Anal. Chem. 2007, 79, 6152-6158
Escherichia coli Genosensor Based on Polyaniline Kavita Arora,†,‡ Nirmal Prabhakar,† Subhash Chand,‡ and B. D. Malhotra*,†
Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi-110012, India, and Department of Biochemical Engineering & Biotechnology, Indian Institute of Technology Delhi, Hauzkhas, New Delhi-110016, India
Avidin-modified polyaniline (PANI) electrochemically deposited onto a Pt disk electrode has been utilized for direct detection of Escherichia coli by immobilizing a 5′-biotin-labeled E. coli probe (BdE) using a differential pulse voltammetric technique in the presence of methylene blue as a DNA hybridization indicator. Depending on the target sample and the sonication time, this BdEavidin-PANI bioelectrode can be utilized to electrochemically detect a complementary target probe (0.009 ng/µL), E. coli genomic DNA (0.01 ng/µL) and 11 E. coli cells/ mL in 60 s to 14 min (hybridization time) without using PCR and can be used 5-7 times at temperatures of 3045 °C. Detection of microbes is considered important for environmental monitoring, food industry, detection of infectious diseases, biological warfare, and various laboratory applications.1 Microbes like Escherichia coli, Campylobacter, Salmonella, and Listeria monocytogenes are responsible for the majority of food-borne outbreaks.2-4 Contemporary methods (microbiological/biochemical/immunological detection kits, southern hybridization, PCR, etc.) are slow and labor intensive. Biosensors have emerged as promising alternatives for faster, low-cost, and simpler methods of microbial detection using nucleic acids-based affinity detection of complementary target sequences. Electrochemical transducers have been considered as a simple and sensitive method of DNA hybridization detection.5-11 A wide range of conducting/semiconducting biomaterials including carbon paste, gold, screen-printed electrodes, self-assembled * Corresponding author. Phone: +91 11 25734273. Fax: +91 11 25726938. E-mail:
[email protected]. † National Physical Laboratory. ‡ Indian Institute of Technology Delhi. (1) Arora, K.; Chand, S.; Malhotra, B. D. Anal. Chim. Acta 2006, 568, 259274. (2) Alocilja, E. C.; Radke, S. M. Biosens. Bioelectron. 2003, 18, 841-846. (3) Brooks, B. W.; Robertson, R. H.; Wallace, C. L. L.; Pfahler, W. Vet. Microbiol. 2002, 87, 37-49. (4) Brown, M. H. Biodeterior. Biodegrad. 2002, 50, 155-160. (5) Kerman, K.; Kobayashi, M.; Tamiya, E. Meas. Sci. Technol. 2004, 15, R1R11. (6) Palecek, E. Talanta 2002, 56, 809-819. (7) Palecek, E.; Fojta, M.; Jelen, F. Bioelectrochemistry 2002, 56, 85-90. (8) Palecek, E.; Fojta, M. In Bioelectronics; Willner, I., Katz, E., Eds.; WileyVCH: New York, 2005; pp 127-193. (9) Panke, O.; Kirbs, A.; Lisdat, F. Biosens. Bioelectron. 2007, 22, 2656-2662. (10) Brazda, V.; Jagelska, E. V.; Fojta, M.; Palecek, E. Biochem. Biophys. Res. Commun. 2006, 341, 470-477. (11) Kourilova, A.; Babkina, S.; Cahova, K.; Havran, L.; Jelen, F.; Palecek, E.; Fojta, M. Anal. Lett. 2005, 38, 2493-2507.
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monolayers, and conducting polymers have been utilized for development of genosensors (DNA electrochemical biosensors). Electrochemical DNA biosensors have been reported to detect complementary sequences of HIV DNA,12 A. ostenfeldii,13 C. parvum,14 Microcystis sp.,15 etc. A nucleic acid sequence-based, amplification-based RNA biosensor has been fabricated for detection of E. coli up to 40 cfu within 15-20 min by measuring changes in reflected light.16 An electrochemical RNA assay based on a pencil electrode has been reported for detection of E. coli in fecal waste.17 Another method reports E. coli detection using sequential flow PCR products up to 23 cells using a piezoelectric quartz crystal.18 Mercury and carbon electrode-based electrochemical DNA biosensors have been used to distinguish single-stranded and double-stranded DNA using methylene blue (MB) as the electrochemical indicator.19 A carbon paste electrode-based DNA sensor having an electrochemically adsorbed probe sequence specific to hepatitis B virus has been reported.20 An electrochemical DNA biosensor based on single-stranded DNA immobilized onto a carboxyl-terminated alkanethiol self-assembled monolayer immobilized with single-stranded (ss) DNA for recognition of complementary DNA hybridization has been reported.21 The DNAimmobilized 2,5-bis(2-thienyl)-N-(3-phosphorylpropyl)pyrrole film has recently been used to detect complementary targets up to 0.16 fmol for an 18-base-long probe and 3.5 fmol for a 27-baselong probe using cyclic voltammetry within 30 min.22 Xiao et al. have reported detection of DNA hybridization via a covalently immobilized thiolated DNA probe onto a gold surface by covalently linked MB as redox indicator using differential pulse (12) Fu, Y.; Yuan, R.; Chai, Y.; Zhu, L.; Zhang, Y. Anal. Lett. 2006, 39, 467482. (13) Metfies, K.; Huljic, S.; Lange, M.; Medlin, L. K. Biosens. Bioelectron. 2005, 20, 1349-1357. (14) Wang, J.; Rivas, G.; Parrado, C.; Cai, X.; Flair, M. N. Talanta 1997, 44, 2003-2010. (15) Yan, F.; Erdem, A.; Meric, B.; Kerman, K.; Ozsoz, M.; Sadik, S. Electrochem. Commun. 2001, 3, 224-228. (16) Baeumner, A. J.; Cohen, R. N.; Miksic, V.; Min, J. Biosens. Bioelectron. 2003, 18, 405-413. (17) LaGier, M. J.; Scholin, C. A.; Fell, J. W.; Wang, J.; Goodwin, K. D. Mar. Pollut. Bull. 2005, 50, 1251-1261. (18) Sun, H.; Zhang, Y.; Fung, Y. Biosens. Bioelectron. 2006, 22, 506-512. (19) Ozkan, D.; Kara, P.; Kerman, K.; Meric, B.; Erdem, A.; Jelen, F.; Nielsen, P. E.; Ozsoz, M. Bioelectrochemistry 2002, 58, 119-126. (20) Erdem, A.; Kerman, K.; Meric, B.; Akarca, U. S.; Osos, M. Anal. Chim. Acta 2000, 422, 139-149. (21) Kerman, K.; Ozkan, D.; Kara, P.; Meric, B.; Gooding, J. J.; Ozsoz. M. Anal. Chim. Acta 2002, 462, 39-47. (22) Riccardi, C. S.; Yamanak, K.; Josowicz, M.; Kowalik, M.; Mizaikoff, B.; Kranz, C. Anal. Chem. 2006, 78, 1139-1145. 10.1021/ac070403i CCC: $37.00
© 2007 American Chemical Society Published on Web 07/14/2007
voltammetry up to 400 fmol of complementary target.23 It may be noted that even though the electrode can be regenerated by stringent washing for 20 min, there is a need to reintroduce a new signaling probe (i.e., MB-tagged DNA probe). Panke et al. have reported electrochemical DNA hybridization detection of a complementary target up to 3 pmol using covalently immobilized thiolated ssDNA onto a gold surface within 10 min of hybridization time.9 Also, detection of single-base-pair mismatches and quantification of label-free target ssDNA can be performed by competitive binding assay using methylene blue-labeled ssDNA probes. Conducting polymer-based genosensors have recently gained wider attention.24-27 Polypyrroles, polyanilines (PANI), and polythiophenes have been reported for application to biosensors.28-35 Polyaniline for its two redox couples, mechanical flexibility, functionalization, and environmental stability can be complexed with double-stranded (ds) DNA immobilized onto a silicon surface for fabrication of polyaniline nanowires.36,37 Direct guanine oxidation, and redox DNA hybridization indicators, like daunomycin, MB, metal complexes (e.g., Ru/Co complexes), enzyme labels, and nanoparticles,1,38-40 have been used to differentiate singlestranded and double-stranded DNA immobilized onto the desired electrochemical electrode. We report results of the studies relating to fabrication of a polyaniline-based genosensor to directly detect E. coli complementary target sequence in complementary oligonuleotide, E. coli genomic DNA, and E. coli cell lysate using MB as redox hybridization indicator. EXPERIMENTAL SECTION Chemicals and Reagents. Aniline, Tris buffer, ethylenediaminetetraacetic acid (EDTA), potassium monohydrogen phosphate, potassium dihydrogen phosphate, MB, N-(3-dimethylaminoproyl)-N-ethylcarbodiimide hybrochloride (EDC), oligonucleotide probes, N-hydroxysuccinimide (NHS), avidin, E. coli genomic DNA, and E. coli cells (DH5R) were procured from Sigma-Aldrich, Milwaukee, WI. All chemicals (molecular biology grade) used (23) Xiao, Yi.; Lubin, A. A.; Baker, B. R.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 16677-16680. (24) Xu, X.; Yang, H.; Mallouk, T. E.; Bard, A. J. J. Am. Chem. Soc. 1994, 116, 8386-8387. (25) Sadik, O. A. Electroanalysis 1999, 11, 839-844. (26) Mikkelsen, S. R. Electroanalysis 1996, 8, 15-19. (27) Wang, J.; Jiang, M. Langmuir 2002, 16, 2269-2274. (28) Malhotra, B. D.; Chaubey, A.; Singh, S. P. Anal. Chim. Acta 2006, 578, 59-74. (29) Gerard, M.; Ramanathan, K; Chaubey, A.; Malhotra, B. D. Electroanalysis 1999, 1, 450-452. (30) Ramanathan, K.; Pandey, S. S.; Kumar, R.; Malhotra, B. D.; Murthy, A. S. N. J. Appl. Polym. Sci. 2000, 78, 662-667. (31) Pandey, S. S.; Ram, M. K.; Srivastava, V. K.; Malhotra, B. D. J. Appl. Polym. Sci. 1997, 65, 2745-2748. (32) Gerard, M.; Chaubey, A.; Malhotra, B. D. Biosens. Bioelectron. 2002, 17, 345-359. (33) Rodriguez, M. I.; Alocilja, E.C. IEEE Sens. J. 2005, 5, 733-736. (34) Youssoufi, H. K.; Yassar, A. Biomacromolcules 2001, 2, 58-64. (35) Garnier, F.; Youssoufi, H. K.; Srivastave, P.; Mandrand, B.; Delair, T. Synth. Met. 1999, 100, 89-94. (36) Ma, Y.; Zhang, J.; Zhang, G.; He, H. J. Am. Chem. Soc. 2004, 126, 70977101. (37) Xiao, Y.; Kharitonov, A. B.; Patolsky, F.; Weizmann, Y.; Willner, I. Chem. Commun. 2003, 1540-1541. (38) Arora, K.; Chaubey, A.; Singhal, R.; Singh, R. P.; Samanta, S. B.; Malhotra, B. D.; Chand, S. Biosens. Bioelectron. 2006, 21, 1777-1784. (39) Prabhakar, N.; Arora, K.; Singh, S. P.; Singh, H.; Malhotra, B. D. Anal. Chim. Acta 2007, 589, 6-13. (40) Zhu, P.; Shelton, D. R.; Karns, J. S.; Sunaram, A.; Li, S.; Amstutz, P.; Tang, C. M. Biosens. Bioelectron. 2005, 21, 678-683.
were prepared in deionized water (Milli Q 10 TS), and the solutions and glassware were autoclaved prior to being used. E. coli specific probes (17 bases) identified from the 16s rRNA coding region of the E. coli genome are as follows: E. coli probe (BdE), biotin-5′-GGT CCG CTT GCT CTC GC-3′; complementary (dE′), 5′-GCG AGA GCA AGC GGA CC-3′; one-base mismatch (dE1′), 5′-GCG AGA GAA AGC GGA CC-3′; noncomplementary (dEnc), 5′-CTA GTC GTA TAG TAG GC-3′. Electrochemical Preparation of BdE-Avidin-PANI Bioelectrodes. Monomer solution containing aniline (0.1 M) in 1 M HCl was subject to 15 µA for 600 s in a three-electrode electrochemical cell having Ag/AgCl as reference, platinum as counter, and Pt disk (∼0.03 cm2) as working electrode using a potentiostat/galvanostat, (model 273A, Princeton Applied Research) to deposit PANI. EDC (15 mM) and NHS (30 mM) solutions, prepared in deionized water, were used to activate 10 µL avidin solution (1 mg/mL) suspended in water after a ∼2-h incubation at 25 °C. Activated avidin (2 µg) was immobilized onto the PANI surface for 2 h. After washing with autoclaved deionized water, the avidin-PANI surface was immobilized with 20-mer biotinylated oligonucleotide specific to E. coli (BdE probe) (0.648 ng) for incubation (5 min) at 25 °C in a humid chamber. These BdE-avidin-PANI bioelectrodes, washed with autoclaved phosphate buffer 0.05 M, (pH 7.0), were subject to 1-min incubation in biotin solution (1 mg/mL) to cover unbound sites of avidin (Schemes 1 and 2). These bioelectrodes have been characterized using cyclic voltammetry and differential pulse voltammetry, respectively. About 85 genoelectrodes were fabricated for the desired studies. DNA Hybridization Detection Using Differential Pulse Voltametry (DPV). DNA hybridization detection studies of BdEavidin-PANI bioelectrodes were carried out after incubation in complementary (dE′), noncomplementary (dEnc), and one-basemismatched (dE1′) oligonucleotides of desired concentration at 30 °C in a humid chamber (Scheme 2). One-minute incubation with target sequence was found to be sufficient for hybridization detection. For hybridization detection using a hybridization indicator, DPV measurements were conducted after 20 µM MB (redox indicator) pretreatment in phosphate buffer, 0.05 M, pH 7.0, at 0.1 V for 10 s. Sample Preparation of E. coli Genomic DNA and E. coli Cells for Direct Detection of Complementary Target. E. coli genomic DNA solution (10 µg/mL) in phosphate buffer was denatured by heating in a water bath (80 °C) for 1 min and was immediately chilled in ice to obtain denatured single-stranded DNA. Another aliquot of genomic DNA solution (without heat denaturation) was subject to sonication (1-6 min at 120 and 2 A using ultrasonic cleaner, Vibronics Pvt. Ltd., Mumbai, India). Similarly, heat-denatured and sonicated (1-8 min) samples were prepared for E. coli cells (∼386 cells/mL) suspended in phosphate buffer, 0.05 M, pH 7.0. These samples (genomic DNA and lysed E. coli cells) were utilized for hybridization studies using BdEavidin-PANI bioelectrodes for different hybridization times (0.514 min). RESULTS AND DISCUSSION Electrochemical Characterization of Bioelectrodes. Figure 1 shows results of the electrochemical characterization (cyclic voltametry, differential pulse voltammetry) of PANI, avidin-PANI, Analytical Chemistry, Vol. 79, No. 16, August 15, 2007
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Scheme 1. Avidin Immobilization onto Polyaniline Coated onto a Pt Disk Electrode
Scheme 2. DNA Hybridization Detection Using a BdE-Avidin-PANI Bioelectrode
and BdE-avidin-PANI genoelectrodes in phosphate buffer, 0.05 M, pH 7.0. Figure 1a shows two redox peaks at about 0.27 and 0.59 V describing oxidation of aniline to the first radical cation and then oxidation of the radical cation to the radical dication.41 It can be seen that there is considerable decrease in the intensity of these redox peaks after the immobilization of avidin, confirming the binding of avidin onto the PANI surface. Further, the observed enhancement in the redox peaks can be attributed to the binding of the probe (BdE) onto the avidin-PANI surface in agreement with the literature.42 The observed decrease in the oxidation peak current (Figure 1b) indicates the immobilization of avidin onto the PANI surface,43,44 and the enhancement in the oxidation current due to immobilization of the probe (BdE) onto the avidinmodified PANI surface is in agreement with the observed differential pulse voltammograms (Figure 1b).45 (41) Ram, M. K.; Sundaresan, N. S.; Malhotra, B. D. J. Phys. Chem. 1993, 97, 11580-11582. (42) Jiang, M.; Wang, J. J. Electroanal. Chem. 2001, 500, 584-589. (43) Prakash, R. J. Appl. Polym. Sci. 2001, 83, 378-386. (44) Chaubey, A.; Pandey, K. K.; Pandey, M. K.; Singh, V. S. Appl. Biochem. Biotechnol. 2001, 96, 239-302.
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DPV Studies. Figure 2a shows guanine oxidation at 1.1 V in the DPV of a BdE-avidin-PANI bioelectrode before and after hybridization with 92 ng/µL complementary target (dE′).1,37 The guanine oxidation current (Figure 2a) decreases on hybridization with complementary target (dE′), indicating formation of the DNA double helix at the BdE-avidin-PANI surface. This decrease can be attributed to the nonavailability of nitrogenous bases for oxidation as compared to that of unpaired nitrogenous bases in single-stranded DNA. It may be noted that there is no change in the guanine oxidation current upon hybridization with noncomplementary (dEnc) and with the one-base-mismatched (dE1′) target, indicating no duplex formation at the BdE-avidin-PANI surface. Figure 2b exhibits the DPV of the BdE-avidin-PANI bioelectrode using MB as the redox DNA hybridization indicator. MB is known to associate with the unpaired nitrogenous bases of ss DNA as compared to the ds DNA.20,21 The MB oxidation peak can be clearly seen at -0.35 V on hybridization with the (45) Stilwell, D. E.; Park, S. M. J. Electrochem. Soc. 1988, 135, 2254-2262.
Figure 1. (a) Cyclic voltametric characterization of PANI, avidinPANI, and BdE-avidin-PANI bioelectrode at a scan rate of 30 mV/ s, (b) DPV characterization of PANI, avidin-PANI, and BdE-avidinPANI bioelectrode at pulse height of 50 mV and pulse width of 70 ms; in 0.05 M phosphate buffer pH 7.0.
one-base-mismatched (dE1′) and noncomplementary (dEnc) targets. It may be noted that no MB oxidation peak is seen on hybridization with the complementary target (dE′). These results reveal that no duplex/hybrid is formed at the BdE-avidin-PANI surface on hybridization with both dEnc and dE1′ targets. This may be attributed to the positively charged PANI molecules that may repel the positively charged MB molecules and become associated with both partially or unpaired nitrogenous bases (on hybridization with one-base-mismatched target) of the DNA probes stationed at the BdE-avidin-PANI surface. The observed -200-mV peak (Figure 2b and 2c) may perhaps be assigned to the reduction of unpaired nitrogenous bases or to the polyaniline matrix. Figure 2c shows the DPV of the BdE-avidin-PANI bioelectrodes obtained on hybridization with complementary target (0.0064-2.3 ng/µL) using MB. MB oxidation current decreases with increase in concentration of the complementary target (dE′) e1.15 ng/µL, indicating saturation of the BdE-avidin-PANI surface. No changes in MB oxidation current are observed for concentration of dE′
