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A novel aptasensor for the colorimetric detection of S. typhimurium based on gold nanoparticles
International Journal of Food Microbiology 245 (2017) 1–5
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
A novel aptasensor for the colorimetric detection of S. typhimurium based on gold nanoparticles Xiaoyuan Ma a,b, Liangjing Song a, Nixin Zhou c, Yu Xia a,b, Zhouping Wang a,b,⁎ a b c
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, PR China Department of Health Management and Basic Education, Jiangsu Jiankang Vocational College, Nanjing 211800, PR China
a r t i c l e
i n f o
Article history: Received 16 October 2016 Received in revised form 28 December 2016 Accepted 29 December 2016 Available online 30 December 2016 Keywords: Gold nanoparticles Aptamer Colorimetric detection S. typhimurium
a b s t r a c t A simple, fast and convenient colorimetric aptasensor was fabricated for the detection of Salmonella typhimurium (S. typhimurium) which was based on the color change effect of gold nanoparticles (GNPs). S. typhimurium is one of the most common causes of food-associated disease. Aptamers with specific recognition toward S. typhimurium was modified to the surface of prepared GNPs. They play a role for the protection of GNPs from aggregation toward high concentrations of NaCl. With the addition of S. typhimurium, aptamers preferably combined to S. typhimurium and the protection effect was broken. With more S. typhimurium, more aptamers detached from GNPs. In such a situation, the exposed GNPs would aggregated to some extent with the addition of NaCl. The color changed from red, purple to blue which could be characterized by UV–Vis spectrophotometer. The absorbance spectra of GNPs redshifted constantly and the intensity ratio of A700/A521 changed regularly. This could be calculated for the basis of quantitative detection of S. typhimurium from 102 cfu/mL to 107 cfu/mL. The obtained linear correlation equation was y = 0.1946x–0.2800 (R2 = 0.9939) with a detection limit as low as 56 cfu/mL. This method is simple and rapid, results in high sensitivity and specificity, and can be used to detect actual samples. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Gold nanoparticles (GNPs) have been widely explored in the field of analytical chemistry, biology, diagnosis and treatment of cancer owing to their specific optical, chemical, electrochemical and catalytical properties. They could combine with a variety of biological macromolecules and will not affect the biological activity. GNPs could be synthesized by several reductants, such as sodium citrate, sodium borohydride, HEPESNaOH, et al. (Grabar et al., 1996; Phadtare et al., 2003; Xie et al., 2007). In recent years, the application of GNPs for the visual detection has attracted more and more attention. These targets mainly include foodborne pathogens, antibiotics, toxins, metal ions, pesticide residues, illegal food additives and so on (Su et al., 2012; Kalidasan et al., 2013; Kim et al., 2010; Yang et al., 2011; Wu et al., 2013; Xu et al., 2010). Tanga has used GNPs labeling and silver staining method to achieve the visual detection of trachoma Chlamydia (Tanga et al., 2010). The detection limit can reach 2 ng/mL. Shyu has successfully realized the lateral chromatography detection of Staphylococcus aureus enterotoxin B using GNPs and silver enhancement technique with a detection limit ⁎ Corresponding author. E-mail address: [email protected] (Z. Wang).
http://dx.doi.org/10.1016/j.ijfoodmicro.2016.12.024 0168-1605/© 2017 Elsevier B.V. All rights reserved.
of 10 pg/mL (Rong-Hwa et al., 2010). Besides, in the treatment of diseases, Nam has designed a kind of “smart” GNPs. The exposure to acidic environment or cancer cells could result in the aggregation of the “smart” GNPs (Nam et al., 2009). The absorption peaks shift to the near infrared wavelength and it can be applied to the photo-thermal cancer therapy. Aptamers are specific oligonucleotide sequences that bind to the target substance (Tombelli et al., 2005). They are usually screened by SELEX technique from a DNA or RNA library (Hamula et al., 2006). Due to their high specificity and affinity with its corresponding target materials, aptamers are widely applied to the analysis and detection field especially in food security detecting (Suh et al., 2014; Kim et al., 2014; Guo et al., 2014; Luo et al., 2014a, 2014b; Ragavan et al., 2013). Girolamo has fixed the OTA aptamer to the surface of the coupled gel (Girolamo et al., 2011). It has been used as a solid phase extraction column to remove OTA from wheat samples. Bai has proposed an array sensor based on the aptamer to detect kanamycin (Bai et al., 2014). It is fast and without any labeling. Xue has established an electrochemical aptasensor for the detection of bisphenol A in water samples within 30 min (Xue et al., 2013). The detection limit can reach 0.284 pg/mL. S. typhimurium is a common anaerobic gram-negative bacteria, which is widely distributed in the environment. It is first isolated by
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X. Ma et al. / International Journal of Food Microbiology 245 (2017) 1–5
Salmon since the 1885 cholera epidemic and has been found more than 2500 serum types and variants so far. Almost all of these serum types may be parasitic in the intestines of human body or animals. S. typhimurium can be transmitted to humans through contaminated poultry, eggs, milk, fish and meat products. With the increasing awareness of food safety, the design of fast, efficient, simple and specific method for the detection of S. typhimurium has become an inevitable trend in the field of food safety and health inspection. The traditional culture method for S. typhimurium detection is tedious and time-consuming which includes the sequential steps of pre-enrichment, selective enrichment and selective differential plating (Patel et al., 2006). It is difficult to meet the needs of the current detection of foodborne bacteria. A variety of detection methods have been reported based on the convenient and fast detection demand, such as immunofluorescence detection, immunodiffusion method, enzyme linked immunoassay (ELISA), gene chip techniques, resistance detection method and latex agglutination test, et al. (Falkenhorst et al., 2013; Jain et al., 2012; Ma et al., 2014; Luo et al., 2014a, 2014b). In this study, a new colorimetric detection of S. typhimurium based on GNPs and aptamer is discussed. The specific S. typhimurium aptamer was first modified at the surface of prepared GNPs. They protect GNPs from aggregation in high NaCl solution. With the addition of S. typhimurium, aptamer preferably connected to S. typhimurium and detached from GNPs. Without the protection effect of aptamer, GNPs will be aggregated. The color that changed from red, purple to blue could be observed by naked eyes and characterized by UV–Vis spectrophotometer. S. typhimurium could thus be quantified. It is a simple, fast, and convenient method without complex detecting instrument. Moreover, it is expected to be applied in other food safety detection fields.
2. Materials and methods 2.1. Materials Chloroauric acid tetrahydrate (HAuCl4·4H2O), Trisodium citrate (Na3C6H5O7), sodium chloride (NaCl) were purchased from the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The S. typhimurium aptamer sequence is 5′ - TAT GGC GGC GTC ACC CGA CGG GGA CTT GAC ATT ATG ACA G - 3′ which is synthesized by Shanghai Sangon Biological Science & Technology Company (Shanghai, China). The ultrapure water used in the experiments was prepared using a Millipore Direct-Q® 3 system (Merck Millipore, MA, USA) and had a resistivity of 18.2 MΩ cm.
2.2. Preparation of GNPs GNPs were prepared with some modifications as described by Grabar et al. (1996). First, 4.2 mL HAuCl4·4H2O (1%, w/w) and 95.8 mL ultrapure water were added to a flask with three necks. The mixture was heated to boil until 10 min under uniform magnetic stirring with oil bath. Then, 10 mL sodium citrate (1%, w/w) was rapidly injected and reacted for another 15 min. The obtained wine-red solution was GNPs. The resulting GNPs were purified by three times of centrifugation (10,000 rpm, 25 min) and were redispersed in 40 mL of ultrapure water. The GNPs were stored at 4 °C for further use and characterization. 2.3. Preparation of GNPs-aptamer complex (aptasensor) The S. typhimurium aptamer solution was centrifuged at 10,000 rpm for 5 min (4 °C). Then it was diluted to 70 pM by PBS. 1 mL GNPs was added to 100 μL 70 pM S. typhimurium aptamer solution and incubated at 37 °C for 30 h. The mixture was centrifuged at 10,000 rpm for 10 min (4 °C) to remove the excessive aptamer. Thus, the GNPs-aptamer complex (aptasensor) was prepared and stored at 4 °C for further use. 2.4. Detection of S. typhimurium based on the aptasensor 100 μL gradient dilutions of S. typhimurium (10 cfu/mL, 102 cfu/mL, 103 cfu/mL, 104 cfu/mL, 105 cfu/mL, 106 cfu/mL, 107 cfu/mL, 108 cfu/mL) was added to 100 μL aptasensor and incubated at 37 °C for 1 h. Then, 1.5 M NaCl was added and mixed. The resulting solution was characterized by UV–Vis spectrophotometer. 2.5. Recovery experiments for milk sample In this experiment, the commercial milk was used as realistic samples for recovery experiments in the detection of S. typhimurium. Gradient dilutions of S. typhimurium were added to the milk sample. Then the aptasensor based detection method was conducted to calculate the detectable amount of S. typhimurium. The results were compared with the traditional plate counting method and the recovery rate was calculated. 3. Results and discussion 3.1. Aptasensor based detection for S. typhimurium The aptasensor based detection process using GNPs for S. typhimurium is shown in Fig. 1. Firstly, the S. typhimurium specific aptamer was attached to GNPs via the effect of thiol group. When the S. typhimurium
Fig. 1. Schematic illustration for the colorimetric detection of S. typhimurium based on GNPs and aptamers.
X. Ma et al. / International Journal of Food Microbiology 245 (2017) 1–5
3
Fig. 2. (A) TEM image and (B) UV–visible absorption spectra of GNPs.
was added, the aptamer sequences would preferentially combined to S. typhimurium and split away off GNPs. Then, with the addition of NaCl, free GNPs would be aggregated. On the contrary, if the GNPs were decorated with aptamer, they would not aggregate and still show a single scattered state owing to the protective effect of a large number of negative charges on the surfaces. The aggregation degree could be characterized by the UV–Vis spectrophotometer. So, the S. typhimurium could be quantified using the GNPs based aptasensor.
followed by the increase of time. At the same incubation time, the binding rate increased when the reaction temperature was increased. When the reaction temperature was at 37 °C, the binding rate was above 50%. If the temperature is further increased, the aptamer sequence will be changed and the properties of GNPs will be destroyed. Therefore, we use 37 °C as the optimal incubation temperature.
3.2. Preparation of GNPs
It is reported by Li and Rothberg (2004) that the single stranded ssDNA in solution showed irregular curl state. They could be adsorbed to GNPs by electrostatic force effect of the exposed bases, the Van der Waals force and hydrophobic interactions. The adsorption process is related to the temperature and time. The adsorption of aptamer and GNPs were conducted at different temperatures for different times. Results were shown in Fig. 3. The binding rate was calculated by characterizing the absorption intensity of aptamer at 270 nm before and after connecting to GNPs. At the same temperature, the binding rate increased with the increase of incubation time. It reached the maximum value at 30 h and no longer changed
Generally, GNPs without any decoration will be aggregated at high concentration of salt solution. They form an irregular network structure other than monodispersion. The color changes from red to purple or blue. When GNPs were decorated with aptamer, the negative charge of the phosphate backbone could effectively protect the GNPs from the salt ions. The GNPs will not be aggregated and change color in a certain concentration range of salt solution. Therefore, it is the key factor to find the maximum NaCl solution concentration for the aptamer protected GNPs solution without changing color. The optimization experiment was conducted as follows. 100 μL 10−5 M aptamer was added to 500 μL GNPs in order to ensure that the aptamer was absolutely excessive. In other words, all GNPs were adsorbed by the aptamer and the protective effect was produced. After adding different concentrations of NaCl, the color change could be observed by naked eyes and characterized by UV–Vis spectrophotometer shown in Fig. 4. When the NaCl concentration changed from 0.5 M to 1.5 M, the absorption intensity at 521 nm decreased slightly. The color of GNPs remained red wine. When it further reached to 1.75 M, the color of GNPs changed to purple observed by naked eyes.
Fig. 3. The influence of incubation time and reaction temperature for the binding rate of aptamers at the surface of GNPs.
Fig. 4. UV–visible absorption spectra of GNPs at different concentrations of NaCl solution (1–6: 0.50 M, 0.75 M, 1.00 M, 1.25 M, 1.50 M, 1.75 M).
Fig. 2 is the TEM image and UV–visible absorption spectrum of GNPs. It shows that the prepared GNPs have an absorption peak at approximately 521 nm and the size is ~15 nm. 3.3. Optimization for the decoration of aptamer to GNPs
3.4. Optimization of NaCl concentration
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concentration of aptamer was selected as 80 pM and used for further experiment. 3.6. Analytical performance
Fig. 5. Optimization for the concentration of aptamer (1–8: 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM).
It can be seen that the absorption intensity at 521 nm obviously decreased along with the redshift of the absorption peak. The absorption intensity at 700 nm increased. All these illustrated that GNPs aggregated together. The NaCl concentration had already exceeded the suitable aptamer protected GNPs from aggregation discoloration. So the optimal concentration of NaCl was 1.5 M.
3.5. Optimization of aptamer concentration In order to explore the protective effect of the aptamer in the high salt concentration solution, the suitable aptamer amount is very important to ensure the experiment operation. 100 μL different dilutions of aptamer (10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM) were added to 500 μL GNPs and incubated at 37 °C for 30 h. Then, 1.5 M NaCl was mixed and the solution color was observed by naked eyes and characterized by UV–Vis spectrophotometer. Results were shown in Fig. 5. When the concentration of aptamer increased from 10 pM to 70 pM, the protective effect toward GNPs increased. The GNPs were not easy to change their monodispersed state. As for the absorption peak, it remained closer to the pure GNPs solution. While the concentration of aptamer increased to 80 pM, the absorption peak remained almost the same as the addition of 70 pM aptamer, which illustrated that the amount of adapter was saturated. So the optimal
Under the optimal experimental conditions, a series of concentrations of S. typhimurium (0 cfu/mL, 10 cfu/mL, 102 cfu/mL, 103 cfu/mL, 104 cfu/mL, 105 cfu/mL, 106 cfu/mL, 107 cfu/mL, 108 cfu/mL) were detected. Results were shown in Fig. 6. When the amount was 10 cfu/mL, the absorption peak almost overlapped with the control peak. The reason is that the concentration of S. typhimurium was too low, rarely aptamers were split away off GNPs. The NaCl solution was not enough to cause the aggregation of GNPs color change. The absorbance spectra redshifted constantly with the increased amount of S. typhimurium. The absorbance intensity at 521 nm gradually decreased while the absorbance intensity at 700 nm increased (Fig. 6A). While the concentration reached to 107 cfu/mL, the absorption peak change was no longer evident. That's because all GNPs had been separated from the aptamers, and the concentration of S. typhimurium had reached saturation. The ratio of A700/A521 was calculated as the basis for quantitative analysis. As depicted in Fig. 6B, there was a good linear correlation between the ratio of A700/A521 and the amount of S. typhimurium ranged from 102 cfu/mL to 107 cfu/mL. The linear correlation equation obtained was y = 0.1946x–0.2800 (R2 = 0.9939). The statistical analysis revealed that the detection limit of S. typhimurium was 56 cfu/mL. The detection limit is based on the calculation formula D = 3 N/S (N is the standard deviation of blank sample signal. S is the slope of standard curve). 3.7. Specificity Some other pathogenic bacteria were selected to do the specificity detection. These included S. aureus, V. parahemolyticus, B. cereus, S. dysenteriae. The concentration of pathogenic bacteria was maintained at 105 cfu/mL. Experimental results shown in Fig. 7 clearly showed that the ratio of A700/A521 for the other pathogenic bacteria were much lower than that of the S. typhimurium. 3.8. Milk sample detection The utility for the colorimetric detection of S. typhimurium was examined using milk sample obtained from a supermarket. The sample was tested using the new method and the classical plate counting
Fig. 6. (A) UV–visible absorption spectra of GNPs at different concentrations of S. typhimurium (0–8: 0 cfu/mL, 10 cfu/mL, 102 cfu/mL, 103 cfu/mL, 104 cfu/mL, 105 cfu/mL, 106 cfu/mL, 107 cfu/mL, 108 cfu/mL). (B) The corresponding linear relationship between different concentrations of S. typhimurium and A700/A521 ratio.
X. Ma et al. / International Journal of Food Microbiology 245 (2017) 1–5
Fig. 7. Specificity result for the detection of (a) S. typhimurium, (b) S. aureus, (c) V. parahemolyticus, (d) B. cereus, (e) S. dysenteriae. Table 1 Comparison of the milk sample results obtained from the colorimetric detection and classical plate counting method (all results were repeated three times and shown as average ± SD). Milk sample
Plate counting (cfu/mL)
Colorimetric detection (cfu/mL)
Recovery ratio (%)
1 2 3 4 5
28 ± 1 69 ± 3 251 ± 5 704 ± 10 1176 ± 12
ND 72 ± 3 242 ± 6 699 ± 9 1183 ± 13
ND 104.3% 96.4% 99.5% 100.5%
methods. The analytical results were shown in Table 1. The results obtained using the colorimetric detection method were similar to those obtained using the plate counting method. The recoveries were between 96.4% and 104.3%, indicating good accuracy of the proposed test for S. typhimurium detection. 4. Conclusion The colorimetric detection of S. typhimurium using GNPs and aptamer was investigated. It is simple, fast and without complex detection procedure or instrument. Aptamers immobilized at the surface of GNPs could protect GNPs from aggregation toward high concentration of NaCl. The addition of S. typhimurium broke the protection layer due to preferable connections between aptamers and S. typhimurium. Under the optimal experimental conditions, a good linear correlation between the ratio of A700/A521 and the amount of S. typhimurium ranged from 102 cfu/mL to 107 cfu/mL was achieved. The obtained linear correlation equation was y = 0.1946x–0.2800 (R2 = 0.9939) with a detection limit of 56 cfu/mL. In addition, this developed method was successfully used to analyze milk samples, and there was no significant difference between a classical plate counting method and the developed method. So it has the potential for wide use in the detection of other foodborne pathogenic bacteria in food samples. Acknowledgments This work was supported by the NSFC (31401665, 21375049), National S&T Support Program of China (2015BAD17B02), and program of “Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province”. References Bai, X.J., Hou, H., Zhang, B.L., Tang, J.L., 2014. Label-free detection of kanamycin using aptamer-based cantilever array sensor. Biosens. Bioelectron. 56, 112–116.
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Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
A novel aptasensor for the colorimetric detection of S. typhimurium based on gold nanoparticles Xiaoyuan Ma a,b, Liangjing Song a, Nixin Zhou c, Yu Xia a,b, Zhouping Wang a,b,⁎ a b c
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, PR China Department of Health Management and Basic Education, Jiangsu Jiankang Vocational College, Nanjing 211800, PR China
a r t i c l e
i n f o
Article history: Received 16 October 2016 Received in revised form 28 December 2016 Accepted 29 December 2016 Available online 30 December 2016 Keywords: Gold nanoparticles Aptamer Colorimetric detection S. typhimurium
a b s t r a c t A simple, fast and convenient colorimetric aptasensor was fabricated for the detection of Salmonella typhimurium (S. typhimurium) which was based on the color change effect of gold nanoparticles (GNPs). S. typhimurium is one of the most common causes of food-associated disease. Aptamers with specific recognition toward S. typhimurium was modified to the surface of prepared GNPs. They play a role for the protection of GNPs from aggregation toward high concentrations of NaCl. With the addition of S. typhimurium, aptamers preferably combined to S. typhimurium and the protection effect was broken. With more S. typhimurium, more aptamers detached from GNPs. In such a situation, the exposed GNPs would aggregated to some extent with the addition of NaCl. The color changed from red, purple to blue which could be characterized by UV–Vis spectrophotometer. The absorbance spectra of GNPs redshifted constantly and the intensity ratio of A700/A521 changed regularly. This could be calculated for the basis of quantitative detection of S. typhimurium from 102 cfu/mL to 107 cfu/mL. The obtained linear correlation equation was y = 0.1946x–0.2800 (R2 = 0.9939) with a detection limit as low as 56 cfu/mL. This method is simple and rapid, results in high sensitivity and specificity, and can be used to detect actual samples. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Gold nanoparticles (GNPs) have been widely explored in the field of analytical chemistry, biology, diagnosis and treatment of cancer owing to their specific optical, chemical, electrochemical and catalytical properties. They could combine with a variety of biological macromolecules and will not affect the biological activity. GNPs could be synthesized by several reductants, such as sodium citrate, sodium borohydride, HEPESNaOH, et al. (Grabar et al., 1996; Phadtare et al., 2003; Xie et al., 2007). In recent years, the application of GNPs for the visual detection has attracted more and more attention. These targets mainly include foodborne pathogens, antibiotics, toxins, metal ions, pesticide residues, illegal food additives and so on (Su et al., 2012; Kalidasan et al., 2013; Kim et al., 2010; Yang et al., 2011; Wu et al., 2013; Xu et al., 2010). Tanga has used GNPs labeling and silver staining method to achieve the visual detection of trachoma Chlamydia (Tanga et al., 2010). The detection limit can reach 2 ng/mL. Shyu has successfully realized the lateral chromatography detection of Staphylococcus aureus enterotoxin B using GNPs and silver enhancement technique with a detection limit ⁎ Corresponding author. E-mail address: [email protected] (Z. Wang).
http://dx.doi.org/10.1016/j.ijfoodmicro.2016.12.024 0168-1605/© 2017 Elsevier B.V. All rights reserved.
of 10 pg/mL (Rong-Hwa et al., 2010). Besides, in the treatment of diseases, Nam has designed a kind of “smart” GNPs. The exposure to acidic environment or cancer cells could result in the aggregation of the “smart” GNPs (Nam et al., 2009). The absorption peaks shift to the near infrared wavelength and it can be applied to the photo-thermal cancer therapy. Aptamers are specific oligonucleotide sequences that bind to the target substance (Tombelli et al., 2005). They are usually screened by SELEX technique from a DNA or RNA library (Hamula et al., 2006). Due to their high specificity and affinity with its corresponding target materials, aptamers are widely applied to the analysis and detection field especially in food security detecting (Suh et al., 2014; Kim et al., 2014; Guo et al., 2014; Luo et al., 2014a, 2014b; Ragavan et al., 2013). Girolamo has fixed the OTA aptamer to the surface of the coupled gel (Girolamo et al., 2011). It has been used as a solid phase extraction column to remove OTA from wheat samples. Bai has proposed an array sensor based on the aptamer to detect kanamycin (Bai et al., 2014). It is fast and without any labeling. Xue has established an electrochemical aptasensor for the detection of bisphenol A in water samples within 30 min (Xue et al., 2013). The detection limit can reach 0.284 pg/mL. S. typhimurium is a common anaerobic gram-negative bacteria, which is widely distributed in the environment. It is first isolated by
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X. Ma et al. / International Journal of Food Microbiology 245 (2017) 1–5
Salmon since the 1885 cholera epidemic and has been found more than 2500 serum types and variants so far. Almost all of these serum types may be parasitic in the intestines of human body or animals. S. typhimurium can be transmitted to humans through contaminated poultry, eggs, milk, fish and meat products. With the increasing awareness of food safety, the design of fast, efficient, simple and specific method for the detection of S. typhimurium has become an inevitable trend in the field of food safety and health inspection. The traditional culture method for S. typhimurium detection is tedious and time-consuming which includes the sequential steps of pre-enrichment, selective enrichment and selective differential plating (Patel et al., 2006). It is difficult to meet the needs of the current detection of foodborne bacteria. A variety of detection methods have been reported based on the convenient and fast detection demand, such as immunofluorescence detection, immunodiffusion method, enzyme linked immunoassay (ELISA), gene chip techniques, resistance detection method and latex agglutination test, et al. (Falkenhorst et al., 2013; Jain et al., 2012; Ma et al., 2014; Luo et al., 2014a, 2014b). In this study, a new colorimetric detection of S. typhimurium based on GNPs and aptamer is discussed. The specific S. typhimurium aptamer was first modified at the surface of prepared GNPs. They protect GNPs from aggregation in high NaCl solution. With the addition of S. typhimurium, aptamer preferably connected to S. typhimurium and detached from GNPs. Without the protection effect of aptamer, GNPs will be aggregated. The color that changed from red, purple to blue could be observed by naked eyes and characterized by UV–Vis spectrophotometer. S. typhimurium could thus be quantified. It is a simple, fast, and convenient method without complex detecting instrument. Moreover, it is expected to be applied in other food safety detection fields.
2. Materials and methods 2.1. Materials Chloroauric acid tetrahydrate (HAuCl4·4H2O), Trisodium citrate (Na3C6H5O7), sodium chloride (NaCl) were purchased from the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The S. typhimurium aptamer sequence is 5′ - TAT GGC GGC GTC ACC CGA CGG GGA CTT GAC ATT ATG ACA G - 3′ which is synthesized by Shanghai Sangon Biological Science & Technology Company (Shanghai, China). The ultrapure water used in the experiments was prepared using a Millipore Direct-Q® 3 system (Merck Millipore, MA, USA) and had a resistivity of 18.2 MΩ cm.
2.2. Preparation of GNPs GNPs were prepared with some modifications as described by Grabar et al. (1996). First, 4.2 mL HAuCl4·4H2O (1%, w/w) and 95.8 mL ultrapure water were added to a flask with three necks. The mixture was heated to boil until 10 min under uniform magnetic stirring with oil bath. Then, 10 mL sodium citrate (1%, w/w) was rapidly injected and reacted for another 15 min. The obtained wine-red solution was GNPs. The resulting GNPs were purified by three times of centrifugation (10,000 rpm, 25 min) and were redispersed in 40 mL of ultrapure water. The GNPs were stored at 4 °C for further use and characterization. 2.3. Preparation of GNPs-aptamer complex (aptasensor) The S. typhimurium aptamer solution was centrifuged at 10,000 rpm for 5 min (4 °C). Then it was diluted to 70 pM by PBS. 1 mL GNPs was added to 100 μL 70 pM S. typhimurium aptamer solution and incubated at 37 °C for 30 h. The mixture was centrifuged at 10,000 rpm for 10 min (4 °C) to remove the excessive aptamer. Thus, the GNPs-aptamer complex (aptasensor) was prepared and stored at 4 °C for further use. 2.4. Detection of S. typhimurium based on the aptasensor 100 μL gradient dilutions of S. typhimurium (10 cfu/mL, 102 cfu/mL, 103 cfu/mL, 104 cfu/mL, 105 cfu/mL, 106 cfu/mL, 107 cfu/mL, 108 cfu/mL) was added to 100 μL aptasensor and incubated at 37 °C for 1 h. Then, 1.5 M NaCl was added and mixed. The resulting solution was characterized by UV–Vis spectrophotometer. 2.5. Recovery experiments for milk sample In this experiment, the commercial milk was used as realistic samples for recovery experiments in the detection of S. typhimurium. Gradient dilutions of S. typhimurium were added to the milk sample. Then the aptasensor based detection method was conducted to calculate the detectable amount of S. typhimurium. The results were compared with the traditional plate counting method and the recovery rate was calculated. 3. Results and discussion 3.1. Aptasensor based detection for S. typhimurium The aptasensor based detection process using GNPs for S. typhimurium is shown in Fig. 1. Firstly, the S. typhimurium specific aptamer was attached to GNPs via the effect of thiol group. When the S. typhimurium
Fig. 1. Schematic illustration for the colorimetric detection of S. typhimurium based on GNPs and aptamers.
X. Ma et al. / International Journal of Food Microbiology 245 (2017) 1–5
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Fig. 2. (A) TEM image and (B) UV–visible absorption spectra of GNPs.
was added, the aptamer sequences would preferentially combined to S. typhimurium and split away off GNPs. Then, with the addition of NaCl, free GNPs would be aggregated. On the contrary, if the GNPs were decorated with aptamer, they would not aggregate and still show a single scattered state owing to the protective effect of a large number of negative charges on the surfaces. The aggregation degree could be characterized by the UV–Vis spectrophotometer. So, the S. typhimurium could be quantified using the GNPs based aptasensor.
followed by the increase of time. At the same incubation time, the binding rate increased when the reaction temperature was increased. When the reaction temperature was at 37 °C, the binding rate was above 50%. If the temperature is further increased, the aptamer sequence will be changed and the properties of GNPs will be destroyed. Therefore, we use 37 °C as the optimal incubation temperature.
3.2. Preparation of GNPs
It is reported by Li and Rothberg (2004) that the single stranded ssDNA in solution showed irregular curl state. They could be adsorbed to GNPs by electrostatic force effect of the exposed bases, the Van der Waals force and hydrophobic interactions. The adsorption process is related to the temperature and time. The adsorption of aptamer and GNPs were conducted at different temperatures for different times. Results were shown in Fig. 3. The binding rate was calculated by characterizing the absorption intensity of aptamer at 270 nm before and after connecting to GNPs. At the same temperature, the binding rate increased with the increase of incubation time. It reached the maximum value at 30 h and no longer changed
Generally, GNPs without any decoration will be aggregated at high concentration of salt solution. They form an irregular network structure other than monodispersion. The color changes from red to purple or blue. When GNPs were decorated with aptamer, the negative charge of the phosphate backbone could effectively protect the GNPs from the salt ions. The GNPs will not be aggregated and change color in a certain concentration range of salt solution. Therefore, it is the key factor to find the maximum NaCl solution concentration for the aptamer protected GNPs solution without changing color. The optimization experiment was conducted as follows. 100 μL 10−5 M aptamer was added to 500 μL GNPs in order to ensure that the aptamer was absolutely excessive. In other words, all GNPs were adsorbed by the aptamer and the protective effect was produced. After adding different concentrations of NaCl, the color change could be observed by naked eyes and characterized by UV–Vis spectrophotometer shown in Fig. 4. When the NaCl concentration changed from 0.5 M to 1.5 M, the absorption intensity at 521 nm decreased slightly. The color of GNPs remained red wine. When it further reached to 1.75 M, the color of GNPs changed to purple observed by naked eyes.
Fig. 3. The influence of incubation time and reaction temperature for the binding rate of aptamers at the surface of GNPs.
Fig. 4. UV–visible absorption spectra of GNPs at different concentrations of NaCl solution (1–6: 0.50 M, 0.75 M, 1.00 M, 1.25 M, 1.50 M, 1.75 M).
Fig. 2 is the TEM image and UV–visible absorption spectrum of GNPs. It shows that the prepared GNPs have an absorption peak at approximately 521 nm and the size is ~15 nm. 3.3. Optimization for the decoration of aptamer to GNPs
3.4. Optimization of NaCl concentration
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concentration of aptamer was selected as 80 pM and used for further experiment. 3.6. Analytical performance
Fig. 5. Optimization for the concentration of aptamer (1–8: 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM).
It can be seen that the absorption intensity at 521 nm obviously decreased along with the redshift of the absorption peak. The absorption intensity at 700 nm increased. All these illustrated that GNPs aggregated together. The NaCl concentration had already exceeded the suitable aptamer protected GNPs from aggregation discoloration. So the optimal concentration of NaCl was 1.5 M.
3.5. Optimization of aptamer concentration In order to explore the protective effect of the aptamer in the high salt concentration solution, the suitable aptamer amount is very important to ensure the experiment operation. 100 μL different dilutions of aptamer (10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM) were added to 500 μL GNPs and incubated at 37 °C for 30 h. Then, 1.5 M NaCl was mixed and the solution color was observed by naked eyes and characterized by UV–Vis spectrophotometer. Results were shown in Fig. 5. When the concentration of aptamer increased from 10 pM to 70 pM, the protective effect toward GNPs increased. The GNPs were not easy to change their monodispersed state. As for the absorption peak, it remained closer to the pure GNPs solution. While the concentration of aptamer increased to 80 pM, the absorption peak remained almost the same as the addition of 70 pM aptamer, which illustrated that the amount of adapter was saturated. So the optimal
Under the optimal experimental conditions, a series of concentrations of S. typhimurium (0 cfu/mL, 10 cfu/mL, 102 cfu/mL, 103 cfu/mL, 104 cfu/mL, 105 cfu/mL, 106 cfu/mL, 107 cfu/mL, 108 cfu/mL) were detected. Results were shown in Fig. 6. When the amount was 10 cfu/mL, the absorption peak almost overlapped with the control peak. The reason is that the concentration of S. typhimurium was too low, rarely aptamers were split away off GNPs. The NaCl solution was not enough to cause the aggregation of GNPs color change. The absorbance spectra redshifted constantly with the increased amount of S. typhimurium. The absorbance intensity at 521 nm gradually decreased while the absorbance intensity at 700 nm increased (Fig. 6A). While the concentration reached to 107 cfu/mL, the absorption peak change was no longer evident. That's because all GNPs had been separated from the aptamers, and the concentration of S. typhimurium had reached saturation. The ratio of A700/A521 was calculated as the basis for quantitative analysis. As depicted in Fig. 6B, there was a good linear correlation between the ratio of A700/A521 and the amount of S. typhimurium ranged from 102 cfu/mL to 107 cfu/mL. The linear correlation equation obtained was y = 0.1946x–0.2800 (R2 = 0.9939). The statistical analysis revealed that the detection limit of S. typhimurium was 56 cfu/mL. The detection limit is based on the calculation formula D = 3 N/S (N is the standard deviation of blank sample signal. S is the slope of standard curve). 3.7. Specificity Some other pathogenic bacteria were selected to do the specificity detection. These included S. aureus, V. parahemolyticus, B. cereus, S. dysenteriae. The concentration of pathogenic bacteria was maintained at 105 cfu/mL. Experimental results shown in Fig. 7 clearly showed that the ratio of A700/A521 for the other pathogenic bacteria were much lower than that of the S. typhimurium. 3.8. Milk sample detection The utility for the colorimetric detection of S. typhimurium was examined using milk sample obtained from a supermarket. The sample was tested using the new method and the classical plate counting
Fig. 6. (A) UV–visible absorption spectra of GNPs at different concentrations of S. typhimurium (0–8: 0 cfu/mL, 10 cfu/mL, 102 cfu/mL, 103 cfu/mL, 104 cfu/mL, 105 cfu/mL, 106 cfu/mL, 107 cfu/mL, 108 cfu/mL). (B) The corresponding linear relationship between different concentrations of S. typhimurium and A700/A521 ratio.
X. Ma et al. / International Journal of Food Microbiology 245 (2017) 1–5
Fig. 7. Specificity result for the detection of (a) S. typhimurium, (b) S. aureus, (c) V. parahemolyticus, (d) B. cereus, (e) S. dysenteriae. Table 1 Comparison of the milk sample results obtained from the colorimetric detection and classical plate counting method (all results were repeated three times and shown as average ± SD). Milk sample
Plate counting (cfu/mL)
Colorimetric detection (cfu/mL)
Recovery ratio (%)
1 2 3 4 5
28 ± 1 69 ± 3 251 ± 5 704 ± 10 1176 ± 12
ND 72 ± 3 242 ± 6 699 ± 9 1183 ± 13
ND 104.3% 96.4% 99.5% 100.5%
methods. The analytical results were shown in Table 1. The results obtained using the colorimetric detection method were similar to those obtained using the plate counting method. The recoveries were between 96.4% and 104.3%, indicating good accuracy of the proposed test for S. typhimurium detection. 4. Conclusion The colorimetric detection of S. typhimurium using GNPs and aptamer was investigated. It is simple, fast and without complex detection procedure or instrument. Aptamers immobilized at the surface of GNPs could protect GNPs from aggregation toward high concentration of NaCl. The addition of S. typhimurium broke the protection layer due to preferable connections between aptamers and S. typhimurium. Under the optimal experimental conditions, a good linear correlation between the ratio of A700/A521 and the amount of S. typhimurium ranged from 102 cfu/mL to 107 cfu/mL was achieved. The obtained linear correlation equation was y = 0.1946x–0.2800 (R2 = 0.9939) with a detection limit of 56 cfu/mL. In addition, this developed method was successfully used to analyze milk samples, and there was no significant difference between a classical plate counting method and the developed method. So it has the potential for wide use in the detection of other foodborne pathogenic bacteria in food samples. Acknowledgments This work was supported by the NSFC (31401665, 21375049), National S&T Support Program of China (2015BAD17B02), and program of “Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province”. References Bai, X.J., Hou, H., Zhang, B.L., Tang, J.L., 2014. Label-free detection of kanamycin using aptamer-based cantilever array sensor. Biosens. Bioelectron. 56, 112–116.
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