Biosensors and Bioelectronics 57 (2014) 133–138
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Highly sensitive and rapid bacteria detection using molecular beacon–Au nanoparticles hybrid nanoprobes Jing Cao a, Chao Feng b, Yan Liu c, Shouyu Wang d, Fei Liu a,n a
Single Molecule Nanometry Laboratory, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China Department of Pathology, The First People's Hospital of Shangqiu, Henan, Shangqiu 476000, China c Clinical Laboratory, The First People's Hospital of Shangqiu, Henan, Shangqiu 476000, China d Department of Information Physics and Engineering, Nanjing University of Science and Technology, Nanjing, Jiangsu 210094, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 7 November 2013 Received in revised form 30 January 2014 Accepted 10 February 2014 Available online 19 February 2014
Since many diseases are caused by pathogenic bacterial infections, accurate and rapid detection of pathogenic bacteria is in urgent need to timely apply appropriate treatments and to reduce economic costs. To end this, we designed molecular beacon–Au nanoparticle hybrid nanoprobes to improve the bacterial detection efficiency and sensitivity. Here, we show that the designed molecular beacon modified Au nanoparticles could specifically recognize synthetic DNAs targets and can readily detect targets in clinical samples. Moreover, the hybrid nanoprobes can recognize Escherichia coli within an hour at a concentration of 102 cfu/ml, which is 1000-folds sensitive than using molecular beacon directly. Our results show that the molecular beacon–Au nanoparticle hybrid nanoprobes have great potential in medical and biological applications. & 2014 Elsevier B.V. All rights reserved.
Keywords: Molecular beacon Au nanoparticles Sensitive and rapid bacterial detection
1. Introduction Pathogenic bacterial infections occur widespread in the world and lead to many diseases, such as pneumonia (Prass et al., 2006), pulmonary tuberculosis (Bhat et al., 1999) and urinary tract infection (UTI) (Morens et al., 2004), etc., which cause millions of cases every year in the world and many other problems, such as high morbidity, large economic costs and productivity decrease. Therefore, accurate and rapid detections of pathogenic bacteria are in urgent needs to identify bacterial infections in order to provide appropriate antibiotic treatments and prevent the spread of diseases. Most traditional diagnoses of bacterial infections are complex, expensive and special environment limited. As a clinical gold standard, bacterial culture can provide extremely accurate measurements; however, it often costs several days. In order to accelerate the detection efficiency, developed molecular detection technologies, such as polymerase chain reaction (PCR) (Tunney et al., 1999), real-time PCR (Corless et al., 2000), microarray-based tests (Peplies et al., 2003) and enzyme-linked immunosorbent assay (ELISA) (Boehme et al., 2005), etc., have been adopted. While, PCR and real-time PCR do not only require amplification of the target sequence, but also need the extraction and purification of nucleic acids (Mariella, 2008). Besides,
n
Corresponding author. E-mail address: [email protected] (F. Liu). URLS: http://www.feiliugroup.com (J. Cao), http://www.feiliugroup.com (F. Liu).
http://dx.doi.org/10.1016/j.bios.2014.02.020 0956-5663 & 2014 Elsevier B.V. All rights reserved.
there are always complex procedures in ELISA detections which are not simple and rapid (Harms et al., 2003). Therefore, there is still a need of a more rapid, sensitive, convenient and cost effective bacteria detection approach, which would be enormously beneficial for patients to receive rational therapy in the health care system (Bercovici et al., 2011). Molecular beacons are considered as one of the most promising technologies in biological detection. DNA beacons have been widely used for qualitative and quantitative detections of bacteria, viruses, single nucleotide polymorphisms, etc., with high speed and sensitivity (Fang et al., 2000; Fujimoto et al., 2004; Peyerl et al., 2005). As a well-characterized specific bio-signature, 16S rRNA, because of its great conservation and high copy numbers (Cai and Zhang, 2013; Hu et al., 2013), is often used as the target for beacons to detect bacteria, such as Lactobacilli in Thai fermented sausage (Rungrassamee et al., 2012), Escherichia coli in UTI (Bercovici et al., 2011), predominant bacteria in human feces (Harms et al., 2003; Loy et al., 2002; Matsuki et al., 2002), etc. However, one major limitation of conventional beacons is a lack of sufficient sensitivity in detection. Au nanoparticles (AuNPs) with unique optical properties, facile surface chemistry and appropriate size scale are very attractive in molecular biological applications, such as sensitive and real-time detection of infectious viruses, as well as the real-time visualization of cell-to-cell virus spreading (Yeh et al., 2010). Moreover, AuNPs could induce molecular aggregation. Combining with AuNPs and DNA beacons, beacon–AuNPs can highly improve detection sensitivity
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though signal amplification (Beni et al., 2010; Li et al., 2009; Selvaraju et al., 2008), since there will be extremely high DNA beacon density in the local region. Here, in this paper, molecular beacons (Chen et al., 2011) combined with AuNPs as molecular beacon–Au nanoparticles hybrid nanoprobes were designed to detect both synthetic DNA targets and 16S rRNA of E. coli (DH5α) in cell cultures. The DNA beacon used is for a wide range of urinary pathogens which consists of a 27-mer probe sequence (Liao et al., 2006), a 7 base pair self-complementary stem, the fluorophore of FAM in the 50 terminus and the quencher of BHQ1 in the 30 terminus (Tyagi and Kramer, 1996) as shown in Fig. 1(a) (more information is in Supplementary information, Table S1). The stem sequences keep the hairpin-shaped structure and fluorescence of FAM is quenched by BHQ1 when no targets exist. With the presence of targets, hybridization of targets with complementary beacon sequences results in significant fluorescent signals since the quencher is separated from fluorophore. Therefore, the existence of targets can be recorded by the intensity of fluorescent signals. By conjugating DNA beacons with modified 13 nm AuNPs as shown in Fig. 1(b), the sensitivity is enhanced by three orders of magnitude, thus overall this approach has the advantage that there is no need of amplification and the fluorescent signals could be directly measured and analyzed. Moreover, the whole process could be finished within 1 h. To the best of our knowledge, the current study is the first demonstration of the molecular beacon–AuNP hybrid nanoprobes for sensitive and rapid bacteria detection. Because of its high accuracy and efficiency, easy preparation and low cost, this method could be widely used in clinical test, biological and chemical research, and food safety inspection, etc.
2. Materials and methods 2.1. Chemicals and reagents Tri (2-carboxyethyl) phosphine hydrochloride (C9H15O6PdHCl), lysozyme, Triton X-100, 2 mM EDTA and Tris–HCl are analytical grade and used without further purification. These reagents were purchased from Sigma-Aldrich (USA). Other chemicals, chloroauric acid (HAuCl4), sodium citrate (Na3C6H5O7), NaCl, Na2HPO4d2H2O, KH2PO4, KCl and NaOH employed were all of analytical grade, and ultrapure water in the experiment was provided by Milli-Q Academic system (USA). All glassware used was ultrasonically cleaned. The 10 mM phosphate-buffered saline (PBS) solutions (pH¼7.4) containing 0.01 M sodium phosphate, 0.137 M NaCl, 0.01 M Na2HPO4d2H2O, 0.002 M KH2PO4 and 0.0027 M KCl were prepared. Lysis buffer (pH ¼8.0) contained 0.1% Triton X-100, 2 mM EDTA and 1 mg/ml lysozyme in 20 mM Tris–HCl. The fetal bovine serum was purchased from Gibico (Australia). The linearized pBAsi-mU6 DNA plasmids were obtained from Takara (Japan). 2.2. DNA beacon design DNA beacon used for a wide range of urinary pathogens detection consists of a 27-mer probe sequence (Tyagi and Kramer, 1996), a 7 base pair self-complementary stem, the fluorophore of FAM (Carboxyfluorescein) in the 50 terminus and the quencher of BHQ1 (Black Hole Quencher 1) in the 30 terminus. The sequences of target, target with four mismatches and mismatched target are designed as control experiments in order to
Fig. 1. (A) The principle of targets detection with DNA beacons. (B) The schematic design of beacon–AuNPs for targets detection.
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demonstrate the specificity of DNA beacons. More characteristics of beacon, target, target with four mismatches and mismatched target are shown in Table S1 of Supplementary information.
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temperature of the reaction was decreased from 80 1C to 40 1C. With temperature control of real-time PCR instrument, the whole process could be finished in 1 h.
2.3. Preparation and characterization of AuNPs 3. Results and discussion AuNPs were prepared with the sodium citrate reduction method (Frens, 1973). Firstly, 200 μl HAuCl4 (50 mM) was brought to 9.8 ml boiling ddH2O with vigorous stirring. 1 ml sodium citrate (38.8 mM) was added to the vortex of the solution. The boiling solution turned dark in 1 min. After 3 min, the dark color changed into burgundy. After boiling for 10 min, ultrasonication is needed for 30 min at room temperature (25 1C). In order to characterize the prepared AuNPs, UV–vis extinction spectra (TU1900, Persee, China) is applied to test absorption band of AuNPs. Dynamic light scattering (DLS) (Zetasizer Nano ZS90, USA) is for detecting hydrodynamic diameter of AuNPs and beacon–AuNPs. The prepared AuNPs are observed by the TEM (H7650, Hitachi, Japan). 2.4. Conjugation between DNA beacons and AuNPs
3.1. TEM and dynamic light scattering (DLS) characteristics of AuNPs Firstly, prepared AuNPs were detected by UV–vis extinction spectra. The details of UV–vis extinction spectra are shown in Fig. S2 of Supplementary information. Besides, AuNPs were observed via TEM as shown in Fig. 2. The results indicate that most AuNPs exhibited uniform particle size with an average diameter of 15 nm. Since thiolated DNA beacons on the surface of the AuNPs cannot be observed by TEM, DLS is applied to measure the connection condition between AuNPs and DNA beacons. The hydrodynamic diameter of AuNPs measured by DLS would be enlarged when DNA beacons are attached to the AuNPs surfaces (Miao et al., 2011). Fig. 3 demonstrates the hydrodynamic
In order to link DNA beacons with AuNPs, firstly, 30 μl thiolated beacons (20 μM) were activated by 30 μl TCEP (50 mM). The pH value of the mixture was adjusted to 5 using 0.5 M NaOH. Then, the solution was incubated at room temperature (25 1C) overnight. 100 μl AuNPs (10 nM) and 33 μl PBS (50 mM) were added, followed by sonication for 10 s, heated at 50 1C for 10 min and gently shaking for 24 h. Afterwards, 0.01% SDS and NaCl (3 M) were mixed in the solution. The solution was processed by sonication for 10 s and then heated at 50 1C for 10 min. The process was repeated for three times. Hereafter, the reaction solution was incubated for 48 h at room temperature. The free beacons and beacon–AuNPs were separated by centrifugation at 12,000 rpm for 30 min at 4 1C. The precipitation was washed by 10 mM PBS and then recentrifuged. The process was repeated twice. Finally the beacon–AuNPs were dispersed in 100 μl PBS (20 mM, pH ¼7.4) and stored at 4 1C. The connection between beacons and AuNPs could be analyzed by the DLS. 2.5. Sample preparation The samples included E. coli (DH5α) and clinical urine. DH5α were inoculated with Luria broth (LB) in a shaker at 37 1C and grew to 108 cfu/ml. The traditional plate colony-counting method was used to calculate the density of DH5α (Fig. S1 in the Supplementary information). One milliliter bacteria sample was centrifuged at 10,000 rpm for 5 min and then the supernatant was discarded. The pellets were kept frozen at 80 1C. The urine sample was collected from patient with approval of biological ethics. The bacterial concentration of the sample was 3 108 cfu/ ml (Supplementary information). 1 ml sample was pelleted by centrifugation for 5 min at 10,000 rpm. After removing supernatant, the sample was stored at 80 1C. The samples were lysed by resuspension of the pellet in 10 μl of 0.5 M NaOH and 10 μl of lysis buffer, and then incubated at room temperature (25 1C) for 5 min.
Fig. 2. Transmission Electron Microscopy (TEM) images of AuNPs, the white bar indicates 100 nm.
2.6. Fluorescence detection Real-time PCR instrument (Bio-rad, iQ2, USA) was applied for detecting fluorescence and a 20 μl reaction system was prepared. Firstly, 1 ml bacteria solution was collected and centrifuged for 5 min, 10 μl NaOH and 10 μl lysis buffer was added and the final concentration of beacons was 100 nM (10 mM PBS, pH ¼ 7.4). After an initial denaturation step (3 min at 80 1C), melting curves were determined by measuring the fluorescence signals when the
Fig. 3. Dynamic light scattering (DLS) analysis of AuNPs and beacon–AuNPs.
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diameter of AuNPs and beacon–AuNPs. With Gaussian fitting, the average diameter of the AuNPs is 16.82 nm with full width half maximum of 14.18 nm, while the average diameter of the beacon– AuNPs is 33.68 nm with full width half maximum of 24.85 nm. The results illustrate that the hydrodynamic diameter of beacon– AuNPs are significantly larger than AuNPs (p o0.05), indicating successful conjunction of beacons and AuNPs. 3.2. Specificity of DNA beacons and beacon–AuNPs In order to demonstrate the specificity of DNA beacon–AuNPs, firstly, synthetic DNA target, four mismatched target and mismatched target were tested. As shown in Fig. 4(A) and (B), the normalized
fluorescent intensities of mismatched target and four mismatched target were significantly lower than target for both DNA beacon and DNA beacon–AuNPs. To further demonstrate the specificity of the DNA beacon and DNA beacon–AuNPs to the target, we tested an unrelated 3008 bp DNA, a linearized pBAsi-mU6 DNA plasmid. As shown in Fig. 4(C) and (D), with unrelated large DNA, both DNA beacon and beacon–AuNPs show fluorescent signals similar to background level. Moreover, clinical urine samples collected from UTI patient with 107 cfu/ml bacteria were also tested by the proposed method (Fig. 4 (E) and (F), and Supplementary information). These experimental results indicate that the DNA beacon–Au nanoprobe can detect the target sequence with high specificity and is also promising for pathological diagnosis.
Fig. 4. (A, B) Normalized fluorescent intensities of target, four mismatch targets and mismatch target tested by beacon and beacon–AuNPs, respectively. (C, D) Normalized fluorescent intensities with 108 cfu/ml E. coli concentration and unrelated 3008 bp DNA tested by beacon and beacon–AuNPs, respectively. (E, F) Clinical urine samples with 107 cfu/ml bacteria detected via DNA beacons and beacon–AuNPs. Each error bar represents 7 SEM of separate experiments. The p values of different cases are all less than 0.04.
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Fig. 5. Titration curve of the DNA beacons (A) and beacon–AuNPs (B) determined using E. coli bacteria (DH5α) with the concentration range of DH5α from 108 cfu/ml to 10 cfu/ml and ddH2O. Each error bar represents 7 SEM of separate experiments. The p values of different E. coli concentrations (108–10 cfu/ml) are all less than 0.05.
3.4. Sensitivity of detection of E. coli 16S rRNA by DNA beacons– AuNPs
Fig. 6. Normalized fluorescent intensities with and without blood serum tested by beacon–AuNPs. Each error bar represents 7 SEM of separate experiments. The p values of E. coli detection are all less than 0.03.
3.3. Sensitivity of detection of E. coli 16S rRNA by DNA beacons Since the molecular beacons could determine the existence of the bacteria, here, the sensitivity of both beacons only and beacon–AuNPs are discussed, respectively. We used E. coli as model bacteria for quantification. Firstly, experiments were conducted to use the DNA beacons alone to detect E. coli 16S rRNA without target amplification. Fig. 5(A) shows the titration curves of the DNA beacon for detecting E. coli 16S rRNA at a large range of concentrations. With the decrease of the bacterial density, the fluorescent intensity generally decreases. In order to determine the existence of bacteria, here, we define that bacteria exists if the fluorescent intensity of the sample is at least 1.5 times as that of the control sample which is ddH2O. As shown in Fig. 5(A), when the bacterial concentration is above 105 cfu/ml, the fluorescent intensity is obviously larger compared to the control group. When the bacterial concentration is between 0 and 104 cfu/ ml, the fluorescent intensity is not significantly higher than the control group. In addition, when the bacterial concentration changes from 108 cfu/ml to 107 cfu/ml and from 107 cfu/ml to 106 cfu/ml, the fluorescent intensity decreases significantly. However, when the bacterial concentration changes from 106 cfu/ml to 105 cfu/ml, the decrease of fluorescent intensity is not as large. Collectively, the effective detection range should be no smaller than 105 cfu/ml which means there are at least 20,000 copies of 16S rRNA or 3.5 pM bacterium (Neidhardt and Umbarger, 1987). And the most sensitive bacterial concentration detection range using DNA beacon alone is above 106 cfu/ml with detection limit at 105 cfu/ml.
Next we evaluate whether DNA beacon coated AuNPs could improve the detection sensitivity of E. coli Fig. 5(B) shows the titration curves of the beacon–AuNPs for detecting E. coli 16S rRNA, the concentration range of DH5α tested is the same as shown in Fig. 5(A). With the decrease of the bacterial concentration, the fluorescent intensity generally decreases as well. When the bacterial concentration is among 0 and 10 cfu/ml, the fluorescent intensity difference compared to control group is not obvious. While as bacterial concentration is 102 cfu/ml, the fluorescent intensity is more than 1.5 fold larger than that for control group. Therefore beacon–AuNPs could effectively detect the sample with a bacterial concentration no smaller than 102 cfu/ml which is 1000-fold better than using DNA beacons alone. In addition, as the bacterial concentration decreases, the fluorescent intensity decreases gradually, which is quite different from the trend shown in Fig. 5(A). Thus for beacon–AuNPs, it appears to have much larger sensitive detection range, and it may also have the potential for quasi-quantitative measurements for the bacterial concentration. 3.5. Anti-interference of DNA beacon–AuNPs for bacteria detection Finally, the anti-interference of DNA beacon–AuNPs for bacteria detection was evaluated. For gold nanoparticle based application, blood serum was often used to mimic the complex physiological conditions (Huang and Liu, 2010), thus experiments were conducted to use the DNA beacon–AuNPs to detect E. coli 16S rRNA with and without serum. The fetal bovine serum (10%) was prepared by dissolving in 10 mM PBS as background solvent. According to the above experiments, we chose the bacterial concentrations at 108 cfu/ml and 102 cfu/ml, in which 102 cfu/ml is the detection limit for DNA beacon–AuNPs. As shown in Fig. 6 there is no obvious difference with and without blood serum. These results demonstrate that the DNA beacon–AuNPs have high sensitivity and robustness for bacteria detection.
4. Conclusions In this paper, we have designed a new molecular beacon–Au nanoparticle hybrid nanoprobe which could detect bacteria with high accuracy, sensitivity, efficiency and low cost. Compared with traditional beacon probes, the beacon–AuNPs improve the sensitivity by
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more than 103 times, which could achieve detection sensitivity of bacterial concentration at 102 cfu/ml. Besides, we also have shown that beacon–AuNPs have a good diagnostic capacity for UTI samples. Moreover, this method does not need sample amplification or cell culture, which could accelerate the whole detection time into less than 1 h. We believe that by changing the sequence of the molecular beacons, the principles presented here are also true for other bacteriaspecific sequences. This promising method could be widely applied in many fields as clinical testing, biological and chemical research, food safety inspection, etc., to achieve rapid and sensitive detection. Acknowledgments This work was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the National Natural Science Foundation of China (Grant no. 31372399) and Natural Science Foundation of Jiangsu Province (Grant no. BK20130699) for F.L. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.02.020. References Beni, V., Hayes, K., Lerga, T.M., O’Sullivan, C.K., 2010. Biosens. Bioelectron. 26 (2), 307–313. Bercovici, M., Kaigala, G., Mach, K., Han, C., Liao, J., Santiago, J., 2011. Anal. Chem. 83 (11), 4110–4117. Bhat, S., Singal, N., Aggarwal, C.S., Jain, R.C., 1999. J. Commun. Dis. 31 (4), 247–252. Boehme, C., Molokova, E., Minja, F., Geis, S., Loscher, T., Maboko, L., Koulchin, V., Hoelscher, M., 2005. Trans. R. Soc. Trop. Med. Hyg. 99 (12), 893–900.
Cai, L., Zhang, T., 2013. Environ. Sci. Technol. 47 (10), 5433–5441. Chen, C.-K., Shiang, Y.-C., Huang, C.-C., Chang, H.-T., 2011. Biosens. Bioelectron. 26 (8), 3464–3468. Corless, C.E., Guiver, M., Borrow, R., Edwards-Jones, V., Kaczmarski, E.B., Fox, A.J., 2000. J. Clin. Microbiol. 38 (5), 1747–1752. Fang, X., Li, J.J., Perlette, J., Tan, W., Wang, K., 2000. Anal. Chem. 72 (23), 747A–753A. Frens, G., 1973. Nature 241 (105), 20–22. Fujimoto, K., Shimizu, H., Inouye, M., 2004. J. Org. Chem. 69 (10), 3271–3275. Harms, G., Layton, A.C., Dionisi, H.M., Gregory, I.R., Garrett, V.M., Hawkins, S.A., Robinson, K.G., Sayler, G.S., 2003. Environ. Sci. Technol. 37 (2), 343–351. Hu, X., Xu, B., Yang, Y., Liu, D., Yang, M., Wang, J., Shen, H., Zhou, X., Ma, X., 2013. BMC Microbiol. 13 (1), 58. Huang, P.J.J., Liu, J.W., 2010. Anal. Chem. 82 (10), 4020–4026. Li, G., Li, X., Wan, J., Zhang, S., 2009. Biosens. Bioelectron. 24 (11), 3281–3287. Liao, J.C., Mastali, M., Gau, V., Suchard, M.A., Møller, A.K., Bruckner, D.A., Babbitt, J.T., Li, Y., Gornbein, J., Landaw, E.M., 2006. J. Clin. Microbiol. 44 (2), 561–570. Loy, A., Lehner, A., Lee, N., Adamczyk, J., Meier, H., Ernst, J., Schleifer, K.-H., Wagner, M., 2002. Appl. Environ. Microbiol. 68 (10), 5064–5081. Mariella , R., 2008. Biomed. Microdev. 10 (6), 777–784. Matsuki, T., Watanabe, K., Fujimoto, J., Miyamoto, Y., Takada, T., Matsumoto, K., Oyaizu, H., Tanaka, R., 2002. Appl. Environ. Microbiol. 68 (11), 5445–5451. Miao, X.M., Xiong, C., Wang, W.W., Ling, L.S., Shuai, X.T., 2011. Chem. – A Eur. J. 17 (40), 11230–11236. Morens, D.M., Folkers, G.K., Fauci, A.S., 2004. Nature 430 (6996), 242–249. Neidhardt, F., Umbarger, H.E., 1987. In: Neidhardt, F.C., Ingraham, J.L., Low, K.B., Magasanik, B., Umbarger, H.E. (Eds.), E. coli and S. typhimurium: Cellular and Molecular Biology. American Society for Microbiology, Washington, DC Peplies, J., Glockner, F.O., Amann, R., 2003. Appl. Environ. Microbiol. 69 (3), 1397–1407. Peyerl, F.W., Barouch, D.H., Bazick, H.S., Manuel, E., Letvin, N.L., 2005. J. Clin. Microbiol. 43 (9), 4773–4779. Prass, K., Braun, J.S., Dirnagl, U., Meisel, C., Meisel, A., 2006. Stroke: A J. Cereb. Circ. 37 (10), 2607–2612. Rungrassamee, W., Tosukhowong, A., Klanchui, A., Maibunkaew, S., Plengvidhya, V., Karoonuthaisiri, N., 2012. J. Microbiol. Methods 91 (3), 341–353. Selvaraju, T., Das, J., Jo, K., Kwon, K., Huh, C.-H., Kim, T.K., Yang, H., 2008. Langmuir 24 (17), 9883–9888. Tunney, M.M., Patrick, S., Curran, M.D., Ramage, G., Hanna, D., Nixon, J.R., Gorman, S.P., Davis, R.I., Anderson, N., 1999. J. Clin. Microbiol. 37 (10), 3281–3290. Tyagi, S., Kramer, F.R., 1996. Nat. Biotechnol. 14 (3), 303–308. Yeh, H.-Y., Yates, M.V., Mulchandani, A., Chen, W., 2010. Chem. Commun. 46 (22), 3914–3916.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Highly sensitive and rapid bacteria detection using molecular beacon–Au nanoparticles hybrid nanoprobes Jing Cao a, Chao Feng b, Yan Liu c, Shouyu Wang d, Fei Liu a,n a
Single Molecule Nanometry Laboratory, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China Department of Pathology, The First People's Hospital of Shangqiu, Henan, Shangqiu 476000, China c Clinical Laboratory, The First People's Hospital of Shangqiu, Henan, Shangqiu 476000, China d Department of Information Physics and Engineering, Nanjing University of Science and Technology, Nanjing, Jiangsu 210094, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 7 November 2013 Received in revised form 30 January 2014 Accepted 10 February 2014 Available online 19 February 2014
Since many diseases are caused by pathogenic bacterial infections, accurate and rapid detection of pathogenic bacteria is in urgent need to timely apply appropriate treatments and to reduce economic costs. To end this, we designed molecular beacon–Au nanoparticle hybrid nanoprobes to improve the bacterial detection efficiency and sensitivity. Here, we show that the designed molecular beacon modified Au nanoparticles could specifically recognize synthetic DNAs targets and can readily detect targets in clinical samples. Moreover, the hybrid nanoprobes can recognize Escherichia coli within an hour at a concentration of 102 cfu/ml, which is 1000-folds sensitive than using molecular beacon directly. Our results show that the molecular beacon–Au nanoparticle hybrid nanoprobes have great potential in medical and biological applications. & 2014 Elsevier B.V. All rights reserved.
Keywords: Molecular beacon Au nanoparticles Sensitive and rapid bacterial detection
1. Introduction Pathogenic bacterial infections occur widespread in the world and lead to many diseases, such as pneumonia (Prass et al., 2006), pulmonary tuberculosis (Bhat et al., 1999) and urinary tract infection (UTI) (Morens et al., 2004), etc., which cause millions of cases every year in the world and many other problems, such as high morbidity, large economic costs and productivity decrease. Therefore, accurate and rapid detections of pathogenic bacteria are in urgent needs to identify bacterial infections in order to provide appropriate antibiotic treatments and prevent the spread of diseases. Most traditional diagnoses of bacterial infections are complex, expensive and special environment limited. As a clinical gold standard, bacterial culture can provide extremely accurate measurements; however, it often costs several days. In order to accelerate the detection efficiency, developed molecular detection technologies, such as polymerase chain reaction (PCR) (Tunney et al., 1999), real-time PCR (Corless et al., 2000), microarray-based tests (Peplies et al., 2003) and enzyme-linked immunosorbent assay (ELISA) (Boehme et al., 2005), etc., have been adopted. While, PCR and real-time PCR do not only require amplification of the target sequence, but also need the extraction and purification of nucleic acids (Mariella, 2008). Besides,
n
Corresponding author. E-mail address: [email protected] (F. Liu). URLS: http://www.feiliugroup.com (J. Cao), http://www.feiliugroup.com (F. Liu).
http://dx.doi.org/10.1016/j.bios.2014.02.020 0956-5663 & 2014 Elsevier B.V. All rights reserved.
there are always complex procedures in ELISA detections which are not simple and rapid (Harms et al., 2003). Therefore, there is still a need of a more rapid, sensitive, convenient and cost effective bacteria detection approach, which would be enormously beneficial for patients to receive rational therapy in the health care system (Bercovici et al., 2011). Molecular beacons are considered as one of the most promising technologies in biological detection. DNA beacons have been widely used for qualitative and quantitative detections of bacteria, viruses, single nucleotide polymorphisms, etc., with high speed and sensitivity (Fang et al., 2000; Fujimoto et al., 2004; Peyerl et al., 2005). As a well-characterized specific bio-signature, 16S rRNA, because of its great conservation and high copy numbers (Cai and Zhang, 2013; Hu et al., 2013), is often used as the target for beacons to detect bacteria, such as Lactobacilli in Thai fermented sausage (Rungrassamee et al., 2012), Escherichia coli in UTI (Bercovici et al., 2011), predominant bacteria in human feces (Harms et al., 2003; Loy et al., 2002; Matsuki et al., 2002), etc. However, one major limitation of conventional beacons is a lack of sufficient sensitivity in detection. Au nanoparticles (AuNPs) with unique optical properties, facile surface chemistry and appropriate size scale are very attractive in molecular biological applications, such as sensitive and real-time detection of infectious viruses, as well as the real-time visualization of cell-to-cell virus spreading (Yeh et al., 2010). Moreover, AuNPs could induce molecular aggregation. Combining with AuNPs and DNA beacons, beacon–AuNPs can highly improve detection sensitivity
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though signal amplification (Beni et al., 2010; Li et al., 2009; Selvaraju et al., 2008), since there will be extremely high DNA beacon density in the local region. Here, in this paper, molecular beacons (Chen et al., 2011) combined with AuNPs as molecular beacon–Au nanoparticles hybrid nanoprobes were designed to detect both synthetic DNA targets and 16S rRNA of E. coli (DH5α) in cell cultures. The DNA beacon used is for a wide range of urinary pathogens which consists of a 27-mer probe sequence (Liao et al., 2006), a 7 base pair self-complementary stem, the fluorophore of FAM in the 50 terminus and the quencher of BHQ1 in the 30 terminus (Tyagi and Kramer, 1996) as shown in Fig. 1(a) (more information is in Supplementary information, Table S1). The stem sequences keep the hairpin-shaped structure and fluorescence of FAM is quenched by BHQ1 when no targets exist. With the presence of targets, hybridization of targets with complementary beacon sequences results in significant fluorescent signals since the quencher is separated from fluorophore. Therefore, the existence of targets can be recorded by the intensity of fluorescent signals. By conjugating DNA beacons with modified 13 nm AuNPs as shown in Fig. 1(b), the sensitivity is enhanced by three orders of magnitude, thus overall this approach has the advantage that there is no need of amplification and the fluorescent signals could be directly measured and analyzed. Moreover, the whole process could be finished within 1 h. To the best of our knowledge, the current study is the first demonstration of the molecular beacon–AuNP hybrid nanoprobes for sensitive and rapid bacteria detection. Because of its high accuracy and efficiency, easy preparation and low cost, this method could be widely used in clinical test, biological and chemical research, and food safety inspection, etc.
2. Materials and methods 2.1. Chemicals and reagents Tri (2-carboxyethyl) phosphine hydrochloride (C9H15O6PdHCl), lysozyme, Triton X-100, 2 mM EDTA and Tris–HCl are analytical grade and used without further purification. These reagents were purchased from Sigma-Aldrich (USA). Other chemicals, chloroauric acid (HAuCl4), sodium citrate (Na3C6H5O7), NaCl, Na2HPO4d2H2O, KH2PO4, KCl and NaOH employed were all of analytical grade, and ultrapure water in the experiment was provided by Milli-Q Academic system (USA). All glassware used was ultrasonically cleaned. The 10 mM phosphate-buffered saline (PBS) solutions (pH¼7.4) containing 0.01 M sodium phosphate, 0.137 M NaCl, 0.01 M Na2HPO4d2H2O, 0.002 M KH2PO4 and 0.0027 M KCl were prepared. Lysis buffer (pH ¼8.0) contained 0.1% Triton X-100, 2 mM EDTA and 1 mg/ml lysozyme in 20 mM Tris–HCl. The fetal bovine serum was purchased from Gibico (Australia). The linearized pBAsi-mU6 DNA plasmids were obtained from Takara (Japan). 2.2. DNA beacon design DNA beacon used for a wide range of urinary pathogens detection consists of a 27-mer probe sequence (Tyagi and Kramer, 1996), a 7 base pair self-complementary stem, the fluorophore of FAM (Carboxyfluorescein) in the 50 terminus and the quencher of BHQ1 (Black Hole Quencher 1) in the 30 terminus. The sequences of target, target with four mismatches and mismatched target are designed as control experiments in order to
Fig. 1. (A) The principle of targets detection with DNA beacons. (B) The schematic design of beacon–AuNPs for targets detection.
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demonstrate the specificity of DNA beacons. More characteristics of beacon, target, target with four mismatches and mismatched target are shown in Table S1 of Supplementary information.
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temperature of the reaction was decreased from 80 1C to 40 1C. With temperature control of real-time PCR instrument, the whole process could be finished in 1 h.
2.3. Preparation and characterization of AuNPs 3. Results and discussion AuNPs were prepared with the sodium citrate reduction method (Frens, 1973). Firstly, 200 μl HAuCl4 (50 mM) was brought to 9.8 ml boiling ddH2O with vigorous stirring. 1 ml sodium citrate (38.8 mM) was added to the vortex of the solution. The boiling solution turned dark in 1 min. After 3 min, the dark color changed into burgundy. After boiling for 10 min, ultrasonication is needed for 30 min at room temperature (25 1C). In order to characterize the prepared AuNPs, UV–vis extinction spectra (TU1900, Persee, China) is applied to test absorption band of AuNPs. Dynamic light scattering (DLS) (Zetasizer Nano ZS90, USA) is for detecting hydrodynamic diameter of AuNPs and beacon–AuNPs. The prepared AuNPs are observed by the TEM (H7650, Hitachi, Japan). 2.4. Conjugation between DNA beacons and AuNPs
3.1. TEM and dynamic light scattering (DLS) characteristics of AuNPs Firstly, prepared AuNPs were detected by UV–vis extinction spectra. The details of UV–vis extinction spectra are shown in Fig. S2 of Supplementary information. Besides, AuNPs were observed via TEM as shown in Fig. 2. The results indicate that most AuNPs exhibited uniform particle size with an average diameter of 15 nm. Since thiolated DNA beacons on the surface of the AuNPs cannot be observed by TEM, DLS is applied to measure the connection condition between AuNPs and DNA beacons. The hydrodynamic diameter of AuNPs measured by DLS would be enlarged when DNA beacons are attached to the AuNPs surfaces (Miao et al., 2011). Fig. 3 demonstrates the hydrodynamic
In order to link DNA beacons with AuNPs, firstly, 30 μl thiolated beacons (20 μM) were activated by 30 μl TCEP (50 mM). The pH value of the mixture was adjusted to 5 using 0.5 M NaOH. Then, the solution was incubated at room temperature (25 1C) overnight. 100 μl AuNPs (10 nM) and 33 μl PBS (50 mM) were added, followed by sonication for 10 s, heated at 50 1C for 10 min and gently shaking for 24 h. Afterwards, 0.01% SDS and NaCl (3 M) were mixed in the solution. The solution was processed by sonication for 10 s and then heated at 50 1C for 10 min. The process was repeated for three times. Hereafter, the reaction solution was incubated for 48 h at room temperature. The free beacons and beacon–AuNPs were separated by centrifugation at 12,000 rpm for 30 min at 4 1C. The precipitation was washed by 10 mM PBS and then recentrifuged. The process was repeated twice. Finally the beacon–AuNPs were dispersed in 100 μl PBS (20 mM, pH ¼7.4) and stored at 4 1C. The connection between beacons and AuNPs could be analyzed by the DLS. 2.5. Sample preparation The samples included E. coli (DH5α) and clinical urine. DH5α were inoculated with Luria broth (LB) in a shaker at 37 1C and grew to 108 cfu/ml. The traditional plate colony-counting method was used to calculate the density of DH5α (Fig. S1 in the Supplementary information). One milliliter bacteria sample was centrifuged at 10,000 rpm for 5 min and then the supernatant was discarded. The pellets were kept frozen at 80 1C. The urine sample was collected from patient with approval of biological ethics. The bacterial concentration of the sample was 3 108 cfu/ ml (Supplementary information). 1 ml sample was pelleted by centrifugation for 5 min at 10,000 rpm. After removing supernatant, the sample was stored at 80 1C. The samples were lysed by resuspension of the pellet in 10 μl of 0.5 M NaOH and 10 μl of lysis buffer, and then incubated at room temperature (25 1C) for 5 min.
Fig. 2. Transmission Electron Microscopy (TEM) images of AuNPs, the white bar indicates 100 nm.
2.6. Fluorescence detection Real-time PCR instrument (Bio-rad, iQ2, USA) was applied for detecting fluorescence and a 20 μl reaction system was prepared. Firstly, 1 ml bacteria solution was collected and centrifuged for 5 min, 10 μl NaOH and 10 μl lysis buffer was added and the final concentration of beacons was 100 nM (10 mM PBS, pH ¼ 7.4). After an initial denaturation step (3 min at 80 1C), melting curves were determined by measuring the fluorescence signals when the
Fig. 3. Dynamic light scattering (DLS) analysis of AuNPs and beacon–AuNPs.
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diameter of AuNPs and beacon–AuNPs. With Gaussian fitting, the average diameter of the AuNPs is 16.82 nm with full width half maximum of 14.18 nm, while the average diameter of the beacon– AuNPs is 33.68 nm with full width half maximum of 24.85 nm. The results illustrate that the hydrodynamic diameter of beacon– AuNPs are significantly larger than AuNPs (p o0.05), indicating successful conjunction of beacons and AuNPs. 3.2. Specificity of DNA beacons and beacon–AuNPs In order to demonstrate the specificity of DNA beacon–AuNPs, firstly, synthetic DNA target, four mismatched target and mismatched target were tested. As shown in Fig. 4(A) and (B), the normalized
fluorescent intensities of mismatched target and four mismatched target were significantly lower than target for both DNA beacon and DNA beacon–AuNPs. To further demonstrate the specificity of the DNA beacon and DNA beacon–AuNPs to the target, we tested an unrelated 3008 bp DNA, a linearized pBAsi-mU6 DNA plasmid. As shown in Fig. 4(C) and (D), with unrelated large DNA, both DNA beacon and beacon–AuNPs show fluorescent signals similar to background level. Moreover, clinical urine samples collected from UTI patient with 107 cfu/ml bacteria were also tested by the proposed method (Fig. 4 (E) and (F), and Supplementary information). These experimental results indicate that the DNA beacon–Au nanoprobe can detect the target sequence with high specificity and is also promising for pathological diagnosis.
Fig. 4. (A, B) Normalized fluorescent intensities of target, four mismatch targets and mismatch target tested by beacon and beacon–AuNPs, respectively. (C, D) Normalized fluorescent intensities with 108 cfu/ml E. coli concentration and unrelated 3008 bp DNA tested by beacon and beacon–AuNPs, respectively. (E, F) Clinical urine samples with 107 cfu/ml bacteria detected via DNA beacons and beacon–AuNPs. Each error bar represents 7 SEM of separate experiments. The p values of different cases are all less than 0.04.
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Fig. 5. Titration curve of the DNA beacons (A) and beacon–AuNPs (B) determined using E. coli bacteria (DH5α) with the concentration range of DH5α from 108 cfu/ml to 10 cfu/ml and ddH2O. Each error bar represents 7 SEM of separate experiments. The p values of different E. coli concentrations (108–10 cfu/ml) are all less than 0.05.
3.4. Sensitivity of detection of E. coli 16S rRNA by DNA beacons– AuNPs
Fig. 6. Normalized fluorescent intensities with and without blood serum tested by beacon–AuNPs. Each error bar represents 7 SEM of separate experiments. The p values of E. coli detection are all less than 0.03.
3.3. Sensitivity of detection of E. coli 16S rRNA by DNA beacons Since the molecular beacons could determine the existence of the bacteria, here, the sensitivity of both beacons only and beacon–AuNPs are discussed, respectively. We used E. coli as model bacteria for quantification. Firstly, experiments were conducted to use the DNA beacons alone to detect E. coli 16S rRNA without target amplification. Fig. 5(A) shows the titration curves of the DNA beacon for detecting E. coli 16S rRNA at a large range of concentrations. With the decrease of the bacterial density, the fluorescent intensity generally decreases. In order to determine the existence of bacteria, here, we define that bacteria exists if the fluorescent intensity of the sample is at least 1.5 times as that of the control sample which is ddH2O. As shown in Fig. 5(A), when the bacterial concentration is above 105 cfu/ml, the fluorescent intensity is obviously larger compared to the control group. When the bacterial concentration is between 0 and 104 cfu/ ml, the fluorescent intensity is not significantly higher than the control group. In addition, when the bacterial concentration changes from 108 cfu/ml to 107 cfu/ml and from 107 cfu/ml to 106 cfu/ml, the fluorescent intensity decreases significantly. However, when the bacterial concentration changes from 106 cfu/ml to 105 cfu/ml, the decrease of fluorescent intensity is not as large. Collectively, the effective detection range should be no smaller than 105 cfu/ml which means there are at least 20,000 copies of 16S rRNA or 3.5 pM bacterium (Neidhardt and Umbarger, 1987). And the most sensitive bacterial concentration detection range using DNA beacon alone is above 106 cfu/ml with detection limit at 105 cfu/ml.
Next we evaluate whether DNA beacon coated AuNPs could improve the detection sensitivity of E. coli Fig. 5(B) shows the titration curves of the beacon–AuNPs for detecting E. coli 16S rRNA, the concentration range of DH5α tested is the same as shown in Fig. 5(A). With the decrease of the bacterial concentration, the fluorescent intensity generally decreases as well. When the bacterial concentration is among 0 and 10 cfu/ml, the fluorescent intensity difference compared to control group is not obvious. While as bacterial concentration is 102 cfu/ml, the fluorescent intensity is more than 1.5 fold larger than that for control group. Therefore beacon–AuNPs could effectively detect the sample with a bacterial concentration no smaller than 102 cfu/ml which is 1000-fold better than using DNA beacons alone. In addition, as the bacterial concentration decreases, the fluorescent intensity decreases gradually, which is quite different from the trend shown in Fig. 5(A). Thus for beacon–AuNPs, it appears to have much larger sensitive detection range, and it may also have the potential for quasi-quantitative measurements for the bacterial concentration. 3.5. Anti-interference of DNA beacon–AuNPs for bacteria detection Finally, the anti-interference of DNA beacon–AuNPs for bacteria detection was evaluated. For gold nanoparticle based application, blood serum was often used to mimic the complex physiological conditions (Huang and Liu, 2010), thus experiments were conducted to use the DNA beacon–AuNPs to detect E. coli 16S rRNA with and without serum. The fetal bovine serum (10%) was prepared by dissolving in 10 mM PBS as background solvent. According to the above experiments, we chose the bacterial concentrations at 108 cfu/ml and 102 cfu/ml, in which 102 cfu/ml is the detection limit for DNA beacon–AuNPs. As shown in Fig. 6 there is no obvious difference with and without blood serum. These results demonstrate that the DNA beacon–AuNPs have high sensitivity and robustness for bacteria detection.
4. Conclusions In this paper, we have designed a new molecular beacon–Au nanoparticle hybrid nanoprobe which could detect bacteria with high accuracy, sensitivity, efficiency and low cost. Compared with traditional beacon probes, the beacon–AuNPs improve the sensitivity by
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more than 103 times, which could achieve detection sensitivity of bacterial concentration at 102 cfu/ml. Besides, we also have shown that beacon–AuNPs have a good diagnostic capacity for UTI samples. Moreover, this method does not need sample amplification or cell culture, which could accelerate the whole detection time into less than 1 h. We believe that by changing the sequence of the molecular beacons, the principles presented here are also true for other bacteriaspecific sequences. This promising method could be widely applied in many fields as clinical testing, biological and chemical research, food safety inspection, etc., to achieve rapid and sensitive detection. Acknowledgments This work was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the National Natural Science Foundation of China (Grant no. 31372399) and Natural Science Foundation of Jiangsu Province (Grant no. BK20130699) for F.L. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.02.020. References Beni, V., Hayes, K., Lerga, T.M., O’Sullivan, C.K., 2010. Biosens. Bioelectron. 26 (2), 307–313. Bercovici, M., Kaigala, G., Mach, K., Han, C., Liao, J., Santiago, J., 2011. Anal. Chem. 83 (11), 4110–4117. Bhat, S., Singal, N., Aggarwal, C.S., Jain, R.C., 1999. J. Commun. Dis. 31 (4), 247–252. Boehme, C., Molokova, E., Minja, F., Geis, S., Loscher, T., Maboko, L., Koulchin, V., Hoelscher, M., 2005. Trans. R. Soc. Trop. Med. Hyg. 99 (12), 893–900.
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