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J. Phys. Chem. B 2008, 112, 4808-4816
Polyaniline Based Nucleic Acid Sensor Nirmal Prabhakar,†,‡ Kavita Arora,† Harpal Singh,‡ and Bansi D. Malhotra*,† Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi-110012, India, and Centre for Biomedical Engineering, Indian Institute of Technology Delhi, Hauz Khas, New Delhi-110016, India ReceiVed: December 18, 2007
Twenty-bases long NH2-modified DNA and PNA probes specific to a pathogen (Mycobacterium tuberculosis) were covalently immobilized onto a polyaniline (PANI)/Au electrode to detect nucleic acid hybridization with complementary, one-base mismatch and noncomplementary targets within 30 s using Methylene Blue. The PNA-PANI/Au electrode exhibits improved specificity (1000 times) and detection limit (0.125 × 10-18 M) as compared to that of the DNA-PANI/Au electrode (2.5 × 10-18 M). These PNA-PANI/Au electrodes can be utilized for detection of hybridization with the complementary sequence in 5 min sonicated M. tuberculosis genomic DNA within 1 min of hybridization time. These DNA-PANI/Au and PNA-PANI/Au electrodes can be used 6-7 and 13-15 times, respectively.
Introduction There is increased interest toward the application of conducting polymer based nucleic acid sensors for the detection of desired pathogens. This is because various other matrices1 including glassy carbon electrodes2 and CNT-modified disposable graphite electrodes3 have a longer response time, poor sensitivity, and insufficient reusability. Among various conducting polymers, polyaniline (PANI), having a good electrochemical activity and chemical stability, has been widely used for nucleic acid based sensors providing advantages such as lowtemperature synthesis, tunable conductivity, and no need for purification, end-opening, or catalytic processing.4 Highly organized conducting PANI nanotube arrays deposited onto graphite electrodes were used to covalently immobilize 21-mer oligonucleotides for the hybridization detection of a complementary target sequence up to 0.756 fM within 2 h of hybridization time after daunorubicin pretreatment of approximately 15 min using differential pulse voltammetry.5 A polyaniline based electrochemical Escherichia coli genosensor for direct detection of complementary targets in E. coli genomic DNA (0.01 ng/µL) and E. coli cell lysate (11 cells/mL) within 1-14 min of hybridization time using Methylene Blue (MB) has been reported.6 Among the various pathogens, Mycobacterium tuberculosis and other environmental Mycobacteria are of special interest since these are known to cause tuberculosis in individuals with altered, local or systematic immunity. Tuberculosis (TB) is primarily an illness of the respiratory system and is spread by coughing and sneezing; each year, approximately 1.6 million people die from this disease.7 Chronic obstructive pulmonary disease, emphysema, pneumoconiosis, bronchiectasis, cystic fibrosis, thoracic scoliosis, previous gastrectomy, and chronic alcoholism are some of the common conditions that have been linked to the diseases due to M. tuberculosis and nontuberculosis mycobacterium (NTM).8 Widely used contemporary * Corresponding author. Tel.: +91 11 25734273; fax: +91 11 25726938; e-mail:
[email protected]. † National Physical Laboratory. ‡ Indian Institute of Technology.
methods9 for the detection of pathogens such as M. tuberculosis including biochemical tests,10 animal inoculation, chemical tests (including lipid profiling, protein/isozyme patterning, and antigen profiling),11 and molecular techniques such as polymerase chain reaction (PCR),12,13 restriction fragment length polymorphism (RFLP),14,15 DNA fingerprinting,16,17 rapid amplified polymorphic DNA (RAPD),18 and amplified fragment length polymorphism (AFLP)19,20 are time-consuming and cannot be used to determine accurately the drug resistant strains of M. tuberculosis. An electronic nose based on a 14 sensor conducting polymer array has been developed to sense the volatile gas patterns produced by a pathogen (M. tuberculosis) and associated bacterial infections from sputum samples in vitro and in situ.21 An amorphous/nanocrystalline silicon biosensor using gold nanoparticles as the immobilization matrix for the probe from the rpo B gene of M. tuberculosis for the identification of complementary sequences based on colorimetric detection methods has been reported.22 This sensor has, however, an insufficient detection limit (10.0 × 10-9 M, formation of a hybrid (DNAPNA duplex) occurs at the PNA-PANI/Au electrode in 30 s. It may be noted that no hybridization is observed at a concentration of 10.0 × 10-12 M, with the MB current (321 µA), that is almost equal to that of the PNA-PANI/Au electrode (326 µA), when it is not hybridized with any target (Figure 6a). However, the DNA-PANI/Au electrode shows nonspecific binding (presence of a DNA-DNA duplex) with the probe III at concentration g5.0 × 10-12 M (Figure 6b). The improved performance of the PNA-PANI/Au electrode is attributed to the higher affinity of neutral PNA probe molecules present at the surface for nucleic acid hybridization23 and the much higher melting temperature of PNA-DNA than the DNA-DNA hybrid.50 These results indicate that PNA-PANI/Au electrodes show an improved detection limit (20 times) as compared to that of the DNA-PANI/Au electrode. It is interesting to find that the PNA-PANI/Au electrode shows increased specificity (1000 times) as compared to the DNA-PANI/Au electrode as there is no hybridization with the 10 × 10-12 M concentration of probe III, while the DNA-PANI/Au electrode exhibits complete
hybridization (Figure 6). Besides this, the PNA-PANI/Au electrode can be regenerated and used 13-15 times as compared to 6-7 times for the DNA-PANI/Au electrode, resulting in about a 19% decrease in the peak height (data not shown). Keeping this in mind (the improved characteristics of the PNA-PANI/ Au electrode), further investigations regarding the hybridization detection with complementary sequence present in M. tuberculosis genomic DNA have been carried out using PNA-PANI/ Au electrode. Hybridization Detection of Complementary Target Sequence in Genomic DNA of M. tuberculosis. Figure 7 shows SWV images of the PNA-PANI/Au electrode upon hybridization with 5 min sonicated M. tuberculosis genomic DNA (5.0 to 150 pg/µL) after 60 s of hybridization time. With increase in the M. tuberculosis genomic DNA concentration (5.0 to 150 pg/µL), the MB peak current decreases, indicating an increased number of PNA-DNA duplexes formed at the PNA-PANI/Au electrode surface. These results reveal 10.0 pg/µL as the saturating concentration and 5.0 pg/µL ( 3% as the detection limit. The inset in Figure 7 shows the plot of MB current as a function of genomic DNA concentration (pg/µL) and obeys eq 3 I µA ) 35.16 - 0.205 lngenomic DNA concentration(3) Table 1 shows the characteristics of the DNA-PANI/Au and PNA-PANI/Au electrodes including those reported in the literature. Conclusions PNA-PANI/Au and DNA-PANI/Au electrodes can be used to detect the presence of a complementary target of M. tuberculosis using SWV up to 2.5 × 10-18 and 0.125 × 10-18 M, respectively, within 30 s of hybridization time. As compared to DNA-PANI/Au electrodes, PNA-PANI/Au electrodes have been shown to have an improved detection limit (20 times), higher specificity (1000 times) when hybridized with one-base mismatch targets, and increased reusability (13-15 times). It
this work 30 s, 60 s 0.125 × 5.0 pg/µL of M. tuberculosis genomic DNA +0.1 V for 10 s SWV
this work 30 s +0.1 V for 10 s DNA-PANI/Au bioelectrode
PNA-PANI/Au electrode 6
4
5
covalent binding using glutaraldehyde covalent binding using glutaraldehyde
SWV
10-18 mol,
6
0.001 fmol of complementary target, 0.01 ng of E. coli genomic DNA 2.5 × 10-18 mol +0.1 V for 10 s in MB
1 30 min amperometrically
HCV virus specific probes onto polystyrene surface coated onto carbon based disc electrode BdE-avidin-PANI/Pt bioelectrode 3
streptavidin biotin
DPV
2 60 min at 40 °C
dipped in 6.7 × mol/L [CdL2]2+ for 10 min at 40 °C with stirring [OsIII(bpy)2pyCl]2+ carbodiimide chemistry 2
21-mer oligonucleotide immobilized PANI nanoarray onto graphite electrode 21-mer oligonucleotide probe for HBV DNA onto glassy carbon electrode 1
biotin avidin coupling
5
10-5
electrode no.
carbodiimide chemistry
differential pulse voltammetry (DPV) DPV
15 min in 22.5 µM daunorubicin
up to 0.756 fM complementary target up to 7.19 × 10-9 mol/L and detection range of 1.01 × 10-8 to 1.62 × 10-6 mol/L up to 200 pM and 1 nM for longer (406 bases) targets, respectively
120 min
ref hybridization time detection limit electroactive hybridization indicator electrochemical technique used method of immobilization
TABLE 1: Characteristics of PNA-PANI/Au Electrode and DNA-PANI/Au Electrode
J. Phys. Chem. B, Vol. 112, No. 15, 2008 4815
1 min, 1-5 min
Polyaniline Based Nucleic Acid Sensor
was revealed that PNA electrodes have a much higher affinity for the complementary sequences than that of the DNA electrode. The PNA-PANI/Au electrodes can be used to detect the complementary sequence in genomic DNA without PCR amplification obviating the need for stringent washings. These polyaniline based nucleic acid sensors can be used for faster and reagentless detection of pathogens (M. tuberculosis). To obtain increased sensitivity, reusability, and better detection limit, efforts should be made to unravel the detailed role of MB and to fabricate new nucleic acid sensors using nanocomposites and functionalized conducting polymers for the detection of other pathogens including Salmonella typhimurium and Nesseria gonorrhea. Acknowledgments. We are grateful to Dr. Vikram Kumar, Director, NPL, New Delhi, India for his interest in this work. N.P. is grateful to the Council of Scientific Industrial Research, India for the award of a Senior Research Fellowship. Thanks are due to all members of BECPRG for interesting discussions and Dr. K. N. Sood for the SEM characterization. Financial support received under the DST sponsored projects DST/TSG/ ME/2002/19 and DST/TSG/CLP041332 and the MLP0004 project are gratefully acknowledged. Note Added after ASAP Publication. This paper was published ASAP on March 12, 2008. Due to production error, the authors’ requested text changes were not incorporated. The revised paper was reposted on March 21, 2008. References and Notes (1) Djellouli, N.; Rochelet-Dequaire, M.; Limoges, B.; Druet, M.; Brossier, P. Biosens. Bioelectron. 2007, 22, 2906-2913. (2) Zhang, S.; Tan, Q.; Feng, Li.; Zhang, X. Sens. Actuators, B 2007, 24, 290-296. (3) Erdem, A.; Papakonstantinou, P.; Murphy, H. Anal. Chem. 2006, 78, 6656-6659. (4) Arora, K.; Prabhakar, N.; Chand, S.; Malhotra, B. D. Biosens. Bioelectron. 2007, 23, 613-620. (5) Chang, H.; Yuan, Y.; Shi, N.; Guan, Y. Anal. Chem. 2007, 79, 5111-5115. (6) (a) Arora, K.; Prabhakar, N.; Chand, S.; Malhotra, B. D. Anal. Chem. 2007, 79, 6152-6158. (b) Arora, K.; Prabhakar, N.; Chand, S.; Malhotra, B. D. Anal. Chem. 2008, 80, 1833. (7) World Health Organization. Global Tuberculosis Report; WHO: Geneva, Switzerland, 2007. (8) Smith, I. Clin. Microbiol. ReV. 2003, 16, 463-496. (9) Anie, Y.; Sumi, S.; Varghese, P.; Madhavi, L. G. K.; Sathish, M.; Radhakrishnan, V. V. Diagn. Microbiol. Infect. Dis. 2007, 59, 389-394. (10) Vestal, A. L. Procedures for the isolation and identification of Mycobacteria; U.S. Department of Health, Education, and Welfare, Centers for Disease Control: Atlanta, GA, 1975. (11) Minnikin, D. E.; Hutchinson, I. G.; Caldicot, A. B.; Goodfellow, M. J. Chromatogr. 1980, 188, 221-233. (12) Cheng, X.; Zhang, J.; Yang, L.; Xu, X.; Liu, J.; Yu, W.; Su, M.; Hao, X. J. Microbiol. Met. 2007, 70, 301-305. (13) Cho, S.; Patrick, J. B. Tuberculosis 2007, 87, 14-17. (14) Collins, D. M.; Gabric, D. M.; Lisle, G. W. D. J. Clin. Microbiol. 1990, 28, 1591-1596. (15) Collins, D. M.; De Lisle, G. W. J. Clin. Microbiol. 1985, 21, 562564. (16) Chauhan, A.; Chauhan, D. S.; Parashar, D.; Gupta, P.; Sharma, V. D., Katoch, V. M. Indian J. Med. Microbiol. 2004, 22, 238-240. (17) van Soolingen, D.; de Haas, P. E. W.; Hermans, P. W. M.; Groenen, P. M. A.; van Embden, J. D. A. J. Clin. Microbiol. 1993, 31, 1987-1995. (18) Linton, C. J.; Jalal, H.; Leeming, J. P.; Millar, M. R. J. Clin. Microbiol. 1994, 32, 2169-2174. (19) Janssen, P.; Coopman, R.; Huys, G.; Swings, J.; Bleeker, M.; Vos, P.; Zabeau, M.; Kersters, K. Microbiology 1996, 142, 1881-1893. (20) Ripabelli, G.; McLauchlin, J.; Mithani, V.; Threlfall, E. J. Lett. Appl. Microbiol. 2000, 30, 358-363. (21) Pavlou, A. K.; Magan, N.; Jones, J. M.; Brown, J.; Klatser, P.; Turner, A. P. F. Biosens. Bioelectron. 2004, 20, 538-544. (22) Martins, R.; Baptista, P.; Raniero, L.; Doria, G.; Silva, L.; Franco, R.; Fortunato, E. Appl. Phys. Lett. 2007, 90, 23903-1-23903-3. (23) Wang, J. Biosens. Bioelectron. 1998, 13, 757-762.
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