Biosensors and Bioelectronics 78 (2016) 67–72
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Engineered “hot” core–shell nanostructures for patterned detection of chloramphenicol Wenjing Yan a,n, Longping Yang a, Hong Zhuang b, Haizhou Wu a, Jianhao Zhang a,n a b
National Center of Meat Quality & Safety Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China Quality and Safety Assessment Research Unit, Agricultural Research Service, USDA, Athens, GA 30605, USA
art ic l e i nf o
a b s t r a c t
Article history: Received 31 August 2015 Received in revised form 25 October 2015 Accepted 2 November 2015 Available online 4 November 2015
In this study, we described a novel method for highly sensitive and specific detection of chloramphenicol (CAP) based on engineered “hot” Au core-Ag shell nanostructures (Au@Ag NSs). Cy5-labeled DNA aptamer was embedded between the Au and Ag layers as a signal generator and target-recognition element, to fabricate uniform Au@Ag NSs with unexpected strong and stable SERS signals. The presented CAP can specifically bind to the DNA aptamer by forming an aptamer-CAP conjugate, and cause greatly decreased SERS signals of Au@Ag NSs. By using this method, we were able to detect as low as 0.19 pg mL 1 of CAP with high selectivity, which is much lower than those previously reported biosensors. Compared with the other SERS sensors that attached a dye in the outer layer of nanoparticles, this method exhibits excellent sensitivity and has the potential to significantly improve stability and reproducibility of SERS-based detection techniques. & 2015 Elsevier B.V. All rights reserved.
Keywords: Chloramphenicol Core–shell nanostructure Aptamer SERS Cy5 dye
1. Introduction Chloramphenicol (CAP) is a broad spectrum antibiotic secreted by the bacterium Streptomyces venezuelae. Because it can inhibit bacterial activities by preventing elongation of the peptide chains, CAP has been widely used in the field of animal breeding and clinical to prevent and treat a variety of bacterial infections (Cao et al., 2012; Feng et al., 2015; Malmasi et al., 2015). However, this drug is easy to accumulate in liver once it was absorbed into the body. Even after it is discharged into the environment, it cannot be degraded in a short period of time (Hanekamp and Bast, 2015; Li et al., 2013). In turn, the drug residues can get into the body through livestock products or environment and eventually causes aplastic anemia, bone marrow suppression and other adverse effects (Jain and Tripathi, 2015; Shukla et al., 2011). For these reasons, CAP has been strictly banned in China, USA, and Canada in all food producing animals. In the past decades, the content of the drug residues in animal source food have triggered a series of trade disputes between China and EU, resulting in about seven hundred million dollars losses in each year. Thereupon, it is of considerable significance to develop a specific and ultrasensitive approach for the detection of CAP in food. Traditional methods for CAP detection, such as liquid n
Corresponding authors. E-mail addresses: [email protected] (W. Yan), [email protected] (J. Zhang). http://dx.doi.org/10.1016/j.bios.2015.11.011 0956-5663/& 2015 Elsevier B.V. All rights reserved.
chromatography mass spectrometer (LC–MS) (Tian et al., 2013), gas chromatography (GC) (Liu et al., 2014) and high performance liquid chromatography (HPLC) (Vosough and Esfahani, 2013), usually need professional technology and rich experience, and suffer from the expensive, time-consuming and complex analytical procedures. In recent years, metal nanoparticle-based methods have attracted the most attention in the field of rapid and highly sensitive detection, due to their unique optical and chemical properties (Saha et al., 2012). Various methods have been developed based on metal nanoparticles, such as colorimetric (F Gao et al., 2015; H Gao et al., 2015), fluorescence (Berlina et al., 2013), electrochemical (Feng et al., 2015) and surface-enhanced Raman scattering (F Gao et al., 2015; H Gao et al., 2015; Gao et al., 2013). Among these approaches, surface-enhanced Raman scattering (SERS), as a molecular fingerprint spectrum, is one of the most powerful techniques for ultrasensitive detection of specific analyte in complex matrices (Osberg et al., 2012; Shin et al., 2015). By designing the construction of substrate materials, SERS signal can be amplified to 106–1014 orders of magnitude depending on the particle sizes, shapes and gap widths, reaching single molecule detection (Cecchini et al., 2013; Lim et al., 2010). Most importantly, SERS provides a non-destructive approach for identification of molecular species by the unique fingerprint, which effectively avoids the interference of background signal and improves the specificity of detection. Based all of these features, SERS has been widely used in various areas, such as food safety, environment, bio-diagnosis, medicine, chemistry, etc. (Li et al., 2010; Zhang et al., 2013).
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Substrate materials are of great importance for the fabrication of a SERS sensor, which significantly affects the stability, repeatability and enhancement of SERS signals (Hugall and Baumberg, 2015). So far, aggregated metal nanoparticles (NPs) is the most common substrate material, because the hot junction formed between aggregated NPs can strongly enhance SERS signals (Wang et al., 2013a, 2013b, 2013c). However, it is difficult to control the aggregation degree of NPs. The random distribution of hotspots and the poor repeatability of SERS signals remain challenging (Alvarez-Puebla and Liz-Marzan, 2010). Recently, some progresses have been made in the fabrication of self-assembled SERS-active nanostructures, such as dimers (Ma et al., 2013), trimers (Chen et al., 2010), pyramids (Xu et al., 2015a, 2015b), satellites (Gandra et al., 2012) and chains (Xu et al., 2015a, 2015b). Some of these discrete assemblies show strong SERS signals with high enhancement factor (EF) values range from 1.0 103 to 1.0 109, but still face challenges in the complex assembly process, low production yield, variability in particle distance and poor repeatability (Halas et al., 2011). Thus, developing a stable substrate material with high SERS enhancement is highly advantageous for ultrasensitive detection. Gold core-silver shell nanostructures (Au@Ag NSs), made of an inner layer gold NPs encapsulated with silver materials, has been proven to be one of the most promising artificial SERS-active substrates (Lim et al., 2011). On the one hand, embedding Raman reporters insides between Au and Ag layers strongly enhance SERS compared to pure Au or Ag NPs by themselves, due to enhanced electric field formation between two closely metal layers (Wang et al., 2013a, 2013b, 2013c). On the other hand, a thin silver shell not only prevents the aggregation induced SERS enhancement, but also limits the diffusion of the Raman dye and the changes of hotspot regions (Shen et al., 2015). These factors are crucial for achieving a reliable, reproducible and ultrasensitive SERS nanoprobe. In the past decades, most attentions have been focused on the fabrication of SERS “tags” based on SERS-active Au@Ag NSs (Wang et al., 2013a, 2013b, 2013c). However, there are few studies on the utilization of the high SERS enhancement of Au@Ag NSs for direct detection of contaminants in food. Here, we demonstrate that an Au@Ag NSs-based SERS active platform for CAP detection can be easily prepared by taking advantage of the high specific recognition between CAP and aptamer, and the strong SERS enhancement of core–shell nanostructures. Au NPs modified with Cy5-labeled ds DNA (aptamers inserted) were used as a seed to synthesize Au core-Ag shell nanostructures with strong SERS signal from Cy5 dye. The presented CAP can competitively bind with the aptamer, and cause the dissociation of aptamer from the surface of Au NPs, and further lead to a drastically decreased Raman signal intensity. Based on the relationship between the concentration of CAP and the SERS signals intensity, a high sensitive and selective sensing platform for CAP detection was achieved. The feasibility of the approach for real-world applications was also demonstrated by the detection of CAP in spiked milk samples.
2. Materials and methods 2.1. Materials and reagents Chloroauric acid (HAuCl4), sodium citrate, silver nitrate, ascorbic acid, polyvinylpyrrolidone; chloramphenicol (CAP), kanamycin (KAN), gentamicin (GEN), tetracycline (TC), thiamphenicol (TAP), streptomycin (SM) were purchased from Sigma-Aldrich (Shanghai, China). Thiolated DNA oligonucleotides were purchased from Shanghai Sangon Biological Engineering Technology & Services Co. Ltd, and were purified by polyacrylamide gel electrophoresis
(PAGE). The DNA sequences used in this work are as follows: DNA1: 5′ SH-TTT TAC CAC CGA CTC GCC-3′ DNA2 (aptamer): 5′-ACT TCA GTG AGT TGT CCC ACG GTC GGC GAG TCG GTG GTA G-Cy5 3′
2.2. Synthesis of Au NPs Au NPs with a diameter of 10 nm were synthesized by reduction of HAuCl4 using trisodium citrate combined with tannic acid. Briefly, 1 mL of 1% HAuCl4 was added to 79 mL of deionized water. The solution was heated to 60 °C for 40 min (solution A). Four mL of 1% sodium citrate, 0.1 mL of 1% tannic acid, and 0.1 mL of 25 mM K2CO3 were added to 15.8 mL of deionized water. The solution was heated to 60 °C for 40 min (solution B). Then, solution B was quickly added to solution A under high speed stirring. The mixture was heated at 60 °C for 50 min. After the heat source was removed, it was cooled down to room temperature.
2.3. Fabrication of Au–DNA conjugates DNA1 and DNA2 at the concentration of 2 mM were mixed in a 1:1 ratio in 10 mM PBS buffer. The solution was heated at 90 °C for 5 min and then slowly cooled to room temperature to obtain a ds DNA, it was stored at 4 °C before using. Five hundred mL of Au NP solution was centrifuged at 13,000 rpm for 10 min, the supernatant was removed, and the pellet was resuspended in 500 mL ds DNA solution. The mixture of Au and ds DNA synthesized above was incubated for 30 min at room temperature and was salted by increasing the concentration of NaCl. Every 3 h, the salt concentration was increased by 0.05 M to reach the final concentration of NaCl at 0.3 M in 24 h. The Au–DNA conjugates were centrifuged (13,000 rpm, 10 min) to remove the free (unattached) ds DNA. The precipitate was re-suspended in 10 mM PBS buffer.
2.4. Synthesis of SERS-active Au@Ag NSs Fifty mL of 5 fold concentrated Au–DNA conjugates were dispersed into a solution containing 200 mL of 10 mM PBS, 100 mL of 1% PVP and 50 mL of 0.1 M sodium ascorbate. Then, different amounts of 1 mM AgNO3 solution (0 mL, 30 mL, 50 mL, 70 mL and 100 mL) were added to synthesis of Au@Ag NSs with different thickness of Ag shells.
2.5. Fabrication of SERS SENSOR for CAP detection Au–DNA conjugates prepared in 2.3 were transferred to eight separated tubes (200 mL in each tube), and 5 mL of the CAP standard solution with different concentrations were added to make a final concentration of 0, 1, 5, 10, 50, 100, 500, and 1000 pg mL 1. The mixtures were incubated for 40 min at room temperature with constant shaking and then was centrifuged at 1300 rpm for 10 min. The precipitates were resuspended in 10 mM PBS buffer and then coated with a Ag shell based on Section 2.4. The samples were characterized by LabRam-HR800 Micro-Raman spectrometer. An air-cooled He–Ne laser giving 514 nm excitation was used as the excitation source with an acquisition time of 20 s, the laser power used for SERS detection is 10 mW. A standard curve was established based on the logarithmic relationship between CAP concentration and SERS signal intensity.
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3. Results and discussion 3.1. Principles of SERS-based CAP sensor As shown in Fig. 1, DNA1 with a thiol group at the 5′-end is hybridized with DNA2 (aptamer) containing a Cy5 dye at its 3′-end to form a functional ds DNA, the ds DNA is then attached to a 10nm Au NPs through S–Au covalent bonds to obtain a Au–DNA conjugates. The Cy5 dye located near the surface of Au NPs provides a detectable SERS signal and the DNA aptamer is used as a probe for molecular recognition. In order to obtain strong SERS enhancement, a Ag shell is deposited outside the Au core to form a Au core-Ag shell nanostructure (Au@Ag NS). Because the large electromagnetic enhancement between two metal layers of Au@Ag NSs, the core–shell nanostructures exhibit a unexpected strong SERS signal (Feng et al., 2012). Aptamers for CAP are specific DNA molecules generated by exponential enrichment (SELEX) in vitro, which can specially bind to CAP with high affinity (Mehta et al., 2011). In presence of CAP, CAP and the complementary sequence of the aptamer competitively bind the aptamer, because the high affinity to CAP, the crucial region of aptamer selectively binds to CAP by forming a stable stem-loop structure, and causes the dissociation of ds DNA. With Cy5-aptamers separate from the surface of the Au core, the SERS signals exhibit a significant decrease in intensity. Based on the relationship between the concentration of CAP and the SERS intensity, CAP can be sensitively, quantitatively, and specifically detected at a low concentration. 3.2. Characterizations of SERS-active Au@Ag NSs To synthesize Au@Ag NSs, firstly, pre-prepared Au NPs with the diameter of 10 nm were modified with ds DNA through S–Au bonding. The concentration of Au NPs was estimated to be 10 nM, according to the previous report (Haiss et al., 2007). Au NPs were incubated with ds DNA at a ratio of 1:200, the number of DNA attached to the Au NPs surface was estimated to be 63 74, according to the fluorescence method (Hurst et al., 2006). Fig. S1 shows the zeta potentials of synthesized Au@Ag NSs (70 μL of 1 mM AgNO3) and its intermediate products. Obviously, Au–DNA
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conjugates displayed a more negative charge ( 38.3 mV) compared with the Au NPs ( 29.4 mV), confirming a successful modification of Au NPs with ds DNA. Au@Ag NSs exhibited a more neutral charge ( 1.0 mV) compared with Au NPs or Au–DNA conjugates, which was attributed to the influence of polyvinylpyrrolidone modified on the surface of Ag shells. Besides, agarose gel electrophoresis was used to characterize the differences in size and surface charge between Au and Au@Ag NSs (Fig. S2), demonstrating that the Au @Ag NSs were synthesized as expected. In order to obtain Au@Ag NSs with varying thickness of Ag shells, different amount of AgNO3 solution (1 mM) were added during the synthesis process. Transmission electron microscope (TEM) and high-resolution TEM were used to directly characterize the heterogeneous structures of Au@Ag NSs. As shown in Fig. 2 A– E, the outside Ag layer had a lighter color compared to the Au core (Zhao et al., 2014), and with the increase volume of Ag þ ions (from 0 to 100 mL), the sizes of Au@Ag NSs and the thickness of Ag shell were gradually increased. Based on the dynamic light scattering (DLS) measurement, the diameters of NPs were found to increase from 14.2 71.2 nm to 28.3 72.3 nm, with the increases in the volume of AgNO3 solution from 0 to 100 mL (Fig. 3A), and no DLS signal was observed virtually above 30 nm, indicating the absence of aggregation. The thickness of Ag shell was estimated to increase from 0 nm to 6.17 0.4 nm (Fig. 3B) based on DLS spectrum, which is in good agreement with the statistical results of TEM images. Fig. 3C shows representative photographs of Au@Ag NSs with different shell thickness. The color of the solution changed from the original pink to yellow with increasing shell thickness, indicating the dominance of Ag plasmons in Au@Ag. UV–vis absorption spectra of Au@Ag NSs (Fig. 3D) shows that, with increasing amount of AgNO3 solution, the absorption peak of Au NPs increased in intensity and shifted from 520 nm to 501 nm, and a new absorption peak of metal Ag showed at 400 nm and gradually enhanced, which demonstrated the formation of Ag shell (Lim et al., 2010; Zhao et al., 2014).
Fig. 1. Schematic illustration of SERS-active Au@Ag NSs for CAP detection.
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Fig. 2. (A–E) TEM and (inset) high-resolution TEM images of Au@Ag NSs with varying shell thickness by addition of 0, 30, 50, 70 and 100 μL solution of 1 mM AgNO3.
3.3. SERS activities of Au@AG NSs
3.4. Feasibility of CAP detection
To estimate the SERS enhancement of Au@Ag NSs, SERS actives of four different subtract nanomaterials were tested at the beginning of the study. Cy5 labeled thiol- terminated DNA as a signal generator were attached on the surface of Au NPs (namely, AuCy5), Ag NPs (namely, Ag-Cy5), outer layer of Au@Ag NSs (namely, Au@Ag-Cy5), and inner layer of Au@Ag NSs (namely, Au-Cy5@Ag), respectively. In the SERS spectra shown in Fig. S3, it can be clearly seen that, Au-Cy5@Ag NSs exhibited the strongest SERS signals compared with Au or Ag or Au@Ag-Cy5 NSs. SERS signal from AuCy5@Ag was approximately 9 times higher than that for Au@AgCy5. This unexpected enhancement is attributed to electromagnetic enhancement localized in the hot junctions between a Au core and a Ag shell, this result is in good agreement with the previous report (Feng et al., 2012). For the control, SERS active of Au@Ag NSs in the absence of Cy5 was also examined under the same condition, and no SERS signal was observed on Au@Ag NSs, which indicated that Au@Ag NSs can greatly enhance the SERS signal of Cy5, when it was embedded between Au and Ag layer. Thereafter, the influences of the shell thickness on the SERS enhancement of Au@Ag NSs were studied. As shown in Fig. 4, with the volume of Ag þ ions (1 mM) increased from 0 to 100 mL, SERS signals of the system first increased and then decreased when the amount of Ag þ was over 70 mL. The SERS intensity reached a maximum when 70 mL of 1 mM AgNO3 was added to the system. It is understandable that if the Ag shell is too thick, it is difficult for light to pass through the metal layer and interact with Raman active molecules, and more scattering light was trapped in the metal layers (Feng et al., 2012; Tang et al., 2015). Therefore, 70 mL of 1 mM AgNO3 was used in the subsequent experiments. Besides, the reproducibility of the SERS signals was evaluated in this work, as shown in Fig. S4, the substrate materials exhibited extremely good reproducibility.
The high specificity and affinity between CAP and the DNA aptamer makes the approach feasible for CAP detection (Mehta et al., 2011). As illustrated in Fig. S5, the SERS intensity of Au@Ag NSs was significantly decreased in the presence of 1 ng mL 1 CAP solution, compared with that in the absence of CAP, suggesting that this approach has great potential for sensitive determination of CAP. Meanwhile, in order to confirm the state of DNA aptamer after binding to CAP (attached to or separated from the surface of Au NPs), the supernatant liquid of Au–DNA conjugates with or without CAP were measured by fluorescence and UV–vis spectrophotometer. As shown in Fig. S6, before the addition of CAP, Cy5 was attached on the surface of Au NPs, so no signal was observed in the fluorescence or UV–vis spectra of the supernatant liquid. After the addition of CAP, the absorption peak at 648 nm and the fluorescence peak at 664 nm were obviously enhanced, corresponding to the wavelengths of maximum absorption and emission of Cy5. It is demonstrated that after CAP binding to aptamer, Cy5-aptamers were separated from the surface of Au core and dispersed in solution. Besides, incubation time of aptamer bonding to CAP was optimized in this study, which is an important factor for biosensor evaluation. As shown in Fig. S7, specific recognition between CAP and DNA aptamer can be completed within 40 min. Thus, 40 min was chosen for subsequent analyses. 3.5. Fabricated SERS sensor for CAP detection The quantitative detection of CAP was investigated by adding different amounts of CAP to the constructed Au–DNA conjugates, and then depositing Ag to form Au@Ag NSs. Fig. 5 presents SERS spectra of Au@Ag NSs with different concentrations of CAP, in which the SERS signals decreased along with increased CAP contents. The linear correlation between the intensity of SERS peak at
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Fig. 3. (A) DLS analysis and (B) Statistical analysis of Ag shell thickness of Au@Ag NSs. (C)Visual color change and (D) UV–vis spectra of Au@Ag NSs with different shell thickness. Number 1–5: Au@Ag NSs with (1 mM) AgNO3 solution of 0, 30, 50, 70 and 100 mL, respectively.
Fig. 4. SERS spectra of Au@Ag NSs with different thickness of Ag shells, number 1– 5: Au@Ag NSs with (1 mM) AgNO3 solution of 0, 30, 50, 70 and 100 mL, respectively.
1465 cm 1 and CAP concentrations ranging from 0 to 1000 pg mL 1 was represented by logarithmic curve with a correlation coefficient of 0.995 (Fig. 6). The limit of detection was estimated to be 0.19 pg mL 1, which is much lower than those detected by using other conventional biosensors (Table S1).
Fig. 5. SERS spectra of Au@Ag NSs with different concentrations of CAP, the concentration is 0 pg mL 1, 1 pg mL 1, 5 pg mL 1, 10 pg mL 1, 50 pg mL 1 100 pg mL 1, 500 pg mL 1and 1000 pg mL 1.
3.6. Selective detection The selectivity of the developed approach was investigated by the detection of five other antibiotics (KAN, GEN, TC, TAP and SM) under the same condition. As Fig. S8 indicates, the other similar drugs showed almost negligible changes in SERS intensity even at
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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.2015.11.011.
References
Fig. 6. Linear plots of SERS intensity at 1465 cm 1 of Au@Ag NSs vs. CAP concentration.
a much higher concentration (10 ng mL 1). This demonstrates that a high selectivity can be achieved for the determination of CAP in solution with this newly developed SERS sensor. 3.7. Practical application In order to confirm the practicability of the SERS sensing platform, milk samples were spiked with different concentrations of CAP and then tested with our developed sensor. Table S2 shows excellent recoveries (96.6–110.2%) of the concentration in the spiked milk samples. The variation coefficients (CV) were in the range of 1.8–4.9%, demonstrating a great potential in the real word application.
4. Conclusions In summary, this study demonstrates an effective SERS sensing platform for CAP detection through the use of SERS-active Au@Ag NSs, in which Cy5-labeled aptamer is embedded between two metal layers as a signal generator and molecular recognition probe. A thin Ag shell can not only greatly enhance SERS signals of Cy5, but also effectively prevent the loss of the Raman dye and the aggregation of NPs, providing a stable and reversible SERS sensor. Using the developed SERS-active Au@Ag NSs system, as low as 0.19 pg mL 1 of CAP concentration can be detected. This approach develops a simply pattern without complex self-assembly process to achieve a reliable, ultrasensitive and selective detection. We foresee a widespread application of this kind of SERS platforms in multiple areas, especially in food safety.
Acknowledgments This study was sponsored by the Fundamental Research Funds for the Central Universities (Grant no. 0806J0452); the National Science & Technology Pillar Program (Grant no. 2015BAD16B00); the International Technology Cooperation Program of Jiangsu province, China (Grant no. BZ2014034).
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Engineered “hot” core–shell nanostructures for patterned detection of chloramphenicol Wenjing Yan a,n, Longping Yang a, Hong Zhuang b, Haizhou Wu a, Jianhao Zhang a,n a b
National Center of Meat Quality & Safety Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China Quality and Safety Assessment Research Unit, Agricultural Research Service, USDA, Athens, GA 30605, USA
art ic l e i nf o
a b s t r a c t
Article history: Received 31 August 2015 Received in revised form 25 October 2015 Accepted 2 November 2015 Available online 4 November 2015
In this study, we described a novel method for highly sensitive and specific detection of chloramphenicol (CAP) based on engineered “hot” Au core-Ag shell nanostructures (Au@Ag NSs). Cy5-labeled DNA aptamer was embedded between the Au and Ag layers as a signal generator and target-recognition element, to fabricate uniform Au@Ag NSs with unexpected strong and stable SERS signals. The presented CAP can specifically bind to the DNA aptamer by forming an aptamer-CAP conjugate, and cause greatly decreased SERS signals of Au@Ag NSs. By using this method, we were able to detect as low as 0.19 pg mL 1 of CAP with high selectivity, which is much lower than those previously reported biosensors. Compared with the other SERS sensors that attached a dye in the outer layer of nanoparticles, this method exhibits excellent sensitivity and has the potential to significantly improve stability and reproducibility of SERS-based detection techniques. & 2015 Elsevier B.V. All rights reserved.
Keywords: Chloramphenicol Core–shell nanostructure Aptamer SERS Cy5 dye
1. Introduction Chloramphenicol (CAP) is a broad spectrum antibiotic secreted by the bacterium Streptomyces venezuelae. Because it can inhibit bacterial activities by preventing elongation of the peptide chains, CAP has been widely used in the field of animal breeding and clinical to prevent and treat a variety of bacterial infections (Cao et al., 2012; Feng et al., 2015; Malmasi et al., 2015). However, this drug is easy to accumulate in liver once it was absorbed into the body. Even after it is discharged into the environment, it cannot be degraded in a short period of time (Hanekamp and Bast, 2015; Li et al., 2013). In turn, the drug residues can get into the body through livestock products or environment and eventually causes aplastic anemia, bone marrow suppression and other adverse effects (Jain and Tripathi, 2015; Shukla et al., 2011). For these reasons, CAP has been strictly banned in China, USA, and Canada in all food producing animals. In the past decades, the content of the drug residues in animal source food have triggered a series of trade disputes between China and EU, resulting in about seven hundred million dollars losses in each year. Thereupon, it is of considerable significance to develop a specific and ultrasensitive approach for the detection of CAP in food. Traditional methods for CAP detection, such as liquid n
Corresponding authors. E-mail addresses: [email protected] (W. Yan), [email protected] (J. Zhang). http://dx.doi.org/10.1016/j.bios.2015.11.011 0956-5663/& 2015 Elsevier B.V. All rights reserved.
chromatography mass spectrometer (LC–MS) (Tian et al., 2013), gas chromatography (GC) (Liu et al., 2014) and high performance liquid chromatography (HPLC) (Vosough and Esfahani, 2013), usually need professional technology and rich experience, and suffer from the expensive, time-consuming and complex analytical procedures. In recent years, metal nanoparticle-based methods have attracted the most attention in the field of rapid and highly sensitive detection, due to their unique optical and chemical properties (Saha et al., 2012). Various methods have been developed based on metal nanoparticles, such as colorimetric (F Gao et al., 2015; H Gao et al., 2015), fluorescence (Berlina et al., 2013), electrochemical (Feng et al., 2015) and surface-enhanced Raman scattering (F Gao et al., 2015; H Gao et al., 2015; Gao et al., 2013). Among these approaches, surface-enhanced Raman scattering (SERS), as a molecular fingerprint spectrum, is one of the most powerful techniques for ultrasensitive detection of specific analyte in complex matrices (Osberg et al., 2012; Shin et al., 2015). By designing the construction of substrate materials, SERS signal can be amplified to 106–1014 orders of magnitude depending on the particle sizes, shapes and gap widths, reaching single molecule detection (Cecchini et al., 2013; Lim et al., 2010). Most importantly, SERS provides a non-destructive approach for identification of molecular species by the unique fingerprint, which effectively avoids the interference of background signal and improves the specificity of detection. Based all of these features, SERS has been widely used in various areas, such as food safety, environment, bio-diagnosis, medicine, chemistry, etc. (Li et al., 2010; Zhang et al., 2013).
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Substrate materials are of great importance for the fabrication of a SERS sensor, which significantly affects the stability, repeatability and enhancement of SERS signals (Hugall and Baumberg, 2015). So far, aggregated metal nanoparticles (NPs) is the most common substrate material, because the hot junction formed between aggregated NPs can strongly enhance SERS signals (Wang et al., 2013a, 2013b, 2013c). However, it is difficult to control the aggregation degree of NPs. The random distribution of hotspots and the poor repeatability of SERS signals remain challenging (Alvarez-Puebla and Liz-Marzan, 2010). Recently, some progresses have been made in the fabrication of self-assembled SERS-active nanostructures, such as dimers (Ma et al., 2013), trimers (Chen et al., 2010), pyramids (Xu et al., 2015a, 2015b), satellites (Gandra et al., 2012) and chains (Xu et al., 2015a, 2015b). Some of these discrete assemblies show strong SERS signals with high enhancement factor (EF) values range from 1.0 103 to 1.0 109, but still face challenges in the complex assembly process, low production yield, variability in particle distance and poor repeatability (Halas et al., 2011). Thus, developing a stable substrate material with high SERS enhancement is highly advantageous for ultrasensitive detection. Gold core-silver shell nanostructures (Au@Ag NSs), made of an inner layer gold NPs encapsulated with silver materials, has been proven to be one of the most promising artificial SERS-active substrates (Lim et al., 2011). On the one hand, embedding Raman reporters insides between Au and Ag layers strongly enhance SERS compared to pure Au or Ag NPs by themselves, due to enhanced electric field formation between two closely metal layers (Wang et al., 2013a, 2013b, 2013c). On the other hand, a thin silver shell not only prevents the aggregation induced SERS enhancement, but also limits the diffusion of the Raman dye and the changes of hotspot regions (Shen et al., 2015). These factors are crucial for achieving a reliable, reproducible and ultrasensitive SERS nanoprobe. In the past decades, most attentions have been focused on the fabrication of SERS “tags” based on SERS-active Au@Ag NSs (Wang et al., 2013a, 2013b, 2013c). However, there are few studies on the utilization of the high SERS enhancement of Au@Ag NSs for direct detection of contaminants in food. Here, we demonstrate that an Au@Ag NSs-based SERS active platform for CAP detection can be easily prepared by taking advantage of the high specific recognition between CAP and aptamer, and the strong SERS enhancement of core–shell nanostructures. Au NPs modified with Cy5-labeled ds DNA (aptamers inserted) were used as a seed to synthesize Au core-Ag shell nanostructures with strong SERS signal from Cy5 dye. The presented CAP can competitively bind with the aptamer, and cause the dissociation of aptamer from the surface of Au NPs, and further lead to a drastically decreased Raman signal intensity. Based on the relationship between the concentration of CAP and the SERS signals intensity, a high sensitive and selective sensing platform for CAP detection was achieved. The feasibility of the approach for real-world applications was also demonstrated by the detection of CAP in spiked milk samples.
2. Materials and methods 2.1. Materials and reagents Chloroauric acid (HAuCl4), sodium citrate, silver nitrate, ascorbic acid, polyvinylpyrrolidone; chloramphenicol (CAP), kanamycin (KAN), gentamicin (GEN), tetracycline (TC), thiamphenicol (TAP), streptomycin (SM) were purchased from Sigma-Aldrich (Shanghai, China). Thiolated DNA oligonucleotides were purchased from Shanghai Sangon Biological Engineering Technology & Services Co. Ltd, and were purified by polyacrylamide gel electrophoresis
(PAGE). The DNA sequences used in this work are as follows: DNA1: 5′ SH-TTT TAC CAC CGA CTC GCC-3′ DNA2 (aptamer): 5′-ACT TCA GTG AGT TGT CCC ACG GTC GGC GAG TCG GTG GTA G-Cy5 3′
2.2. Synthesis of Au NPs Au NPs with a diameter of 10 nm were synthesized by reduction of HAuCl4 using trisodium citrate combined with tannic acid. Briefly, 1 mL of 1% HAuCl4 was added to 79 mL of deionized water. The solution was heated to 60 °C for 40 min (solution A). Four mL of 1% sodium citrate, 0.1 mL of 1% tannic acid, and 0.1 mL of 25 mM K2CO3 were added to 15.8 mL of deionized water. The solution was heated to 60 °C for 40 min (solution B). Then, solution B was quickly added to solution A under high speed stirring. The mixture was heated at 60 °C for 50 min. After the heat source was removed, it was cooled down to room temperature.
2.3. Fabrication of Au–DNA conjugates DNA1 and DNA2 at the concentration of 2 mM were mixed in a 1:1 ratio in 10 mM PBS buffer. The solution was heated at 90 °C for 5 min and then slowly cooled to room temperature to obtain a ds DNA, it was stored at 4 °C before using. Five hundred mL of Au NP solution was centrifuged at 13,000 rpm for 10 min, the supernatant was removed, and the pellet was resuspended in 500 mL ds DNA solution. The mixture of Au and ds DNA synthesized above was incubated for 30 min at room temperature and was salted by increasing the concentration of NaCl. Every 3 h, the salt concentration was increased by 0.05 M to reach the final concentration of NaCl at 0.3 M in 24 h. The Au–DNA conjugates were centrifuged (13,000 rpm, 10 min) to remove the free (unattached) ds DNA. The precipitate was re-suspended in 10 mM PBS buffer.
2.4. Synthesis of SERS-active Au@Ag NSs Fifty mL of 5 fold concentrated Au–DNA conjugates were dispersed into a solution containing 200 mL of 10 mM PBS, 100 mL of 1% PVP and 50 mL of 0.1 M sodium ascorbate. Then, different amounts of 1 mM AgNO3 solution (0 mL, 30 mL, 50 mL, 70 mL and 100 mL) were added to synthesis of Au@Ag NSs with different thickness of Ag shells.
2.5. Fabrication of SERS SENSOR for CAP detection Au–DNA conjugates prepared in 2.3 were transferred to eight separated tubes (200 mL in each tube), and 5 mL of the CAP standard solution with different concentrations were added to make a final concentration of 0, 1, 5, 10, 50, 100, 500, and 1000 pg mL 1. The mixtures were incubated for 40 min at room temperature with constant shaking and then was centrifuged at 1300 rpm for 10 min. The precipitates were resuspended in 10 mM PBS buffer and then coated with a Ag shell based on Section 2.4. The samples were characterized by LabRam-HR800 Micro-Raman spectrometer. An air-cooled He–Ne laser giving 514 nm excitation was used as the excitation source with an acquisition time of 20 s, the laser power used for SERS detection is 10 mW. A standard curve was established based on the logarithmic relationship between CAP concentration and SERS signal intensity.
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3. Results and discussion 3.1. Principles of SERS-based CAP sensor As shown in Fig. 1, DNA1 with a thiol group at the 5′-end is hybridized with DNA2 (aptamer) containing a Cy5 dye at its 3′-end to form a functional ds DNA, the ds DNA is then attached to a 10nm Au NPs through S–Au covalent bonds to obtain a Au–DNA conjugates. The Cy5 dye located near the surface of Au NPs provides a detectable SERS signal and the DNA aptamer is used as a probe for molecular recognition. In order to obtain strong SERS enhancement, a Ag shell is deposited outside the Au core to form a Au core-Ag shell nanostructure (Au@Ag NS). Because the large electromagnetic enhancement between two metal layers of Au@Ag NSs, the core–shell nanostructures exhibit a unexpected strong SERS signal (Feng et al., 2012). Aptamers for CAP are specific DNA molecules generated by exponential enrichment (SELEX) in vitro, which can specially bind to CAP with high affinity (Mehta et al., 2011). In presence of CAP, CAP and the complementary sequence of the aptamer competitively bind the aptamer, because the high affinity to CAP, the crucial region of aptamer selectively binds to CAP by forming a stable stem-loop structure, and causes the dissociation of ds DNA. With Cy5-aptamers separate from the surface of the Au core, the SERS signals exhibit a significant decrease in intensity. Based on the relationship between the concentration of CAP and the SERS intensity, CAP can be sensitively, quantitatively, and specifically detected at a low concentration. 3.2. Characterizations of SERS-active Au@Ag NSs To synthesize Au@Ag NSs, firstly, pre-prepared Au NPs with the diameter of 10 nm were modified with ds DNA through S–Au bonding. The concentration of Au NPs was estimated to be 10 nM, according to the previous report (Haiss et al., 2007). Au NPs were incubated with ds DNA at a ratio of 1:200, the number of DNA attached to the Au NPs surface was estimated to be 63 74, according to the fluorescence method (Hurst et al., 2006). Fig. S1 shows the zeta potentials of synthesized Au@Ag NSs (70 μL of 1 mM AgNO3) and its intermediate products. Obviously, Au–DNA
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conjugates displayed a more negative charge ( 38.3 mV) compared with the Au NPs ( 29.4 mV), confirming a successful modification of Au NPs with ds DNA. Au@Ag NSs exhibited a more neutral charge ( 1.0 mV) compared with Au NPs or Au–DNA conjugates, which was attributed to the influence of polyvinylpyrrolidone modified on the surface of Ag shells. Besides, agarose gel electrophoresis was used to characterize the differences in size and surface charge between Au and Au@Ag NSs (Fig. S2), demonstrating that the Au @Ag NSs were synthesized as expected. In order to obtain Au@Ag NSs with varying thickness of Ag shells, different amount of AgNO3 solution (1 mM) were added during the synthesis process. Transmission electron microscope (TEM) and high-resolution TEM were used to directly characterize the heterogeneous structures of Au@Ag NSs. As shown in Fig. 2 A– E, the outside Ag layer had a lighter color compared to the Au core (Zhao et al., 2014), and with the increase volume of Ag þ ions (from 0 to 100 mL), the sizes of Au@Ag NSs and the thickness of Ag shell were gradually increased. Based on the dynamic light scattering (DLS) measurement, the diameters of NPs were found to increase from 14.2 71.2 nm to 28.3 72.3 nm, with the increases in the volume of AgNO3 solution from 0 to 100 mL (Fig. 3A), and no DLS signal was observed virtually above 30 nm, indicating the absence of aggregation. The thickness of Ag shell was estimated to increase from 0 nm to 6.17 0.4 nm (Fig. 3B) based on DLS spectrum, which is in good agreement with the statistical results of TEM images. Fig. 3C shows representative photographs of Au@Ag NSs with different shell thickness. The color of the solution changed from the original pink to yellow with increasing shell thickness, indicating the dominance of Ag plasmons in Au@Ag. UV–vis absorption spectra of Au@Ag NSs (Fig. 3D) shows that, with increasing amount of AgNO3 solution, the absorption peak of Au NPs increased in intensity and shifted from 520 nm to 501 nm, and a new absorption peak of metal Ag showed at 400 nm and gradually enhanced, which demonstrated the formation of Ag shell (Lim et al., 2010; Zhao et al., 2014).
Fig. 1. Schematic illustration of SERS-active Au@Ag NSs for CAP detection.
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Fig. 2. (A–E) TEM and (inset) high-resolution TEM images of Au@Ag NSs with varying shell thickness by addition of 0, 30, 50, 70 and 100 μL solution of 1 mM AgNO3.
3.3. SERS activities of Au@AG NSs
3.4. Feasibility of CAP detection
To estimate the SERS enhancement of Au@Ag NSs, SERS actives of four different subtract nanomaterials were tested at the beginning of the study. Cy5 labeled thiol- terminated DNA as a signal generator were attached on the surface of Au NPs (namely, AuCy5), Ag NPs (namely, Ag-Cy5), outer layer of Au@Ag NSs (namely, Au@Ag-Cy5), and inner layer of Au@Ag NSs (namely, Au-Cy5@Ag), respectively. In the SERS spectra shown in Fig. S3, it can be clearly seen that, Au-Cy5@Ag NSs exhibited the strongest SERS signals compared with Au or Ag or Au@Ag-Cy5 NSs. SERS signal from AuCy5@Ag was approximately 9 times higher than that for Au@AgCy5. This unexpected enhancement is attributed to electromagnetic enhancement localized in the hot junctions between a Au core and a Ag shell, this result is in good agreement with the previous report (Feng et al., 2012). For the control, SERS active of Au@Ag NSs in the absence of Cy5 was also examined under the same condition, and no SERS signal was observed on Au@Ag NSs, which indicated that Au@Ag NSs can greatly enhance the SERS signal of Cy5, when it was embedded between Au and Ag layer. Thereafter, the influences of the shell thickness on the SERS enhancement of Au@Ag NSs were studied. As shown in Fig. 4, with the volume of Ag þ ions (1 mM) increased from 0 to 100 mL, SERS signals of the system first increased and then decreased when the amount of Ag þ was over 70 mL. The SERS intensity reached a maximum when 70 mL of 1 mM AgNO3 was added to the system. It is understandable that if the Ag shell is too thick, it is difficult for light to pass through the metal layer and interact with Raman active molecules, and more scattering light was trapped in the metal layers (Feng et al., 2012; Tang et al., 2015). Therefore, 70 mL of 1 mM AgNO3 was used in the subsequent experiments. Besides, the reproducibility of the SERS signals was evaluated in this work, as shown in Fig. S4, the substrate materials exhibited extremely good reproducibility.
The high specificity and affinity between CAP and the DNA aptamer makes the approach feasible for CAP detection (Mehta et al., 2011). As illustrated in Fig. S5, the SERS intensity of Au@Ag NSs was significantly decreased in the presence of 1 ng mL 1 CAP solution, compared with that in the absence of CAP, suggesting that this approach has great potential for sensitive determination of CAP. Meanwhile, in order to confirm the state of DNA aptamer after binding to CAP (attached to or separated from the surface of Au NPs), the supernatant liquid of Au–DNA conjugates with or without CAP were measured by fluorescence and UV–vis spectrophotometer. As shown in Fig. S6, before the addition of CAP, Cy5 was attached on the surface of Au NPs, so no signal was observed in the fluorescence or UV–vis spectra of the supernatant liquid. After the addition of CAP, the absorption peak at 648 nm and the fluorescence peak at 664 nm were obviously enhanced, corresponding to the wavelengths of maximum absorption and emission of Cy5. It is demonstrated that after CAP binding to aptamer, Cy5-aptamers were separated from the surface of Au core and dispersed in solution. Besides, incubation time of aptamer bonding to CAP was optimized in this study, which is an important factor for biosensor evaluation. As shown in Fig. S7, specific recognition between CAP and DNA aptamer can be completed within 40 min. Thus, 40 min was chosen for subsequent analyses. 3.5. Fabricated SERS sensor for CAP detection The quantitative detection of CAP was investigated by adding different amounts of CAP to the constructed Au–DNA conjugates, and then depositing Ag to form Au@Ag NSs. Fig. 5 presents SERS spectra of Au@Ag NSs with different concentrations of CAP, in which the SERS signals decreased along with increased CAP contents. The linear correlation between the intensity of SERS peak at
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Fig. 3. (A) DLS analysis and (B) Statistical analysis of Ag shell thickness of Au@Ag NSs. (C)Visual color change and (D) UV–vis spectra of Au@Ag NSs with different shell thickness. Number 1–5: Au@Ag NSs with (1 mM) AgNO3 solution of 0, 30, 50, 70 and 100 mL, respectively.
Fig. 4. SERS spectra of Au@Ag NSs with different thickness of Ag shells, number 1– 5: Au@Ag NSs with (1 mM) AgNO3 solution of 0, 30, 50, 70 and 100 mL, respectively.
1465 cm 1 and CAP concentrations ranging from 0 to 1000 pg mL 1 was represented by logarithmic curve with a correlation coefficient of 0.995 (Fig. 6). The limit of detection was estimated to be 0.19 pg mL 1, which is much lower than those detected by using other conventional biosensors (Table S1).
Fig. 5. SERS spectra of Au@Ag NSs with different concentrations of CAP, the concentration is 0 pg mL 1, 1 pg mL 1, 5 pg mL 1, 10 pg mL 1, 50 pg mL 1 100 pg mL 1, 500 pg mL 1and 1000 pg mL 1.
3.6. Selective detection The selectivity of the developed approach was investigated by the detection of five other antibiotics (KAN, GEN, TC, TAP and SM) under the same condition. As Fig. S8 indicates, the other similar drugs showed almost negligible changes in SERS intensity even at
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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.2015.11.011.
References
Fig. 6. Linear plots of SERS intensity at 1465 cm 1 of Au@Ag NSs vs. CAP concentration.
a much higher concentration (10 ng mL 1). This demonstrates that a high selectivity can be achieved for the determination of CAP in solution with this newly developed SERS sensor. 3.7. Practical application In order to confirm the practicability of the SERS sensing platform, milk samples were spiked with different concentrations of CAP and then tested with our developed sensor. Table S2 shows excellent recoveries (96.6–110.2%) of the concentration in the spiked milk samples. The variation coefficients (CV) were in the range of 1.8–4.9%, demonstrating a great potential in the real word application.
4. Conclusions In summary, this study demonstrates an effective SERS sensing platform for CAP detection through the use of SERS-active Au@Ag NSs, in which Cy5-labeled aptamer is embedded between two metal layers as a signal generator and molecular recognition probe. A thin Ag shell can not only greatly enhance SERS signals of Cy5, but also effectively prevent the loss of the Raman dye and the aggregation of NPs, providing a stable and reversible SERS sensor. Using the developed SERS-active Au@Ag NSs system, as low as 0.19 pg mL 1 of CAP concentration can be detected. This approach develops a simply pattern without complex self-assembly process to achieve a reliable, ultrasensitive and selective detection. We foresee a widespread application of this kind of SERS platforms in multiple areas, especially in food safety.
Acknowledgments This study was sponsored by the Fundamental Research Funds for the Central Universities (Grant no. 0806J0452); the National Science & Technology Pillar Program (Grant no. 2015BAD16B00); the International Technology Cooperation Program of Jiangsu province, China (Grant no. BZ2014034).
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