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Ultrasensitive SERS detection of VEGF based on a self-assembled Ag ornamented–AU pyramid superstructure
Biosensors and Bioelectronics 68 (2015) 593–597
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Ultrasensitive SERS detection of VEGF based on a self-assembled Ag ornamented–AU pyramid superstructure Sen Zhao, Wei Ma, Liguang Xu n, Xiaoling Wu, Hua Kuang, Libing Wang, Chuanlai Xu State Key Lab of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, JiangSu 214122, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 4 November 2014 Received in revised form 13 January 2015 Accepted 23 January 2015 Available online 24 January 2015
For the first time, we demonstrated the fabrication of silver nanoparticle ornamented–gold nanoparticle pyramids (Ag–Au Pys) using an aptamer-based self-assembly process and investigated their surfaceenhanced Raman scattering (SERS) properties in the detection of vascular endothelial growth factor (VEGF). Under optimized conditions, the SERS signal was negatively related to VEGF concentration over the range 0.01–1.0 fM and the limit of detection (LOD) was as low as 22.6 aM. The matrix effect and the specificity of this developed method were further examined, and the results showed that the superstructure sensor was ultrasensitive and highly selective. This developed aptamer-based SERS detection method suggests that it may be a promising strategy for a variety of sensing applications. & 2015 Elsevier B.V. All rights reserved.
Keywords: Surface-enhanced Raman scattering Vascular endothelial growth factor Detection Pyramid Self-assembly Ultrasensitive
1. Introduction Cancer is a major disease with high mortality, thus early diagnosis is essential to prevent cancer progression (Wulfkuhle et al., 2003). Vascular endothelial growth factor (VEGF) is a critical growth regulator of both physiologic and pathologic angiogenesis. VEGF stimulates early embryogenesis through vasculogenesis (Freeman et al., 2012) and angiogenesis (Augustin et al., 2009), and plays a crucial role in many diseases, such as lung cancer (Kaya et al., 2004), breast cancer (Carmeliet and Jain, 2000), colorectal cancer (Rugo, 2004), psoriasis (Crawshaw et al., 2012; Prabhulkar et al., 2009), proliferating retinopathy (Ray et al., 2004) and rheumatoid arthritis (Nakahara et al., 2003; Strunk et al., 2013). In addition, VEGF is also germane to age-related macular degeneration (Wang and Si, 2013). Therefore, the vital role of VEGF makes it an important biomarker for human diseases and clinical disorders (Carmeliet and Jain, 2000). To date, a variety of VEGF detection methods have been reported and the traditional method is immunoassay, including immunohistochemistry, radioimmunoassay and others. However, as these techniques are time-consuming, labor-intensive and require expensive reagents and sophisticated instrumentation, they are not ideal for clinical diagnostics. Therefore, the development of new, simple, rapid, inexpensive and sensitive assays is desirable. n
Corresponding author. E-mail address: [email protected] (L. Xu).
http://dx.doi.org/10.1016/j.bios.2015.01.056 0956-5663/& 2015 Elsevier B.V. All rights reserved.
Aptamers are nucleic acid-based molecules or peptide molecules which have high affinity, specificity and selectivity, and can be selected to bind to specific target molecules (Ellington and Szostak, 1990; Tuerk and Gold, 1990). It is well-known that the systematic evolution of ligands by exponential enrichment (SELEX), an efficient method for identifying aptamers, sometimes fails to identify the best aptamer to bind to the target (Nonaka et al., 2013). Fortunately, post-SELEX optimization of aptamers is now available to improve their binding affinity (Nonaka et al., 2013). The post optimization aptamers possess more advantages than antibodies such as chemical synthesis, easy modification, target versatility, high stability and resistance to degradation and denaturation. Moreover, biosensors with various aptamers based on nanomaterials (Guo et al., 2007; Kwon et al., 2010; Wu et al., 2009), which may potentially be designed for a lower concentration of biomolecules, have attracted wide attention (Ma et al., 2014; Zhu et al., 2012). Surface-enhanced Raman scattering (SERS) is a promising technique for chemical and biological sensing and imaging (Alvarez-Puebla and Liz-Marzán, 2010). The characteristic spectral signals are derived from Raman reporters adsorbed around the surface of SERS substrates, and the enhanced intensity is largely associated with the high electromagnetic enhancement in the hot spot between metal nanoparticles (Theiss et al., 2010; Wustholz et al., 2010). In addition, compared with the widely used fluorescence techniques, specific fingerprint spectra can be obtained by lasers of nonspecific wavelength excitation. These advantages
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S. Zhao et al. / Biosensors and Bioelectronics 68 (2015) 593–597
V1: 5′-SH-TTTGCCTGGAGATACATGCACATTACGGCTTTCCCTATTAGAAGGTCTCAGGTGCGCGTTTCGGTAAGTAGACG-3′ V2: 5′-SH-TTTCGCGCACCTGAGACCTTCTAATAGGGTTTCAGTCCACCCCCACAAACTAGAATGCCCTTTGGGCTGTTCCGGG-3′ V3: 5′-SH-TTTGGCCGAGGACTCCTGCTCCGCTGCGGTTTCAGTCCACCCCCACACGTCTACTTACCGTTTCAGTCCACCCCCACACCCGGAACAGCCC-3′ V4: 5′-SH-TTTGCCGTAATGTGCATGTATCTCCAGGCTTTCCGCAGCGGAGCAGGAGTCCTCGGCCTTTGGGCATTCTAGTT-3′ V5: 5′-SH-TTTTTTTGTGGGGGTGGACTGGGTGGGTACC-3′ (italics represent the VEGF165 aptamer) (Nonaka et al., 2013).
2.2. Apparatus
Fig. 1. Scheme for SERS detection of VEGF based on self-assembled Ag ornamented–Au pyramid superstructure.
make SERS a suitable alternative to fluorescence, particularly in bio-sensing detection. Here, a novel Ag ornamented–Au pyramid superstructure comprised of a pyramid-like structure of 10 nm gold nanoparticles and 6 nm Ag nanoparticles was designed. With the advantages of SERS in mind, we assumed that the as-designed superstructure (Fig. 1) may have intense electromagnetic hot-spots and may be used as an ideal SERS substrate. Considering the simplicity and the stability of the superstructure, we chose three Ag NPs ornamented in one Au pyramid structure using the principle of complementary base pairing. In this study, a specific aptamer (Nonaka et al., 2013) for VEGF and its partial complementary sequence was used to assemble Ag ornamented–Au pyramid nanostructures. As shown in Fig. 1, the aptamer modified Ag NPs combined with the pyramid structure in the absence of VEGF. Following the addition of VEGF, the aptamer bound VEGF, resulting in the release of Ag NPs from the scaffold of the Au pyramid. In accordance with our assumption, the SERS intensity had a good relationship with the number of hot-spots between Ag NPs and Au NPs in the pyramid-like superstructure. Given these findings, the concentration of VEGF could be quantified by the SERS intensity.
2. Experimental 2.1. Materials and regents Chloroauric acid, AgNO3, trisodium citrate, 4-nitrothiophenol (4-NTP) and bis (p-sulfonatophenyl) phenylphosphine dihydrate dipotassium salt (BPS) were obtained from Sigma-Aldrich (Shanghai, China). Water used during the procedure was purchased from a Milli-Q device (18.2 MΩ, Millipore, Molsheim, France). Other chemicals such as sodium chloride (NaCl) and 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) were obtained from Shanghai Chemical Reagent Company (Shanghai, China). DNA oligonucleotides purified by high performance liquid chromatography (HPLC) were purchased from Shanghai Sangon Biological Engineering Technology&Services Co. Ltd. (Shanghai, P.R. China) and suspended in deionized (DI) water to a final concentration of 50 mM. The DNA sequences are as follows (Mastroianni et al., 2009):
Transmission electron microscopy images were obtained with a JEOL JEM-2100 transmission electron microscope operating at an acceleration voltage of 200 kV. Assembly sizes were achieved using dynamic light scattering (DLS; Malvern Zetasizer Nano-ZS, 632.8 nm laser). All UV–vis results were acquired on a UNICO 2100PC UV/vis spectrophotometer and processed using Origin Lab software. Raman scattering spectra were measured in a liquid cell using a LabRam-HR800 Micro-Raman spectrometer with Lab-spec 6.0 software. The slit and pinhole were set at 100 mm and 400 mm, respectively. An air-cooled He–Ne laser for 532 nm excitation was used at a power of 8 mW.
2.3. Synthesis and modification of gold and silver nanoparticles Au NPs (10 7 2 nm) were synthesized according to the protocol of Slot (Slot and Geuze, 1985). Phosphine ligand [bis (p-sulfonatophenyl) phenylphosphine dihydrate dipotassium salt (BPS)] was used to ensure that the gold nanoparticles were well-dispersed in high ionic strength solution, following the modified Loweth method (Loweth et al., 1999; Yan et al., 2012). Ag NPs (6 72 nm) were synthesized following a previously described method with modifications (Liu et al., 2009; Sun and Xia, 2003). Previously prepared BPS-coated Au NPs were divided into four equal parts, each part was corresponding modified with thiolated single strand DNA (mole ratio 1:5), respectively, according to a previously reported method (Yan et al., 2012). 4-NTP (final concentration 4 mM) was added to the DNA-modified Ag nanoparticles solution to produce Raman reporter coated Ag nanoparticles.
2.4. Self-assembly of Ag ornamented–AU pyramids Au pyramids were assembled according to the method of Yan (Yan et al., 2012). Ag NP@NTP-Aptamer (VEGF aptamer-modified 4-NTP coated Ag NPs) and the pyramids (mole ratio 3:1) were mixed in Tris–HCl (pH 7.4 50 mM and 50 mM NaCl). The mixture was incubated for 20 h at room temperature with gentle stirring to yield Ag ornamented Au pyramids.
2.5. Detection of VEGF The prepared Ag–Au Pys solution was divided into six equal parts and VEGF was added to the solution to obtain final concentrations of 0 fM, 0.01 fM, 0.05 fM,0.1 fM, 0.5 fM, and 1.0 fM, respectively. After incubation at room temperature for 4 h under gentle shaking, the samples were processed by UV–visible spectrophotometry, TEM, and SERS.
S. Zhao et al. / Biosensors and Bioelectronics 68 (2015) 593–597
3. Results and discussion 3.1. Characterization The gold and silver nanoparticles were characterized by TEM and UV–vis spectrum (Figs. S1 and S2). The diameter of the Au and Ag nanoparticles were 10 72 nm and 6 72 nm, respectively. To estimate the change in diameter of the proposed assemblies, the hydrodynamic sizes of the different states of the assemblies were evaluated using DLS. The typical size distribution is presented in Fig. S3. The DLS data provided a guide for the hydrodynamic diameter of assemblies at different states. The diameter of Ag nanoparticles was measured to be 15 nm, which was larger than the diameter obtained by TEM. Bearing in mind that PVP, a surfactant which maintained the stability of the silver nanoparticles solution, coated the silver nanoparticles, we regarded this result as acceptable. It was not difficult to distinguish the DNA-modified nanoparticles from the bare nanoparticles, using hydrodynamic diameter (Fig. S3). In addition, the diameter of the Ag–Au Pys superstructure was larger than the Au Pys, which is further evidence for the assemblies we proposed. Transmission electron microscopy was also employed to characterize the developed assembly. Fig. S4 shows that the silver nanoparticles ornamented the scaffold of the gold pyramid in a variety of states, which were consistent with the structure we successfully proposed previously. Metallic nanoparticles play an important role in the fabrication of sensitive biosensors due to their localized surface plasmon resonance, which arises from resonant oscillation of the conducting electrons around the metallic nanoparticles. In this study, as shown in Fig. 1, three Ag nanoparticles (6 nm) were ornamented between the Au nanoparticles (10 nm) which served as the scaffold of the superstructure. Considering the length of the DNA sequence, we calculated the gap between the Ag and Au particles to be around 1 nm. Based on our proposal, this intriguing structure included six hot spots, and a Raman scattering signal was generated and enhanced in the hot spot of plasmonic nanostructures. Computer simulation of the
595
E-fields of the developed superstructure was conducted to clarify the origin of the Raman signal (Fig. 2). When the number of Ag nanoparticles ornamented in the pyramid increased (from 0 to 3), the maximum E-fields were significantly enhanced from 3.77 to 5.18. Moreover, the number of enhanced regions was also increased when the quantity of Ag nanoparticles increased. Thus, we applied the proposed structure in the detection of VEGF. 3.2. The scheme and detection of VEGF Fig. 1 illustrates the mechanism of the developed method to detect VEGF. The aptamer modified Ag NPs combined with the pyramid structure in the absence of VEGF. Following the addition of VEGF, the aptamer bound VEGF competitively, leading to the release of Ag NPs from the scaffold of the Au pyramid. In accordance with our assumption, the SERS intensity had a direct relationship with the number of hot spots between Ag NPs and Au NPs in the pyramid-like superstructure. Therefore, the higher the concentration of VEGF, the weaker the Raman intensity became. The solutions with different final concentrations (0, 0.01, 0.05, 0.1, 0.5, and 1.0 fM) of VEGF were characterized by TEM. As seen from the TEM images (Fig. S5), the strong SERS intensity was primarily attributed to the hot spots between the nanoparticles. In addition, the UV–vis spectrum was employed to monitor the aptamer binding to its target, VEGF (Fig. S6). The characteristic peak of silver nanoparticles showed a little blue shift when the VEGF concentration increased and the characteristic peak of gold nanoparticles showed almost no change compared with the original superstructure spectrum. We attributed this phenomenon to the release of Ag nanoparticles from the scaffold of the Au pyramid. Thereafter, the SERS intensity at 1333 cm 1 (Fig. 3A) was chosen to plot the calibration curve based on the logarithmic VEGF concentration. A calibration curve for VEGF detection with an excellent correlation (R2 of 0.9975) was achieved by SERS signals in the range of 0.01–1.0 fM, and the LOD was calculated to be 22.6 aM (Fig. 3B). To date, various detection systems have been developed.
Fig. 2. Representative TEM images (Up) of Ag ornamented–Au pyramids in the state of Ag3–Au Py (A), Ag2–Au Py (B), Ag1–Au Py (C), Au Py (D) and their calculated maps of the E-field (Down). Scale bars were 10 nm.
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Fig. 3. SERS spectra of 4-NTP responses for Ag NPs ornamented Au pyramids-based SERS analysis of VEGF (A). Calibration curve for VEGF detection, the corresponding peak intensities were at 1333 cm 1 (B). The error bars represent the standard deviations of three independent measurements.
Traditionally, the techniques for VEGF detection include Luminex (Albani and Prakken, 2006) and enzyme linked immunosorbent assays (ELISA) (Rifai et al., 2006). However, ELISA is time consuming and labor intensive. The spectral overlap limits the sensitivity of the Luminex method (Mohammad Al-Ameen, 2013). Other analytical platforms proposed for the quantification of VEGF include electrical detection of VEGF using Si nanowire field effect transistors (Lee et al., 2009), rolling circle amplification (Cheng et al., 2010a, 2010b; Suzuki and Yokoyama, 2011), enhanced resonance light scattering (Chen et al. 2012), aptasensor based on fluorescence polarization (Wang et al., 2014), fluorescent peptide conjugated nanopillar chip (Suzuki and Yokoyama, 2011), porosity induced hydrogel microspheres (Mohammad Al-Ameen, 2013) and optical and electrochemical sensors based on an aptasensor (Abe et al., 2012; Cho et al., 2012; Freeman et al., 2012). However, expensive instrumentation is involved in these methods. In addition, low sensitivity and high background signal or noise is often observed. Recently, aptamer-based immunoassay or aptamer-based surface-enhanced Raman scattering detection method has been employed in cancer marker detection or in food hazard factor measuring (He et al., 2011; Ko et al., 2013). The obvious advantages have been presented by SERS in these works. Compared with other assays (Table S1), the superstructure-based sensor developed in our work possesses the highest sensitivity and low background signal.
Au Pys) were assembled using a DNA-driven self-assembly process and an ultrasensitive method for VEGF detection was developed using SERS signals. It was found that the Ag–Au Pys aptasensor exhibited strong SERS signals and the SERS intensity was directly related to the number of Ag NPs in the scaffold of the Au pyramid over the VEGF concentration range of 0.01–1.0 fM. We achieved specific detection of 22.6 aM VEGF165. Moreover, the Ag–Au Pys superstructure developed in this study could be used to detect many other important biomarkers for human diseases and clinical disorders.
Acknowledgments This work is financially supported by the National Natural Science Foundation of China (21371081 and 21301073), the Key Programs from MOST (2012YQ09019410 and 2012BAK17B10), and Grants from Natural Science Foundation of Jiangsu Province, MOF and MOE (BE2013613 and BE2013611).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.01.056.
3.3. Matrix effect and specificity analysis To examine the matrix effect of the proposed method, SERS intensities were obtained after treatment with 0.1 fM and 1.0 fM VEGF in Tris–HCl buffer, 50% cell culture fluid, and 50% fetal bovine serum. The results indicated almost no interference of complex matrices on the developed strategy (Fig. S7A). Moreover, the specificity of the proposed method was further confirmed (Fig. S7B). The aptasensor was challenged with nonspecific biomolecules such as glutamic acid, L-cysteine, glycine, alanine, glucose, human serum albumin, bull serum albumin, immunoglobulin G, alpha fetoprotein, and prostate specific antigen. Fig. S7B shows the aptamer-based sensor following incubation with 10 interference components at high concentration (1 mM) and VEGF (1.0 fM). The results show that no significant SERS intensity change was observed following the addition of nonspecific biomolecules. 4. Conclusions In summary, Ag nanoparticles ornamented–Au pyramids (Ag–
References Abe, K., Hasegawa, H., Ikebukuro, K., 2012. Electrochemistry 80 (5), 348–352. Albani, S., Prakken, B., 2006. Arthritis Rheum. 54 (1), 19–25. Alvarez-Puebla, R.A., Liz-Marzán, L.M., 2010. Small 6 (5), 604–610. Augustin, H.G., Young Koh, G., Thurston, G., Alitalo, K., 2009. Nat. Rev. Mol. Cell. Biol. 10 (3), 165–177. Carmeliet, P., Jain, R.K., 2000. Nature 407 (6801), 249–257. Chen, Z., Lei, Y., Gao, W., Liu, J., 2012. Plasmonics 8 (2), 605–611. Cheng, W., Ding, L., Chen, Y., Yan, F., Ju, H., Yin, Y., 2010a. Chem. Commun. (Camb.) 46 (36), 6720–6722. Cheng, W., Yan, F., Ding, L., Ju, H., Yin, Y., 2010b. Anal. Chem. 82 (8), 3337–3342. Cho, H., Yeh, E.-C., Sinha, R., Laurence, T.A., Bearinger, J.P., Lee, L.P., 2012. ACS Nano 6 (9), 7607–7614. Crawshaw, A., Griffiths, C., Young, H., 2012. Expert Opin. Investig. Drugs 21 (1), 33–43. Ellington, A.D., Szostak, J.W., 1990. Nature 346 (6287), 818–822. Freeman, R., Girsh, J., Jou, A.F.-j, Ho, J.-aA., Hug, T., Dernedde, J., Willner, I., 2012. Anal. Chem. 84 (14), 6192–6198. Guo, K.-T., Scharnweber, D., Schwenzer, B., Ziemer, G., Wendel, H.P., 2007. Biomaterials 28 (3), 468–474. He, L., Lamont, E., Veeregowda, B., Sreevatsan, S., Haynes, C.L., Diez-Gonzalez, F., Labuza, T.P., 2011. Chem. Sci. 2 (8), 1579–1582.
S. Zhao et al. / Biosensors and Bioelectronics 68 (2015) 593–597
Kaya, A., Çiledag, A., Gulbay, B.E., Poyraz, B.M., Çelik, G., Sen, E., Savas, H., Savas, I., 2004. Respir. Med. 98 (7), 632–636. Ko, J., Lee, S., Lee, E.K., Chang, S.-I., Chen, L., Yoon, S.-Y., Choo, J., 2013. Phys. Chem. Chem. Phys. 15 (15), 5379–5385. Kwon, O.S., Park, S.J., Jang, J., 2010. Biomaterials 31 (17), 4740–4747. Lee, H.-S., Kim, K.S., Kim, C.-J., Hahn, S.K., Jo, M.-H., 2009. Biosens. Bioelectron. 24 (6), 1801–1805. Liu, L., Wei, T., Guan, X., Zi, X., He, H., Dai, H., 2009. J. Phys. Chem. C 113 (20), 8595–8600. Loweth, C.J., Caldwell, W.B., Peng, X., Alivisatos, A.P., Schultz, P.G., 1999. Angew. Chem. Int. Ed. 38 (12), 1808–1812. Ma, W., Yin, H., Xu, L., Wu, X., Kuang, H., Wang, L., Xu, C., 2014. Chem. Commun. (Camb.) 50 (68), 9737–9740. Mastroianni, A.J., Claridge, S.A., Alivisatos, A.P., 2009. J. Am. Chem. Soc. 131 (24), 8455–8459. Al-Ameen, G.G., Mohammad Ali, 2013. Biosens. Bioelectron. 49, 105–110. Nakahara, H., Song, J., Sugimoto, M., Hagihara, K., Kishimoto, T., Yoshizaki, K., Nishimoto, N., 2003. Arthritis Rheum. 48 (6), 1521–1529. Nonaka, Y., Yoshida, W., Abe, K., Ferri, S., Schulze, H., Bachmann, T.T., Ikebukuro, K., 2013. Anal. Chem. 85 (2), 1132–1137. Prabhulkar, S., Alwarappan, S., Liu, G., Li, C.-Z., 2009. Biosens. Bioelectron. 24 (12), 3524–3530. Ray, D., Mishra, M., Ralph, S., Read, I., Davies, R., Brenchley, P., 2004. Diabetes 53 (3), 861–864.
597
Rifai, N., Gillette, M.A., Carr, S.A., 2006. Nat. Biotechnol. 24 (8), 971–983. Rugo, H.S., 2004. Oncologist 9 (Suppl. 1), 43S–49S. Slot, J.W., Geuze, H.J., 1985. Eur. J. Cell Biol. 38 (1), 87–93. Strunk, J., Rumbaur, C., Albrecht, K., Neumann, E., Müller-Ladner, U., 2013. Joint Bone Spine 80 (3), 270–273. Sun, Y., Xia, Y., 2003. Adv. Mater. 15 (9), 695–699. Suzuki, Y., Yokoyama, K., 2011. Biosens. Bioelectron. 26 (8), 3696–3699. Theiss, J., Pavaskar, P., Echternach, P.M., Muller, R.E., Cronin, S.B., 2010. Nano Lett. 10 (8), 2749–2754. Tuerk, C., Gold, L., 1990. Science 249 (4968), 505–510. Wang, S.-E., Huang, Y., Hu, K., Tian, J., Zhao, S., 2014. Anal. Methods 6 (1), 62. Wang, S.-E., Si, S., 2013. Appl. Spectrosc. 67 (11), 1270–1274. Wu, Z.-S., Zheng, F., Shen, G.-L., Yu, R.-Q., 2009. Biomaterials 30 (15), 2950–2955. Wulfkuhle, J.D., Liotta, L.A., Petricoin, E.F., 2003. Nat. Rev. Cancer 3 (4), 267–275. Wustholz, K.L., Henry, A.-I., McMahon, J.M., Freeman, R.G., Valley, N., Piotti, M.E., Natan, M.J., Schatz, G.C., Duyne, R.P.V., 2010. J. Am. Chem. Soc. 132 (31), 10903–10910. Yan, W., Xu, L., Xu, C., Ma, W., Kuang, H., Wang, L., Kotov, N.A., 2012. J. Am. Chem. Soc. 134 (36), 15114–15121. Zhu, Y., Xu, L., Ma, W., Xu, Z., Kuang, H., Wang, L., Xu, C., 2012. Chem. Commun. (Camb.) 48 (97), 11889–11891.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Ultrasensitive SERS detection of VEGF based on a self-assembled Ag ornamented–AU pyramid superstructure Sen Zhao, Wei Ma, Liguang Xu n, Xiaoling Wu, Hua Kuang, Libing Wang, Chuanlai Xu State Key Lab of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, JiangSu 214122, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 4 November 2014 Received in revised form 13 January 2015 Accepted 23 January 2015 Available online 24 January 2015
For the first time, we demonstrated the fabrication of silver nanoparticle ornamented–gold nanoparticle pyramids (Ag–Au Pys) using an aptamer-based self-assembly process and investigated their surfaceenhanced Raman scattering (SERS) properties in the detection of vascular endothelial growth factor (VEGF). Under optimized conditions, the SERS signal was negatively related to VEGF concentration over the range 0.01–1.0 fM and the limit of detection (LOD) was as low as 22.6 aM. The matrix effect and the specificity of this developed method were further examined, and the results showed that the superstructure sensor was ultrasensitive and highly selective. This developed aptamer-based SERS detection method suggests that it may be a promising strategy for a variety of sensing applications. & 2015 Elsevier B.V. All rights reserved.
Keywords: Surface-enhanced Raman scattering Vascular endothelial growth factor Detection Pyramid Self-assembly Ultrasensitive
1. Introduction Cancer is a major disease with high mortality, thus early diagnosis is essential to prevent cancer progression (Wulfkuhle et al., 2003). Vascular endothelial growth factor (VEGF) is a critical growth regulator of both physiologic and pathologic angiogenesis. VEGF stimulates early embryogenesis through vasculogenesis (Freeman et al., 2012) and angiogenesis (Augustin et al., 2009), and plays a crucial role in many diseases, such as lung cancer (Kaya et al., 2004), breast cancer (Carmeliet and Jain, 2000), colorectal cancer (Rugo, 2004), psoriasis (Crawshaw et al., 2012; Prabhulkar et al., 2009), proliferating retinopathy (Ray et al., 2004) and rheumatoid arthritis (Nakahara et al., 2003; Strunk et al., 2013). In addition, VEGF is also germane to age-related macular degeneration (Wang and Si, 2013). Therefore, the vital role of VEGF makes it an important biomarker for human diseases and clinical disorders (Carmeliet and Jain, 2000). To date, a variety of VEGF detection methods have been reported and the traditional method is immunoassay, including immunohistochemistry, radioimmunoassay and others. However, as these techniques are time-consuming, labor-intensive and require expensive reagents and sophisticated instrumentation, they are not ideal for clinical diagnostics. Therefore, the development of new, simple, rapid, inexpensive and sensitive assays is desirable. n
Corresponding author. E-mail address: [email protected] (L. Xu).
http://dx.doi.org/10.1016/j.bios.2015.01.056 0956-5663/& 2015 Elsevier B.V. All rights reserved.
Aptamers are nucleic acid-based molecules or peptide molecules which have high affinity, specificity and selectivity, and can be selected to bind to specific target molecules (Ellington and Szostak, 1990; Tuerk and Gold, 1990). It is well-known that the systematic evolution of ligands by exponential enrichment (SELEX), an efficient method for identifying aptamers, sometimes fails to identify the best aptamer to bind to the target (Nonaka et al., 2013). Fortunately, post-SELEX optimization of aptamers is now available to improve their binding affinity (Nonaka et al., 2013). The post optimization aptamers possess more advantages than antibodies such as chemical synthesis, easy modification, target versatility, high stability and resistance to degradation and denaturation. Moreover, biosensors with various aptamers based on nanomaterials (Guo et al., 2007; Kwon et al., 2010; Wu et al., 2009), which may potentially be designed for a lower concentration of biomolecules, have attracted wide attention (Ma et al., 2014; Zhu et al., 2012). Surface-enhanced Raman scattering (SERS) is a promising technique for chemical and biological sensing and imaging (Alvarez-Puebla and Liz-Marzán, 2010). The characteristic spectral signals are derived from Raman reporters adsorbed around the surface of SERS substrates, and the enhanced intensity is largely associated with the high electromagnetic enhancement in the hot spot between metal nanoparticles (Theiss et al., 2010; Wustholz et al., 2010). In addition, compared with the widely used fluorescence techniques, specific fingerprint spectra can be obtained by lasers of nonspecific wavelength excitation. These advantages
594
S. Zhao et al. / Biosensors and Bioelectronics 68 (2015) 593–597
V1: 5′-SH-TTTGCCTGGAGATACATGCACATTACGGCTTTCCCTATTAGAAGGTCTCAGGTGCGCGTTTCGGTAAGTAGACG-3′ V2: 5′-SH-TTTCGCGCACCTGAGACCTTCTAATAGGGTTTCAGTCCACCCCCACAAACTAGAATGCCCTTTGGGCTGTTCCGGG-3′ V3: 5′-SH-TTTGGCCGAGGACTCCTGCTCCGCTGCGGTTTCAGTCCACCCCCACACGTCTACTTACCGTTTCAGTCCACCCCCACACCCGGAACAGCCC-3′ V4: 5′-SH-TTTGCCGTAATGTGCATGTATCTCCAGGCTTTCCGCAGCGGAGCAGGAGTCCTCGGCCTTTGGGCATTCTAGTT-3′ V5: 5′-SH-TTTTTTTGTGGGGGTGGACTGGGTGGGTACC-3′ (italics represent the VEGF165 aptamer) (Nonaka et al., 2013).
2.2. Apparatus
Fig. 1. Scheme for SERS detection of VEGF based on self-assembled Ag ornamented–Au pyramid superstructure.
make SERS a suitable alternative to fluorescence, particularly in bio-sensing detection. Here, a novel Ag ornamented–Au pyramid superstructure comprised of a pyramid-like structure of 10 nm gold nanoparticles and 6 nm Ag nanoparticles was designed. With the advantages of SERS in mind, we assumed that the as-designed superstructure (Fig. 1) may have intense electromagnetic hot-spots and may be used as an ideal SERS substrate. Considering the simplicity and the stability of the superstructure, we chose three Ag NPs ornamented in one Au pyramid structure using the principle of complementary base pairing. In this study, a specific aptamer (Nonaka et al., 2013) for VEGF and its partial complementary sequence was used to assemble Ag ornamented–Au pyramid nanostructures. As shown in Fig. 1, the aptamer modified Ag NPs combined with the pyramid structure in the absence of VEGF. Following the addition of VEGF, the aptamer bound VEGF, resulting in the release of Ag NPs from the scaffold of the Au pyramid. In accordance with our assumption, the SERS intensity had a good relationship with the number of hot-spots between Ag NPs and Au NPs in the pyramid-like superstructure. Given these findings, the concentration of VEGF could be quantified by the SERS intensity.
2. Experimental 2.1. Materials and regents Chloroauric acid, AgNO3, trisodium citrate, 4-nitrothiophenol (4-NTP) and bis (p-sulfonatophenyl) phenylphosphine dihydrate dipotassium salt (BPS) were obtained from Sigma-Aldrich (Shanghai, China). Water used during the procedure was purchased from a Milli-Q device (18.2 MΩ, Millipore, Molsheim, France). Other chemicals such as sodium chloride (NaCl) and 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) were obtained from Shanghai Chemical Reagent Company (Shanghai, China). DNA oligonucleotides purified by high performance liquid chromatography (HPLC) were purchased from Shanghai Sangon Biological Engineering Technology&Services Co. Ltd. (Shanghai, P.R. China) and suspended in deionized (DI) water to a final concentration of 50 mM. The DNA sequences are as follows (Mastroianni et al., 2009):
Transmission electron microscopy images were obtained with a JEOL JEM-2100 transmission electron microscope operating at an acceleration voltage of 200 kV. Assembly sizes were achieved using dynamic light scattering (DLS; Malvern Zetasizer Nano-ZS, 632.8 nm laser). All UV–vis results were acquired on a UNICO 2100PC UV/vis spectrophotometer and processed using Origin Lab software. Raman scattering spectra were measured in a liquid cell using a LabRam-HR800 Micro-Raman spectrometer with Lab-spec 6.0 software. The slit and pinhole were set at 100 mm and 400 mm, respectively. An air-cooled He–Ne laser for 532 nm excitation was used at a power of 8 mW.
2.3. Synthesis and modification of gold and silver nanoparticles Au NPs (10 7 2 nm) were synthesized according to the protocol of Slot (Slot and Geuze, 1985). Phosphine ligand [bis (p-sulfonatophenyl) phenylphosphine dihydrate dipotassium salt (BPS)] was used to ensure that the gold nanoparticles were well-dispersed in high ionic strength solution, following the modified Loweth method (Loweth et al., 1999; Yan et al., 2012). Ag NPs (6 72 nm) were synthesized following a previously described method with modifications (Liu et al., 2009; Sun and Xia, 2003). Previously prepared BPS-coated Au NPs were divided into four equal parts, each part was corresponding modified with thiolated single strand DNA (mole ratio 1:5), respectively, according to a previously reported method (Yan et al., 2012). 4-NTP (final concentration 4 mM) was added to the DNA-modified Ag nanoparticles solution to produce Raman reporter coated Ag nanoparticles.
2.4. Self-assembly of Ag ornamented–AU pyramids Au pyramids were assembled according to the method of Yan (Yan et al., 2012). Ag NP@NTP-Aptamer (VEGF aptamer-modified 4-NTP coated Ag NPs) and the pyramids (mole ratio 3:1) were mixed in Tris–HCl (pH 7.4 50 mM and 50 mM NaCl). The mixture was incubated for 20 h at room temperature with gentle stirring to yield Ag ornamented Au pyramids.
2.5. Detection of VEGF The prepared Ag–Au Pys solution was divided into six equal parts and VEGF was added to the solution to obtain final concentrations of 0 fM, 0.01 fM, 0.05 fM,0.1 fM, 0.5 fM, and 1.0 fM, respectively. After incubation at room temperature for 4 h under gentle shaking, the samples were processed by UV–visible spectrophotometry, TEM, and SERS.
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3. Results and discussion 3.1. Characterization The gold and silver nanoparticles were characterized by TEM and UV–vis spectrum (Figs. S1 and S2). The diameter of the Au and Ag nanoparticles were 10 72 nm and 6 72 nm, respectively. To estimate the change in diameter of the proposed assemblies, the hydrodynamic sizes of the different states of the assemblies were evaluated using DLS. The typical size distribution is presented in Fig. S3. The DLS data provided a guide for the hydrodynamic diameter of assemblies at different states. The diameter of Ag nanoparticles was measured to be 15 nm, which was larger than the diameter obtained by TEM. Bearing in mind that PVP, a surfactant which maintained the stability of the silver nanoparticles solution, coated the silver nanoparticles, we regarded this result as acceptable. It was not difficult to distinguish the DNA-modified nanoparticles from the bare nanoparticles, using hydrodynamic diameter (Fig. S3). In addition, the diameter of the Ag–Au Pys superstructure was larger than the Au Pys, which is further evidence for the assemblies we proposed. Transmission electron microscopy was also employed to characterize the developed assembly. Fig. S4 shows that the silver nanoparticles ornamented the scaffold of the gold pyramid in a variety of states, which were consistent with the structure we successfully proposed previously. Metallic nanoparticles play an important role in the fabrication of sensitive biosensors due to their localized surface plasmon resonance, which arises from resonant oscillation of the conducting electrons around the metallic nanoparticles. In this study, as shown in Fig. 1, three Ag nanoparticles (6 nm) were ornamented between the Au nanoparticles (10 nm) which served as the scaffold of the superstructure. Considering the length of the DNA sequence, we calculated the gap between the Ag and Au particles to be around 1 nm. Based on our proposal, this intriguing structure included six hot spots, and a Raman scattering signal was generated and enhanced in the hot spot of plasmonic nanostructures. Computer simulation of the
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E-fields of the developed superstructure was conducted to clarify the origin of the Raman signal (Fig. 2). When the number of Ag nanoparticles ornamented in the pyramid increased (from 0 to 3), the maximum E-fields were significantly enhanced from 3.77 to 5.18. Moreover, the number of enhanced regions was also increased when the quantity of Ag nanoparticles increased. Thus, we applied the proposed structure in the detection of VEGF. 3.2. The scheme and detection of VEGF Fig. 1 illustrates the mechanism of the developed method to detect VEGF. The aptamer modified Ag NPs combined with the pyramid structure in the absence of VEGF. Following the addition of VEGF, the aptamer bound VEGF competitively, leading to the release of Ag NPs from the scaffold of the Au pyramid. In accordance with our assumption, the SERS intensity had a direct relationship with the number of hot spots between Ag NPs and Au NPs in the pyramid-like superstructure. Therefore, the higher the concentration of VEGF, the weaker the Raman intensity became. The solutions with different final concentrations (0, 0.01, 0.05, 0.1, 0.5, and 1.0 fM) of VEGF were characterized by TEM. As seen from the TEM images (Fig. S5), the strong SERS intensity was primarily attributed to the hot spots between the nanoparticles. In addition, the UV–vis spectrum was employed to monitor the aptamer binding to its target, VEGF (Fig. S6). The characteristic peak of silver nanoparticles showed a little blue shift when the VEGF concentration increased and the characteristic peak of gold nanoparticles showed almost no change compared with the original superstructure spectrum. We attributed this phenomenon to the release of Ag nanoparticles from the scaffold of the Au pyramid. Thereafter, the SERS intensity at 1333 cm 1 (Fig. 3A) was chosen to plot the calibration curve based on the logarithmic VEGF concentration. A calibration curve for VEGF detection with an excellent correlation (R2 of 0.9975) was achieved by SERS signals in the range of 0.01–1.0 fM, and the LOD was calculated to be 22.6 aM (Fig. 3B). To date, various detection systems have been developed.
Fig. 2. Representative TEM images (Up) of Ag ornamented–Au pyramids in the state of Ag3–Au Py (A), Ag2–Au Py (B), Ag1–Au Py (C), Au Py (D) and their calculated maps of the E-field (Down). Scale bars were 10 nm.
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Fig. 3. SERS spectra of 4-NTP responses for Ag NPs ornamented Au pyramids-based SERS analysis of VEGF (A). Calibration curve for VEGF detection, the corresponding peak intensities were at 1333 cm 1 (B). The error bars represent the standard deviations of three independent measurements.
Traditionally, the techniques for VEGF detection include Luminex (Albani and Prakken, 2006) and enzyme linked immunosorbent assays (ELISA) (Rifai et al., 2006). However, ELISA is time consuming and labor intensive. The spectral overlap limits the sensitivity of the Luminex method (Mohammad Al-Ameen, 2013). Other analytical platforms proposed for the quantification of VEGF include electrical detection of VEGF using Si nanowire field effect transistors (Lee et al., 2009), rolling circle amplification (Cheng et al., 2010a, 2010b; Suzuki and Yokoyama, 2011), enhanced resonance light scattering (Chen et al. 2012), aptasensor based on fluorescence polarization (Wang et al., 2014), fluorescent peptide conjugated nanopillar chip (Suzuki and Yokoyama, 2011), porosity induced hydrogel microspheres (Mohammad Al-Ameen, 2013) and optical and electrochemical sensors based on an aptasensor (Abe et al., 2012; Cho et al., 2012; Freeman et al., 2012). However, expensive instrumentation is involved in these methods. In addition, low sensitivity and high background signal or noise is often observed. Recently, aptamer-based immunoassay or aptamer-based surface-enhanced Raman scattering detection method has been employed in cancer marker detection or in food hazard factor measuring (He et al., 2011; Ko et al., 2013). The obvious advantages have been presented by SERS in these works. Compared with other assays (Table S1), the superstructure-based sensor developed in our work possesses the highest sensitivity and low background signal.
Au Pys) were assembled using a DNA-driven self-assembly process and an ultrasensitive method for VEGF detection was developed using SERS signals. It was found that the Ag–Au Pys aptasensor exhibited strong SERS signals and the SERS intensity was directly related to the number of Ag NPs in the scaffold of the Au pyramid over the VEGF concentration range of 0.01–1.0 fM. We achieved specific detection of 22.6 aM VEGF165. Moreover, the Ag–Au Pys superstructure developed in this study could be used to detect many other important biomarkers for human diseases and clinical disorders.
Acknowledgments This work is financially supported by the National Natural Science Foundation of China (21371081 and 21301073), the Key Programs from MOST (2012YQ09019410 and 2012BAK17B10), and Grants from Natural Science Foundation of Jiangsu Province, MOF and MOE (BE2013613 and BE2013611).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.01.056.
3.3. Matrix effect and specificity analysis To examine the matrix effect of the proposed method, SERS intensities were obtained after treatment with 0.1 fM and 1.0 fM VEGF in Tris–HCl buffer, 50% cell culture fluid, and 50% fetal bovine serum. The results indicated almost no interference of complex matrices on the developed strategy (Fig. S7A). Moreover, the specificity of the proposed method was further confirmed (Fig. S7B). The aptasensor was challenged with nonspecific biomolecules such as glutamic acid, L-cysteine, glycine, alanine, glucose, human serum albumin, bull serum albumin, immunoglobulin G, alpha fetoprotein, and prostate specific antigen. Fig. S7B shows the aptamer-based sensor following incubation with 10 interference components at high concentration (1 mM) and VEGF (1.0 fM). The results show that no significant SERS intensity change was observed following the addition of nonspecific biomolecules. 4. Conclusions In summary, Ag nanoparticles ornamented–Au pyramids (Ag–
References Abe, K., Hasegawa, H., Ikebukuro, K., 2012. Electrochemistry 80 (5), 348–352. Albani, S., Prakken, B., 2006. Arthritis Rheum. 54 (1), 19–25. Alvarez-Puebla, R.A., Liz-Marzán, L.M., 2010. Small 6 (5), 604–610. Augustin, H.G., Young Koh, G., Thurston, G., Alitalo, K., 2009. Nat. Rev. Mol. Cell. Biol. 10 (3), 165–177. Carmeliet, P., Jain, R.K., 2000. Nature 407 (6801), 249–257. Chen, Z., Lei, Y., Gao, W., Liu, J., 2012. Plasmonics 8 (2), 605–611. Cheng, W., Ding, L., Chen, Y., Yan, F., Ju, H., Yin, Y., 2010a. Chem. Commun. (Camb.) 46 (36), 6720–6722. Cheng, W., Yan, F., Ding, L., Ju, H., Yin, Y., 2010b. Anal. Chem. 82 (8), 3337–3342. Cho, H., Yeh, E.-C., Sinha, R., Laurence, T.A., Bearinger, J.P., Lee, L.P., 2012. ACS Nano 6 (9), 7607–7614. Crawshaw, A., Griffiths, C., Young, H., 2012. Expert Opin. Investig. Drugs 21 (1), 33–43. Ellington, A.D., Szostak, J.W., 1990. Nature 346 (6287), 818–822. Freeman, R., Girsh, J., Jou, A.F.-j, Ho, J.-aA., Hug, T., Dernedde, J., Willner, I., 2012. Anal. Chem. 84 (14), 6192–6198. Guo, K.-T., Scharnweber, D., Schwenzer, B., Ziemer, G., Wendel, H.P., 2007. Biomaterials 28 (3), 468–474. He, L., Lamont, E., Veeregowda, B., Sreevatsan, S., Haynes, C.L., Diez-Gonzalez, F., Labuza, T.P., 2011. Chem. Sci. 2 (8), 1579–1582.
S. Zhao et al. / Biosensors and Bioelectronics 68 (2015) 593–597
Kaya, A., Çiledag, A., Gulbay, B.E., Poyraz, B.M., Çelik, G., Sen, E., Savas, H., Savas, I., 2004. Respir. Med. 98 (7), 632–636. Ko, J., Lee, S., Lee, E.K., Chang, S.-I., Chen, L., Yoon, S.-Y., Choo, J., 2013. Phys. Chem. Chem. Phys. 15 (15), 5379–5385. Kwon, O.S., Park, S.J., Jang, J., 2010. Biomaterials 31 (17), 4740–4747. Lee, H.-S., Kim, K.S., Kim, C.-J., Hahn, S.K., Jo, M.-H., 2009. Biosens. Bioelectron. 24 (6), 1801–1805. Liu, L., Wei, T., Guan, X., Zi, X., He, H., Dai, H., 2009. J. Phys. Chem. C 113 (20), 8595–8600. Loweth, C.J., Caldwell, W.B., Peng, X., Alivisatos, A.P., Schultz, P.G., 1999. Angew. Chem. Int. Ed. 38 (12), 1808–1812. Ma, W., Yin, H., Xu, L., Wu, X., Kuang, H., Wang, L., Xu, C., 2014. Chem. Commun. (Camb.) 50 (68), 9737–9740. Mastroianni, A.J., Claridge, S.A., Alivisatos, A.P., 2009. J. Am. Chem. Soc. 131 (24), 8455–8459. Al-Ameen, G.G., Mohammad Ali, 2013. Biosens. Bioelectron. 49, 105–110. Nakahara, H., Song, J., Sugimoto, M., Hagihara, K., Kishimoto, T., Yoshizaki, K., Nishimoto, N., 2003. Arthritis Rheum. 48 (6), 1521–1529. Nonaka, Y., Yoshida, W., Abe, K., Ferri, S., Schulze, H., Bachmann, T.T., Ikebukuro, K., 2013. Anal. Chem. 85 (2), 1132–1137. Prabhulkar, S., Alwarappan, S., Liu, G., Li, C.-Z., 2009. Biosens. Bioelectron. 24 (12), 3524–3530. Ray, D., Mishra, M., Ralph, S., Read, I., Davies, R., Brenchley, P., 2004. Diabetes 53 (3), 861–864.
597
Rifai, N., Gillette, M.A., Carr, S.A., 2006. Nat. Biotechnol. 24 (8), 971–983. Rugo, H.S., 2004. Oncologist 9 (Suppl. 1), 43S–49S. Slot, J.W., Geuze, H.J., 1985. Eur. J. Cell Biol. 38 (1), 87–93. Strunk, J., Rumbaur, C., Albrecht, K., Neumann, E., Müller-Ladner, U., 2013. Joint Bone Spine 80 (3), 270–273. Sun, Y., Xia, Y., 2003. Adv. Mater. 15 (9), 695–699. Suzuki, Y., Yokoyama, K., 2011. Biosens. Bioelectron. 26 (8), 3696–3699. Theiss, J., Pavaskar, P., Echternach, P.M., Muller, R.E., Cronin, S.B., 2010. Nano Lett. 10 (8), 2749–2754. Tuerk, C., Gold, L., 1990. Science 249 (4968), 505–510. Wang, S.-E., Huang, Y., Hu, K., Tian, J., Zhao, S., 2014. Anal. Methods 6 (1), 62. Wang, S.-E., Si, S., 2013. Appl. Spectrosc. 67 (11), 1270–1274. Wu, Z.-S., Zheng, F., Shen, G.-L., Yu, R.-Q., 2009. Biomaterials 30 (15), 2950–2955. Wulfkuhle, J.D., Liotta, L.A., Petricoin, E.F., 2003. Nat. Rev. Cancer 3 (4), 267–275. Wustholz, K.L., Henry, A.-I., McMahon, J.M., Freeman, R.G., Valley, N., Piotti, M.E., Natan, M.J., Schatz, G.C., Duyne, R.P.V., 2010. J. Am. Chem. Soc. 132 (31), 10903–10910. Yan, W., Xu, L., Xu, C., Ma, W., Kuang, H., Wang, L., Kotov, N.A., 2012. J. Am. Chem. Soc. 134 (36), 15114–15121. Zhu, Y., Xu, L., Ma, W., Xu, Z., Kuang, H., Wang, L., Xu, C., 2012. Chem. Commun. (Camb.) 48 (97), 11889–11891.