Synthesis and Characterization of Silver Nanoparticle

Analytical Letters

ISSN: 0003-2719 (Print) 1532-236X (Online) Journal homepage: http://www.tandfonline.com/loi/lanl20

Synthesis and Characterization of Silver Nanoparticle Modified 3-Aminophenol Resin Microspheres with Application for Determination of Carcinoembryonic Antigens by SurfaceEnhanced Raman Scattering Lin Tao, Wenbo Lu, Man Wang, Ying Wang, Jian Dong & Weiping Qian To cite this article: Lin Tao, Wenbo Lu, Man Wang, Ying Wang, Jian Dong & Weiping Qian (2015) Synthesis and Characterization of Silver Nanoparticle Modified 3-Aminophenol Resin Microspheres with Application for Determination of Carcinoembryonic Antigens by Surface-Enhanced Raman Scattering, Analytical Letters, 48:14, 2245-2257, DOI: 10.1080/00032719.2015.1027899 To link to this article: http://dx.doi.org/10.1080/00032719.2015.1027899

Accepted author version posted online: 14 Jun 2015.

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Date: 23 October 2015, At: 18:43

Analytical Letters, 48: 2245–2257, 2015 Copyright # Taylor & Francis Group, LLC ISSN: 0003-2719 print/1532-236X online DOI: 10.1080/00032719.2015.1027899

Biosensors

Downloaded by [Fudan University] at 18:43 23 October 2015

SYNTHESIS AND CHARACTERIZATION OF SILVER NANOPARTICLE MODIFIED 3-AMINOPHENOL RESIN MICROSPHERES WITH APPLICATION FOR DETERMINATION OF CARCINOEMBRYONIC ANTIGENS BY SURFACE-ENHANCED RAMAN SCATTERING Lin Tao, Wenbo Lu, Man Wang, Ying Wang, Jian Dong, and Weiping Qian State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, PR China Uniform phenolic resin microspheres were prepared by the polycondensation of 3-aminophenol and formaldehyde. On the surface of the 3-aminophenol resin microspheres, silver nanoparticles were synthesized in situ and immobilized by simple heating. The composite was employed as a substrate for surface-enhanced Raman scattering (SERS). The SERS enhancement factor was evaluated using 4-mercaptobenzoic acid and Nile blue A as signal molecules. A highly sensitive SERS immunoassay that combined labeled antibody conjugated silver nanoparticle modified 3-aminophenol resin microspheres and coating antibody conjugated magnetic nanoparticles was fabricated to determine carcinoembryonic antigen. A linear relationship was obtained between the Raman intensity and the concentration of carcinoembryonic antigen. The limit of detection was 1.2 picograms per milliliter at a signal-to-noise ratio of three. This is believed to be the first report of a SERS immunoassay using silver nanoparticle modified 3-aminophenol resin microspheres as substrates. Keywords: Carcinoembryonic antigen; Immunoassay; Phenolic resin microspheres; Surface-enhanced Raman scattering

INTRODUCTION Lung cancer is the most common form of the disease accounting for approximately 13 percent of deaths each year (M. Yang and Gong 2010; Arya and Bhansali 2011), ranking it second in cancer related deaths. Consequently, an effective strategy to conduct clinical cancer screening, disease diagnosis, and monitoring is of great Received 20 October 2014; accepted 1 March 2015. Address correspondence to Weiping Qian, State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Sipailou #2, Nanjing, 210096, PR China. E-mail: [email protected] Color versions of one or more of the figures in this article can be found online at www.tandfonline. com/lanl. 2245


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significance (M. Yang and Gong 2010; Wan et al. 2013). A biomarker is a substance that can be measured in serum or tissue, and may be of diagnostic, prognostic, or predictive value to cancer (Grunnet and Sorensen 2012). The most commonly used lung cancer associated biomarkers include carcinoembryonic antigen, neuronspecific enolase, and squamous cell carcinoma antigen. Carcinoembryonic antigen is a glycoprotein involved in cell adhesion, and some researchers have reported it to be a prognostic marker in lung cancer (Grunnet and Sorensen 2012). Conventional approaches for biomarker detection include enzyme-linked immunosorbent assays (ELISA) (Ambrosi, Airo, and Merkoci 2010), radioimmunoassays (Grange, Thompson, and Lambert 2014), mass spectrometry (K.-Y. Wang et al. 2008), and electrochemical methods (Lu et al. 2014). However, these techniques suffer from drawbacks such as time-consuming analysis, laboratory-based protocols, or the requirement of experienced personnel. Surfaceenhanced Raman scattering (SERS) is a powerful technique for chemical and biological sensing (Penn, Drake, and Driskel 2013; Y. Wang, Yan, and Chen 2013) that provides spectral fingerprints of analytes with few interferences in complex biological materials (He et al. 2012). Recently, SERS based immunoassays have been reported that employed enhanced substrates such as aggregates of gold nanostars (Pei et al. 2013), flower-like gold nanoparticles (Song et al. 2014), and Au@SiO2 core/shell nanorods (Quyen et al. 2014). Silver provides higher SERS enhancement compared to gold (Song et al. 2011), so an easily prepared silver nanoparticle substrate with highly stability and good enhancement factor is significant. Phenolic resins were recently applied in cell imaging (Guo et al. 2008), photothermal therapy (P. Yang et al. 2012), and gas adsorption (J. Liu et al. 2011) because of their excellent biocompatibility, low toxicity, and porosity. In our previous reports (Lu et al. 2014), an electrochemical immunoassay based on gold nanoparticles loaded with biomolecule-based formaldehyde resin microspheres was reported. The silver nanoparticles were prepared without additional reductant on the surface of 3-aminophenol resin microspheres. The rough silver nanoparticle modified 3-aminophenol resin microspheres were considered to be appropriate for a SERS substrate. The SERS enhancement factor was evaluated by using 4-mercaptobenzoic acid and Nile blue A as reporter molecules. A highly sensitive SERS immunoassay was developed for carcinoembryonic antigen detection.

MATERIAL AND METHODS Materials The 3-Aminophenol, ammonium hydroxide (28 wt% in water), silver nitrate, formaldehyde (37 wt%), glutaraldehyde (40 wt%), and bovine serum albumin were purchased from Aladin (Shanghai, China). NaH2PO4, Na2HPO4, and ethanol were purchased from Beijing Chemical Reagent (Beijing, China). Phosphate buffer saline was prepared by mixing stock solutions of NaH2PO4 and Na2HPO4. Carcinoembryonic standard grade antigens, and anti-carcinoembryonic antibodies I and II were purchased from Linc-Bio Science Shanghai, China). The water used throughout all

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experiments was purified through a Millipore system. All chemicals were used as received without further purification.


Instruments Scanning electron microscopy (SEM) measurements were obtained on an Xl30 Esem Feg instrument at an accelerating potential of 20 kilovolts. Fourier transform infrared spectroscopy (FT-IR) measurements were made on a Tensor 27 (Bruker Optik GmbH, Ettlingen, Germany). The Raman experiments were collected with a Renishaw Invia Reflex system equipped with Peltier-cooled charge-coupled device detectors and a Leica microscope. Samples were excited with a diode laser (785 nanometers) with a spot size of approximately 2 millimeters and power density of approximately 3 × 104 watts per square centimeter. In each sample, five random spots were selected for Raman and the signals were averaged. Synthesis of 3-Aminophenol Resin Microspheres The 0.0828 gram 3-aminophenol were dissolved in 12 milliliters ethanol and 4.8 milliliters distilled water. After adding 56 microliters ammonium hydroxide solution and reacting for thirty minutes at 30 degree Celsius, 58 microliters formaldehyde solution were added and reacted for four hours at 30 degree Celsius. The resulting suspension was autoclaved in a continuous reaction at 110 degree Celsius for twenty-four hours. The synthetic resin microspheres were rinsed twice with distilled water and ethanol by centrifugation at 5,000 revolutions per minute. Preparation of Silver Nanoparticles Modified 3-Aminophenol Resin The 100 microliters of 3-aminophenol resin microspheres were dispersed into 2.9 milliliters of distilled water and 3 milliliters of 0.1 molar silver nitrate were added and reacted for thirty minutes at 90 degree Celsius. The synthetic silver nanoparticle modified 3-aminophenol resin microspheres were purified rinsed twice with distilled water and ethanol by centrifugation at 4,000 revolutions per minute. Construction of SERS Immunoassay for Carcinoembryonic Antigen Detection Construction of the coating probe. Magnetic nanoparticles were obtained through a solvothermal method as described by Zhai et al. (2009). Briefly, ferric sulfate and manganese sulfate were dissolved in ethylene glycol, followed by the addition of anhydrous sodium acetate and polyvinyl pyrrolidone. After stirring for thirty minutes, the clear solution was sealed in a Teflon-lined autoclave and reacted at 200 degree Celsius for eight hours. The magnetic nanoparticles were washed with distilled water and ethanol twice and 50 microliters of the paramagnetic nanoparticles were dispersed in 450 microliters distilled water and coincubated with 500 microliters glutaraldehyde (2 percent, wt) for thirty minutes and washed to remove the remaining glutaraldehyde. The glutaraldehyde-decorated

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paramagnetic nanoparticles were redispersed in 500 microliters of pH 7.5 phosphate buffer saline and coincubated with 20 microliters anti-carcinoembryonic antibodies I (10 micrograms per milliliter) for one hour. The modified paramagnetic nanoparticles were sealed with bovine serum albumin (1 percent, wt) to cover the positive antibody site. Construction of the labeling probe. The 50 microliters of silver nanoparticle modified 3-aminophenol resin microspheres were dispersed in 500 microliters distilled water and coincubated with 50 microliters of 105 moles per liter Nile blue A aqueous solution for thirty minutes. The Nile blue A adsorbed silver nanoparticle modified 3-aminophenol resin microspheres were redispersed in 500 microliters of pH 7.5 phosphate buffer saline and coincubated with 20 microliters carcinoembryonic antigen labeling antibody (10 micrograms per milliliter) for one hour. The silver nanoparticle modified 3-aminophenol resin microspheres were sealed with bovine serum albumin (1 percent, wt) to cover the positive antibody site. Detection of carcinoembryonic antigen in phosphate buffer saline. Equivalent amounts of coating and labeling probes were mixed to form 1000 microliters of solution. The following volumes of carcinoembryonic antigen antigens were added: 100 nanograms per milliliter, 10 nanograms per milliliter, 1 nanogram per milliliter, 100 picograms per meter, and 10 picograms per meter. After coincubated for two hours, the materials were isolated by a magnet, washed with phosphate buffer saline, deposited on clear quartz glass, and dried at room temperature for Raman analysis. RESULTS AND DISCUSSION Figure 1 shows the scanning electron micrographs of the 3-aminophenol resin microspheres, demonstrating regular spherical morphology and uniform size distribution with diameters of 300–400 nanometers. Figure 1D shows a fluffy surface that may be attributed to the branched chains of amino and hydroxyl groups. These functional groups derived from 3-aminophenol and formaldehyde are crucial for the synthesis of silver nanoparticles. Chemical reduction is the most frequently applied method for the preparation of silver nanoparticles. The commonly used reductants include borohydride, citrate, ascorbate, and elemental hydrogen (El-Nour et al. 2010). However, using additional reductant cannot eliminate impurities. Due to the functional groups of 3-aminophenol resin microspheres, Agþ was reduced to silver nanoparticles with a simple procedure using reductant-free condition. Figure 2 shows scanning electron micrographs of products obtained with different molar ratios of silver nitrate and 3-aminophenol. The silver nanoparticles were more dense with increasing amounts of silver nitrate. When the amount of silver nitrate was low, the nanoparticles were preferentially reduced and immobilized in sites of the 3-aminophenol resin microspheres where more functional groups were present. With increasing silver nitrate concentration, other parts of the resin microsphere participated in the reaction and formed a layer of rough silver nanoparticles. Surface plasmons may interact strongly with incident electromagnetic fields, because collective oscillations of electrons are confined to a metal/dielectric boundary



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Figure 1. Representative scanning electron micrographs of 3-aminophenol resin microspheres: (A) 10 micrometers, (B) 1 micrometer, (C) 500 nanometers, and (D) 200 nanometers.

such as a metal nanoparticle surface. The surface plasmon behavior of metal nanoparticles is directly influenced by the nanoparticle structure, shape, size, and material that determine electron confinement by the metal (Khoury and Vo-Dinh 2008; Li, Ma, and

Figure 2. Scanning electron micrographs of silver nanoparticle modified 3-aminophenol using (A) 1:1, (B) 2:1, (C) 4:1, and (D) 8:1 molar ratios of silver nitrate and 3-aminophenol.


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Ma 2013). This property affects the optical properties of the nanoparticles, which can be characterized by ultraviolet-visible absorption. Figure 3 shows absorption spectra of the 3-aminophenol resin microspheres and the silver nanoparticle modified 3-aminophenol resin microsphere dispersion. No characteristic peak is observed in the 3-aminophenol resin microspheres (Figure 3a). When the silver nanoparticles were immobilized on the surface of 3-aminophenol resin microspheres, a peak at 534 nanometers was observed. As the concentration of silver increased, the surface plasmon bands of the silver nanoparticle modified 3-aminophenol resin microspheres exhibited a red shift, as shown in Figures 3b–e. Figure 4 shows infrared spectra for the silver nanoparticle modified 3-aminophenol resin microspheres and 3-aminophenol resin microspheres. The 1445 per centimeter band is attributed to the C-H stretching (Liu et al. 2011), whereas the absorption band at 2920 per centimeter is due to CH2 asymmetric stretching (Hu et al. 2006). The appearance of a weak and broad band at 3357 per centimeter and a strong and narrow band at 1602 per centimeter are respectively attributed to the presence of -OH (Ramanathan et al. 2005) and primary amines (-NH2) (Morlieras et al. 2013). The bonds of the -OH groups and primary amines (-NH2) were retained in the silver nanoparticle modified 3-aminophenol resin microspheres, but the intensity decreased after reducing and immobilizing the silver nanoparticles. These changes are typical of complexation of the carboxylate anion functional group by coordination with metals (Lin et al. 2005). Because of the rough surface of silver nanoparticles, the composite was employed as a SERS active substrate. Two dyes (4-mercaptobenzoic acid and Nile blue A) were used to characterize the SERS activity of the substrate. The silver nanoparticle modified 3-aminophenol resin microspheres prepared with an 8:1 molar ratio of silver nitrate and 3-aminophenol were chosen as the standard substrate for their

Figure 3. Ultraviolet-visible absorption spectra of the (a) 3-aminophenol resin microspheres and the silver nanoparticles modified 3-aminophenol resin microspheres obtained with (b) 1:1, (c) 2:1, (d) 4:1, and (e) 8:1 molar ratios of silver nitrate and 3-aminophenol.



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Figure 4. Infrared spectra of (a) silver nanoparticles modified 3-aminophenol resin microspheres and (b) 3-aminophenol resin microspheres.

best Raman results (Figure 5). Figure 6 shows SERS spectra of 4-mercaptobenzoic acid at various concentrations (1 × 108 to 1 × 104 molar). The SERS spectra of 4-mercaptobenzoic acid were characterized by two strong bands at 1076 and 1583 per centimeter, assigning to the aromatic benzene ring breathing and stretching vibrations (Michota and Bukowska 2003). Figure 7 shows the spectra of Nile blue A absorbed on the SERS substrate. The peak at 592 per centimeter was chosen as the reference in this study.

Figure 5. Raman spectra of Nile blue A based on the silver nanoparticle modified 3-aminophenol resin microspheres with (a) 1:1, (b) 2:1, (c) 4:1, and (d) 8:1 molar ratios of silver nitrate and 3-aminophenol.


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Figure 6. Representative 785 nanometers excited SERS spectra as a function of 4-mercaptobenzoic acid concentration: (a) 104 molar, (b) 105 molar, (c) 106 molar, (d) 107 molar, and (e) 108 molar on the silver nanoparticle modified 3-aminophenol resin microsphere substrate.

The Raman spectra of 1 × 103 molar 4-mercaptobenzoic acid were characterized when quartz glass was used as the non-SERS substrate. The spectrum was very weak but essential for the evaluation of the surface enhancement factor (EF), which has several definitions in the literature (Ru et al. 2007; Su et al. 2011). In this paper, it was calculated as the analytical enhancement factor (AEF) defined as: AEF ¼ (ISERS/ CSERS)/(IRS/CRS), where ISERS corresponds to the Raman intensity obtained with the SERS substrate at a specific concentration and CSERS and IRS correspond to the Raman intensity obtained with non-SERS conditions with an analyte concentration equal to CRS. In the SERS spectra of 4-mercaptobenzoic acid, the 1583 per

Figure 7. Representative 785 nanometers excited SERS spectra as a function of Nile blue A concentration: (a) 104 molar, (b) 105 molar, (c) 106 molar, (d) 107 molar, and (e) 108 molar on the silver nanoparticle modified 3-aminophenol resin microsphere substrate.



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centimeter Raman band was chosen to determine the analytical enhancement factor of the substrate. The analytical enhancement factor was 1.2 × 106 for 4-mercaptobenzoic acid and 1.8 × 106 for Nile blue A (CSERS ¼ 1.0 × 108 molar and CRS ¼ 1 × 103 molar). The Nile blue A was more suitable as a reporter molecule for the quantitative measurements, due to its higher analytical enhancement factor and favorable correlation coefficient. These results indicate that the silver nanoparticle modified 3-aminophenol resin microspheres possess high activity and potential as an SERS active substrate. The silver nanoparticle modified 3-aminophenol resin microsphere-based SERS active substrate was further used to construct an immunoassay to determine carcinoembryonic antigen. The immunoassay involved formation of a coating probe, a labeling probe, and the analyte carcinoembryonic antigen. The coating probe was constructed by conjugating coating antibodies on the surface of the magnetic nanoparticles with a spherical morphology and average size of 150 nanometers, as shown in Figure 8. The labeling probe was constructed by conjugating labeling antibodies on the surface of silver nanoparticle modified 3-aminophenol resin microsphere based SERS active substrate. When carcinoembryonic antigen was present in phosphate buffer saline, the coating and the labeling probes formed a sandwich complex by antibody–antigen–antibody interactions; however, they did not form a sandwich in the absence of antigen. Figure 9 shows SERS spectra of Nile blue A with various concentrations of carcinoembryonic antigen. The peak at 592 per centimeter was used for the determination of carcinoembryonic antigen. A linear relationship was obtained between the Raman signals and the concentrations of carcinoembryonic antigen from 10 picograms per milliliter to 100 nanograms per milliliter with a correlation coefficient of 0.997. The limit of detection was estimated to be 1.2 picograms per milliliter at a signal-to-noise rate of three. The selectivity of the SERS immunoassay is important in biological sample analysis. Consequently, an interference study was conducted using bovine serum

Figure 8. Scanning electron micrographs of magnetic nanoparticles at 500 nanometers and (Inset) 100 nanometers.


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Figure 9. Representative 785 nanometers SERS spectra as a function of carcinoembryonic antigen concentration: (a) 100 nanograms per milliliter, (b) 10 nanograms per milliliter, (c) 1 nanogram per milliliter, (d) 100 picograms per milliliter, and (e) 10 picograms per milliliter based on the silver nanoparticle modified 3-aminophenol resin microsphere immunoassay.

albumin (BSA), glucose, alpha fetal protein (AFP), and dopamine. The Raman signals of the SERS immunoassay with 1 nanogram per milliliter of carcinoembryonic antigen and 5 nanograms per milliliter of the aforementioned interfering substances were measured. Figure 10 shows that the change in Raman intensity with the interfering substances was 2.3 percent less compared to solutions only containing analyte. These results demonstrate that the immunoassay exhibited acceptable selectivity for the determination of carcinoembryonic antigen.

Figure 10. Raman intensity of the SERS immunoassay with 1 nanogram per milliliter carcinoembryonic antigen (CEA) þ 5 nanogram per milliliter interferent. BSA is bovine serum albumin and AFP is alpha fetal protein.


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CONCLUSIONS Uniform phenolic resin microspheres were prepared by the polycondensation of 3-aminophenol and formaldehyde. Silver nanoparticles were synthesized in situ and immobilized on the surface of the 3-aminophenol resin microspheres. The composite was employed as a SERS active substrate and good enhancement was obtained. A highly sensitive SERS immunoassay was fabricated to determine carcinoembryonic antigen; the limit of detection was estimated to be 1.2 picograms per milliliter. The silver nanoparticle modified 3-aminophenol resin microspheres were prepared by a facile and reductant-free method and had application as a SERS substrate for a novel immunoassay.


FUNDING This work was supported by the National Key Basic Research Program of China (973 Program) (Grant: 2012CB933302), the National Natural Science Foundation of China (Grant: 21175022), and the National High Technology Research and Development Program of China (863 Program) (Grant: 2012AA022703). REFERENCES Ambrosi, A., F. Airo, and A. Merkoci. 2010. Enhanced gold nanoparticle based ELISA for a breast cancer biomarker. Analytical Chemistry 82: 1151–56. doi:10.1021/ac902492c Arya, S. K., and S. Bhansali. 2011. Lung cancer and its early detection using biomarker-based biosensors. Chemical Reviews 111: 6783–809. doi:10.1021/cr100420s El-Nour, K. M. M. A., A. Eftaiha, A. Al-Warthan, and R. A. A. Ammar. 2010. Synthesis and applications of silver nanoparticles. Arabian Journal of Chemistry 3: 135–40. doi:10.1016/j. arabjc.2010.04.008 Grange, R. D., J. P. Thompson, and D. G. Lambert. 2014. Radioimmunoassay, enzyme and non-enzyme-based immunoassays. British Journal of Anaesthesia 112(2): 213–16. doi:10.1093/bja/aet293 Grunnet, M., and J. B. Sorensen. 2012. Carcinoembryonic antigen (CEA) as tumor marker in lung cancer. Lung Cancer 76: 138–43. doi:10.1016/j.lungcan.2011.11.012 Guo, S.-R., J.-Y. Gong, P. Jiang, M. Wu, Y. Lu, and S.-H. Yu. 2008. Biocompatible, luminescent silver@phenol formaldehyde resin core/shell nanospheres: Large-scale synthesis and application for in vivo bioimaging. Advanced Functional Materials 18: 872–79. doi:10.1002/adfm.200701440 He, S., K.-K. Liu, S. Su, J. Yan, X. Mao, D. Wang, Y. He, L.-J. Li, S. Song, and C. Fan. 2012. Graphene-based high-efficiency surface-enhanced Raman scattering-active platform for sensitive and multiplex DNA detection. Analytical Chemistry 84: 4622–27. doi:10.1021/ac300577d Hu, Z. G., P. Prunici, P. Patzner, and P. Hess. 2006. Infrared spectroscopic ellipsometry of n-Alkylthiol (C5-C18) self-assembled monolayers on gold. The Journal of Physical Chemistry B 110: 14824–31. doi:10.1021/jp060596y Khoury, C. G., and T. Vo-Dinh. 2008. Gold nanostars for surface-enhanced Raman scattering: Synthesis, characterization and optimization. The Journal of Physical Chemistry C 112: 18849–59. doi:10.1021/jp8054747 Li, Y., J. Ma, and Z. Ma. 2013. Synthesis of gold nanostars with tunable morphology and their electrochemical application for hydrogen peroxide sensing. Electrochimica Acta 108: 435–40. doi:10.1016/j.electacta.2013.06.141


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