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Salmonella typhimurium detection using a surface-enhanced Raman scattering-based aptasensor
International Journal of Food Microbiology 218 (2016) 38–43
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Salmonella typhimurium detection using a surface-enhanced Raman scattering-based aptasensor Nuo Duan a, Boya Chang a, Hui Zhang b, Zhouping Wang a, Shijia Wu a,c,⁎ a b c
State Key Laboratory of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China China Rural Technology Development Center, Beijing 100045, China School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China
a r t i c l e
i n f o
Article history: Received 28 July 2015 Received in revised form 19 October 2015 Accepted 14 November 2015 Available online xxxx Keywords: SERS Au@Ag core/shell NPs Aptamer S. typhimurium
a b s t r a c t Surface-enhanced Raman spectroscopy (SERS) has been used in a variety of biological applications due to its high sensitivity and specificity. Here, we report a SERS-based aptasensor approach for quantitative detection of pathogenic bacteria. A SERS substrate bearing Au@Ag core/shell nanoparticles (NPs) is functionalized with aptamer 1 (apt 1) for the capture of target molecules. X-rhodamine (ROX)-modified aptamer 2 (apt 2) is used as recognition element and Raman reporter. Salmonella typhimurium specifically interacted with the aptamers to form Au@Ag-apt 1-target-apt 2-ROX sandwich-like complexes. As a result, the concentration of S. typhimurium was determined using this developed aptasensor structure, and a calibration curve is obtained in the range of 15 to 1.5 × 106 cfu/mL with a limit of detection of 15 cfu/mL. Our method was successfully applied to real food samples, and the results are consistent with the results obtained using plate counting methods. We believe that the developed method shows potential for the rapid and sensitive detection of pathogenic bacteria in food safety assurance. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The identification of pathogenic bacteria is an important task in light of the fact that many serious and even fatal medical conditions result from bacterial infection. Salmonella typhimurium, a Gram-negative bacteria pathogen, is one of the leading causes of food-borne illness in human and animal hosts worldwide (Brandt et al., 2013). S. typhimurium is transmitted primarily through the consumption of raw or uncooked vegetables, poultry, eggs, and fruits (Crum-Cianflone, 2008; Mrema et al., 2006). In China, approximately 80% of food-borne bacteria outbreaks are thought to be caused by Salmonella (Yang et al., 2010). This increasing incidence of S. typhimurium in different food products is now attracting the attention of the government. The food safety regulations of some countries (e.g., China, USA) require no tolerance of S. typhimurium in ready-to-eat food. Thus, sensitive detection methods for S. typhimurium play a critical role in many aspects of life, especially in food quality. The existing common detection methods include culture-based methods, molecular methods of regular and real-time polymerase chain reaction (PCR) (Zheng et al., 2014; Park et al., 2013; Silva et al., 2011), and immunoassays (Mantzila et al., 2008; Shim et al., 2014). Despite many advances in these fields, it is still challenging to find new approaches that could improve the simplicity, selectivity, stability and sensitivity of these analytical methods.
⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (S. Wu).
http://dx.doi.org/10.1016/j.ijfoodmicro.2015.11.006 0168-1605/© 2015 Elsevier B.V. All rights reserved.
Currently, SERS is suggested as a powerful tool for characterization of chemical and biological analytes due to its high fingerprint information content, its extreme sensitivity and its obliviousness to the aqueous environment intrinsic to biological systems (Chen et al., 2008; Cialla et al., 2012). Since its discovery in the 1970s, SERS has found increasing application in different fields, such as the biomedical field (Haynes et al., 2005; Kaminska et al., 2015), environmental monitoring (Bhandari et al., 2009; Muller et al., 2014), and food quality assurance (Ma et al., 2015; Aoki et al., 2013). Many efforts have been devoted to the SERS detection of bacterial cells (Sundaram et al., 2013a; Banerjee et al., 2010). Among these, some detection methods were designed based on the spectrum of bacteria themselves (Sundaram et al., 2013b). Kowalska et al. (2015) prepared a Cu-based SERS platform for Staphylococcus aureus detection. The spectrum of S. aureus bacteria exhibits characteristic band vibrations. The band located at 727 cm–1 is assigned to the C–N stretching mode of the adenine moiety of the lipid layer in the cell wall and/or to the purine ring breathing mode. The spectral features at 840, 1027, 1037 and 1084 cm–1 arise from tyrosine, phenylalanine, C–C oscillations, and phosphate binding in DNA, respectively. Additionally, there are many reports of SERS-based assays that used antibodies as recognition agents (Knauer et al., 2012; Wang et al., 2011). Lin and Hamme (2014) combined monoclonal antibody-conjugated sphere-shaped gold nanoparticles with single-walled carbon nanotubes to create a nanohybrid system to selectively detect S. typhimurium DT104 bacteria. The Raman signal intensity was from Rhodamine 6G, with a detection limit of 105 cfu/mL. However, the preparation of the antibodies via animal immunization is time-consuming (several months), and the antibodies may become
N. Duan et al. / International Journal of Food Microbiology 218 (2016) 38–43
susceptible to stability or modification issues. Aptamers with high affinity and selectivity are beginning to emerge as an alternative to antibodies in these applications. To the best of our knowledge, there have been few reports of using SERS coupled with aptamers for the sensitive and rapid detection of bacteria. Therefore, in the present study, we report a sensitive method for the detection of pathogenic bacteria using a SERS sensing platform based on an aptamer. Our strategy exploits the Au@Ag core/shell NPs as the enhanced substrate for SERS. To selectively detect S. typhimurium, we employed apt 1 immobilized on the surface of Au@Ag core/shell NPs. The Raman signal intensity was generated from ROX-modified apt 2, which would bind with the target in the same way as Au@Ag-apt 1. Important factors such as the concentration of Au@Ag-apt 1 and the concentration of ROX-apt 2 were evaluated. The sensitive and selective SERS assay was validated by the analysis of S. typhimurium in food samples, showing its great potential for food quality application. 2. Materials and methods 2.1. Materials Chloroauric acid (HAuCl4), L-ascorbic acid, and AgNO3 were obtained from Sigma-Aldrich (U.S.A.). Trisodium citrate was of analytical grade and was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Milli-Q water with a resistivity of 18.2 MΩ/cm was used throughout the experiments. The S. typhimurium aptamers were prepared in our laboratory (Duan et al., 2013) and synthesized and purified by high-performance liquid chromatography (Sangon Biotechnology, Inc., Shanghai, China). The sequences of the S. typhimurium aptamers are 5′-SH-AGTAATGCCCGGTAGTTATTCAAAGATGAGTAGGAAAAGA-3′ (apt 1) and 5′-ROX-AGTAATGCCCGGTAGTTATTCAAAGATGAGTAGGAAAAGA3′ (apt 2). 2.2. Instrumentation The size and shape of the nanoparticles were tested using transmission electron microscopy (TEM) (TEM, JEOL Ltd., Japan). Absorption spectra were recorded on a UV-1800 spectrophotometer (Shimadzu Co., Japan). The Raman spectra were measured using a Jobin Yvon microRaman spectroscope (Super LabRam II) with a mode of 50 × objective (8 mm), a holographic grating (1800 g/mm), a charge-coupled device detector with 1024 × 256 pixels and a 785 nm excitation laser. 2.3. Bacteria culture The S. typhimurium ATCC 14028 was kindly donated by the Animal, Plant and Food Inspection Centre, Jiangsu Entry-Exit Inspection and Quarantine Bureau (Nanjing, China). S. typhimurium was cultivated in Luria-Bertani medium and then incubated in a shaker at 37 °C for 24 h. One hundred microliters of the bacterial culture was diluted with medium and coated on the agar plates and cultured at 37 °C for 18 h to count colony forming units. The rest of the bacteria were harvested by centrifugation at 3000 rpm and 4 °C and washed twice in 1 × binding buffer (1 × BB 50 mM Tris–HCl at pH 7.4, 5 mM KCl, 100 mM NaCl, and 1 mM MgCl2) at room temperature. 2.4. Preparation of Au@Ag core/shell NPs AuNPs were first synthesized using the citrate reduction method. In brief, all glassware used in the experiment were cleaned with aqua regia (HNO3/HCl, 3:1, v/v), rinsed thoroughly in ultrapure water, and dried prior to use. Then, 500 μL of 1% HAuCl4 solution and 49.5 mL Milli-Q water were heated to boiling with vigorously stirring. Then, 1.5 mL of 1% trisodium citrate was rapidly injected into the boiling reaction mixture. After the mixture was boiled for 15 min, the heating source was removed, and the colloid was cooled to room temperature. The
39
prepared Au colloids were used as seeds for the synthesis of Au@Ag core/shell NPs (Olson et al., 2008). A 0.4-mL aliquot of 0.1 M L-ascorbic acid was added to 2 mL of as-prepared AuNPs and stirred for 5 min at room temperature. Then, 1.2 mL of 1 mM AgNO3 was added dropwise and reacted for 1 h under stirring. The solution color changed from wine red to orange during this period. The mixture was centrifuged (10,000 rpm, 15 min) and washed with Milli-Q water three times. The final deposition was suspended in 200 μL of PBS and stored at 4 °C for further use.
2.5. Preparation of aptamer functionalized Au@Ag core/shell NPs Au@Ag core/shell NPs modified by aptamer were prepared according to the literature with some modification (Wang et al., 2007). The immobilization of aptamer onto Au@Ag core/shell NPs occurs through covalent bonding between Ag and the terminal thiol group. Briefly, 10 μL of 10 μM apt 1 was added to 200 μL of the already prepared Au@Ag core/ shell NPs solution and reacted at 4 °C with gentle shaking for 12 h. The Au@Ag-apt 1 complex was then aged with salts (0.1 M NaCl, 10 mM phosphate, pH 7.0) for 40 h. The prepared complex was centrifuged at 12,000 rpm for 15 min twice to remove the free aptamer. The Au@Agapt 1 was then dispersed in 100 μL of binding buffer for subsequent experiments.
2.6. SERS analysis of sample Various concentrations of samples (10 μL) were mixed with 175 μL of Au@Ag-apt 1 complex and allowed to incubate for 45 min at room temperature. Then, 15 μL of ROX-labeled apt 2 (10 μM) was added and incubated for another 45 min. Following incubation, the mixtures were centrifuged at 3000 rpm and 4 °C for 5 min to remove the extra unbound ROX-apt 2, and the precipitated mixtures were washed twice and resuspended in 200 μL of 1 × BB. The 50 μL of suspend were dropped onto the microscope glass slide and measured. A quantitative analysis of S. typhimurium was performed based on the measured peak area at 1628 cm–1 in the SERS spectrum.
2.7. Preparation of milk samples Five milliliters of the milk sample was pretreated by centrifugation separation (10 °C, 7000 rpm) for 10 min, and the upper cream layer was removed. Subsequently, the supernatant was filtered through a 0.45-μm filtration membrane and diluted with ultrapure water at a 1:20 ratio. A series of known quantities of S. typhimurium were then added to the prepared samples for the experiments.
3. Results and discussion 3.1. Sensing strategy A schematic illustration of the SERS-based aptasensor process for S. typhimurium determination is shown in Fig. 1. Au@Ag core/shell NPs were first synthesized as SERS-active substrates. Then, apt 1 bound with S. typhimurium was conjugated with Au@Ag core/shell NPs and utilized as capture probes. In the presence of the S. typhimurium target, Au@Ag-apt 1 recognized and bonded to S. typhimurium. After the subsequent addition of ROX-modified apt 2, Au@Ag-apt 1-target-apt 2-ROX sandwich-like complexes formed based on the high affinity and specificity of aptamer and S. typhimurium. As more S. typhimurium was added, the surface loading of bound ROX-apt 2 increased, resulting in increased SERS intensity. By monitoring the SERS signal increase along with changes in concentration, highly sensitive quantification of S. typhimurium could be realized.
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N. Duan et al. / International Journal of Food Microbiology 218 (2016) 38–43
Fig. 1. Schematic illustration of SERS-based aptasensor for quantification of S. typhimurium.
Fig. 2. TEM image of AuNPs (A) and Au@Ag core/shell NPs (B), UV/vis absorption spectra of AuNPs and Au@Ag core/shell NPs (C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
N. Duan et al. / International Journal of Food Microbiology 218 (2016) 38–43
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3.2. Characteristics of AuNPs and Au@Ag core–shell NPs Fig. 2 shows the representative TEM images of prepared AuNPs and Au@Ag core/shell NPs that provide evidence for the formation of silver shells on the surface of AuNPs. As shown in Fig. 2A, the AuNPs are monodisperse and spherical with an average size of 15 nm. The size was increased after coating with Ag shell (Fig. 2B). The Ag shell thickness is estimated to be 4 nm (inset Fig. 2B). The effect of silver encapsulation on the UV/vis absorption spectra of Ag NPs is shown in Fig. 2C. The UV/vis spectrum of the AuNPs solution (black line) exhibited a characteristic plasmon absorption peak at 520 nm. Following silver coating, the Au@Ag core/shell NPs gave rise to a new surface plasmon band at 400 nm (red line). 3.3. Characteristics of the nanoparticles conjugated to aptamer Metallic nanoparticles of gold and silver are conventionally used for functionalization of thiol-terminated DNA sequences. To characterize the successful conjugation of the Au@Ag core/shell NPs to the aptamer, UV/vis spectra of Au@Ag core/shell NPs and Au@Ag-apt 1 were recorded. As presented in Fig. 3, Au@Ag show an absorption maximum at 400 nm (curve a). Following the conjugation of the Au@Ag to aptamers, a characteristic aptamer absorption band at approximately 260 nm is observed, indicating successful formation of Au@Ag-apt 1 bioconjugates (curve b). 3.4. Optimization for the assay The effect of Au@Ag-apt 1 on the SERS performance was investigated, and experimental results are shown in Fig. 4. Fig. 4A shows that the SERS intensity of the ROX is gradually enhanced with increasing concentrations of Au@Ag-apt 1 in the 75–200 μL range. The SERS intensity reaches a maximum for the Au@Ag-apt 1 concentration of 175 μL and remains unchanged when the concentration increased to 200 μL. The effect of ROX-apt 2 concentration on SERS spectra was measured experimentally, as shown in Fig. 4B. The maximum and constant SERS intensity of the system occurs at 0.75 μM. Therefore, 175 μL of Au@Ag-apt 1 was selected as the optimum volume, and 0.75 μM of ROX-apt 2 was selected as the optimum concentration for achieving the best SERS performance. 3.5. Analytical performance The Raman peak at 1638 cm–1 is the strongest peak of ROX in the SERS spectra and was used to quantitatively evaluate the amount of S. typhimurium. The calibration curve was constructed by plotting the
Fig. 4. Effect of Au@Ag-apt 1 volume (A) ROX-apt 2 concentration (B) on SERS signals. (Concentration of S. typhimurium was 104 cfu/mL).
peak intensity of the vibrational band at 1638 cm–1 versus the concentration of the analyte. Fig. 5 shows SERS spectra for various concentrations of S. typhimurium and their corresponding calibration curves. The relative Raman intensity of ROX at 1638 cm–1 was monitored and used for the quantitative evaluation of the S. typhimurium cells. As shown in Fig. 5A, a very weak SERS signal was observed (blank) in the absence of S. typhimurium. However, with the increase in the S. typhimurium concentration from 15 to 1.5 × 106 cfu/mL, more sandwich aptamer complexes are formed, and the SERS intensity increased accordingly. Fig. 5B displays the calibration curve resulting from the relative intensity of the SERS signal at 1638 cm–1. There is a good linear relationship between the SERS intensity and the S. typhimurium concentration in the range from 15 to 1.5 × 106 cfu/mL. The linear regression equation is described as Y = 592.54x − 768.98, the corresponding correlation coefficient is 0.996, and the detection limit was found to be 15 cfu/mL.
3.6. Specificity and the analytical performance of the assay
Fig. 3. UV/vis absorption spectra of Au@Ag core/shell NPs and Au@Ag-apt 1 bioconjugates.
To determine the selectivity of the developed method, the response of the SERS assay to other pathogenic bacteria that might affect the detection of S. typhimurium in real sample analysis, including Vibrio parahaemolyticus, S. aureus, Shigella dysenteriae, and Listeria monocytogenes, were examined individually in aqueous buffer. As shown in Fig. 6, it was clearly observed that S. typhimurium resulted in a significantly enhanced Raman signal, while the presence of the other species, even at much high concentrations, influenced the Raman signal only slightly. Thus, the present method could be successfully applied for
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N. Duan et al. / International Journal of Food Microbiology 218 (2016) 38–43 Table 1 Analysis of S. typhimurium cells in the spiked milk samples by the developed method. Original value(cfu/mL) 0 0 0 0 0
Spiked concentration (cfu/mL)
Measured concentration (cfu/mL)
Recovery (%)
6.2 × 10 3.1 × 102 6.2 × 102 3.1 × 103 6.2 × 103
(6.0 ± 0.025) × 10 (3.04 ± 0.017) × 102 (6.15 ± 0.021) × 102 (3.09 ± 0.031) × 103 (6.19 ± 0.011) × 103
96.7 98.1 99.2 99.7 99.8
results obtained via the developed method and those obtained through the standard plate method. 4. Conclusion In the present study, we have developed a SERS-based aptasensor for highly sensitive detection of S. typhimurium. Aptamer-conjugated Au@Ag core/shell nanoparticles were used as sensitive sensing probes. ROX-modified aptamer was used as a recognition element and Raman reporter. S. typhimurium specifically interacted with the aptamer to form Au@Ag-aptamer-target-aptamer-ROX sandwich-like complexes. The obtained SERS aptasensor shows a quite broad linear scope and an ultralow detection limit for S. typhimurium detection and exhibits excellent specificity. This technique shows that it can potentially be used for the rapid and sensitive detection of pathogenic bacteria for food safety assurance. Acknowledgments This work was partially supported by BK20140155, NSFC (31401576, 31401575), JUSRP11547, JUSRP11436, and China Postdoctoral Science Foundation (2015M580402). Fig. 5. SERS responses of the system with increasing concentrations of S. typhimurium (A), and linear correlation between the SERS intensity for the peak centered at 1638 cm–1 and S. typhimurium concentration (B).
the selective detection of S. typhimurium, due to the inherent specificity of aptamers to S. typhimurium. For a realistic application test, different amounts of S. typhimurium were spiked into milk samples. The concentration of S. typhimurium was measured by plate counting methods, which is a standard method for bacteria counting in microbiology. The results are presented in Table 1, indicating that there is no significant difference between the
Fig. 6. Specificity of aptamer-mediated SERS assay. The concentration of S. typhimurium was 104 cfu/mL, and those of the others were 105 cfu/mL.
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Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Salmonella typhimurium detection using a surface-enhanced Raman scattering-based aptasensor Nuo Duan a, Boya Chang a, Hui Zhang b, Zhouping Wang a, Shijia Wu a,c,⁎ a b c
State Key Laboratory of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China China Rural Technology Development Center, Beijing 100045, China School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China
a r t i c l e
i n f o
Article history: Received 28 July 2015 Received in revised form 19 October 2015 Accepted 14 November 2015 Available online xxxx Keywords: SERS Au@Ag core/shell NPs Aptamer S. typhimurium
a b s t r a c t Surface-enhanced Raman spectroscopy (SERS) has been used in a variety of biological applications due to its high sensitivity and specificity. Here, we report a SERS-based aptasensor approach for quantitative detection of pathogenic bacteria. A SERS substrate bearing Au@Ag core/shell nanoparticles (NPs) is functionalized with aptamer 1 (apt 1) for the capture of target molecules. X-rhodamine (ROX)-modified aptamer 2 (apt 2) is used as recognition element and Raman reporter. Salmonella typhimurium specifically interacted with the aptamers to form Au@Ag-apt 1-target-apt 2-ROX sandwich-like complexes. As a result, the concentration of S. typhimurium was determined using this developed aptasensor structure, and a calibration curve is obtained in the range of 15 to 1.5 × 106 cfu/mL with a limit of detection of 15 cfu/mL. Our method was successfully applied to real food samples, and the results are consistent with the results obtained using plate counting methods. We believe that the developed method shows potential for the rapid and sensitive detection of pathogenic bacteria in food safety assurance. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The identification of pathogenic bacteria is an important task in light of the fact that many serious and even fatal medical conditions result from bacterial infection. Salmonella typhimurium, a Gram-negative bacteria pathogen, is one of the leading causes of food-borne illness in human and animal hosts worldwide (Brandt et al., 2013). S. typhimurium is transmitted primarily through the consumption of raw or uncooked vegetables, poultry, eggs, and fruits (Crum-Cianflone, 2008; Mrema et al., 2006). In China, approximately 80% of food-borne bacteria outbreaks are thought to be caused by Salmonella (Yang et al., 2010). This increasing incidence of S. typhimurium in different food products is now attracting the attention of the government. The food safety regulations of some countries (e.g., China, USA) require no tolerance of S. typhimurium in ready-to-eat food. Thus, sensitive detection methods for S. typhimurium play a critical role in many aspects of life, especially in food quality. The existing common detection methods include culture-based methods, molecular methods of regular and real-time polymerase chain reaction (PCR) (Zheng et al., 2014; Park et al., 2013; Silva et al., 2011), and immunoassays (Mantzila et al., 2008; Shim et al., 2014). Despite many advances in these fields, it is still challenging to find new approaches that could improve the simplicity, selectivity, stability and sensitivity of these analytical methods.
⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (S. Wu).
http://dx.doi.org/10.1016/j.ijfoodmicro.2015.11.006 0168-1605/© 2015 Elsevier B.V. All rights reserved.
Currently, SERS is suggested as a powerful tool for characterization of chemical and biological analytes due to its high fingerprint information content, its extreme sensitivity and its obliviousness to the aqueous environment intrinsic to biological systems (Chen et al., 2008; Cialla et al., 2012). Since its discovery in the 1970s, SERS has found increasing application in different fields, such as the biomedical field (Haynes et al., 2005; Kaminska et al., 2015), environmental monitoring (Bhandari et al., 2009; Muller et al., 2014), and food quality assurance (Ma et al., 2015; Aoki et al., 2013). Many efforts have been devoted to the SERS detection of bacterial cells (Sundaram et al., 2013a; Banerjee et al., 2010). Among these, some detection methods were designed based on the spectrum of bacteria themselves (Sundaram et al., 2013b). Kowalska et al. (2015) prepared a Cu-based SERS platform for Staphylococcus aureus detection. The spectrum of S. aureus bacteria exhibits characteristic band vibrations. The band located at 727 cm–1 is assigned to the C–N stretching mode of the adenine moiety of the lipid layer in the cell wall and/or to the purine ring breathing mode. The spectral features at 840, 1027, 1037 and 1084 cm–1 arise from tyrosine, phenylalanine, C–C oscillations, and phosphate binding in DNA, respectively. Additionally, there are many reports of SERS-based assays that used antibodies as recognition agents (Knauer et al., 2012; Wang et al., 2011). Lin and Hamme (2014) combined monoclonal antibody-conjugated sphere-shaped gold nanoparticles with single-walled carbon nanotubes to create a nanohybrid system to selectively detect S. typhimurium DT104 bacteria. The Raman signal intensity was from Rhodamine 6G, with a detection limit of 105 cfu/mL. However, the preparation of the antibodies via animal immunization is time-consuming (several months), and the antibodies may become
N. Duan et al. / International Journal of Food Microbiology 218 (2016) 38–43
susceptible to stability or modification issues. Aptamers with high affinity and selectivity are beginning to emerge as an alternative to antibodies in these applications. To the best of our knowledge, there have been few reports of using SERS coupled with aptamers for the sensitive and rapid detection of bacteria. Therefore, in the present study, we report a sensitive method for the detection of pathogenic bacteria using a SERS sensing platform based on an aptamer. Our strategy exploits the Au@Ag core/shell NPs as the enhanced substrate for SERS. To selectively detect S. typhimurium, we employed apt 1 immobilized on the surface of Au@Ag core/shell NPs. The Raman signal intensity was generated from ROX-modified apt 2, which would bind with the target in the same way as Au@Ag-apt 1. Important factors such as the concentration of Au@Ag-apt 1 and the concentration of ROX-apt 2 were evaluated. The sensitive and selective SERS assay was validated by the analysis of S. typhimurium in food samples, showing its great potential for food quality application. 2. Materials and methods 2.1. Materials Chloroauric acid (HAuCl4), L-ascorbic acid, and AgNO3 were obtained from Sigma-Aldrich (U.S.A.). Trisodium citrate was of analytical grade and was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Milli-Q water with a resistivity of 18.2 MΩ/cm was used throughout the experiments. The S. typhimurium aptamers were prepared in our laboratory (Duan et al., 2013) and synthesized and purified by high-performance liquid chromatography (Sangon Biotechnology, Inc., Shanghai, China). The sequences of the S. typhimurium aptamers are 5′-SH-AGTAATGCCCGGTAGTTATTCAAAGATGAGTAGGAAAAGA-3′ (apt 1) and 5′-ROX-AGTAATGCCCGGTAGTTATTCAAAGATGAGTAGGAAAAGA3′ (apt 2). 2.2. Instrumentation The size and shape of the nanoparticles were tested using transmission electron microscopy (TEM) (TEM, JEOL Ltd., Japan). Absorption spectra were recorded on a UV-1800 spectrophotometer (Shimadzu Co., Japan). The Raman spectra were measured using a Jobin Yvon microRaman spectroscope (Super LabRam II) with a mode of 50 × objective (8 mm), a holographic grating (1800 g/mm), a charge-coupled device detector with 1024 × 256 pixels and a 785 nm excitation laser. 2.3. Bacteria culture The S. typhimurium ATCC 14028 was kindly donated by the Animal, Plant and Food Inspection Centre, Jiangsu Entry-Exit Inspection and Quarantine Bureau (Nanjing, China). S. typhimurium was cultivated in Luria-Bertani medium and then incubated in a shaker at 37 °C for 24 h. One hundred microliters of the bacterial culture was diluted with medium and coated on the agar plates and cultured at 37 °C for 18 h to count colony forming units. The rest of the bacteria were harvested by centrifugation at 3000 rpm and 4 °C and washed twice in 1 × binding buffer (1 × BB 50 mM Tris–HCl at pH 7.4, 5 mM KCl, 100 mM NaCl, and 1 mM MgCl2) at room temperature. 2.4. Preparation of Au@Ag core/shell NPs AuNPs were first synthesized using the citrate reduction method. In brief, all glassware used in the experiment were cleaned with aqua regia (HNO3/HCl, 3:1, v/v), rinsed thoroughly in ultrapure water, and dried prior to use. Then, 500 μL of 1% HAuCl4 solution and 49.5 mL Milli-Q water were heated to boiling with vigorously stirring. Then, 1.5 mL of 1% trisodium citrate was rapidly injected into the boiling reaction mixture. After the mixture was boiled for 15 min, the heating source was removed, and the colloid was cooled to room temperature. The
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prepared Au colloids were used as seeds for the synthesis of Au@Ag core/shell NPs (Olson et al., 2008). A 0.4-mL aliquot of 0.1 M L-ascorbic acid was added to 2 mL of as-prepared AuNPs and stirred for 5 min at room temperature. Then, 1.2 mL of 1 mM AgNO3 was added dropwise and reacted for 1 h under stirring. The solution color changed from wine red to orange during this period. The mixture was centrifuged (10,000 rpm, 15 min) and washed with Milli-Q water three times. The final deposition was suspended in 200 μL of PBS and stored at 4 °C for further use.
2.5. Preparation of aptamer functionalized Au@Ag core/shell NPs Au@Ag core/shell NPs modified by aptamer were prepared according to the literature with some modification (Wang et al., 2007). The immobilization of aptamer onto Au@Ag core/shell NPs occurs through covalent bonding between Ag and the terminal thiol group. Briefly, 10 μL of 10 μM apt 1 was added to 200 μL of the already prepared Au@Ag core/ shell NPs solution and reacted at 4 °C with gentle shaking for 12 h. The Au@Ag-apt 1 complex was then aged with salts (0.1 M NaCl, 10 mM phosphate, pH 7.0) for 40 h. The prepared complex was centrifuged at 12,000 rpm for 15 min twice to remove the free aptamer. The Au@Agapt 1 was then dispersed in 100 μL of binding buffer for subsequent experiments.
2.6. SERS analysis of sample Various concentrations of samples (10 μL) were mixed with 175 μL of Au@Ag-apt 1 complex and allowed to incubate for 45 min at room temperature. Then, 15 μL of ROX-labeled apt 2 (10 μM) was added and incubated for another 45 min. Following incubation, the mixtures were centrifuged at 3000 rpm and 4 °C for 5 min to remove the extra unbound ROX-apt 2, and the precipitated mixtures were washed twice and resuspended in 200 μL of 1 × BB. The 50 μL of suspend were dropped onto the microscope glass slide and measured. A quantitative analysis of S. typhimurium was performed based on the measured peak area at 1628 cm–1 in the SERS spectrum.
2.7. Preparation of milk samples Five milliliters of the milk sample was pretreated by centrifugation separation (10 °C, 7000 rpm) for 10 min, and the upper cream layer was removed. Subsequently, the supernatant was filtered through a 0.45-μm filtration membrane and diluted with ultrapure water at a 1:20 ratio. A series of known quantities of S. typhimurium were then added to the prepared samples for the experiments.
3. Results and discussion 3.1. Sensing strategy A schematic illustration of the SERS-based aptasensor process for S. typhimurium determination is shown in Fig. 1. Au@Ag core/shell NPs were first synthesized as SERS-active substrates. Then, apt 1 bound with S. typhimurium was conjugated with Au@Ag core/shell NPs and utilized as capture probes. In the presence of the S. typhimurium target, Au@Ag-apt 1 recognized and bonded to S. typhimurium. After the subsequent addition of ROX-modified apt 2, Au@Ag-apt 1-target-apt 2-ROX sandwich-like complexes formed based on the high affinity and specificity of aptamer and S. typhimurium. As more S. typhimurium was added, the surface loading of bound ROX-apt 2 increased, resulting in increased SERS intensity. By monitoring the SERS signal increase along with changes in concentration, highly sensitive quantification of S. typhimurium could be realized.
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Fig. 1. Schematic illustration of SERS-based aptasensor for quantification of S. typhimurium.
Fig. 2. TEM image of AuNPs (A) and Au@Ag core/shell NPs (B), UV/vis absorption spectra of AuNPs and Au@Ag core/shell NPs (C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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3.2. Characteristics of AuNPs and Au@Ag core–shell NPs Fig. 2 shows the representative TEM images of prepared AuNPs and Au@Ag core/shell NPs that provide evidence for the formation of silver shells on the surface of AuNPs. As shown in Fig. 2A, the AuNPs are monodisperse and spherical with an average size of 15 nm. The size was increased after coating with Ag shell (Fig. 2B). The Ag shell thickness is estimated to be 4 nm (inset Fig. 2B). The effect of silver encapsulation on the UV/vis absorption spectra of Ag NPs is shown in Fig. 2C. The UV/vis spectrum of the AuNPs solution (black line) exhibited a characteristic plasmon absorption peak at 520 nm. Following silver coating, the Au@Ag core/shell NPs gave rise to a new surface plasmon band at 400 nm (red line). 3.3. Characteristics of the nanoparticles conjugated to aptamer Metallic nanoparticles of gold and silver are conventionally used for functionalization of thiol-terminated DNA sequences. To characterize the successful conjugation of the Au@Ag core/shell NPs to the aptamer, UV/vis spectra of Au@Ag core/shell NPs and Au@Ag-apt 1 were recorded. As presented in Fig. 3, Au@Ag show an absorption maximum at 400 nm (curve a). Following the conjugation of the Au@Ag to aptamers, a characteristic aptamer absorption band at approximately 260 nm is observed, indicating successful formation of Au@Ag-apt 1 bioconjugates (curve b). 3.4. Optimization for the assay The effect of Au@Ag-apt 1 on the SERS performance was investigated, and experimental results are shown in Fig. 4. Fig. 4A shows that the SERS intensity of the ROX is gradually enhanced with increasing concentrations of Au@Ag-apt 1 in the 75–200 μL range. The SERS intensity reaches a maximum for the Au@Ag-apt 1 concentration of 175 μL and remains unchanged when the concentration increased to 200 μL. The effect of ROX-apt 2 concentration on SERS spectra was measured experimentally, as shown in Fig. 4B. The maximum and constant SERS intensity of the system occurs at 0.75 μM. Therefore, 175 μL of Au@Ag-apt 1 was selected as the optimum volume, and 0.75 μM of ROX-apt 2 was selected as the optimum concentration for achieving the best SERS performance. 3.5. Analytical performance The Raman peak at 1638 cm–1 is the strongest peak of ROX in the SERS spectra and was used to quantitatively evaluate the amount of S. typhimurium. The calibration curve was constructed by plotting the
Fig. 4. Effect of Au@Ag-apt 1 volume (A) ROX-apt 2 concentration (B) on SERS signals. (Concentration of S. typhimurium was 104 cfu/mL).
peak intensity of the vibrational band at 1638 cm–1 versus the concentration of the analyte. Fig. 5 shows SERS spectra for various concentrations of S. typhimurium and their corresponding calibration curves. The relative Raman intensity of ROX at 1638 cm–1 was monitored and used for the quantitative evaluation of the S. typhimurium cells. As shown in Fig. 5A, a very weak SERS signal was observed (blank) in the absence of S. typhimurium. However, with the increase in the S. typhimurium concentration from 15 to 1.5 × 106 cfu/mL, more sandwich aptamer complexes are formed, and the SERS intensity increased accordingly. Fig. 5B displays the calibration curve resulting from the relative intensity of the SERS signal at 1638 cm–1. There is a good linear relationship between the SERS intensity and the S. typhimurium concentration in the range from 15 to 1.5 × 106 cfu/mL. The linear regression equation is described as Y = 592.54x − 768.98, the corresponding correlation coefficient is 0.996, and the detection limit was found to be 15 cfu/mL.
3.6. Specificity and the analytical performance of the assay
Fig. 3. UV/vis absorption spectra of Au@Ag core/shell NPs and Au@Ag-apt 1 bioconjugates.
To determine the selectivity of the developed method, the response of the SERS assay to other pathogenic bacteria that might affect the detection of S. typhimurium in real sample analysis, including Vibrio parahaemolyticus, S. aureus, Shigella dysenteriae, and Listeria monocytogenes, were examined individually in aqueous buffer. As shown in Fig. 6, it was clearly observed that S. typhimurium resulted in a significantly enhanced Raman signal, while the presence of the other species, even at much high concentrations, influenced the Raman signal only slightly. Thus, the present method could be successfully applied for
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N. Duan et al. / International Journal of Food Microbiology 218 (2016) 38–43 Table 1 Analysis of S. typhimurium cells in the spiked milk samples by the developed method. Original value(cfu/mL) 0 0 0 0 0
Spiked concentration (cfu/mL)
Measured concentration (cfu/mL)
Recovery (%)
6.2 × 10 3.1 × 102 6.2 × 102 3.1 × 103 6.2 × 103
(6.0 ± 0.025) × 10 (3.04 ± 0.017) × 102 (6.15 ± 0.021) × 102 (3.09 ± 0.031) × 103 (6.19 ± 0.011) × 103
96.7 98.1 99.2 99.7 99.8
results obtained via the developed method and those obtained through the standard plate method. 4. Conclusion In the present study, we have developed a SERS-based aptasensor for highly sensitive detection of S. typhimurium. Aptamer-conjugated Au@Ag core/shell nanoparticles were used as sensitive sensing probes. ROX-modified aptamer was used as a recognition element and Raman reporter. S. typhimurium specifically interacted with the aptamer to form Au@Ag-aptamer-target-aptamer-ROX sandwich-like complexes. The obtained SERS aptasensor shows a quite broad linear scope and an ultralow detection limit for S. typhimurium detection and exhibits excellent specificity. This technique shows that it can potentially be used for the rapid and sensitive detection of pathogenic bacteria for food safety assurance. Acknowledgments This work was partially supported by BK20140155, NSFC (31401576, 31401575), JUSRP11547, JUSRP11436, and China Postdoctoral Science Foundation (2015M580402). Fig. 5. SERS responses of the system with increasing concentrations of S. typhimurium (A), and linear correlation between the SERS intensity for the peak centered at 1638 cm–1 and S. typhimurium concentration (B).
the selective detection of S. typhimurium, due to the inherent specificity of aptamers to S. typhimurium. For a realistic application test, different amounts of S. typhimurium were spiked into milk samples. The concentration of S. typhimurium was measured by plate counting methods, which is a standard method for bacteria counting in microbiology. The results are presented in Table 1, indicating that there is no significant difference between the
Fig. 6. Specificity of aptamer-mediated SERS assay. The concentration of S. typhimurium was 104 cfu/mL, and those of the others were 105 cfu/mL.
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