Anal. Chem. 2006, 78, 835-843
Biosensor Based on Self-Assembling Acetylcholinesterase on Carbon Nanotubes for Flow Injection/Amperometric Detection of Organophosphate Pesticides and Nerve Agents Guodong Liu and Yuehe Lin*
Pacific Northwest National Laboratory, Richland, Washington 99352
A highly sensitive flow injection amperometric biosensor for organophosphate pesticides and nerve agents based on self-assembled acetylcholinesterase (AChE) on a carbon nanotube (CNT)-modified glassy carbon (GC) electrode is described. AChE is immobilized on the negatively charged CNT surface by alternatively assembling a cationic poly(diallyldimethylammonium chloride) (PDDA) layer and an AChE layer. Transmission electron microscopy images confirm the formation of layer-by-layer nanostructures on carboxyl-functionalized CNTs. Fourier transform infrared reflectance spectrum indicates the AChE was immobilized successfully on the CNT/PDDA surface. The unique sandwich-like structure (PDDA/AChE/PDDA) on the CNT surface formed by self-assembling provides a favorable microenvironment to keep the bioactivity of AChE. The electrocatalytic activity of CNT leads to a greatly improved electrochemical detection of the enzymatically generated thiocholine product, including a low oxidation overvoltage (+150 mV), higher sensitivity, and stability. The developed PDDA/AChE/PDDA/CNT/GC biosensor integrated into a flow injection system was used to monitor organophosphate pesticides and nerve agents, such as paraoxon. The sensor performance, including inhibition time and regeneration conditions, was optimized with respect to operating conditions. Under the optimal conditions, the biosensor was used to measure as low as 0.4 pM paraoxon with a 6-min inhibition time. The biosensor had excellent operational lifetime stability with no decrease in the activity of enzymes for more than 20 repeated measurements over a 1-week period. The developed biosensor system is an ideal tool for online monitoring of organophosphate pesticides and nerve agents. Because of the high toxicity of organophosphate pesticides (OPs) and nerve agents, the rapid detection of these toxic agents in the environment, public places, or workplaces and the biomonitoring of individual exposures to chemical warfare agents have become increasingly important for homeland security and health * Corresponding author. Tel: 01-509-376-0529. Fax: 01-509-376-5106. E-mail:
[email protected]. 10.1021/ac051559q CCC: $33.50 Published on Web 01/06/2006
© 2006 American Chemical Society
protection.1,2 Early detection of OPs may also give an indication of terrorist activity, and this would allow proper procedures to be followed to mitigate dangers. Analyzing OPs in environmental and biological samples is routinely carried out using analytical techniques, such as gas or liquid chromatography and mass spectrometry.3 Such analysis is generally performed at centralized laboratories, requiring extensive labor and analytical resources, and often results in a lengthy turnaround time. However, these analysis methods have a number of disadvantages, limiting their applications primarily to laboratory settings and prohibiting their use for rapid analyses under field conditions. Searching for new, simple, and sensitive analytical methods with real-time output and a low cost is of considerable interest. Enzyme-based biosensors have emerged the past few years as the most promising alternative for direct monitoring of pesticides.4,5 Various inhibition and noninhibition biosensor systems, based on the immobilization of acetylcholinesterase (AChE) or OP hydrolase (OPH) onto various electrochemical or optical transducers, have been proposed for field screening of OP neurotoxins.6-10 Although OPH-based noninhibition biosensors provide a direct biosensing route (OPH catalyzes the hydrolysis of OP compounds, resulting in electroactive species, such as p-nitrophenol), OPH is not commercially available, which limits widespread applications. A preferred indirect electrochemical biosensing route based on the inhibition of target enzymes has been widely developed by different groups.11-25 Because of their (1) Compton, J. A. Military Chemical and Biological Agents; Telford Press: Caldwell, NJ, 1988; p 135. (2) United States Department of Agriculture. Agricultural Statistics; United States Government Printing Office: Washington, DC, 1992; p 395. (3) Sherma, J. Anal. Chem. 1993, 65, 40R-54R (4) Evtugyn, G. A.; Budnikov, H. C.; Nikolskaya, E. B. Talanta 1998, 46, 465484. (5) Sadik, O.; Land, W.; Wang, J. Electroanalysis 2003, 15, 1149-1159. (6) Mulchandani, A.; Kaneva, I.; Chen, W. Anal. Chem. 1998, 70, 5042-5046. (7) Wang, J.; Mulchandani, A.; Chen, L.; Mulchandani, P.; Chen, W. Anal. Chem. 1999, 71, 2246-2249. (8) Mulchandani, A.; Mulchandani, P.; Kaneva, I.; Chen, W. Anal. Chem. 1998, 70, 4140-4145. (9) Deo, R. P.; Wang, J.; Block, I.; Mulchandani, A., Joshi, K. A.; Trojanowicz, M.; Scholz, F.; Chen, W.; Lin, Y. Anal. Chim. Acta 2005, 530, 185-189. (10) Wang, J.; Mulchandani, A.; Chen, L.; Mulchandani, P.; Chen, W. Electroanalysis 1999, 11, 866-869. (11) Sole, S.; Merkoci, A.; Alegret, S. Crit. Rev. Anal. Chem. 2003, 33, 89-96. (12) Sotiropoulou, S.; Fournier, D.; Chaniotakis, N. A. Biosens. Bioelectron, 2005, 20, 2347-2352. (13) Lin, Y.; Lu, F.; Wang, J. Electroanalysis 2004, 16, 145-149.
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high sensitivity, amperometric transducers have been the transduction principle of choice in many of these biosensors.11 These biosensors have used either AChE alone or combined with choline oxidase (ChO). The AChE inhibition in the single and bienzyme system is monitored by measuring the oxidation current of the product of the enzyme reaction. In the following bienzyme system (eqs 1 and 2), AChE
acetylcholine + H2O 98 choline + acetate ChO
choline + O2 98 betaine aldehyde + H2O2
(1) (2)
AChE catalyzes the hydrolysis of the neurotransmitter acetylcholine into acetate and choline. The choline is subsequently converted by ChO, producing hydrogen peroxide in the presence of oxygen. Hydrogen peroxide can be detected amperometrically with different electrochemical transducers. In a single enzyme system (eq 3), thiocholine (TCh) ester, acetylthiocholine (ATCh), AChE
acetylthiocholine + H2O 98 thiocholine + acetate acid (3) is preferred as substrate. Acetylthiocholine can be enzymatically hydrolyzed by AChE to TCh, which in turn is oxidized at constant potential at the electrochemical transducer, producing the initial biosensing response. Despite many efforts to develop these inhibition biosensors, certain analytical characteristics of these systems require further improvement to meet the required specifications, such as higher sensitivity, selectivity, and stability. One approach to improve the performance characteristics of the AChE-based inhibitor biosensors would be to design and produce appropriate enzymes with characteristics more suitable for biosensor applications. Initial biochemical studies revealed that Drosophila melanogaster acetylcholinesterase (Dm. AChE) is the most sensitive enzyme toward OPs.12 The Dm. AChE-based inhibitor biosensors show great promise to improve the sensitivity of the biosensor system. Sotiropoulou et al. reported a Dm. AChEbased inhibitor biosensor for the detection of dichlorvos with a detection limit of 10-17 M, which is 5 orders of magnitude lower than the Electropharus electricus AChE-based biosensor.12 However, this approach has been hampered by the fact that AChE (14) Joshi, K.; Tang, J.; Haddon, R.; Wang, J.; Chen, W.; Mulchandani, A. Electroanalysis 2005, 17, 54-58. (15) Sotiropoulou, S.; Chaniotakis, N. A. Anal. Chim. Acta 2005, 530, 199-204. (16) Jeanty, G.; Ghommidh, Ch.; Marty, J. L. Anal. Chim. Acta 2001, 436, 119128. (17) Jeanty, G.; Marty, J. L. Biosens. Bioelectron. 1998, 13, 213-218. (18) Marty, J. L.; Mionetto, Noguer, T.; Ortega, F.; Roux, C. Biosens. Bioelectron. 1993, 8, 273-280. (19) Marty, J. L.; Mionetto, N.; Lacote, S.; Barcelo, D. Anal. Chim. Acta 1995, 311, 265-271. (20) Skladal, P. Anal. Chim. Acta 1991, 252, 11-15. (21) Hart, J. P.; Hartley, I. C. Analyst 1994, 119, 259-263. (22) Halbert, M. K.; Baldwin, R. P. Anal. Chem. 1985, 57, 591-595. (23) Ricci, F.; Aeduini, F.; Amine, A.; Moscone, D.; Palleschi, G. J. Electroanal. Chem. 2004, 563, 229-237. (24) Martorell, D.; Cespedes, F.; Fabreagas, E. M.; Alegret, S. Anal. Chim. Acta 1997, 337, 305-313. (25) Kulys, J.; D’costa, E.; F.; Fabreagas, E. M.; Alegret, S. Biosen. Bioelectron. 1991, 6, 109-115.
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from various sources was not easily available because of difficulties in isolation and purification procedures. Another possible approach for improving the performance characteristics of the AChE-based inhibitor biosensors is to improve the biosensor design and the electrochemical detection of the enzymatic product. Most of the approaches for AChE immobilization on electrode surfaces in the reported literature are covalent binding,13 direct adsorption,14,15 or entrapment in different substrate materials.16,17 A drawback to physical adsorption and entrapment is that the distribution of enzyme molecules is not uniform, is sometimes unstable, and tends to leach with time. The chemical interaction (covalent binding) tends to partially denature the activity of the AChE. These approaches more or less limit the development of AChE biosensors. As the oxidation of enzymatic product thiocholine occurs at a relatively high potential on conventional electrodes,18,19 mediators such as cobalt(II)phthalocyanine,20-22 Prussian blue,23 and tetracyanoquinodimethane24,25 have been used to reduce the overvoltage of oxidation and enhance the sensitivity of the detection. The ideal biosensor fabrication method should employ mild chemical conditions, allow for large quantities of enzyme to be immobilized, provide a favorable microenvironment to maintain the enzyme activity, and provide a large surface area for enzyme-substrate contact within a small total volume. Barriers to mass transport of substrate and product should be minimized, and a chemically and mechanically robust system should be provided. Here we describe a biosensor fabrication method based on self-assembling AChE on the carbon nanotube (CNT) surface, and this is integrated with a flow injection detection system that satisfies all of above criteria. CNTs represent a new class of nanomaterials, composed of graphite carbon with one or several concentric tubules.26 The unique electronic, chemical, and mechanical properties of CNTs make them extremely attractive for electrochemical sensors.27-29 Recent studies demonstrated the capability of CNTs to improve the electrochemical behavior of cytochrome c, NADH, or ascorbic acid and promote the electrontransfer reactions of enzymatically generated species, such as hydrogen peroxide and p-nitrophenol.30-34 The new fabrication method involved layer-by-layer (LBL) self-assembling AChE on a CNT transducer (Figure 1). Here, CNTs play a dual significant role in both the enzyme immobilization and transduction events, namely as carriers for AChE immobilization to provide a suitable microenvironment to retain the AChE activity and as a transducer for amplifying the electrochemical signal of the product of the enzymatic reaction. These novel support and signal amplification functions of CNTs reflect their excellent electrocatalytic characteristics, large specific surface area, and fast electron transfer, and (26) Iijima, S. Nature 1991, 354, 56-58. (27) Lin, Y.; Yantasee, W.; Lu, F.; Wang, J.; Musameh, M.; Tu, Y.; Ren, Z. In Dekker Encyclopedia of Nanoscience and Nanotechnology; Schwarz, J. A., Contescu, C. I., Putyera, K., Eds.; Marcel Dekker Inc.: New York, 2004; p 361. (28) Wang, J. Electroanalysis 2005, 17 (1), 7-14. (29) Zhao, Q.; Gan, Z.; Zhuang, Q. Electroanalysis 2002, 14 (23), 1609-1613. (30) Wang, J.; Musameh, M.; Lin, Y. J. Am. Chem. Soc. 2003, 125, 2408-2409. (31) Wang, J.; Li, M.; Shi, Z.; Li, N.; Guo, Z. Anal. Chem. 2002, 74, 1993-1997. (32) Musameh, M.; Wang, J.; Merkoci, A.; Lin, Y. Electrochem. Commun. 2002, 4, 743-746. (33) Wang, J.; Li, M.; Shi, Z.; Li, N.; Guo, Z. Electroanalysis 2002, 14, 225-230. (34) Wang, Z.; Liu, J.; Liang, Q.; Wang, Y.; Luo, G. Analyst 2002, 127, 653658.
Figure 1. Schematics of layer-by-layer electrostatic self-assembly of AChE on carbon nanotube: (A) assembling positively charged PDDA on negatively charged CNT; (B) assembling negatively charged AChE; (C) assembling the second PDDA layer.
they pave the way to the new AChE-based inhibitor biosensor for OPs. The optimization and advantages of the CNT-based AChE biosensor are reported in the following sections. EXPERIMENTAL SECTION Reagents. AChE, 317 units/mg, ATCh, poly(diallyldimethylammonium chloride) polymer (PDDA), paraoxon, and pyridine 2-aldoxime methiodide (PAM) were purchased from SigmaAldrich (St. Louis, MO). A 10 000 mg/L stock solution of paraoxon was prepared in acetonitrile. The substrate solution of ATCh was prepared in 0.9% NaCl at a final concentration of 0.1 M and kept at -20 °C in aliquots. The TCh solution was prepared by the enzymatic reaction of the AChE and ATCh solution for 30 min in 0.05 M phosphate buffer (pH 7.4). The final concentration of TCh was determined by spectrophotometry after reaction with dithobis(nitrobenzoic acid), as described by Ellman.35,36 Standard solutions of TCh and ATCh for the voltammetric and amperometric studies were prepared by dilution of their stock solution with 0.05 M phosphate buffer. Multiwall CNTs, with ∼95% purity, were obtained from NanoLab (Brighton, MA). Phosphate buffer (0.05 M, pH 7.4) was used as the supporting electrolyte. Other reagents were commercially available and were of analytical reagent grade. Solutions were prepared with ultrapure water from a Millipore Milli-Q water purification system (Billerica, MA). (35) Ellman, G. Arch. Biochem. Biophys. 1958, 74, 443-450. (36) Ellman, G. Arch. Biochem. Biophys. 1959, 82, 70-77.
Instruments. Cyclic voltammetric and amperometric measurements were performed using an electrochemical analyzer CHI 660 (CH Instruments, Austin, TX) connected to a personal computer. A three-electrode configuration was employed, consisting of a PDDA/AChE/PDDA/CNT/glassy carbon (GC) electrode (3-mm diameter) serving as a working electrode, whereas Ag/ AgCl (3 M KCl) and platinum wire served as the reference and counter electrodes, respectively. Batch electrochemical experiments were carried out in a 2-mL voltammetric cell at room temperature (25 °C). All potentials are referred to the Ag/AgCl reference electrode (CH Instruments). The transmission electron microscopy (TEM) images were recorded on a JEOL JEM 2010 microscope with a specified point-to-point resolution of 0.194 nm. The operating voltage on the microscope was 200 keV. The sample was prepared by peeling PDDA/AChE/PDDA/CNT hybrid from the surface of the PDDA/AChE/PDDA/CNT/GC electrode and dispersed on the Cu-C grid. All images were digitally recorded with a slow scan charge-coupled device camera (image size 1024 × 1024 pixels), and image processing was carried out using a Digital Micrograph (Gatan). Fourier transform infrared spectroscopic analysis was performed with a Bruker IFS66v/S Fourier transform infrared spectrometer utilizing a Michelson interferometer. The spectrometer was evacuated to 6 mbar during data collection to remove strong absorbances due to atmospheric carbon dioxide and water vapor. Spectra were initially collected using a Bruker A510 reflectance accessory, which enabled collection of spectra with higher signal-to-noise ratio. LBL Self-Assembling AChE on CNT-Modified GC Electrode. Preparation of CNT-Modified GC Electrode. The multiwall CNT was first shortened and functionalized by sonicating in a mixture of concentrated HNO3 and H2SO4 (v/v, 1:3) for 6 h followed by extensive washing in deionized water until the filtrate was neutral. Then the pH was adjusted to ∼8.0 to achieve net negatively charged carboxylate anions. The negatively charged CNT was centrifuged at 14 000g for 30 min to remove the supernatant and dried in a vacuum. The resulting CNT was dispersed in N,N-dimethylformamide again with a concentration of 5 mg mL-1. We dropped 20 µL of the CNT solution to the cleaned GC electrode surface (3-mm diameter; CH instruments) and allowed the coating to dry at room temperature. The surface was then washed carefully with double-distilled water. Self-Assembling AChE on CNT. AChE was immobilized on the negatively charged CNT surface by alternatively assembling a PDDA layer and an AChE layer (Figure 1). First, the prepared CNT/GC electrode was dipped in 1 M NaOH solution for 5 min to introduce more negative charges on CNT surface and then washed with distilled water twice. The positively charged polycation was adsorbed by dipping the negatively charged CNT/GC electrode in an aqueous solution of 1 mg mL-1 PDDA containing 0.5 M NaCl for 20 min (Figure 1A). Then, the PDDA/CNT/GC electrode was rinsed with distilled water and dried in nitrogen. Using the same procedure, a layer of negatively charged AChE was adsorbed at a Tris-HCl buffer solution (pH 8.0) containing 0.2 unit mL-1 AChE (Figure 1B). The isoelectric point, Ip, of AChE was 4.5, and therefore at pHs of >Ip, its net was negatively charged, whereas at pHs of
