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Cite this: Chem. Commun., 2014, 50, 1848 Received 3rd November 2013, Accepted 18th December 2013
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Detection of single-digit foodborne pathogens with the naked eye using carbon nanotube-based multiple cycle signal amplification† Heng Zhang,ab Yupeng Shi,a Fang Lan,b Yi Pan,a Yankui Lin,b Jingzhang Lv,b Zhihao Zhu,b Qing Jianga and Changqing Yi*a
DOI: 10.1039/c3cc48417c www.rsc.org/chemcomm
A carbon nanotube (CNT)-based multiple cycle signal amplification strategy has been demonstrated for detection of single-digit foodborne pathogens with the naked eye. In the present design, CNTs are used as carriers for loading numerous horseradish peroxidase (HRP) and concanavalin A (ConA) tags, and multiple cycle signal amplification is achieved through the biotinylated anti-HRP antibody and avidin-HRP.
The ultrasensitive detection of pathogens is of critical importance in a variety of societal areas including food safety monitoring, warning against bio-warfare agents, and environmental protection.1 Generally, detecting a pathogen is based on the specific antigen–antibody binding reaction using colorimetric, electrochemical, radioactive and/or fluorometric readouts.2,3 Due to its ability to provide quick and accurate results with low-cost instruments, enzyme-linked immunosorbent assay (ELISA) becomes a popular choice for pathogen detection. However, conventional ELISA methods still suffer from their relatively low sensitivity.4,5 In order to achieve the ultrasensitive detection of pathogens whose concentration is often low in biomedical and food samples, signal amplification strategies such as immuno-polymerase chain reaction (PCR) have been developed.6,7 Our previous study has developed a natural phage nanoparticles-based signal amplification strategy for immuno-PCR to realize the ultrasensitive detection of human hepatitis B surface antigen down to 0.1 pg ml 1.8 Although these methods demonstrate adequate sensitivity, they involve sophisticated instruments and tedious procedures. And there are still key issues such as cost, simplicity, training, and accuracy, which need to be addressed in the development of rapid methods for the pathogen detection, a
Key Laboratory of Sensing Technology and Biomedical Instruments (Guangdong Province), School of Engineering, Sun Yat-Sen University, Guangzhou 510275, China. E-mail: [email protected]; Fax: +86-20-39342380; Tel: +86-20-39342380 b Shenzhen Key Research Laboratory of Detection Technology R&D on Food Safety, Technical Centre for Food Inspection and Quarantine, Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen, Guangdong 518045, China † Electronic supplementary information (ESI) available: Experimental details. See DOI: 10.1039/c3cc48417c
1848 | Chem. Commun., 2014, 50, 1848--1850
especially in resource-constrained countries. In the present communication, we modify the conventional ELISA by integrating a carbon nanotube-based multiple cycle signal amplification strategy to enable the ultrasensitive detection of a few pathogens with the naked eye. The unique properties of nanomaterials in conjugation with the biorecognition abilities of biomolecules offer particularly exciting opportunities to develop ultrasensitive detection strategies.1,3–5,9 We herein functionalize carbon nanotubes (CNTs) with concanavalin A (ConA) and horseradish peroxidases (HRP) to obtain bioconjugates ConA–CNTs–HRP which are subsequently used to develop the multiple cycle signal amplification strategy. In the present design, CNTs were used as carriers for loading numerous enzyme tags and specific recognition elements. The ConA–CNTs– HRP bioconjugates were used as probes to detect the analyte and amplify the detectable signals. ConA, a lectin which is a class of natural nonimmune protein that can specifically recognize the sugar epitope existing on the surface of cells, was employed as the recognition element in the detection probe ConA–CNTs–HRP.10 HRP serve a dual function as the primary signal amplification units and mediators of the secondary signal amplification, where multiple cycle signal amplification is achieved through the biotinylated anti-HRP antibody and avidin-HRP. We demonstrated the feasibility of this novel design by taking Escherichia coli O157:H7 (E. coli O157:H7) as a model analyte which is a leading cause of foodborne illness among all pathogens.11 Details of the preparation of the bioconjugates ConA–CNTs–HRP and immunoassays for E. coli O157:H7 are provided in the ESI† that accompanies the online version of this article (see ESI† Materials and methods). The overall assay procedure is illustrated in Fig. 1. A monoclonal capture antibody against E. coli O157:H7 was coated onto the inner surface of the 96-well polystyrene plates. The target pathogen in the sample was captured by the immobilized capture antibody. Then, the ConA–CNTs–HRP bioconjugates as detection probes are bound to the pathogen through the ConA by glycan– lectin interaction, thereby forming the immuno complex containing numerous HRP tags on the surface. Consequently, the biotinylated anti-HRP antibodies were introduced into the HRP, and the end
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Fig. 1 Schematic diagram of the bioconjugate ConA–CNTs–HRP-based multiple cycle signal amplification strategy.
biotin was further activated by an interaction force between biotin and avidin from avidin-HRP (AV-HRP). The introduction of biotinylated anti-HRP antibodies and AV-HRP resulted in a significantly amplified signal, due to the multiple enzymatic cycles. Color development was produced using H2O2 and tetramethylbenzidine (TMB) to achieve visual detection with the naked eye. Therefore, the ultrasensitive detection of pathogens down to single-digit with the naked eye was realized using the proposed CNTs-based multiple cycle signal amplification strategy. In the present design, CNTs were used as carriers for loading numerous enzyme tags and specific recognition elements.1,12–14 The pristine CNTs were firstly functionalized and shortened with 6 hours’ sonication in 3 : 1 H2SO4 and HNO3 to introduce hydrophilic carboxylic groups on the surface, followed by washing with water to pH neutral and then drying under vacuum before use.14,15 The TEM image showed that the carboxylated CNTs were dispersed well in aqueous solutions with a homogeneous surface (Fig. S1A, ESI†). Afterward, the carboxylic groups on the CNTs surface were converted into active esters via diimide-activation using EDC and NHS, and the active esters subsequently reacted with amine groups on HRP and ConA to form bioconjugates ConA–CNTs–HRP. Surface modification of CNTs with HRP and ConA did not obviously affect the morphology of CNTs, as evidenced by TEM images (Fig. S1B, ESI†). The successful formation of bioconjugates ConA–CNTs–HRP was validated by XPS spectra, where the peaks with the binding energy of 286.8 eV and 400.6 eV in the C1s and N1s spectrum confirmed the presence of amide bonds between proteins and CNTs (Fig. S1C–E, ESI†). Then, the analytical performance of the modified ELISA methods using ConA–CNTs–HRP as detection probes was evaluated. We first choose the low concentration of E. coli O157:H7 at 103 CFU per mL as the target analyte and Salmonella (Sa) at 106 CFU per mL as the control group to analyze the effect of the amplification cycle numbers on the detectable signals. As shown in Fig. 2, the standard sandwich ‘‘immuno’’ reaction can’t detect the 103 CFU per mL E. coli O157:H7, where its pristine signal is similar to the control group. After one cycle amplification, the detectable signal evidently enhanced and can be distinguished from the control group whose signal shows no change before and after one cycle amplification. With the cycle number increasing, the signal gradually boosts up until reaching a plateau. Control experiments performed with the spiking Sa sample showed almost no observable difference, because the capture antibodies against E. coli O157:H7 cannot specifically recognize Sa. Therefore, these results undoubtedly demonstrate that the gradually strengthened signal observed is not produced by nonspecific adsorption. Furthermore, the ability to detect with the
This journal is © The Royal Society of Chemistry 2014
Fig. 2 Histograms of absorbance at 450 nm for detection of Salmonella at 106 CFU per mL (white) and E. coli O157:H7 at 103 CFU per mL (black) via multiple cycle signal amplification: CT0 = no cycle, CT1 = one time cycle, CT2 = two times cycle, and so on. The insets show images of the same sample under visible light.
naked eye can be corroborated after two cycles amplification (the insets of Fig. 2). The whole procedure involved a standard sandwich immuno-reaction and an additional multiple cycle signal amplification. Starting from pathogen capture, the overall time needed was within 3 hours for a batch of 30 samples. The manipulations also became simple as the multiple washing steps could be carried out using an automatic washing machine. To determine the limit of detection (LOD) of this new method, a series of sample solutions containing 101, 102, 103, 104, 105, and 106 CFU per mL of E. coli O157:H7 were prepared and detected in triplicate. In Fig. 3, we observed that the lowest concentration that could be always distinctively detected with the naked eye as the positive signal was 102 CFU per mL, which was therefore set as the LOD of this assay. From 50 mL of reaction volume in each well to calculate, approximately 5 CFU per each well could be observed with the naked eye, indicating the ultra-high sensitivity of the method. To study the impact of real sample matrices on the detection, a series of meat samples with the different concentrations of E. coli O157:H7 by the culture plating method were set up. All of the samples were analysed parallelly by real-time PCR (RT-PCR) and ConA–CNTs–HRP-based ELISA methods for E. coli O157:H7 detection. As shown in Table 1, the results showed that 102 CFU per mL of E. coli O157:H7 in the meat sample can still be detected by the ConA–CNTs–HRP-based ELISA method but not by RT-PCR (see ESI,† Fig. S2). Without doubt, the ConA–CNTs–HRP-based ELISA method was able to detect, at a glance, the ultralow concentration of pathogens that were undetectable by the RT-PCR method. It is obvious that the LOD of the ConA–CNTs–HRP-based ELISA method is approximately 10–100-fold lower compared with a regular RT-PCR method for E. coli O157:H7 detection in meat samples. In Table S1 (ESI†), analytical performance of typical detection methods for E. coli O157:H7 was compared in terms of analysis time and the detection limit. In comparison, the detection limit of ConA–CNTs– HRP-based ELISA methods was found to be lower than or comparable to most of the existing pathogen detection methods, though the proposed method is incapable of quantifying the target analyte concentration.
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Fig. 3 Naked-eye detection of E. coli O157:H7 with multiple cycles assay (three times cycle). E. coli O157:H7 was 10-fold serial diluted from 105 to 101 CFU per mL. Sa was used as negative by spiking the Salmonella of 106 CFU per mL. Error bars indicate the standard deviation of three independent measurements. The insets show images of the same sample under visible light.
Table 1
Detection of E. coli O157:H7 in the meat sample
Samples S1 S2 S3 S4 S5 S6 Note: +, detected;
Concentration (CFU per mL) 6
10 105 104 103 102 101
This work
RT-PCR
+ + + + +
+ + + +/
, undetected.
The CNT-based nanomaterials for loading antibodies and enzymes have been widely used as detection probes in the sandwich immunoassays.13,14 Although those probes provide significant advantages in various respects, they are not sensitive enough for detection of the biomolecule at low levels and require specific conjugation of the different antibodies for the different analytes. It is well-known that the modified antibodies for loading onto nanomaterials result in the adverse effects on their activation, which in turn weaken the detection sensitivity and limit their applications.3 However, previous studies clearly demonstrated that ConA has been successfully conjugated onto the surface of various nanomaterials through covalent and non-covalent linkage and maintains its pristine function as the recognition element.16–18 Therefore, the present work designed a new nanoprobe as the universal detection probe by coupling both ConA and HRP onto CNTs, and integrated a novel multiple cycle signal amplification strategy by repeated reaction between biotinylated anti-HRP antibodies and AV-HRP. The herein described ConA–CNTs–HRP-based ELISA method offers several advantages over current sandwich immunoassays. First, the ConA–CNTs–HRP bioconjugate is used as a universal nanoprobe for the detection of pathogens. The large number of carbohydrates on the surface of target cells makes it a natural harbour for the ConA–CNTs–HRP bioconjugate coupling onto the captured pathogens. Meanwhile, as a detection probe, it can be applied to the different pathogens by changing the
1850 | Chem. Commun., 2014, 50, 1848--1850
corresponding capture antibody immobilized onto the inner surface of 96-well polystyrene plates. Second, the CNTs were used as carriers for not only loading numerous ConA but also numerous HRP tags, which could trigger further signal amplification via the multiple cycle format. Third, the repeated use of the biotinylated anti-HRP antibody and AV-HRP for multiple cycle assays renders much more HRP loading onto the surface thereby amplifying the detectable signal and achieving the naked-eye detection as well. In conclusion, we have demonstrated the detection of pathogens at an ultralow concentration with the naked eye by designing a novel strategy which is based on the universal ConA–CNTs–HRP bioconjugates as the detection probes and the integration of multiple cycle signal amplification using the biotinylated anti-HRP antibody and AV-HRP. It could be envisaged that detection sensitivity could be improved through further experimental condition optimization and integrating with microfluidic devices to realize single pathogen detection in each event. Although detection with the naked eye seems perfectly suited for detecting pathogens in resource-constrained countries, it is inherently difficult to quantify the concentration of target analytes using the multiple cycle signal amplification strategy. Nevertheless, this potential limitation cannot stop it from becoming a promising and versatile toolkit for signal amplification in the detection of a variety of pathogens and other analogues using the naked eye. This work was partially supported by National Scientific Foundation of China (31100723), Guangdong Natural Science Foundation (S2011040001778), Shenzhen CIQ Young Science & Technology Forum Program (SZ2011210), Shenzhen Research Funding Program (JCYJ20120831160213584), Shenzhen City Technology Innovation Program (CXZZ20120831160213589), and Guangzhou Science and Technology and Information Bureau (2013J2200053).
Notes and references 1 J. Lei and H. Ju, Chem. Soc. Rev., 2012, 41, 2122–2134. 2 O. Lazcka, F. Campo and F. X. Munoz, Biosens. Bioelectron., 2007, 22, 1205–1217. 3 D. Ivnitski, I. Abdel-Hamid, P. Atanasov and E. Wilkins, Biosens. Bioelectron., 1999, 14, 599–624. 4 E. Crowley, C. O’Sullivan and G. Guilbault, Analyst, 1999, 124, 295–299. 5 V. Velusamy, K. Arshak, O. Korostynska, K. Oliwa and C. Adley, Biotechnol. Adv., 2010, 28, 232–254. 6 J. B. Ristaino, C. T. Groves and G. R. Parra, Nature, 2001, 411, 695–697. 7 E. Kakizaki, T. Yoshida, H. Kawakami, M. Oseto, T. Sakai and M. Sakai, Lett. Appl. Microbiol., 1996, 23, 101–103. 8 H. Zhang, Y. Xu, Q. Huang, C. Yi, T. Xiao and Q.-G. Li, Chem. Commun., 2013, 49, 3778–3780. 9 R. de La Rica and M. M. Stevens, Nat. Nanotechnol., 2012, 7, 821–824. 10 H. Lis and N. Sharon, Chem. Rev., 1998, 98, 637–674. 11 P. M. Griffin and R. V. Tauxe, Epidemiol. Rev., 1991, 13, 60–98. 12 W. Gao, H. Dong, J. Lei, H. Ji and H. Ju, Chem. Commun., 2011, 47, 5220–5222. 13 X. Yu, B. Munge, V. Patel, G. Jensen, A. Bhirde, J. D. Gong, S. N. Kim, J. Gillespie, J. S. Gutkind and F. Papadimitrakopoulos, J. Am. Chem. Soc., 2006, 128, 11199–11205. 14 J. Wang, G. Liu and M. R. Jan, J. Am. Chem. Soc., 2004, 126, 3010–3011. 15 C. Yi, C.-C. Fong, Q. Zhang, S.-T. Lee and M. Yang, Nanotechnology, 2008, 19, 095102. 16 L. Ding, W. Cheng, X. Wang, S. Ding and H. Ju, J. Am. Chem. Soc., 2008, 130, 7224–7225. 17 E. Han, L. Ding, H. Lian and H. Ju, Chem. Commun., 2010, 46, 5446–5448. 18 L. Ding, Q. Ji, R. Qian, W. Cheng and H. Ju, Anal. Chem., 2010, 82, 1292–1298.
This journal is © The Royal Society of Chemistry 2014
COMMUNICATION
Cite this: Chem. Commun., 2014, 50, 1848 Received 3rd November 2013, Accepted 18th December 2013
View Article Online View Journal | View Issue
Detection of single-digit foodborne pathogens with the naked eye using carbon nanotube-based multiple cycle signal amplification† Heng Zhang,ab Yupeng Shi,a Fang Lan,b Yi Pan,a Yankui Lin,b Jingzhang Lv,b Zhihao Zhu,b Qing Jianga and Changqing Yi*a
DOI: 10.1039/c3cc48417c www.rsc.org/chemcomm
A carbon nanotube (CNT)-based multiple cycle signal amplification strategy has been demonstrated for detection of single-digit foodborne pathogens with the naked eye. In the present design, CNTs are used as carriers for loading numerous horseradish peroxidase (HRP) and concanavalin A (ConA) tags, and multiple cycle signal amplification is achieved through the biotinylated anti-HRP antibody and avidin-HRP.
The ultrasensitive detection of pathogens is of critical importance in a variety of societal areas including food safety monitoring, warning against bio-warfare agents, and environmental protection.1 Generally, detecting a pathogen is based on the specific antigen–antibody binding reaction using colorimetric, electrochemical, radioactive and/or fluorometric readouts.2,3 Due to its ability to provide quick and accurate results with low-cost instruments, enzyme-linked immunosorbent assay (ELISA) becomes a popular choice for pathogen detection. However, conventional ELISA methods still suffer from their relatively low sensitivity.4,5 In order to achieve the ultrasensitive detection of pathogens whose concentration is often low in biomedical and food samples, signal amplification strategies such as immuno-polymerase chain reaction (PCR) have been developed.6,7 Our previous study has developed a natural phage nanoparticles-based signal amplification strategy for immuno-PCR to realize the ultrasensitive detection of human hepatitis B surface antigen down to 0.1 pg ml 1.8 Although these methods demonstrate adequate sensitivity, they involve sophisticated instruments and tedious procedures. And there are still key issues such as cost, simplicity, training, and accuracy, which need to be addressed in the development of rapid methods for the pathogen detection, a
Key Laboratory of Sensing Technology and Biomedical Instruments (Guangdong Province), School of Engineering, Sun Yat-Sen University, Guangzhou 510275, China. E-mail: [email protected]; Fax: +86-20-39342380; Tel: +86-20-39342380 b Shenzhen Key Research Laboratory of Detection Technology R&D on Food Safety, Technical Centre for Food Inspection and Quarantine, Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen, Guangdong 518045, China † Electronic supplementary information (ESI) available: Experimental details. See DOI: 10.1039/c3cc48417c
1848 | Chem. Commun., 2014, 50, 1848--1850
especially in resource-constrained countries. In the present communication, we modify the conventional ELISA by integrating a carbon nanotube-based multiple cycle signal amplification strategy to enable the ultrasensitive detection of a few pathogens with the naked eye. The unique properties of nanomaterials in conjugation with the biorecognition abilities of biomolecules offer particularly exciting opportunities to develop ultrasensitive detection strategies.1,3–5,9 We herein functionalize carbon nanotubes (CNTs) with concanavalin A (ConA) and horseradish peroxidases (HRP) to obtain bioconjugates ConA–CNTs–HRP which are subsequently used to develop the multiple cycle signal amplification strategy. In the present design, CNTs were used as carriers for loading numerous enzyme tags and specific recognition elements. The ConA–CNTs– HRP bioconjugates were used as probes to detect the analyte and amplify the detectable signals. ConA, a lectin which is a class of natural nonimmune protein that can specifically recognize the sugar epitope existing on the surface of cells, was employed as the recognition element in the detection probe ConA–CNTs–HRP.10 HRP serve a dual function as the primary signal amplification units and mediators of the secondary signal amplification, where multiple cycle signal amplification is achieved through the biotinylated anti-HRP antibody and avidin-HRP. We demonstrated the feasibility of this novel design by taking Escherichia coli O157:H7 (E. coli O157:H7) as a model analyte which is a leading cause of foodborne illness among all pathogens.11 Details of the preparation of the bioconjugates ConA–CNTs–HRP and immunoassays for E. coli O157:H7 are provided in the ESI† that accompanies the online version of this article (see ESI† Materials and methods). The overall assay procedure is illustrated in Fig. 1. A monoclonal capture antibody against E. coli O157:H7 was coated onto the inner surface of the 96-well polystyrene plates. The target pathogen in the sample was captured by the immobilized capture antibody. Then, the ConA–CNTs–HRP bioconjugates as detection probes are bound to the pathogen through the ConA by glycan– lectin interaction, thereby forming the immuno complex containing numerous HRP tags on the surface. Consequently, the biotinylated anti-HRP antibodies were introduced into the HRP, and the end
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 19 December 2013. Downloaded by University of Massachusetts - Amherst on 27/10/2014 06:28:59.
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Fig. 1 Schematic diagram of the bioconjugate ConA–CNTs–HRP-based multiple cycle signal amplification strategy.
biotin was further activated by an interaction force between biotin and avidin from avidin-HRP (AV-HRP). The introduction of biotinylated anti-HRP antibodies and AV-HRP resulted in a significantly amplified signal, due to the multiple enzymatic cycles. Color development was produced using H2O2 and tetramethylbenzidine (TMB) to achieve visual detection with the naked eye. Therefore, the ultrasensitive detection of pathogens down to single-digit with the naked eye was realized using the proposed CNTs-based multiple cycle signal amplification strategy. In the present design, CNTs were used as carriers for loading numerous enzyme tags and specific recognition elements.1,12–14 The pristine CNTs were firstly functionalized and shortened with 6 hours’ sonication in 3 : 1 H2SO4 and HNO3 to introduce hydrophilic carboxylic groups on the surface, followed by washing with water to pH neutral and then drying under vacuum before use.14,15 The TEM image showed that the carboxylated CNTs were dispersed well in aqueous solutions with a homogeneous surface (Fig. S1A, ESI†). Afterward, the carboxylic groups on the CNTs surface were converted into active esters via diimide-activation using EDC and NHS, and the active esters subsequently reacted with amine groups on HRP and ConA to form bioconjugates ConA–CNTs–HRP. Surface modification of CNTs with HRP and ConA did not obviously affect the morphology of CNTs, as evidenced by TEM images (Fig. S1B, ESI†). The successful formation of bioconjugates ConA–CNTs–HRP was validated by XPS spectra, where the peaks with the binding energy of 286.8 eV and 400.6 eV in the C1s and N1s spectrum confirmed the presence of amide bonds between proteins and CNTs (Fig. S1C–E, ESI†). Then, the analytical performance of the modified ELISA methods using ConA–CNTs–HRP as detection probes was evaluated. We first choose the low concentration of E. coli O157:H7 at 103 CFU per mL as the target analyte and Salmonella (Sa) at 106 CFU per mL as the control group to analyze the effect of the amplification cycle numbers on the detectable signals. As shown in Fig. 2, the standard sandwich ‘‘immuno’’ reaction can’t detect the 103 CFU per mL E. coli O157:H7, where its pristine signal is similar to the control group. After one cycle amplification, the detectable signal evidently enhanced and can be distinguished from the control group whose signal shows no change before and after one cycle amplification. With the cycle number increasing, the signal gradually boosts up until reaching a plateau. Control experiments performed with the spiking Sa sample showed almost no observable difference, because the capture antibodies against E. coli O157:H7 cannot specifically recognize Sa. Therefore, these results undoubtedly demonstrate that the gradually strengthened signal observed is not produced by nonspecific adsorption. Furthermore, the ability to detect with the
This journal is © The Royal Society of Chemistry 2014
Fig. 2 Histograms of absorbance at 450 nm for detection of Salmonella at 106 CFU per mL (white) and E. coli O157:H7 at 103 CFU per mL (black) via multiple cycle signal amplification: CT0 = no cycle, CT1 = one time cycle, CT2 = two times cycle, and so on. The insets show images of the same sample under visible light.
naked eye can be corroborated after two cycles amplification (the insets of Fig. 2). The whole procedure involved a standard sandwich immuno-reaction and an additional multiple cycle signal amplification. Starting from pathogen capture, the overall time needed was within 3 hours for a batch of 30 samples. The manipulations also became simple as the multiple washing steps could be carried out using an automatic washing machine. To determine the limit of detection (LOD) of this new method, a series of sample solutions containing 101, 102, 103, 104, 105, and 106 CFU per mL of E. coli O157:H7 were prepared and detected in triplicate. In Fig. 3, we observed that the lowest concentration that could be always distinctively detected with the naked eye as the positive signal was 102 CFU per mL, which was therefore set as the LOD of this assay. From 50 mL of reaction volume in each well to calculate, approximately 5 CFU per each well could be observed with the naked eye, indicating the ultra-high sensitivity of the method. To study the impact of real sample matrices on the detection, a series of meat samples with the different concentrations of E. coli O157:H7 by the culture plating method were set up. All of the samples were analysed parallelly by real-time PCR (RT-PCR) and ConA–CNTs–HRP-based ELISA methods for E. coli O157:H7 detection. As shown in Table 1, the results showed that 102 CFU per mL of E. coli O157:H7 in the meat sample can still be detected by the ConA–CNTs–HRP-based ELISA method but not by RT-PCR (see ESI,† Fig. S2). Without doubt, the ConA–CNTs–HRP-based ELISA method was able to detect, at a glance, the ultralow concentration of pathogens that were undetectable by the RT-PCR method. It is obvious that the LOD of the ConA–CNTs–HRP-based ELISA method is approximately 10–100-fold lower compared with a regular RT-PCR method for E. coli O157:H7 detection in meat samples. In Table S1 (ESI†), analytical performance of typical detection methods for E. coli O157:H7 was compared in terms of analysis time and the detection limit. In comparison, the detection limit of ConA–CNTs– HRP-based ELISA methods was found to be lower than or comparable to most of the existing pathogen detection methods, though the proposed method is incapable of quantifying the target analyte concentration.
Chem. Commun., 2014, 50, 1848--1850 | 1849
View Article Online
Published on 19 December 2013. Downloaded by University of Massachusetts - Amherst on 27/10/2014 06:28:59.
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Fig. 3 Naked-eye detection of E. coli O157:H7 with multiple cycles assay (three times cycle). E. coli O157:H7 was 10-fold serial diluted from 105 to 101 CFU per mL. Sa was used as negative by spiking the Salmonella of 106 CFU per mL. Error bars indicate the standard deviation of three independent measurements. The insets show images of the same sample under visible light.
Table 1
Detection of E. coli O157:H7 in the meat sample
Samples S1 S2 S3 S4 S5 S6 Note: +, detected;
Concentration (CFU per mL) 6
10 105 104 103 102 101
This work
RT-PCR
+ + + + +
+ + + +/
, undetected.
The CNT-based nanomaterials for loading antibodies and enzymes have been widely used as detection probes in the sandwich immunoassays.13,14 Although those probes provide significant advantages in various respects, they are not sensitive enough for detection of the biomolecule at low levels and require specific conjugation of the different antibodies for the different analytes. It is well-known that the modified antibodies for loading onto nanomaterials result in the adverse effects on their activation, which in turn weaken the detection sensitivity and limit their applications.3 However, previous studies clearly demonstrated that ConA has been successfully conjugated onto the surface of various nanomaterials through covalent and non-covalent linkage and maintains its pristine function as the recognition element.16–18 Therefore, the present work designed a new nanoprobe as the universal detection probe by coupling both ConA and HRP onto CNTs, and integrated a novel multiple cycle signal amplification strategy by repeated reaction between biotinylated anti-HRP antibodies and AV-HRP. The herein described ConA–CNTs–HRP-based ELISA method offers several advantages over current sandwich immunoassays. First, the ConA–CNTs–HRP bioconjugate is used as a universal nanoprobe for the detection of pathogens. The large number of carbohydrates on the surface of target cells makes it a natural harbour for the ConA–CNTs–HRP bioconjugate coupling onto the captured pathogens. Meanwhile, as a detection probe, it can be applied to the different pathogens by changing the
1850 | Chem. Commun., 2014, 50, 1848--1850
corresponding capture antibody immobilized onto the inner surface of 96-well polystyrene plates. Second, the CNTs were used as carriers for not only loading numerous ConA but also numerous HRP tags, which could trigger further signal amplification via the multiple cycle format. Third, the repeated use of the biotinylated anti-HRP antibody and AV-HRP for multiple cycle assays renders much more HRP loading onto the surface thereby amplifying the detectable signal and achieving the naked-eye detection as well. In conclusion, we have demonstrated the detection of pathogens at an ultralow concentration with the naked eye by designing a novel strategy which is based on the universal ConA–CNTs–HRP bioconjugates as the detection probes and the integration of multiple cycle signal amplification using the biotinylated anti-HRP antibody and AV-HRP. It could be envisaged that detection sensitivity could be improved through further experimental condition optimization and integrating with microfluidic devices to realize single pathogen detection in each event. Although detection with the naked eye seems perfectly suited for detecting pathogens in resource-constrained countries, it is inherently difficult to quantify the concentration of target analytes using the multiple cycle signal amplification strategy. Nevertheless, this potential limitation cannot stop it from becoming a promising and versatile toolkit for signal amplification in the detection of a variety of pathogens and other analogues using the naked eye. This work was partially supported by National Scientific Foundation of China (31100723), Guangdong Natural Science Foundation (S2011040001778), Shenzhen CIQ Young Science & Technology Forum Program (SZ2011210), Shenzhen Research Funding Program (JCYJ20120831160213584), Shenzhen City Technology Innovation Program (CXZZ20120831160213589), and Guangzhou Science and Technology and Information Bureau (2013J2200053).
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