Anal. Chem. 2010, 82, 1117–1122
Carbon Nanohorn Sensitized Electrochemical Immunosensor for Rapid Detection of Microcystin-LR Jing Zhang,† Jianping Lei,† Chuanlai Xu,‡ Lin Ding,† and Huangxian Ju*,† Key Laboratory of Analytical Chemistry for Life Science (Ministry of Education of China), Department of Chemistry, Nanjing University, Nanjing 210093, and School of Food Science and Technology, Jiangnan University, Wuxi 214122, P. R. China A sensitive electrochemical immunosensor was proposed by functionalizing single-walled carbon nanohorns (SWNHs) with analyte for microcystin-LR (MC-LR) detection. The functionalization of SWNHs was performed by covalently binding MC-LR to the abundant carboxylic groups on the cone-shaped tips of SWNHs in the presence of linkage reagents and characterized with Raman spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, and a transmission electron micrograph. Compared with single-walled carbon nanotubes, SWNHs as immobilization matrixes showed a better sensitizing effect. Using home-prepared horseradish peroxidase-labeled MC-LR antibody for the competitive immunoassay, under optimal conditions, the immunosensor exhibited a wide linear response to MC-LR ranging from 0.05 to 20 µg/L with a detection limit of 0.03 µg/L at a signal-to-noise of 3. This method showed good accuracy, acceptable precision, and reproducibility. The assay results of MC-LR in polluted water were in a good agreement with the reference values. The proposed strategy provided a biocompatible immobilization and sensitized recognition platform for analytes as small antigens and possessed promisingapplicationinfoodandenvironmentalmonitoring. Cyanobacterial bloom has significant hazard to public health and the environment due to the release of cyanotoxins into water supplies. One of the most common cyanotoxins is cyclic heptapeptide toxin of microcystins (MCs), which may cause mainly functional and structural disturbances of the liver due to a marked inhibition of protein phosphatases 1 and 2A.1-5 To date, more than 90 congeners of MCs have been identified from lowly toxic to highly toxic with molecular weights from 900 to 1120 Da. * Corresponding author. Tel/Fax: +86-25-83593593. E-mail:
[email protected]. † Nanjing University. ‡ Jiangnan University. (1) Chianella, I.; Lotierzo, M.; Piletsky, S. A.; Tothill, I. E.; Chen, B. N.; Karim, K.; Turner, A. P. F. Anal. Chem. 2002, 74, 1288–1293. (2) Yoshizawa, S.; Matsushima, R.; Watanabe, M. F.; Harada, K.; Ichihara, A.; Carmichael, W. W.; Fujiki, H. J. Cancer Res. Clin. Oncol. 1990, 116, 609– 614. (3) Honkanen, R. E.; Zwiller, J.; Moore, R. E.; Daily, S. L.; Khatra, B. S.; Dukelow, M.; Boynton, A. L. J. Biol. Chem. 1990, 265, 19401–19404. (4) Dietrich, D.; Hoeger, S. Toxicol. Appl. Pharmacol. 2005, 203, 273–289. (5) Codd, G. A.; Morrison, L. F.; Metcalf, J. S. Toxicol. Appl. Pharmacol. 2005, 203, 264–272. 10.1021/ac902914r 2010 American Chemical Society Published on Web 01/07/2010
Microcystin-LR (MC-LR), containing five nonproteinogens and two substitutions of leucine (L) and arginine (R) at positions 2 and 4, is the most toxic species.6-9 In 1998, the World Health Organization (WHO) set up a provisional guideline limit of 1 µg/L for MCLR in drinking water.10 Thus, the recognition and quantification of MC-LR are of great importance in the analysis of environmental samples. The analytical techniques for MC-LR detection usually involve thin-layer chromatography,11 high-performance liquid chromatography(HPLC),12,13 liquidchromatography/massspectrometry,14-16 and protein phosphatase inhibition assays.17,18 Although these methods are well-proven and widely accepted, they require relatively expensive equipment, advanced technical expertise, and high cost and are time-consuming. Thus, immunoassay techniques are of great interest in qualitative and quantitative detection of MC-LR due to their highly specific molecular recognition without the need for prior sample concentration or pretreatment.19-22 The best candidates for the on-site immunoassay of MC-LR are the electrochemical immunosensors due to the high sensitivity, simplicity, ease of miniaturization, and low cost of both the sensors and the instrumentation. The first electrochemical immunosensor (6) Welker, M.; Brunke, M.; Preussel, K.; Lippert, I.; Von Do¨hren, H. Microbiology 2004, 150, 1785–1796. (7) Liu, B. H.; Yu, F. Y.; Huang, X.; Chu, F. S. Toxicon 2000, 38, 619–632. (8) Howard, K. L.; Boyer, G. L. Anal. Chem. 2007, 79, 5980–5986. (9) Christiansen, G.; Yoshida, W. Y.; Blom, J. F.; Portmann, C.; Gadermann, K.; Hemscheidt, T.; Kurmayer, R. J. Nat. Prod. 2008, 71, 1881–1886. (10) WHO. Guidelines for Drinking-Water Quality, Addendum to Volume 2, Health Criteria and Other Supporting Information;World Health Organization: Geneva, Switzerland, 1998. (11) Meisen, I.; Distler, U.; Mu ¨thing, J.; Berkenkamp, S.; Dreisewerd, K.; Mathys, W.; Karch, H.; Mormann, M. Anal. Chem. 2009, 81, 3858–3866. (12) Spoof, L.; Karlsson, K.; Meriluoto, J. J. Chromatogr., A 2001, 909, 225– 236. (13) Shen, P. P.; Shi, Q.; Hua, Z. C.; Kong, F. X.; Wang, Z. G.; Zhuang, S. X.; Chen, D. C. Environ. Int. 2003, 29, 641–647. (14) Maizels, M.; Budde, W. L. Anal. Chem. 2004, 76, 1342–1351. (15) Allis, O.; Dauphard, J.; Hamilton, B.; Shuilleabhain, A. N.; Lehane, M.; James, K. J.; Furey, A. Anal. Chem. 2007, 79, 3436–3447. (16) Draper, W. M.; Xu, D. D.; Perera, S. K. Anal. Chem. 2009, 81, 4153–4160. (17) Campa`s, M.; Szydlowska, D.; Trojanowicz, M.; Marty, J. L. Biosens. Bioelectron. 2005, 20, 1520–1530. (18) Noble, J. E.; Ganju, P.; Cass, A. E. G. Anal. Chem. 2003, 75, 2042–2047. (19) Zeck, A.; Weller, M. G.; Niessner, R. Anal. Chem. 2001, 73, 5509–5517. (20) Metcalf, J. S.; Codd, G. A. Chem. Res. Toxicol. 2003, 16, 103–112. (21) Loyprasert, S.; Thavarungkul, P.; Asawatreratanakul, P.; Wongkittisuksa, B.; Limsakul, C.; Kanatharana, P. Biosens. Bioelectron. 2008, 24, 78–86. (22) Kyprianou, D.; Guerreiro, A. R.; Chianella, I.; Piletska, E. V.; Fowler, S. A.; Karim, K.; Whitcombe, M. J.; Turner, A. P. F.; Piletsky, S. A. Biosens. Bioelectron. 2009, 24, 1365–1371.
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for MC-LR analysis was designed using a screen-printed graphite electrode as support for antibody adsorption in 2007.23 An immunosensor for the electrochemical enzyme immunoassay of MC-LR was also proposed by immobilizing MC-LR antibody on a double-sided microporous gold electrode.24 These immunosensors used enzyme labeled MC-LR as signal element. The preparation and purification of enzyme labeled small antigen molecules are very complicated, and the physical adsorption may cause unacceptable precision, reproducibility, and stability. Alternately, searching a biocompatible nanomaterial for the effective immobilization and presentation of small antigen molecules to construct a highly sensitive immunosensor is of considerable interest for the detection of MC-LR. Single-walled carbon nanohorns (SWNHs), as dahlia flowerlike spherical aggregates (diameters of about 80-120 nm), are composed of thousands of graphitic tubule closed ends with coneshaped horns25,26 and have large surface area, excellent conductivity, plentiful inner nanospaces, and highly defective horns.27,28 The oxidation treatment of SWNHs can produce extensive oxygenfunctionalized sites exposed on the cone-shaped tips29 for immobilization of biomacromolecules30 and drug delivery in cancer phototherapy.31 This work used SWNHs as an immobilization scaffold of small antigen molecules to propose a novel immunosensor for MC-LR. The immobilization of MC-LR on SWNHs could be conveniently performed on an electrode surface by covalently binding MC-LR to the carboxylic groups on the cone-shaped tips of SWNHs. Due to the three-dimensional recognition, SWNHs showed an efficient sensitizing action, leading to a high sensitivity and a wide linear range for rapid immunoassay of MC-LR. The designed immunosensor for MC-LR had acceptable precision and reproducibility. The good biocompatibility of SWNHs and simple preparation resulted in excellent stability of the immunosensor. It could be successfully applied in the detection of MC-LR in Tai Lake water without any pretreatment. These results demonstrated SWNHs are an excellent matrix for immobilization of small toxin residues and then sensitive recognition to labeled antibody for immunoassay of toxins. This strategy provided a useful tool for monitoring the hazard components in biological, food, and environmental fields. EXPERIMENTAL SECTION Materials and Reagents. SWNHs were kindly provided by Professor Sumio Iijima, who leads the carbon nanotube project (23) Campa`s, M.; Marty, J. Biosens. Bioelectron. 2007, 22, 1034–1040. (24) Zhang, F.; Yang, S. H.; Kang, T. Y.; Cha, G. S.; Nam, H.; Meyerhoff, M. E. Biosens. Bioelectron. 2007, 22, 1419–1425. (25) Yuge, R.; Ichihashi, T.; Shimakawa, Y.; Kubo, Y.; Yudasaka, M.; Iijima, S. Adv. Mater. 2004, 16, 1420–1423. (26) Zhang, M. F.; Yamaguchi, T.; Iijima, S.; Yudasaka, M. J. Phys. Chem. C 2009, 113, 11184–11186. (27) Yang, C.; Noguchi, H.; Murada, K.; Yudasaka, M.; Hashimoto, A.; Iijima, S.; Kaneko, K. Adv. Mater. 2005, 17, 866–870. (28) Urita, K. M.; Seki, S.; Utsumi, S.; Noguchi, D.; Kanoh, H.; Tanaka, H.; Hattori, Y.; Ochiai, Y.; Aoki, N.; Yudasaka, M.; Iijima, S.; Kaneko, K. Nano Lett. 2006, 6, 1325–1328. (29) Pagona, G.; Tagmatarchis, N.; Fan, J.; Yudasaka, M.; Iijima, S. Chem. Mater. 2006, 18, 3918–3920. (30) Tu, W. W.; Lei, J. P.; Ding, L.; Ju, H. X. Chem. Commun. 2009, 28, 4227– 4229. (31) Zhang, M.; Murakami, T.; Ajima, K.; Tsuchida, K.; Sandanayaka, A. S. D.; Ito, O.; Iijima, S.; Yudasaka, M. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 14773–14778.
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in the Japan Science and Technology Agency. Single-walled carbon nanotubes (SWNTs) (
