h. The APTMS-functionalized magnetic Fe3O4@SiO2 nanoparticles were magnetically separated from the mixture solution by the use of a magnet and were then washed with ethanol and deionized water. These APTMS-functionalized magnetic Fe3O4@SiO2 nanoparticles were then functionalized again with Au by dispersion in the Au seed solution. The Fe3O4@SiO2@Au-seed nanoparticles were separated and washed with water. Finally, to coat silver onto the Fe3O4@SiO2@Au-seed nanoparticles, silver enhancer kit of A (0.5 mL, 25% in distilled water) and B (0.5 mL, 25% in distilled water) were added to the 1 mL of Fe3O4@SiO2@Au-seed solution (containing 1 mg of Fe3O4 particles). 2.3. Buffer solutions Phosphate-buffered saline (PBS; 0.01 M, pH 7.0) was used as the basis of the blocking buffer in this study. The blocking buffer solution was prepared by dissolving BSA in the PBS buffer (1% BSA) solution. 2.4. Atto610-complex deposition on AgMNPs Avidin (from 10 ng/mL to 5 pg/mL in PBS buffer solution) was immobilized on the AgMNP substrate by immersing the substrate in an avidin solution for one hour at 37 °C. After being rinsed with the PBS buffer solution, the avidin-coated AgMNPs were soaked in a blocking buffer solution. The particles were then immersed in atto610-biotin (1 lg/mL in PBS buffer) solution for one hour at 37 °C.
SERS spectra were measured with a Jobin Yvon/HORIBA LabRam ARAMIS Raman spectrometer equipped with an integral BX 41 confocal microscope. The radiation from the air-cooled HeNe laser (633 nm) was used as an excitation source. Raman scattering was detected with 180° geometry using a multichannel air-cooled ( 70 °C) charge-coupled device (CCD) camera (1024 256 pixels). 3. Results and discussion The fabrication process for the proposed AgMNP is shown in Scheme 1. Magnetic nanoparticles were characterized at each step using TEM, XRD, XPS, magnetization measurements, and UV–Vis absorption spectroscopy. Fig. 1A and B show TEM images of Fe3O4@SiO2 nanoparticles, which have a relatively smooth surface with diameters of approximately 158 ± 10 nm with a magnetic core of 150 ± 10 nm. The surface is covered with 8 nm of SiO2. The silica-coated magnetic particles are stable, possess a large surface area, and easily react with various coupling agents to covalently attach specific ligands for silver coating. The silica-coated magnetic particles were functionalized by APTMS, and these APTMS-functionalized magnetic Fe3O4@SiO2 nanoparticles were again functionalized with 2–3 nm gold nanoparticles, which acted as autocatalytic and nucleation sites for silver reduction. Fig. 1C shows the TEM image of Fe3O4@SiO2@Au-seed nanoparticles. The silver particles were finally decorated onto the Fe3O4@SiO2@Au-seed nanoparticles by the reduction of silver ions using hydroquinone at room temperature. The obtained Ag shell nanoparticles on Fe3O4 were used as the SERS substrate. As shown in Fig. 1D, the TEM image of the AgMNPs illustrates the rough features of the surface, which were due to the silver coated on the surface of the magnetic Fe3O4@SiO2 nanoparticles. Powder XRD was utilized to determine the crystalline structure of the AgMNPs, Fe3O4@SiO2, and Fe3O4 particles. As shown in Fig. 2c, the peak position and relative intensity of all diffraction peaks for the Fe3O4 particles are in good agreement with the results of a previous study [26]. The sharp and strong peaks confirmed that the products were well crystallized. New peaks at 2h = 38.1°, 44.4°, 64.4°, and 77.4° are shown in Fig. 2a. These peaks are indexed to the (1 1 1), (2 0 0), (2 2 0), and (3 1 1) planes of the fcc structures of silver [27]. The XPS analysis gives some insight into the chemical composition of the composite microspheres of the AgMNP, Fe3O4@SiO2, and Fe3O4 particles. Fig. 3 shows the Fe 2p, O 1s, Si 2p, and Ag 3d XPS spectra of the particles (AgMNPs, Fe3O4@SiO2, and Fe3O4) that were obtained. These spectra clearly show the presence of Fe3O4 and the
2.5. Characterization of substrates The UV–Vis spectra of Fe3O4@SiO2 and AgMNP were recorded on a UV-3600 spectrophotometer (Shimadzu) and characterized by transmission electron microscopy (TEM). TEM images of Fe3O4@SiO2, Fe3O4@SiO2@Au-seed, and AgMNP were obtained with a Hitachi H-8100 microscope operated at 200 kV. X-ray diffraction
Scheme 1. The schematic of preparation of AgMNPs.
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L. Chen et al. / Journal of Colloid and Interface Science 368 (2012) 282–286
Fig. 1. TEM images of Fe3O4@SiO2 nanoparticles at (A) lower and (B) higher magnification, (C) gold-seed-decorated Fe3O4@SiO2 nanoparticles at higher magnification, and (D) AgMNPs.
Fig. 2. XRD patterns of AgMNPs (a), Fe3O4@SiO2 (b), and Fe3O4 (c) particles.
surface covered with SiO2 and Ag, which supports the results from the TEM images. Fig. 4 shows the magnetization curves measured at room temperature for the Fe3O4, Fe3O4@SiO2, and AgMNP particles. The curve presents a hysteresis loop, which suggests that all of these particles exhibit ferromagnetic behavior. The magnetic saturation values are 71.9, 53.2, and 36.0 emu/g, respectively, for Fe3O4, Fe3O4@SiO2, and AgMNPs. The decrease in the overall magnetization values indicates that the Fe3O4 surface is covered with nonmagnetic materials such as SiO2 and Ag. The ferromagnetic properties of the magnetic nanoparticles are critical for their applications in the biomedical and bioengineering fields. These ferromagnetic properties prevent aggregation and enable the magnetic nanoparticles to rapidly redisperse when the magnetic field is removed. Fig. 5a and b show the UV–Vis spectra of the Fe3O4@SiO2 and AgMNP, respectively. The absorption at approximately 480 nm is
ascribed to the Fe3O4 nanoparticles themselves, whereas the absorbance at approximately 600 nm is ascribed to the continuous and rough silver particles decorated on the magnetic Fe3O4@SiO2 nanoparticles [28]. According to the surface plasmon resonance (SPR) spectra of the AgMNPs, a 633-nm laser was used as an excitation wavelength for electromagnetic (EM) enhancement [29]. Magnetic nanoparticles with a particular character have received considerable attention because of their potential applications in high-sensitivity bioassays. The proposed method employed AgMNPs as a protein carrier to capture probe-conjugated small molecules for a highly sensitive protein SERRS assay. The AgMNPs were first coated with avidin (from 10 ng/mL to 5 pg/mL) and then soaked in a blocking buffer solution to block the areas lacking nonspecific avidin adsorption on the proposed substrate. The atto610-labeled biotin solution was then added for specific recognition of avidin. When a magnetic field was applied to a capillary tube filled with the dispersed solutions of the protein-coupled AgMNPs, the AgMNPs were easily and densely aggregated. The protein and small-molecule interactions were finally detected on the proposed AgMNPs using SERRS. In this proposed protocol, a magnet was used as an external magnetic field for AgMNP aggregation to induce a strong SERRS effect for a protein assay. Highly enhanced SERS spectra can be obtained when the protein concentration in a unit area is high. The intensities of the bands in the SERS spectra will thus depend on the amount of AgMNPs added to the analyte solution in the proposed study. We conclude from SI_Fig. 1 that the intensities of the bands in the SERRS spectra of atto610 increased as the amount of the AgMNPs were decreased. The intensities of the bands in the SERRS spectra of the protein-conjugated AgMNPs soaked in atto610-biotin increased with an increase in the reaction time for avidin to capture the biotin (SI_Fig. 2). However, no obvious intensity changes in the SERRS spectra were observed after protein-conjugated AgMNPs were soaked in atto610-biotin solution for 60 min. Fig. 6A shows the SERRS spectra of atto610-biotin/avidin (chemical structure of atto610 is shown in SI_Fig. 3) with different
L. Chen et al. / Journal of Colloid and Interface Science 368 (2012) 282–286
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Fig. 3. XPS spectra of the Fe3O4, Fe3O4@SiO2, and AgMNPs: (A) Fe 2p, (B) O 1s, (C) Si 2p, and (D) Ag 3d.
Fig. 5. UV–Vis absorbance spectra of (a) Fe3O4@SiO2 nanoparticles and (b) AgMNPs. Fig. 4. Hysteresis loops of the Fe3O4, Fe3O4@SiO2, and AgMNPs recorded at room temperature.
concentrations of avidin (from 10 ng/mL to 5 pg/mL). It is noted that SERRS intensities of the label significantly increase with the increase in target protein (avidin). The C@O stretching (amide I band) of avidin or avidin/biotin complex and the aromatic C@C stretching vibration of atto610 contribute to the Raman band at 1634 cm 1 [30–34]. Atto610, with the maximum absorption of 610 nm, belongs to a new generation of fluorescent labels for the red spectral region. In SERS and resonance condition with 633 nm laser excitation, the bands at 1634, 855, and 793 cm 1
can be assigned to vibrational modes of atto610. Concentrationdependent SERRS intensities of atto610 are plotted in Fig. 6B. A strong band at 1634 cm 1, which is assigned to the aromatic C@C stretching vibration of atto610, was used for Gaussian curve fitting based on the relative intensities. Error bars are included to indicate the sample-to-sample variability in the SERRS intensities. Fig. 6B shows the remarkable increase in the SERRS intensities of atto610 over a large range of protein concentrations. We successfully detected very low concentrations of avidin (5 pg/mL) with the proposed AgMNPs. This observation implies that the proposed SERRS-based method with its advantages of fast magnet separation
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L. Chen et al. / Journal of Colloid and Interface Science 368 (2012) 282–286
Fig. 6. (A) SERRS spectra of atto610-biotin/avidin complex with different concentrations of avidin (from 10 ng/mL to 5 pg/mL) and (B) concentration-dependent SERRS intensities at 1634 cm 1.
and detection and high sensitivity can be compatible with highthroughput methods. The external magnetic field, which can induce aggregation of the AgMNPs, tunes the aggregated particles for the maximum SERRS signal intensity. To the best of our knowledge, the formation of two AgMNP aggregates results in higher EM enhancement for atto610. It would therefore be interesting to explore whether the as-prepared hybrid magnetic nanospheres that contain Ag aggregates could be used for the fabrication of substrates for intense SERS. Furthermore, the aggregated nanoparticles can provide more surface area for protein to adsorb than can flat substrates in a given unit area. With these results, we have demonstrated the great potential of this methodology for highly sensitive protein detection. 4. Conclusion In this study, AgMNPs, which can be aggregated by an external magnetic field, were used as protein carriers for SERRS-based highly sensitive protein detection. As protein carriers and SERS substrate, these AgMNPs were much more sensitive than other nanoparticles for protein detection. The most important aspect of this study is the potential applications of AgMNPs for quantitative immunoassays because the magnetic nanoparticles can increase the amount of protein in a given unit area. We successfully detected low concentrations of avidin (5 pg/mL) using the proposed AgMNPs because the external magnetic field induced the aggregation of the AgMNPs. Magnetically assisted protein separation and detection by SERRS will provide an effective and sensitive immunoassay technique. Acknowledgments This study was supported by the National Natural Science Foundation (Grant Nos. 20873050, 20921003, 20973074, 21073072) of PR China; the 111 Project (B06009). This work was also supported by the National Research Foundation of Korea (NRF) Grants funded by the Korea government (MEST) (Nos. 2011-C00052 and 20090087013) and the BK 21 program from the Ministry of Education, Science and Technology of Korea. The authors thank the Central Laboratory of Kangwon National University for the measurements of Raman spectra. Appendix A. Supplementary material SERRS intensities of atto610 (1634, 855, and 793 cm 1) with different amounts of magnetic particles; SERRS intensities of atto610
(1634 cm 1) of the protein-conjugated AgMNPs soaked in atto610biotin with different reaction times for avidin to capture the biotin; Chemical structure of atto610-biotin. Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jcis.2011.10.069. References [1] S.M. Nie, S.R. Emory, Science 275 (1997) 1102. [2] K. Kneipp, Y. Wang, H. Kneipp, L.T. Perelman, I. Itzkan, R.R. Dasari, M.S. Feld, Phys. Rev. Lett. 78 (1997) 1667. [3] X.X. Han, B. Zhao, Y. Ozaki, Anal. Bioanal. Chem. 394 (2009) 1719. [4] L.R. Nathaniel, A.M. Chad, Chem. Rev. 105 (2005) 1547. [5] A.W. Katherine, Anal. Bioanal. Chem. 394 (2009) 85. [6] B.H. Jun, M.S. Noh, J. Kim, G. Kim, H. Kang, M.S. Kim, Y.T. Seo, J. Baek, J.H. Kim, J. Park, S. Kim, Y.K. Kim, T. Hyeon, M.H. Cho, D.H. Jeong, Y.S. Lee, Small 6 (2010) 119. [7] X.X. Han, G.G. Huang, B. Zhao, Y. Ozaki, Anal. Chem. 81 (2009) 3329. [8] G. Wang, H.Y. Park, R.J. Lipert, Anal. Chem. 81 (2009) 9643. [9] L. Chen, X.X. Han, J.X. Yang, J. Zhou, W. Song, B. Zhao, W.Q. Xu, Y. Ozaki, J. Colloid Interface Sci. 360 (2011) 482. [10] X.X. Han, L. Chen, J. Guo, B. Zhao, Y. Ozaki, Anal. Chem. 82 (2010) 4102. [11] S.P. Xu, X.H. Ji, W.Q. Xu, X.L. Li, L.Y. Wang, Y.B. Bai, B. Zhao, Y. Ozaki, Analyst 129 (2004) 63. [12] J.L. Gong, Y. Liang, Y. Huang, J.W. Chen, J.H. Jiang, G.L. Shen, R.Q. Yu, Biosensors Bioelectron. 22 (2007) 1501. [13] S. Laurent, D. Forge, M. Port, A. Roch, C. Robic, L.V. Elst, R.N. Muller, Chem. Rev. 108 (2008) 2064. [14] J.H. Gao, H.W. Gu, B. Xu, Acc. Chem. Res. 42 (2009) 1097. [15] D. Bahadur, J. Giri, Sadhana 28 (2003) 639. [16] Y.H. Deng, C.H. Deng, D.W. Qi, C. Liu, J. Liu, X.M. Zhang, D.Y. Zhao, Adv. Mater. 21 (2009) 1377. [17] X.Q. Xu, C.H. Deng, P.Y. Yang, X.M. Zhang, J. Proteome Res. 6 (2007) 3849. [18] I. Koh, X. Wang, B. Varughese, L. Isaacs, S.H. Ehrman, D.S. English, J. Phys. Chem. B 110 (2006) 1553. [19] T.J. Yoon, K.N. Yu, E. Kim, J.S. Kim, B.G. Kim, S.H. Yun, B.H. Sohn, M.H. Cho, J.K. Lee, S.B. Park, Small 2 (2006) 209. [20] T.R. Sathe, A. Agrawal, S.M. Nie, Anal. Chem. 78 (2006) 5627. [21] P.A. Mosier-Boss, S.H. Lieberman, Anal. Chem. 77 (2005) 1031. [22] B.H. Jun, M.S. Noh, G. Kim, H. Kang, J.H. Kim, W.J. Chung, M.S. Kim, Y.K. Kim, M.H. Cho, D.H. Jeong, Y.S. Lee, Anal. Biochem. 391 (2009) 24. [23] I.I.S. Lim, P.N. Njoki, H.Y. Park, X. Wang, L.Y. Wang, D. Mott, C.J. Zhong, Nanotechnology 19 (2008) 305102. [24] F. Bao, J.L. Yao, R.A. Gu, Langmuir 25 (2009) 10782. [25] Y. Deng, D. Qi, C. Deng, X. Zhang, D. Zhao, J. Am. Chem. Soc. 130 (2008) 28. [26] G.C. Xi, C. Wang, X. Wang, Eur. J. Inorg. Chem. (2008) 425. [27] W. Song, Y.F. Wang, H.L. Hu, B. Zhao, J. Raman Spectrosc. 38 (2007) 1320. [28] S.J. Guo, S.J. Dong, E.K. Wang, Chem. Eur. J. 15 (2009) 2416. [29] H. Wang, C.S. Levin, N.J. Halas, J. Am. Chem. Soc. 127 (2005) 14992. [30] A. Torreggiani, G. Battura, G. Fini, J. Raman Spectrosc. 31 (2000) 445. [31] P. Hildebrandt, M. Stockburger, J. Phys. Chem. 88 (1984) 5935. [32] P. Hildebrandt, M. Stockburger, J. Raman Spectrosc. 17 (1986) 55. [33] X.X. Han, Y. Kitahama, T. Itoh, C.X. Wang, B. Zhao, Y. Ozaki, Anal. Chem. 81 (2009) 3350. [34] X.X. Han, Y. Kitahama, Y. Tanaka, J. Guo, W.Q. Xu, B. Zhao, Y. Ozaki, Anal. Chem. 80 (2008) 6567.
Contents lists available at SciVerse ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Magnetic assistance highly sensitive protein assay based on surface-enhanced resonance Raman scattering Lei Chen a,b, Wonjin Hong b, Zhinan Guo a, Youngjo Sa b, Xu Wang a, Young Mee Jung b,⇑, Bing Zhao a,⇑ a b
State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun 130012, PR China Department of Chemistry, Institute for Molecular Science and Fusion Technology, Kangwon National University, Chunchon 200-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 28 September 2011 Accepted 28 October 2011 Available online 6 November 2011 Keywords: SERS AgMNPs Avidin Protein detection
a b s t r a c t A simple and effective surface-enhanced Raman scattering (SERS)-based protocol for the detection of protein–small molecule interactions has been developed. We employed silver-coated magnetic particles (AgMNPs), which can provide high SERS activity as a protein carrier to capture a small molecule. Combining magnetic separation and the SERS method for protein detection, highly reproducible SERS spectra of a protein–small molecule complex can be obtained with high sensitivity. This time-saving method employs an external magnetic field to induce the AgMNPs to aggregate to increase the amount of atto610-biotin/ avidin complex in a unit area with the SERS enhancement. Because of the contribution of the AgMNP aggregation to the SERS, this protocol has great potential for practical high-throughput detection of the protein–small molecule complex and the antigen–antibody immunocomplex. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Surface-enhanced Raman scattering (SERS) and surface-enhanced resonance Raman scattering (SERRS) have potential for the highly selective and sensitive detection of molecules, including single molecules. SERS and SERRS have recently attained considerable prominence as powerful analytical techniques for the detection of biomaterials such as proteins, DNA, and cells [1–5]. Applications of protein detection based on SERS and SERRS can be divided into two major detection modes: directly detected target protein signal (label-free detection protocol) and indirectly employed Raman-label compound detection (label detection protocol). Label detection protocols have been used widely because of the high Raman scattering cross-sections for dye molecules. Raman-label compounds provide much greater enhancement to the SERS spectra than direct detection methods. This enhancement is important for achieving simple, multiple, and sensitive signals in protein detection based on SERS spectra [6–9]. Many SERS-based label protocols have been previously proposed, such as Coomassie Brilliant Blue (CBB) as a SERS probe for protein concentration evaluations [10], SERS-based immunoassay with probe-labeling immunogold nanoparticles [11], and SERS markers embedded into biocompatible materials (e.g., polyelectrolytes and silicon dioxide) for multiplex immunoassay [6,12]. However, most of the previous
⇑ Corresponding authors. E-mail addresses: [email protected] (Y.M. Jung), [email protected] (B. Zhao). 0021-9797/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2011.10.069
methods have focused on the introduction of different kinds of labeling methods for ultrasensitive protein detection. Magnetic nanoparticles have been widely used in protein separation and purification, drug delivery, and medical imaging because of the potential benefits for biomedical applications [13–15]. Silica-coated, iron-oxide-based magnetic nanoparticles are of particular interest because silica-rich surfaces can easily react with various coupling agents to covalently attach specific ligands. Silica-coated magnetic particles combined with different nanoscale materials may lead to the development of multifunctional nano-assembled systems that simultaneously exhibit novel optical, electronic, and magnetic properties. Magnetic nanoparticles that exhibit superparamagnetism can provide a large surface area. More importantly, these magnetic nanoparticles have no magnetic memory and can be easily redispersed by removal of the external magnetic field [16–20]. Magnetic nanoparticles coated with Ag or Au have been applied to protein detection and analysis based on SERS [21–24]. In SERSbased immunoassays, antigen-capped gold nanoparticles were generally labeled with Raman-active molecules such as mercaptobenzoic acid, 4-aminothiophenol, 4-methylbenzenethiol, or benzenethiol. These labeled nanoparticles can be specifically recognized using antibodies conjugated to gold-coated magneticbead nanoparticles [22–24]. In this paper, we introduce silver-coated magnetic nanoparticles (AgMNPs) that exhibited strong magnetic properties as well as high SERS activity in protein assays. High-quality SERS (SERRS) spectra with both high sensitivity and reproducibility were obtained with these particles. Quality, sensitivity, and reproducibility
L. Chen et al. / Journal of Colloid and Interface Science 368 (2012) 282–286
are crucial for any analytical method. We demonstrate that with the use of the magnetic nanoparticles, the avidin–biotin complex separation and SERS assay can be finished in minutes. By decreasing the amount of magnetic nanoparticles and by increasing the incubation time for proteins and small molecules, the SERS intensity of atto610 was increased. Employing the optimal conditions, we could ultimately detect avidin at very low levels (5 pg/mL) using the proposed method.
283
(XRD) using a Siemens D5005 X-ray powder diffractometer with a Cu Ka radiation source at 40 kV and 30 mA was used. X-ray photoelectron spectroscopy (XPS) was performed on an ESCLAB MKII using Al as the excitation source. Magnetization measurements as a function of the applied field at room temperature were performed in a Quantum Design Magnetic Properties Measurement System. 2.6. SERS measurements
2. Experimental section 2.1. Materials Ferric chloride (FeCl36H2O), sodium acetate, ethylene glycol, tetrakis(hydroxymethyl)phosphonium chloride (THPC), tetraethylorthosilicate (TEOS), ammonium hydroxide, gold chloride hydrate, avidin, atto610-biotin (MW = 801.43 g/mol), bovine serum albumin (BSA), silver enhancer kit, solution A and B, (3-aminopropyl)trimethoxysilane (APTMS), and poly(diallyldimethylammonium chloride) (PDDA) were purchased from Sigma–Aldrich at the highest purity available and used as received without further purification. Ultrapure water (18 MX cm 1) was used throughout the present study. 2.2. Preparation of Fe3O4, core–shell Fe3O4@SiO2, and AgMNPs The Fe3O4 and Fe3O4@SiO2 nanoparticles were prepared according to a previously reported method [25]. The magnetic Fe3O4@SiO2 nanoparticles were dispersed in 1% APTMS solution and stirred for 24 h. The APTMS-functionalized magnetic Fe3O4@SiO2 nanoparticles were magnetically separated from the mixture solution by the use of a magnet and were then washed with ethanol and deionized water. These APTMS-functionalized magnetic Fe3O4@SiO2 nanoparticles were then functionalized again with Au by dispersion in the Au seed solution. The Fe3O4@SiO2@Au-seed nanoparticles were separated and washed with water. Finally, to coat silver onto the Fe3O4@SiO2@Au-seed nanoparticles, silver enhancer kit of A (0.5 mL, 25% in distilled water) and B (0.5 mL, 25% in distilled water) were added to the 1 mL of Fe3O4@SiO2@Au-seed solution (containing 1 mg of Fe3O4 particles). 2.3. Buffer solutions Phosphate-buffered saline (PBS; 0.01 M, pH 7.0) was used as the basis of the blocking buffer in this study. The blocking buffer solution was prepared by dissolving BSA in the PBS buffer (1% BSA) solution. 2.4. Atto610-complex deposition on AgMNPs Avidin (from 10 ng/mL to 5 pg/mL in PBS buffer solution) was immobilized on the AgMNP substrate by immersing the substrate in an avidin solution for one hour at 37 °C. After being rinsed with the PBS buffer solution, the avidin-coated AgMNPs were soaked in a blocking buffer solution. The particles were then immersed in atto610-biotin (1 lg/mL in PBS buffer) solution for one hour at 37 °C.
SERS spectra were measured with a Jobin Yvon/HORIBA LabRam ARAMIS Raman spectrometer equipped with an integral BX 41 confocal microscope. The radiation from the air-cooled HeNe laser (633 nm) was used as an excitation source. Raman scattering was detected with 180° geometry using a multichannel air-cooled ( 70 °C) charge-coupled device (CCD) camera (1024 256 pixels). 3. Results and discussion The fabrication process for the proposed AgMNP is shown in Scheme 1. Magnetic nanoparticles were characterized at each step using TEM, XRD, XPS, magnetization measurements, and UV–Vis absorption spectroscopy. Fig. 1A and B show TEM images of Fe3O4@SiO2 nanoparticles, which have a relatively smooth surface with diameters of approximately 158 ± 10 nm with a magnetic core of 150 ± 10 nm. The surface is covered with 8 nm of SiO2. The silica-coated magnetic particles are stable, possess a large surface area, and easily react with various coupling agents to covalently attach specific ligands for silver coating. The silica-coated magnetic particles were functionalized by APTMS, and these APTMS-functionalized magnetic Fe3O4@SiO2 nanoparticles were again functionalized with 2–3 nm gold nanoparticles, which acted as autocatalytic and nucleation sites for silver reduction. Fig. 1C shows the TEM image of Fe3O4@SiO2@Au-seed nanoparticles. The silver particles were finally decorated onto the Fe3O4@SiO2@Au-seed nanoparticles by the reduction of silver ions using hydroquinone at room temperature. The obtained Ag shell nanoparticles on Fe3O4 were used as the SERS substrate. As shown in Fig. 1D, the TEM image of the AgMNPs illustrates the rough features of the surface, which were due to the silver coated on the surface of the magnetic Fe3O4@SiO2 nanoparticles. Powder XRD was utilized to determine the crystalline structure of the AgMNPs, Fe3O4@SiO2, and Fe3O4 particles. As shown in Fig. 2c, the peak position and relative intensity of all diffraction peaks for the Fe3O4 particles are in good agreement with the results of a previous study [26]. The sharp and strong peaks confirmed that the products were well crystallized. New peaks at 2h = 38.1°, 44.4°, 64.4°, and 77.4° are shown in Fig. 2a. These peaks are indexed to the (1 1 1), (2 0 0), (2 2 0), and (3 1 1) planes of the fcc structures of silver [27]. The XPS analysis gives some insight into the chemical composition of the composite microspheres of the AgMNP, Fe3O4@SiO2, and Fe3O4 particles. Fig. 3 shows the Fe 2p, O 1s, Si 2p, and Ag 3d XPS spectra of the particles (AgMNPs, Fe3O4@SiO2, and Fe3O4) that were obtained. These spectra clearly show the presence of Fe3O4 and the
2.5. Characterization of substrates The UV–Vis spectra of Fe3O4@SiO2 and AgMNP were recorded on a UV-3600 spectrophotometer (Shimadzu) and characterized by transmission electron microscopy (TEM). TEM images of Fe3O4@SiO2, Fe3O4@SiO2@Au-seed, and AgMNP were obtained with a Hitachi H-8100 microscope operated at 200 kV. X-ray diffraction
Scheme 1. The schematic of preparation of AgMNPs.
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Fig. 1. TEM images of Fe3O4@SiO2 nanoparticles at (A) lower and (B) higher magnification, (C) gold-seed-decorated Fe3O4@SiO2 nanoparticles at higher magnification, and (D) AgMNPs.
Fig. 2. XRD patterns of AgMNPs (a), Fe3O4@SiO2 (b), and Fe3O4 (c) particles.
surface covered with SiO2 and Ag, which supports the results from the TEM images. Fig. 4 shows the magnetization curves measured at room temperature for the Fe3O4, Fe3O4@SiO2, and AgMNP particles. The curve presents a hysteresis loop, which suggests that all of these particles exhibit ferromagnetic behavior. The magnetic saturation values are 71.9, 53.2, and 36.0 emu/g, respectively, for Fe3O4, Fe3O4@SiO2, and AgMNPs. The decrease in the overall magnetization values indicates that the Fe3O4 surface is covered with nonmagnetic materials such as SiO2 and Ag. The ferromagnetic properties of the magnetic nanoparticles are critical for their applications in the biomedical and bioengineering fields. These ferromagnetic properties prevent aggregation and enable the magnetic nanoparticles to rapidly redisperse when the magnetic field is removed. Fig. 5a and b show the UV–Vis spectra of the Fe3O4@SiO2 and AgMNP, respectively. The absorption at approximately 480 nm is
ascribed to the Fe3O4 nanoparticles themselves, whereas the absorbance at approximately 600 nm is ascribed to the continuous and rough silver particles decorated on the magnetic Fe3O4@SiO2 nanoparticles [28]. According to the surface plasmon resonance (SPR) spectra of the AgMNPs, a 633-nm laser was used as an excitation wavelength for electromagnetic (EM) enhancement [29]. Magnetic nanoparticles with a particular character have received considerable attention because of their potential applications in high-sensitivity bioassays. The proposed method employed AgMNPs as a protein carrier to capture probe-conjugated small molecules for a highly sensitive protein SERRS assay. The AgMNPs were first coated with avidin (from 10 ng/mL to 5 pg/mL) and then soaked in a blocking buffer solution to block the areas lacking nonspecific avidin adsorption on the proposed substrate. The atto610-labeled biotin solution was then added for specific recognition of avidin. When a magnetic field was applied to a capillary tube filled with the dispersed solutions of the protein-coupled AgMNPs, the AgMNPs were easily and densely aggregated. The protein and small-molecule interactions were finally detected on the proposed AgMNPs using SERRS. In this proposed protocol, a magnet was used as an external magnetic field for AgMNP aggregation to induce a strong SERRS effect for a protein assay. Highly enhanced SERS spectra can be obtained when the protein concentration in a unit area is high. The intensities of the bands in the SERS spectra will thus depend on the amount of AgMNPs added to the analyte solution in the proposed study. We conclude from SI_Fig. 1 that the intensities of the bands in the SERRS spectra of atto610 increased as the amount of the AgMNPs were decreased. The intensities of the bands in the SERRS spectra of the protein-conjugated AgMNPs soaked in atto610-biotin increased with an increase in the reaction time for avidin to capture the biotin (SI_Fig. 2). However, no obvious intensity changes in the SERRS spectra were observed after protein-conjugated AgMNPs were soaked in atto610-biotin solution for 60 min. Fig. 6A shows the SERRS spectra of atto610-biotin/avidin (chemical structure of atto610 is shown in SI_Fig. 3) with different
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Fig. 3. XPS spectra of the Fe3O4, Fe3O4@SiO2, and AgMNPs: (A) Fe 2p, (B) O 1s, (C) Si 2p, and (D) Ag 3d.
Fig. 5. UV–Vis absorbance spectra of (a) Fe3O4@SiO2 nanoparticles and (b) AgMNPs. Fig. 4. Hysteresis loops of the Fe3O4, Fe3O4@SiO2, and AgMNPs recorded at room temperature.
concentrations of avidin (from 10 ng/mL to 5 pg/mL). It is noted that SERRS intensities of the label significantly increase with the increase in target protein (avidin). The C@O stretching (amide I band) of avidin or avidin/biotin complex and the aromatic C@C stretching vibration of atto610 contribute to the Raman band at 1634 cm 1 [30–34]. Atto610, with the maximum absorption of 610 nm, belongs to a new generation of fluorescent labels for the red spectral region. In SERS and resonance condition with 633 nm laser excitation, the bands at 1634, 855, and 793 cm 1
can be assigned to vibrational modes of atto610. Concentrationdependent SERRS intensities of atto610 are plotted in Fig. 6B. A strong band at 1634 cm 1, which is assigned to the aromatic C@C stretching vibration of atto610, was used for Gaussian curve fitting based on the relative intensities. Error bars are included to indicate the sample-to-sample variability in the SERRS intensities. Fig. 6B shows the remarkable increase in the SERRS intensities of atto610 over a large range of protein concentrations. We successfully detected very low concentrations of avidin (5 pg/mL) with the proposed AgMNPs. This observation implies that the proposed SERRS-based method with its advantages of fast magnet separation
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Fig. 6. (A) SERRS spectra of atto610-biotin/avidin complex with different concentrations of avidin (from 10 ng/mL to 5 pg/mL) and (B) concentration-dependent SERRS intensities at 1634 cm 1.
and detection and high sensitivity can be compatible with highthroughput methods. The external magnetic field, which can induce aggregation of the AgMNPs, tunes the aggregated particles for the maximum SERRS signal intensity. To the best of our knowledge, the formation of two AgMNP aggregates results in higher EM enhancement for atto610. It would therefore be interesting to explore whether the as-prepared hybrid magnetic nanospheres that contain Ag aggregates could be used for the fabrication of substrates for intense SERS. Furthermore, the aggregated nanoparticles can provide more surface area for protein to adsorb than can flat substrates in a given unit area. With these results, we have demonstrated the great potential of this methodology for highly sensitive protein detection. 4. Conclusion In this study, AgMNPs, which can be aggregated by an external magnetic field, were used as protein carriers for SERRS-based highly sensitive protein detection. As protein carriers and SERS substrate, these AgMNPs were much more sensitive than other nanoparticles for protein detection. The most important aspect of this study is the potential applications of AgMNPs for quantitative immunoassays because the magnetic nanoparticles can increase the amount of protein in a given unit area. We successfully detected low concentrations of avidin (5 pg/mL) using the proposed AgMNPs because the external magnetic field induced the aggregation of the AgMNPs. Magnetically assisted protein separation and detection by SERRS will provide an effective and sensitive immunoassay technique. Acknowledgments This study was supported by the National Natural Science Foundation (Grant Nos. 20873050, 20921003, 20973074, 21073072) of PR China; the 111 Project (B06009). This work was also supported by the National Research Foundation of Korea (NRF) Grants funded by the Korea government (MEST) (Nos. 2011-C00052 and 20090087013) and the BK 21 program from the Ministry of Education, Science and Technology of Korea. The authors thank the Central Laboratory of Kangwon National University for the measurements of Raman spectra. Appendix A. Supplementary material SERRS intensities of atto610 (1634, 855, and 793 cm 1) with different amounts of magnetic particles; SERRS intensities of atto610
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