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Multiple detection of proteins by SERS-based immunoassay with core shell magnetic gold nanoparticles
Vibrational Spectroscopy 72 (2014) 44–49
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Vibrational Spectroscopy journal homepage: www.elsevier.com/locate/vibspec
Multiple detection of proteins by SERS-based immunoassay with core shell magnetic gold nanoparticles夽 Min Hwa Shin a , Wonjin Hong a , Youngjo Sa a , Lei Chen a,b , Yu-Jin Jung c , Xu Wang b , Bing Zhao b , Young Mee Jung a,∗ a
Department of Chemistry, Institute for Molecular Science and Fusion Technology, Kangwon National University, Chunchon 200-701, Republic of Korea State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun 130012, PR China c Department of Biological Sciences, Kangwon National University, Chunchon 200-701, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 29 December 2013 Received in revised form 10 February 2014 Accepted 15 February 2014 Available online 24 February 2014 Keywords: SERS SERS immunoassay Core shell magnetic gold nanoparticles Protein detection Protein separation Multiple detections
a b s t r a c t A highly selective and sensitive surface-enhanced Raman scattering (SERS)-based immunoassay for the multiple detection of proteins has been developed. The proposed core shell magnetic gold (Au) nanoparticles allow for successful protein separation and high SERS enhancement for protein detection. To selectively detect a specific protein in a mixed protein solution, we employed the sandwich type SERS immunoassay with core shell magnetic Au nanoparticles utilizing specific antigen–antibody interactions. Based on this proposed SERS immunoassay, we can successfully detect proteins in very low concentrations (∼800 ag/mL of mouse IgG and ∼5 fg/mL of human IgG) with high reproducibility. Magnetically assisted protein separation and detection by this proposed SERS immunoassay would provide great potential for effective and sensitive multiple protein detection. This technique allows for the straightforward SERS-based bioassays for quantitative protein detections. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Surface-enhanced Raman scattering (SERS), which greatly enhances the Raman intensity of molecules adsorbed on rough precious metal surfaces, has been extensively studied as an ultrasensitive analytical technique [1–22]. SERS has potential for the highly selective and sensitive detection of molecules. This technique is promising for biological applications as a result of its biocompatibility and has been probed to have great potential in a high-throughput detection of biomolecules, especially in the detection of proteins. Specially, SERS-based biosensors have recently been developed for various applications, including immunoassays, chemical–biochemical analyses, nanostructure characterization, and biomedical applications [2,3,11–22]. Labeled and label-free detection methods are most commonly used for applications of SERS-based biosensors. In the label-free detection method, the internal structural information of target protein is directly detected [11,17–19,21–25]. While in the labeled method, the extrinsic
夽 Selected paper presented at 7th International Conference on Advanced Vibrational Spectroscopy, Kobe, Japan, August 25–30, 2013. ∗ Corresponding author. Tel.: +82 33 250 8495; fax: +82 33 259 5667. E-mail address: [email protected] (Y.M. Jung). http://dx.doi.org/10.1016/j.vibspec.2014.02.007 0924-2031/© 2014 Elsevier B.V. All rights reserved.
SERS of labeling molecules, called SERS tags or Raman reporters, is indirectly detected to determine biomolecules [16,20,26–28]. Labeled detection method has been widely used because Ramanlabeled compounds provide much greater SERS enhancement than target molecules in direct detection methods. We have also studied labeled and label-free SERS-based biosensors for protein detections, in situ cell assays, medical diagnostics, and immune recognition [29–34]. SERS-active substrates are of prime importance for successful SERS-based assays. Until now, the most common SERS-active substrates for bioassays have been metal nanoparticles (e.g., gold, silver, and metal composites), core–shell nanoparticles, and magnetic composite nanoparticles. Magnetic nanoparticles, in particular, have been widely used in protein separation and purification, drug delivery, and medical imaging because of their magnetic separation capability and biocompatibility [35–37]. Among these substrates, functionalized core–shell magnetic nanoparticles have recently been used for highly sensitive protein detections. Silica-coated magnetic particles combined with different nanoparticles may lead to the development of multifunctional nano-assembled systems that simultaneously exhibit novel optical, electronic, and magnetic properties [38–41]. In particular, core shell nanocomposites of Fe3 O4 @Au provide enhanced stabilization and biofunctionalization [42,43].
M.H. Shin et al. / Vibrational Spectroscopy 72 (2014) 44–49
In this study, we synthesized a sensitive and biocompatible SERS-active substrate for protein detection based on Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles. The sandwich type SERS-based immunoassay with the synthesized core shell magnetic Au nanoparticles was performed to selectively detect a specific protein in a mixed protein solution with high sensitivity and reproducibility. This proposed SERS-based immunoassay provides potential applications in high-level multiplexing proteins detection.
2. Experimental 2.1. Materials Ferric chloride (FeCl3 ·6H2 O), sodium acetate, ethylene glycol, ethanol, tetrakis(hydroxymethyl)phosphonium chloride (THPC), tetraethylorthosilicate (TEOS), ammonium hydroxide, gold (III) chloride trihydrate (HAuCl4 ·3H2 O), potassium carbonate (K2 CO3 ), formaldehyde, sodium citrate tribasic dihydrate, hydrochloric acid, sodium hydroxide aqueous, sulfuric acid, hydrogen peroxide, (3-aminopropyl)trimethoxysilane (APTMS), poly(diallyldimethylammonium chloride) (PDDA), tris-buffer (0.002 M, pH 7.4), phosphate-buffered saline (PBS: 0.01 M, pH 7.4), 4-mercaptobenzoic acid (4-MBA), 5,5 -dithiobis (2nitrobenzoic acid) (DTNB), hydroxylamine hydrochloride, silver nitrate (AgNO3 ), human IgG, anti-human IgG, mouse IgG and anti-mouse IgG were purchased from Sigma–Aldrich at the highest purity available and used as received without further purification. Ultrapure water (18 M cm−1 ) was used throughout the present study.
2.2. Preparation of Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles The Fe3 O4 and Fe3 O4 @SiO2 nanoparticles were prepared according to a previously reported method [44]. The magnetic Fe3 O4 @SiO2 nanoparticles were dispersed in 1% APTMS solution and stirred for 24 h. The APTMS-functionalized magnetic Fe3 O4 @SiO2 nanoparticles were magnetically separated from the mixture solution using a magnet and then washed with ethanol and deionized water. These APTMS-functionalized magnetic Fe3 O4 @SiO2 nanoparticles were further functionalized with Au by dispersion in an Au seed solution. An Au nanoparticle monolayer was thus formed via self-assembly on the SiO2 surface of the magnetic Fe3 O4 @SiO2 nanoparticles. The Fe3 O4 @SiO2 @Auseed nanoparticles were separated and washed with water [34]. Fe3 O4 @SiO2 @Au-seed nanoparticles were used as seeds to form continuous Au shells. To grow the Au overlayer onto the Fe3 O4 @SiO2 @Au-seed nanoparticles, a solution of gold hydroxide was first prepared. In a reaction flask, 1.5 mL of 1% HAuCl4 solution was added to a 0.002 M K2 CO3 solution. The mixture initially appeared transparent yellow and slowly became colorless after 30 min, indicating the formation of gold hydroxide [45–47]. The resulting Au growth solution was aged for 24 h in the dark before being used in subsequent steps. In a typical preparation, 100 mL of the Au growth solution was added to the Fe3 O4 @SiO2 @Au-seed nanoparticles solution. The amount of Au growth solution was varied according to the intended thickness of the Au shell. A solution of HCHO was then added to this vigorously stirred mixture. The resulting transparent solution became red, indicating that the Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles had finally been synthesized. Scheme 1 depicts the fabrication process of the Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles.
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2.3. Preparation of SERS tags for the SERS immunoassay 4-MBA and DTNB were used as Raman reporters for the SERS immunoassay. 4-MBA and DTNB were immobilized on the Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles by mixing 4MBA and DTNB solutions (0.001 M in ethanol each), and dispersing the Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles in the solution for 3 h. 4-MBA and DTNB, which have SH groups, can be easily self-assembled onto the Au surface of the Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles, respectively. After rinsing with the ethanol solution, 4-MBA- or DTNB-functionalized Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles were then rinsed with PBS buffer solution (0.01 M, pH 7.4) to attach a specific antibody. To deposit IgG antibodies onto the 4-MBA- and/or DTNBfunctionalized Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles, solutions of IgG antibodies of appropriate pH were added to the dispersed solutions of the 4-MBA- and/or DTNB-functionalized Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles. This approach allows for the electrostatic assembly of IgG antibodies onto the 4MBA- and/or DTNB-functionalized Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles [48]. Scheme 2 shows the preparation of the proposed SERS tags, which includes the immobilization of captured antibodies on the Raman reporter-functionalized Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles for SERS immunoassays. Antimouse IgG and anti-human IgG were deposited onto the DTNBand 4-MBA-functionalized Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles, respectively. 2.4. Preparation of silver substrates for the sandwich type SERS immunoassay Si wafers were cleaned by immersion in a boiling solution of H2 O2 and H2 SO4 (volume ratio of 3:7). After cooling, the Si wafers were rinsed repeatedly with distilled water and gently dried with N2 gas. The Si wafers with hydroxyl surfaces were then soaked in a 0.5% PDDA solution for 30 min, rinsed with distilled water, and gently dried with N2 gas. The PDDA-decorated Si wafer were then immersed in a silver nanoparticles solution for 2 h, rinsed with distilled water, and gently dried with N2 gas. The silver nanoparticles were prepared according to a previously reported method [49]. Finally, IgG antibodies were deposited on the silver surfaces by immersing the silver substrate in an IgG antibody solution for 3 h at 37 ◦ C. Then the SERS substrates were rinsed with buffer solution and dried with N2 gas. Scheme 3 shows the preparation of the immobilized layer of antibodies on the silver monolayer substrates for the sandwich type SERS-immunoassay. 2.5. Instruments All SERS spectra were recorded using a Jobin Yvon/HORIBA LabRam ARAMIS Raman spectrometer. Radiation from an air cooled HeNe laser (633 nm) was used as the excitation source. Raman scattering was detected at a 180◦ geometry by a multichannel aircooled (−70 ◦ C) charge-coupled device (CCD) camera (1024 × 256 pixels). The typical exposure time for each SERS measurement in this study was 2 s for one acquisition. The absorbance spectra were recorded on a Scinco S-3100 UV-Visible spectrometer. The magnetic nanoparticles were characterized at each step by the energy filtering transmission electron microscopy (EF-TEM) using a LEO912AB OMEGA instrument with an acceleration voltage of 200 kV. X-ray diffraction (XRD) using a PANalyfical, XPERT-PRO and magnetization measurements using a Lake-shore as a function of the applied field at room temperature were obtained for the magnetic nanoparticles at each step. The silver substrates were characterized by ultra high resolution scanning electron microscopy (UHR-SEM)
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Scheme 1. The preparation of Fe3 O4 @SiO2 @Au nanoparticles.
Scheme 2. The preparation of human IgG-captured 4-MBA-AuMNPs and mouse IgG-captured DTNB-AuMNPs.
using a Hitachi S-4800 UHR-SEM with an acceleration voltage of 30 kV. 3. Results and discussion The fabrication process for the proposed Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles is shown in Scheme 1. The magnetic nanoparticles were characterized at each step using TEM, XRD, magnetization measurements, and UV–vis absorption spectroscopy. Fig. 1(a) shows TEM images of the Fe3 O4 nanoparticles, which are spherical with diameters of approximately 350 ± 20 nm. The silica-coated magnetic particles were functionalized by APTMS and the resulting APTMS-functionalized magnetic Fe3 O4 @SiO2 nanoparticles were further functionalized with 7 nm Au nanoparticles. Fig. 1(b) shows a TEM image of the Fe3 O4 @SiO2 @Au-seed nanoparticles, which have a relatively smooth surface with diameters of approximately 400 nm. Finally, the Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles were synthesized by growing an Au overlayer onto the Fe3 O4 @SiO2 @Au-seed nanoparticles. A TEM image of the Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles is shown in Fig. 1(c). The image illustrates the rough features of the nanoparticle surface, which resulted from the Au overlayer coated on the surface of the magnetic Fe3 O4 @SiO2 nanoparticles. Powder XRD was utilized to determine the crystalline structure of the Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles, Fe3 O4 @SiO2 @Au-seed nanoparticles, Fe3 O4 @SiO2 nanoparticles,
and Fe3 O4 nanoparticles. As shown in Fig. 2, the peak positions and relative intensities of all of the diffraction peaks for the Fe3 O4 particles are in good agreement with the results of a previous study [50]. The sharp and strong peaks confirm that the products were well-crystallized. New peaks at 2 = 38.1, 44.34, 64.58, and 77.50◦ are shown in Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles and Fe3 O4 @SiO2 @Au-seed nanoparticles, which can be indexed to the (1 1 1), (2 0 0), (2 2 0), and (3 1 1) planes of Au in the cubic phase, respectively [51]. Fig. 3 shows the magnetization curves measured at room temperature for the Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles, Fe3 O4 @SiO2 @Au-seed nanoparticles, Fe3 O4 @SiO2 nanoparticles, and Fe3 O4 nanoparticles. The curves present a hysteresis loop, which suggests that all of these particles exhibit ferromagnetic behavior. The magnetic saturation values decrease gradually upon coating the Fe3 O4 nanoparticles with SiO2 and Au, which indicates that the Fe3 O4 surface is covered with the non-magnetic SiO2 and Au materials. The ferromagnetic properties of the magnetic nanoparticles are of great importance for their biomedical applications. Fig. 4(a) and (b) shows the UV–vis spectrum and UHR-SEM image, respectively, of the silver substrate for the sandwich type SERS immunoassay. As shown in Fig. 4(a), the absorption at approximately 410 nm results from the silver nanoparticles on the surface of the PDDA-decorated Si wafer. The UHR-SEM image of the silver substrate shows that the silver nanoparticles were homogeneously assembled on the PDDA-decorated substrate. This silver
Scheme 3. The preparation of an immobilized layer of antibodies on the silver monolayer substrate.
M.H. Shin et al. / Vibrational Spectroscopy 72 (2014) 44–49
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Fig. 1. TEM images of Fe3 O4 nanoparticles (a), Fe3 O4 @SiO2 nanoparticles (b) and Fe3 O4 @SiO2 @Au core shell nanoparticles (c).
Fig. 2. XRD patterns of Fe3 O4 nanoparticles, Fe3 O4 @SiO2 nanoparticles, Fe3 O4 @SiO2 @Au-seed nanoparticles and Fe3 O4 @SiO2 @Au core shell nanoparticles.
nanoparticles monolayer serves as a good substrate for capturing antibodies in this proposed sandwich type SERS immunoassay. Scheme 4 depicts the sandwich type SERS immunoassay process with Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles for the multiple detection of protein. This sandwich type SERS immunoassay involves the conjugation of antigens to the antibodies on the Raman reporter-functionalized Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles via the specific antigen–antibody interaction and the SERS detection of the Raman reporter on which the antigen is selectively conjugated with the antibody. Mouse IgG and human IgG can be selectively captured on DTNB- and 4-MBA-functionalized Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles, respectively. As shown in Scheme 4,
Fig. 3. Hysteresis loops of Fe3 O4 nanoparticles, Fe3 O4 @SiO2 nanoparticles, Fe3 O4 @SiO2 @Au-seed nanoparticles and Fe3 O4 @SiO2 @Au core shell nanoparticles recorded at room temperature.
DTNB-functionalized Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles (DTNB-AuMNPs) were firstly immersed in a mixture of antigens (human IgG and mouse IgG) solution. The corresponding antigen (mouse IgG) in the mixed solution was then selectively conjugated to the DTNB-AuMNPs as a result of the highly specific interaction of the antibody molecules with their corresponding antigens. These mouse IgG-captured DTNB-AuMNPs were selectively separated by a magnet and were utilized in the sandwich type SERS immunoassay. In this proposed sandwich type SERS immunoassay, the SERS tags were assayed by their reaction with the silver substrates captured by the corresponding antibodies. After separation of mouse IgG-captured DTNB-AuMNPs with a magnet, the resulting solution contained only human IgG. 4-MBA-functionalized Fe3 O4 @SiO2 @Au core shell magnetic
Fig. 4. UV–vis spectrum (a) and UHR-SEM image (b) of the silver substrate.
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Scheme 4. The protein separation and sandwich type SERS immunoassay process.
Fig. 5. SERS spectra of 4-MBA and DTNB.
nanoparticles (4-MBA-AuMNPs) were then added to the resulting solution. The remaining antigens (human IgG) in the solution were selectively captured by the 4-MBA-AuMNPs. The resulting human IgG-captured 4-MBA-AuMNPs were subsequently employed in the sandwich type SERS immunoassay. The SERS spectra of the Raman reporters, 4-MBA and DTNB, are shown in Fig. 5. The characteristic bands observed at 1073 and 1334 cm−1 correspond to 4-MBA and DTNB, respectively. These two bands are the index of human IgG and mouse IgG, respectively, for the multiple detection of protein in a mixed proteins solution. In Scheme 4, human IgG-captured 4-MBA-AuMNPs and
mouse IgG-captured DTNB-AuMNPs are selectively captured onto the immobilized layer of anti-human IgG and anti-mouse IgG, respectively, on the silver monolayer substrates. The SERS spectra of human IgG-captured 4-MBA-AuMNPs and mouse IgG-captured DTNB-AuMNPs show characteristic bands at 1073 and 1334 cm−1 , respectively, which are in good agreement with those found in Fig. 5. This technique successfully provides the highly selective multiple protein detection by SERS immunoassay. Fig. 6(a) shows the SERS spectra of mouse IgG-captured DTNBAuMNPs with different concentrations of mouse IgG (from 5 ng/mL to 5 fg/mL). The intensity of SERS bands from DTNB molecules increased concomitantly with the increasing concentration of the mouse IgG. The concentration-dependent SERS spectra were further analyzed by plotting the intensity at 1334 cm−1 as a function of the logarithm of mouse IgG concentration. The plot for the concentration-dependent SERS intensity of the band at 1334 cm−1 shown in Fig. 6 (b) obeys the equation: Y = 0.06912X + 1.042, R2 = 0.9929, where Y is the SERS intensity of the band at 1334 cm−1 of mouse IgG-captured DTNB-AuMNPs, and X is the concentration of mouse IgG. It exhibits a good linear relationship between SERS intensity and the concentration of mouse IgG with high reproducibility. Error bars in the plot are included to indicate the sample-to-sample variability in the SERS intensities. The antigen detection sensitivity was reproducibly obtained as down to very low concentration. Using this strategy, we have successfully detected very low concentrations of mouse IgG (∼800 ag/mL). Fig. 7 shows the corresponding SERS spectra of human IgG-captured 4MBA-AuMNPs with different concentrations of human IgG (from 5 ng/mL to 500 fg/mL). It is noted that the SERS intensity of the Raman report significantly increases with increasing target protein (human IgG) concentration. Based on the concentration-dependent SERS intensities of characteristic band at 1073 cm−1 of human
Fig. 6. (a) SERS spectra of mouse IgG-captured DTNB-AuMNPs with different concentrations of mouse IgG (from 5 ng/mL to 5 fg/mL) and (b) the plot of SERS intensities at 1334 cm−1 as a function of the logarithm of mouse IgG concentration.
M.H. Shin et al. / Vibrational Spectroscopy 72 (2014) 44–49
Fig. 7. SERS spectra of human IgG-captured 4-MBA-AuMNPs with different concentrations of human IgG (from 5 ng/mL to 500 fg/mL).
IgG-captured 4-MBA-AuMNPs, we have also successfully detected very low concentrations of human IgG (∼5 fg/mL) with high reproducibility using SERS immunoassay described herein. From these results, we have demonstrated the great potential of this methodology for highly selective and sensitive multiple protein detection with high reproducibility. 4. Conclusion In this study, we proposed a multiple protein detection method by a sandwich type SERS-based immunoassay with core shell magnetic Au nanoparticles. Two antigens in the mixed solution were successfully separated by the proposed core shell magnetic Au nanoparticles showing a potential application in bioseparation. We have achieved SERS spectra that are proportional to concentration of antigen allowing detection of antigen amounts as low as 800 ag/mL and 5 fg/mL for mouse IgG and human IgG, respectively. Using this proposed sandwich type SERS-based immunoassay with core shell magnetic Au nanoparticles, we can selectively detect the target protein in the mixed antigens solution. Our results suggest that highly sensitive immunoassays are possible using this proposed core shell magnetic Au nanoparticles for quantitative detection of proteins. Therefore, this proposed strategy provides a highly selective and sensitive multiple protein detection method with high reproducibility. Acknowledgments This study supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (MEST) (Nos. 2011-C00052 and 2009-0087013). This study was also supported by 2013 Research Grant from Kangwon National University. The authors thank the Central Laboratory of Kangwon National University for the measurements of Raman spectra. References [1] R. Aroca, Surface-Enhanced Vibrational Spectroscopy, John Wiley & Sons, Ltd., Chichester, 2006.
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Multiple detection of proteins by SERS-based immunoassay with core shell magnetic gold nanoparticles夽 Min Hwa Shin a , Wonjin Hong a , Youngjo Sa a , Lei Chen a,b , Yu-Jin Jung c , Xu Wang b , Bing Zhao b , Young Mee Jung a,∗ a
Department of Chemistry, Institute for Molecular Science and Fusion Technology, Kangwon National University, Chunchon 200-701, Republic of Korea State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun 130012, PR China c Department of Biological Sciences, Kangwon National University, Chunchon 200-701, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 29 December 2013 Received in revised form 10 February 2014 Accepted 15 February 2014 Available online 24 February 2014 Keywords: SERS SERS immunoassay Core shell magnetic gold nanoparticles Protein detection Protein separation Multiple detections
a b s t r a c t A highly selective and sensitive surface-enhanced Raman scattering (SERS)-based immunoassay for the multiple detection of proteins has been developed. The proposed core shell magnetic gold (Au) nanoparticles allow for successful protein separation and high SERS enhancement for protein detection. To selectively detect a specific protein in a mixed protein solution, we employed the sandwich type SERS immunoassay with core shell magnetic Au nanoparticles utilizing specific antigen–antibody interactions. Based on this proposed SERS immunoassay, we can successfully detect proteins in very low concentrations (∼800 ag/mL of mouse IgG and ∼5 fg/mL of human IgG) with high reproducibility. Magnetically assisted protein separation and detection by this proposed SERS immunoassay would provide great potential for effective and sensitive multiple protein detection. This technique allows for the straightforward SERS-based bioassays for quantitative protein detections. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Surface-enhanced Raman scattering (SERS), which greatly enhances the Raman intensity of molecules adsorbed on rough precious metal surfaces, has been extensively studied as an ultrasensitive analytical technique [1–22]. SERS has potential for the highly selective and sensitive detection of molecules. This technique is promising for biological applications as a result of its biocompatibility and has been probed to have great potential in a high-throughput detection of biomolecules, especially in the detection of proteins. Specially, SERS-based biosensors have recently been developed for various applications, including immunoassays, chemical–biochemical analyses, nanostructure characterization, and biomedical applications [2,3,11–22]. Labeled and label-free detection methods are most commonly used for applications of SERS-based biosensors. In the label-free detection method, the internal structural information of target protein is directly detected [11,17–19,21–25]. While in the labeled method, the extrinsic
夽 Selected paper presented at 7th International Conference on Advanced Vibrational Spectroscopy, Kobe, Japan, August 25–30, 2013. ∗ Corresponding author. Tel.: +82 33 250 8495; fax: +82 33 259 5667. E-mail address: [email protected] (Y.M. Jung). http://dx.doi.org/10.1016/j.vibspec.2014.02.007 0924-2031/© 2014 Elsevier B.V. All rights reserved.
SERS of labeling molecules, called SERS tags or Raman reporters, is indirectly detected to determine biomolecules [16,20,26–28]. Labeled detection method has been widely used because Ramanlabeled compounds provide much greater SERS enhancement than target molecules in direct detection methods. We have also studied labeled and label-free SERS-based biosensors for protein detections, in situ cell assays, medical diagnostics, and immune recognition [29–34]. SERS-active substrates are of prime importance for successful SERS-based assays. Until now, the most common SERS-active substrates for bioassays have been metal nanoparticles (e.g., gold, silver, and metal composites), core–shell nanoparticles, and magnetic composite nanoparticles. Magnetic nanoparticles, in particular, have been widely used in protein separation and purification, drug delivery, and medical imaging because of their magnetic separation capability and biocompatibility [35–37]. Among these substrates, functionalized core–shell magnetic nanoparticles have recently been used for highly sensitive protein detections. Silica-coated magnetic particles combined with different nanoparticles may lead to the development of multifunctional nano-assembled systems that simultaneously exhibit novel optical, electronic, and magnetic properties [38–41]. In particular, core shell nanocomposites of Fe3 O4 @Au provide enhanced stabilization and biofunctionalization [42,43].
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In this study, we synthesized a sensitive and biocompatible SERS-active substrate for protein detection based on Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles. The sandwich type SERS-based immunoassay with the synthesized core shell magnetic Au nanoparticles was performed to selectively detect a specific protein in a mixed protein solution with high sensitivity and reproducibility. This proposed SERS-based immunoassay provides potential applications in high-level multiplexing proteins detection.
2. Experimental 2.1. Materials Ferric chloride (FeCl3 ·6H2 O), sodium acetate, ethylene glycol, ethanol, tetrakis(hydroxymethyl)phosphonium chloride (THPC), tetraethylorthosilicate (TEOS), ammonium hydroxide, gold (III) chloride trihydrate (HAuCl4 ·3H2 O), potassium carbonate (K2 CO3 ), formaldehyde, sodium citrate tribasic dihydrate, hydrochloric acid, sodium hydroxide aqueous, sulfuric acid, hydrogen peroxide, (3-aminopropyl)trimethoxysilane (APTMS), poly(diallyldimethylammonium chloride) (PDDA), tris-buffer (0.002 M, pH 7.4), phosphate-buffered saline (PBS: 0.01 M, pH 7.4), 4-mercaptobenzoic acid (4-MBA), 5,5 -dithiobis (2nitrobenzoic acid) (DTNB), hydroxylamine hydrochloride, silver nitrate (AgNO3 ), human IgG, anti-human IgG, mouse IgG and anti-mouse IgG were purchased from Sigma–Aldrich at the highest purity available and used as received without further purification. Ultrapure water (18 M cm−1 ) was used throughout the present study.
2.2. Preparation of Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles The Fe3 O4 and Fe3 O4 @SiO2 nanoparticles were prepared according to a previously reported method [44]. The magnetic Fe3 O4 @SiO2 nanoparticles were dispersed in 1% APTMS solution and stirred for 24 h. The APTMS-functionalized magnetic Fe3 O4 @SiO2 nanoparticles were magnetically separated from the mixture solution using a magnet and then washed with ethanol and deionized water. These APTMS-functionalized magnetic Fe3 O4 @SiO2 nanoparticles were further functionalized with Au by dispersion in an Au seed solution. An Au nanoparticle monolayer was thus formed via self-assembly on the SiO2 surface of the magnetic Fe3 O4 @SiO2 nanoparticles. The Fe3 O4 @SiO2 @Auseed nanoparticles were separated and washed with water [34]. Fe3 O4 @SiO2 @Au-seed nanoparticles were used as seeds to form continuous Au shells. To grow the Au overlayer onto the Fe3 O4 @SiO2 @Au-seed nanoparticles, a solution of gold hydroxide was first prepared. In a reaction flask, 1.5 mL of 1% HAuCl4 solution was added to a 0.002 M K2 CO3 solution. The mixture initially appeared transparent yellow and slowly became colorless after 30 min, indicating the formation of gold hydroxide [45–47]. The resulting Au growth solution was aged for 24 h in the dark before being used in subsequent steps. In a typical preparation, 100 mL of the Au growth solution was added to the Fe3 O4 @SiO2 @Au-seed nanoparticles solution. The amount of Au growth solution was varied according to the intended thickness of the Au shell. A solution of HCHO was then added to this vigorously stirred mixture. The resulting transparent solution became red, indicating that the Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles had finally been synthesized. Scheme 1 depicts the fabrication process of the Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles.
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2.3. Preparation of SERS tags for the SERS immunoassay 4-MBA and DTNB were used as Raman reporters for the SERS immunoassay. 4-MBA and DTNB were immobilized on the Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles by mixing 4MBA and DTNB solutions (0.001 M in ethanol each), and dispersing the Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles in the solution for 3 h. 4-MBA and DTNB, which have SH groups, can be easily self-assembled onto the Au surface of the Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles, respectively. After rinsing with the ethanol solution, 4-MBA- or DTNB-functionalized Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles were then rinsed with PBS buffer solution (0.01 M, pH 7.4) to attach a specific antibody. To deposit IgG antibodies onto the 4-MBA- and/or DTNBfunctionalized Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles, solutions of IgG antibodies of appropriate pH were added to the dispersed solutions of the 4-MBA- and/or DTNB-functionalized Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles. This approach allows for the electrostatic assembly of IgG antibodies onto the 4MBA- and/or DTNB-functionalized Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles [48]. Scheme 2 shows the preparation of the proposed SERS tags, which includes the immobilization of captured antibodies on the Raman reporter-functionalized Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles for SERS immunoassays. Antimouse IgG and anti-human IgG were deposited onto the DTNBand 4-MBA-functionalized Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles, respectively. 2.4. Preparation of silver substrates for the sandwich type SERS immunoassay Si wafers were cleaned by immersion in a boiling solution of H2 O2 and H2 SO4 (volume ratio of 3:7). After cooling, the Si wafers were rinsed repeatedly with distilled water and gently dried with N2 gas. The Si wafers with hydroxyl surfaces were then soaked in a 0.5% PDDA solution for 30 min, rinsed with distilled water, and gently dried with N2 gas. The PDDA-decorated Si wafer were then immersed in a silver nanoparticles solution for 2 h, rinsed with distilled water, and gently dried with N2 gas. The silver nanoparticles were prepared according to a previously reported method [49]. Finally, IgG antibodies were deposited on the silver surfaces by immersing the silver substrate in an IgG antibody solution for 3 h at 37 ◦ C. Then the SERS substrates were rinsed with buffer solution and dried with N2 gas. Scheme 3 shows the preparation of the immobilized layer of antibodies on the silver monolayer substrates for the sandwich type SERS-immunoassay. 2.5. Instruments All SERS spectra were recorded using a Jobin Yvon/HORIBA LabRam ARAMIS Raman spectrometer. Radiation from an air cooled HeNe laser (633 nm) was used as the excitation source. Raman scattering was detected at a 180◦ geometry by a multichannel aircooled (−70 ◦ C) charge-coupled device (CCD) camera (1024 × 256 pixels). The typical exposure time for each SERS measurement in this study was 2 s for one acquisition. The absorbance spectra were recorded on a Scinco S-3100 UV-Visible spectrometer. The magnetic nanoparticles were characterized at each step by the energy filtering transmission electron microscopy (EF-TEM) using a LEO912AB OMEGA instrument with an acceleration voltage of 200 kV. X-ray diffraction (XRD) using a PANalyfical, XPERT-PRO and magnetization measurements using a Lake-shore as a function of the applied field at room temperature were obtained for the magnetic nanoparticles at each step. The silver substrates were characterized by ultra high resolution scanning electron microscopy (UHR-SEM)
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Scheme 1. The preparation of Fe3 O4 @SiO2 @Au nanoparticles.
Scheme 2. The preparation of human IgG-captured 4-MBA-AuMNPs and mouse IgG-captured DTNB-AuMNPs.
using a Hitachi S-4800 UHR-SEM with an acceleration voltage of 30 kV. 3. Results and discussion The fabrication process for the proposed Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles is shown in Scheme 1. The magnetic nanoparticles were characterized at each step using TEM, XRD, magnetization measurements, and UV–vis absorption spectroscopy. Fig. 1(a) shows TEM images of the Fe3 O4 nanoparticles, which are spherical with diameters of approximately 350 ± 20 nm. The silica-coated magnetic particles were functionalized by APTMS and the resulting APTMS-functionalized magnetic Fe3 O4 @SiO2 nanoparticles were further functionalized with 7 nm Au nanoparticles. Fig. 1(b) shows a TEM image of the Fe3 O4 @SiO2 @Au-seed nanoparticles, which have a relatively smooth surface with diameters of approximately 400 nm. Finally, the Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles were synthesized by growing an Au overlayer onto the Fe3 O4 @SiO2 @Au-seed nanoparticles. A TEM image of the Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles is shown in Fig. 1(c). The image illustrates the rough features of the nanoparticle surface, which resulted from the Au overlayer coated on the surface of the magnetic Fe3 O4 @SiO2 nanoparticles. Powder XRD was utilized to determine the crystalline structure of the Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles, Fe3 O4 @SiO2 @Au-seed nanoparticles, Fe3 O4 @SiO2 nanoparticles,
and Fe3 O4 nanoparticles. As shown in Fig. 2, the peak positions and relative intensities of all of the diffraction peaks for the Fe3 O4 particles are in good agreement with the results of a previous study [50]. The sharp and strong peaks confirm that the products were well-crystallized. New peaks at 2 = 38.1, 44.34, 64.58, and 77.50◦ are shown in Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles and Fe3 O4 @SiO2 @Au-seed nanoparticles, which can be indexed to the (1 1 1), (2 0 0), (2 2 0), and (3 1 1) planes of Au in the cubic phase, respectively [51]. Fig. 3 shows the magnetization curves measured at room temperature for the Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles, Fe3 O4 @SiO2 @Au-seed nanoparticles, Fe3 O4 @SiO2 nanoparticles, and Fe3 O4 nanoparticles. The curves present a hysteresis loop, which suggests that all of these particles exhibit ferromagnetic behavior. The magnetic saturation values decrease gradually upon coating the Fe3 O4 nanoparticles with SiO2 and Au, which indicates that the Fe3 O4 surface is covered with the non-magnetic SiO2 and Au materials. The ferromagnetic properties of the magnetic nanoparticles are of great importance for their biomedical applications. Fig. 4(a) and (b) shows the UV–vis spectrum and UHR-SEM image, respectively, of the silver substrate for the sandwich type SERS immunoassay. As shown in Fig. 4(a), the absorption at approximately 410 nm results from the silver nanoparticles on the surface of the PDDA-decorated Si wafer. The UHR-SEM image of the silver substrate shows that the silver nanoparticles were homogeneously assembled on the PDDA-decorated substrate. This silver
Scheme 3. The preparation of an immobilized layer of antibodies on the silver monolayer substrate.
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Fig. 1. TEM images of Fe3 O4 nanoparticles (a), Fe3 O4 @SiO2 nanoparticles (b) and Fe3 O4 @SiO2 @Au core shell nanoparticles (c).
Fig. 2. XRD patterns of Fe3 O4 nanoparticles, Fe3 O4 @SiO2 nanoparticles, Fe3 O4 @SiO2 @Au-seed nanoparticles and Fe3 O4 @SiO2 @Au core shell nanoparticles.
nanoparticles monolayer serves as a good substrate for capturing antibodies in this proposed sandwich type SERS immunoassay. Scheme 4 depicts the sandwich type SERS immunoassay process with Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles for the multiple detection of protein. This sandwich type SERS immunoassay involves the conjugation of antigens to the antibodies on the Raman reporter-functionalized Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles via the specific antigen–antibody interaction and the SERS detection of the Raman reporter on which the antigen is selectively conjugated with the antibody. Mouse IgG and human IgG can be selectively captured on DTNB- and 4-MBA-functionalized Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles, respectively. As shown in Scheme 4,
Fig. 3. Hysteresis loops of Fe3 O4 nanoparticles, Fe3 O4 @SiO2 nanoparticles, Fe3 O4 @SiO2 @Au-seed nanoparticles and Fe3 O4 @SiO2 @Au core shell nanoparticles recorded at room temperature.
DTNB-functionalized Fe3 O4 @SiO2 @Au core shell magnetic nanoparticles (DTNB-AuMNPs) were firstly immersed in a mixture of antigens (human IgG and mouse IgG) solution. The corresponding antigen (mouse IgG) in the mixed solution was then selectively conjugated to the DTNB-AuMNPs as a result of the highly specific interaction of the antibody molecules with their corresponding antigens. These mouse IgG-captured DTNB-AuMNPs were selectively separated by a magnet and were utilized in the sandwich type SERS immunoassay. In this proposed sandwich type SERS immunoassay, the SERS tags were assayed by their reaction with the silver substrates captured by the corresponding antibodies. After separation of mouse IgG-captured DTNB-AuMNPs with a magnet, the resulting solution contained only human IgG. 4-MBA-functionalized Fe3 O4 @SiO2 @Au core shell magnetic
Fig. 4. UV–vis spectrum (a) and UHR-SEM image (b) of the silver substrate.
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Scheme 4. The protein separation and sandwich type SERS immunoassay process.
Fig. 5. SERS spectra of 4-MBA and DTNB.
nanoparticles (4-MBA-AuMNPs) were then added to the resulting solution. The remaining antigens (human IgG) in the solution were selectively captured by the 4-MBA-AuMNPs. The resulting human IgG-captured 4-MBA-AuMNPs were subsequently employed in the sandwich type SERS immunoassay. The SERS spectra of the Raman reporters, 4-MBA and DTNB, are shown in Fig. 5. The characteristic bands observed at 1073 and 1334 cm−1 correspond to 4-MBA and DTNB, respectively. These two bands are the index of human IgG and mouse IgG, respectively, for the multiple detection of protein in a mixed proteins solution. In Scheme 4, human IgG-captured 4-MBA-AuMNPs and
mouse IgG-captured DTNB-AuMNPs are selectively captured onto the immobilized layer of anti-human IgG and anti-mouse IgG, respectively, on the silver monolayer substrates. The SERS spectra of human IgG-captured 4-MBA-AuMNPs and mouse IgG-captured DTNB-AuMNPs show characteristic bands at 1073 and 1334 cm−1 , respectively, which are in good agreement with those found in Fig. 5. This technique successfully provides the highly selective multiple protein detection by SERS immunoassay. Fig. 6(a) shows the SERS spectra of mouse IgG-captured DTNBAuMNPs with different concentrations of mouse IgG (from 5 ng/mL to 5 fg/mL). The intensity of SERS bands from DTNB molecules increased concomitantly with the increasing concentration of the mouse IgG. The concentration-dependent SERS spectra were further analyzed by plotting the intensity at 1334 cm−1 as a function of the logarithm of mouse IgG concentration. The plot for the concentration-dependent SERS intensity of the band at 1334 cm−1 shown in Fig. 6 (b) obeys the equation: Y = 0.06912X + 1.042, R2 = 0.9929, where Y is the SERS intensity of the band at 1334 cm−1 of mouse IgG-captured DTNB-AuMNPs, and X is the concentration of mouse IgG. It exhibits a good linear relationship between SERS intensity and the concentration of mouse IgG with high reproducibility. Error bars in the plot are included to indicate the sample-to-sample variability in the SERS intensities. The antigen detection sensitivity was reproducibly obtained as down to very low concentration. Using this strategy, we have successfully detected very low concentrations of mouse IgG (∼800 ag/mL). Fig. 7 shows the corresponding SERS spectra of human IgG-captured 4MBA-AuMNPs with different concentrations of human IgG (from 5 ng/mL to 500 fg/mL). It is noted that the SERS intensity of the Raman report significantly increases with increasing target protein (human IgG) concentration. Based on the concentration-dependent SERS intensities of characteristic band at 1073 cm−1 of human
Fig. 6. (a) SERS spectra of mouse IgG-captured DTNB-AuMNPs with different concentrations of mouse IgG (from 5 ng/mL to 5 fg/mL) and (b) the plot of SERS intensities at 1334 cm−1 as a function of the logarithm of mouse IgG concentration.
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Fig. 7. SERS spectra of human IgG-captured 4-MBA-AuMNPs with different concentrations of human IgG (from 5 ng/mL to 500 fg/mL).
IgG-captured 4-MBA-AuMNPs, we have also successfully detected very low concentrations of human IgG (∼5 fg/mL) with high reproducibility using SERS immunoassay described herein. From these results, we have demonstrated the great potential of this methodology for highly selective and sensitive multiple protein detection with high reproducibility. 4. Conclusion In this study, we proposed a multiple protein detection method by a sandwich type SERS-based immunoassay with core shell magnetic Au nanoparticles. Two antigens in the mixed solution were successfully separated by the proposed core shell magnetic Au nanoparticles showing a potential application in bioseparation. We have achieved SERS spectra that are proportional to concentration of antigen allowing detection of antigen amounts as low as 800 ag/mL and 5 fg/mL for mouse IgG and human IgG, respectively. Using this proposed sandwich type SERS-based immunoassay with core shell magnetic Au nanoparticles, we can selectively detect the target protein in the mixed antigens solution. Our results suggest that highly sensitive immunoassays are possible using this proposed core shell magnetic Au nanoparticles for quantitative detection of proteins. Therefore, this proposed strategy provides a highly selective and sensitive multiple protein detection method with high reproducibility. Acknowledgments This study supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (MEST) (Nos. 2011-C00052 and 2009-0087013). This study was also supported by 2013 Research Grant from Kangwon National University. The authors thank the Central Laboratory of Kangwon National University for the measurements of Raman spectra. References [1] R. Aroca, Surface-Enhanced Vibrational Spectroscopy, John Wiley & Sons, Ltd., Chichester, 2006.
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