Continuous Colorimetric Assay for Acetylcholinesterase and Inhibitor

Jan 20, 2009 - We report herein a new colorimetric assay method for acetylcholinesterase (AChE) activity and its inhibitor screening by making use of ...
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Langmuir 2009, 25, 2504-2507

Continuous Colorimetric Assay for Acetylcholinesterase and Inhibitor Screening with Gold Nanoparticles Ming Wang, Xinggui Gu, Guanxin Zhang, Deqing Zhang,* and Daoben Zhu Beijing National Laboratory for Molecular Sciences, Organic Solids Laboratory, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China ReceiVed NoVember 22, 2008. ReVised Manuscript ReceiVed December 17, 2008 We report herein a new colorimetric assay method for acetylcholinesterase (AChE) activity and its inhibitor screening by making use of the following facts: (1) the aggregation of gold nanoparticles (Au-NPs) results in the red-shift of the plasmon absorption due to interparticle plasmon interactions and (2) AChE can catalyze the hydrolysis of acetylthiocholine into thiocholine which can induce the aggregation of Au-NPs. With this convenient method, the activity of AChE with a concentration as low as 0.6 mU/mL can be assayed. Moreover, this assay method is also useful for screening inhibitors of AChE. Given its simplicity and easy-operation, this method may extend to high-throughput screening of AChE inhibitors and relevant drug discovery.

Introduction Acetylcholine (ACh) is a neurotransmitter and plays a decisive role in memory and learning.1 The low level of acetylcholine shows high relation with Alzheimer’s disease (AD), since the hydrolysis of acetylcholine catalyzed by acetylcholinesterase (AChE) can accelerate the aggregation of amyloid-β peptide which plays a causative role in the development of AD.2 The relation between the observed cholinergic dysfunction and AD severity provides a rationale for AD therapies by using the acetylcholinesterase inhibitor, and preliminary screening of AChE inhibitors requires reliable methods for AChE activity assay. Therefore, development of a reliable assay for AChE and its inhibitor screening is of obvious importance for AD therapies.3 Furthermore, nerve gases and organophosphorus and carbamate pesticides are inhibitors of AChE; thus, efficient assay methods for AChE activity4 will be useful for detecting these nerve gases and pesticides. Traditional methods for AChE assay include the colorimetric Ellmann method5 or the detection of hydrogen peroxide produced by oxidation of choline.6 Recently, sensitive chemiluminescent7 and fluorescent probes8 have been described for cholinesterase activity assay and inhibitor screening. However, * Corresponding author. E-mail: [email protected]. (1) Whitehouse, P. J.; Price, D. L.; Struble, R. G.; Clark, A. W.; Coyle, J. T.; Delon, M. R. Science. 1982, 215, 1237–1239. (2) (a) Koo, E. H.; Lansbury, P. T.; Kelly, J. W. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 9989–9990. (b) Selkoe, D. J. Physiol. ReV. 2001, 81, 741–766. (c) Inestrosa, N. C.; Alvarez, A.; Perez, C. A.; Moreno, R. D.; Vicente, M.; Linker, C. Neuron 1996, 16, 881–891. (3) Liston, D. R.; Nielsen, J. A.; Villalobos, A.; Chapin, D.; Jones, S. B.; Hubbard, S. T.; Shalaby, I. A.; Ramirez, A.; Nason, D.; White, W. F. Eur. J. Pharmacol. 2004, 486, 9–17. (4) (a) Pauluhn, J.; Machemer, L.; Kimmerle, G. Toxicology 1987, 46, 177– 190. (b) Clement, J. G. Fundam. Appl. Toxicol. 1983, 3, 533–535. (c) Zejli, H.; Hidalgo-Hidalgo de Cisneros, J. L.; Naranjo-Rodriguez, I.; Liu, B.; Temsamani, K. R.; Marty, J.-L. Talanta 2008, 77, 217–221. (d) Chen, H. D.; Zuo, X. L.; Su, S.; Tang, Z. Z.; Wu, A. B.; Song, S. P.; Zhang, D. B.; Fan, C. H. Analyst 2008, 133, 1182–1186. (e) Pohanka, M.; Jun, D.; Kalasz, H.; Kuca, K. Protein Pept. Lett. 2008, 15, 795–798. (f) Liu, S. Q.; Yuan, L.; Yue, X. L.; Zheng, Z. Z.; Tang, Z. Y. AdV. Powder Technol. 2008, 19, 419–441. (5) (a) Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7, 88–90. (b) Rhee, I. K.; van Rijn, R. M.; Verpoorte, R. Phytochem. Anal. 2003, 14, 127–131. (6) Riklin, A.; Willner, I. Anal. Chem. 1995, 67, 4118–4126. (7) Sabelle, S.; Renard, P.-Y.; Pecorella, K.; Suzzoni-De´zard, S.; Cre´minon, C.; Grassi, J.; Mioskowski, C. J. Am. Chem. Soc. 2002, 124, 4874–4880. (8) (a) Maeda, H.; Matsuno, H.; Ushida, M.; Katayama, K.; Saekis, K.; Itoh, N. Angew. Chem., Int. Ed. 2005, 44, 2922–2925. (b) Feng, F. D.; Tang, Y. L.; Wang, S.; Li, Y. L.; Zhu, D. B. Angew. Chem., Int. Ed. 2007, 46, 7882–7886.

some of these assay methods are discontinuous and timeconsuming for AChE activity and inhibition studies. It is reported that the use of Ellman’s reagent may result in a false-positive effect.5 Herein, we report a new continuous colorimetric assay method for AChE activity and inhibitor screening by taking the advantage of the fact that the cross-linking/aggregation of gold nanoparticles (Au-NPs) results in the red-shift of the plasmon absorption due to interparticle plasmon interactions.9 In fact, by making use of this feature of Au-NPs, efficient assay protocols have been developed for phosphatase,10a lactamase,10b protease,10c-e and nuclease.10f Similarly, new analytical methods for detection of nucleic acids,11a,b proteins,11c,d metal ions11e-i (e.g., K+, Hg2+, and Cu2+), and explosive TNT11j have been also described with Au-NPs. Willner et al.12 have demonstrated an acetylcholinesterase inhibitor screening method based on the AChE-stimulated growth of Au-NPs. The design rationale for this new continuous colorimetric assay method for AChE activity and inhibitor screening is illustrated in Scheme 1 and explained as follows: (1) Acetylthiocholine (9) (a) Daniel, M. C.; Astru, D. Chem. ReV. 2004, 104, 293–346. (b) Rossi, N. L.; Mirkin, C. A. Chem. ReV. 2005, 105, 1547–1562. (c) Obare, S. O.; Hollowell, R. E.; Murphy, C. J. Langmuir 2002, 18, 10407–10410. (d) Olofsson, L.; Rindzevicius, T.; Pfeiffer, I.; Ka¨ll, M.; Ho¨o¨k, F. Langmuir 2003, 19, 10414– 10419. (e) Wang, W. X.; Liu, H. J.; Liu, D. S.; Xu, Y.; Yang, Y.; Zhou, D. J. Langmuir 2007, 23, 11956–11959. (f) Gate, A. T.; Fakayode, S. O.; Lowry, M.; Ganea, G. M.; Murugeshu, A.; Robinson, J. W.; Strongin, R. M.; Warner, I. M. Langmuir 2008, 24, 4107–4113. (g) Buecker, P.; Trileva, E.; Himmelhaus, M.; Dahint, R. Langmuir 2008, 24, 8229–8239. (10) (a) Choi, Y.; Ho, N-H.; Tung, C.-H. Angew. Chem., Int. Ed. 2007, 46, 707–709. (b) Liu, R. R.; Liew, R. S.; Zhou, J.; Xing, B. G. Angew. Chem., Int. Ed. 2007, 46, 8799–8803. (c) Guarise, C.; Pasquato, L.; De Fillippis, V.; Scrimm, P. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3978–3982. (d) Wang, Z.; Levy, R.; Fernig, D. G.; Burst, M. J. Am. Chem. Soc. 2006, 128, 2214–2215. (e) Laromaine, A.; Koh, L.; Murugesan, M.; Ulijin, R. V.; Stevens, M. M. J. Am. Chem. Soc. 2007, 129, 4156–4157. (f) Xu, X. Y.; Han, M. S.; Mirkin, C. A. Angew. Chem., Int. Ed. 2007, 46, 3468–3470. (11) (a) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science. 1997, 277, 1078–1081. (b) Li, H. X.; Rothberg, L. J. J. Am. Chem. Soc. 2004, 126, 10958–10961. (c) Li, B.; Wei, H.; Dong, S. Chem. Commun. 2007, 73–75. (d) Chen, Y.-M.; Yu, C.-J.; Cheng, T.-L.; Tseng, W.-L. Langmuir 2008, 24, 3654–3660. (e) Wang, L.; Liu, X.; Hu, X.; Song, S.; Fan, C. Chem. Commun. 2006, 3780–3782. (f) Liu, C.-W.; Huang, C.-C.; Chang, H.-T. Langmuir 2008, 24, 8346–8350. (g) Li, D.; Wieckowska, A.; Willner, I. Angew. Chem., Int. Ed. 2008, 47, 3927–3931. (h) Yu, C.-J.; Tseng, W.-L. Langmuir 2008, 24, 12717– 12722. (i) Zhou, Y.; Wang, S. X.; Zhang, K.; Jiang, X. Y. Angew. Chem., Int. Ed. 2008, 47, 7454–7456. (j) Jiang, Y.; Zhao, H.; Zhu, N. N.; Lin, Y. Q.; Yu, P.; Mao, L. Q. Angew. Chem., Int. Ed. 2008, 47, 8601–8604. (12) Pavlov, V.; Xiao, Y.; Willner, I. Nano. Lett. 2005, 5, 649–653.

10.1021/la803870v CCC: $40.75  2009 American Chemical Society Published on Web 01/20/2009

Continuous Colorimetric Assay for AChE Scheme 1. Illustration of Colorimetric Assay for Acetylcholinesterase (AChE) Assay by Using Cross-Linking/ Aggregation of Gold Nanoparticles (Au-NPs) Based on AChE-Catalyzed Hydrolysis of Acetylthiocholine

Langmuir, Vol. 25, No. 4, 2009 2505 nM), followed by addition of AChE (30 mU). The mixtures were incubated at 25 °C for 20 min. For comparison, sample (a) did not contain AChE. Aggregation of Au-NPs in the Presence of Different Concentrations of Tacrine. To the solution composed of 0.9 mL of AuNPs (2.4 nM), 0.1 mL of PBS (10 mM, pH ) 8.0) and acetylthiocholine (10 µM) were added with different concentrations of tacrine (0, 4, 10, 20, and 40 nM), followed by addition of AChE (1.5 mU). The mixtures were incubated at 25 °C. The absorption spectra were recorded every 1 min during the hydrolysis process. TEM Analysis for Au-NP Aggregation in the Presence of Acetylthiocholine Induced by AChE. To two tubes (1.5 mL) was added 0.9 mL of Au-NPs (2.4 nM), 0.1 mL of PBS (10 mM, pH ) 8.0), and acetylthiocholine (10 µM); AChE (0.6 mU) was added to one of the tubes. Both reaction mixtures were incubated at 25 °C for 20 min TEM samples were prepared by the slow evaporation of one drop of the solutions on a carbon-coated copper mesh grid.

Results and Discussion (see Scheme 1) is an analogue of acetylcholine, and it can be easily hydrolyzed to generate thiocholine in the presence of AChE; thus, acetylthiocholine is a good substrate of AChE and it can be employed for AChE activity studies and inhibitor screening as reported earlier.5,7,12 (2) Au-NPs are initially protected by citrate and possess negative charges, and thiocholine is able to substitute the citrate on the surfaces of Au-NPs; as a result, cross-linking/aggregation of interparticles would occur due to the electrostatic interactions and gold-thiols interaction, leading to the red-shift of the plasmon absorption. Obviously, the degree of cross-linking of Au-NPs is dependent on the concentration of thiocholine in the system, and accordingly, it is possible to construct a colorimetric assay for AChE activity and inhibitor screening with Au-NPs and thiocholine.

Experimental Section Citrate-coated gold nanoparticles (12 nM) were synthesized according to the reported method.13 The Au-NP solution was diluted to half of the concentration of the initial one for taking photos, and further to one-fifth of the concentration of the initial one for absorption spectral measurements. AuCl3 · 3H2O and sodium citrate were purchased from Beijing Chemical Corporation (Beijing, China); acetylthiocholine iodide was provided by Alfa-Aeasr, and acetylcholinesterase from Electrophorus electricus was purchased from Sigma-Aldrich Corporation. Ultrapure water was obtained with a Millipore filtration system before use. UV-vis spectra were measured on a U-3010 (Hitach) instrument, and transmission electron microscopy (TEM) images were recorded on a JEM-1011 (JEOL) microscope operating at 100 kV. Photo Taking for Au-NP Solutions Containing Acetylthiocholine with and without AChE Addition. To four tubes (1.5 mL) was added 0.9 mL of Au-NPs (6.0 nM), 0.1 mL of phosphate buffered saline (PBS, 10 mM, pH ) 8.0), and acetylthiocholine (10 µM). AChE with different concentrations (a, 0 mU/mL; b, 25 mU/mL; c, 50 mU/mL, d, 100 mU/mL) was then added, and the mixtures were incubated at 25 °C for 5 min. Aggregation of Au-NPs Containing Acetylthiocholine in the Presence of Different Concentrations of AChE. To the solution composed of 0.9 mL of Au-NPs (2.4 nM), 0.1 mL of PBS (10 mM, pH ) 8.0) and acetylthiocholine (10 µM) were added with different concentrations of AChE (0, 0.6, 1, 1.5, and 2 mU/mL). The mixtures were then incubated at 25 °C for a certain amount of time (min). The absorption spectra were recorded every 1 min during the hydrolysis of acetylthiocholine (0-15 min). Photo Taking for the Inhibition Assay for AChE Activity with Au-NPs. To five tubes (1.5 mL) was added 0.9 mL of Au-NPs (6.0 nM), 0.1 mL of PBS (10 mM, pH ) 8.0), acetylthiocholine (10 µM), and tacrine with different concentrations (0, 50, 100, 200, and 400 (13) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735–743.

Au-NPs were prepared by citrate reduction of HAuCl4 according to the reported procedure.13 The Au-NP (2.4 nM) solution shows typical plasmon absorption around 520 nm, and addition of acetylthiocholine (10 µM) to the solution of Au-NPs did not induce absorption spectral variation. However, obvious absorption variation was detected after further addition of AChE (0.6 mU/mL) to the mixture solution as shown in Figure 1A. The absorption band around 520 nm decreased gradually, and concomitantly, a new broad absorption above 600 nm emerged and its intensity increased by prolonging the reaction time. The maximum absorption spectral variation was achieved when the mixture solution was incubated for 20 min under this reaction condition. More interestingly, such absorption spectral variation can be visualized by the naked-eye. The initial Au-NP solution (6.0 nM) and that containing acetylthiocholine (10.0 µM) are red in color. After introducing AChE (100 mU/mL), the solution was incubated at 25 °C for 5.0 min, and then the solution changed to gray as displayed in Figure 1B. If less AChE was added, the color change was not so contrasting, but it was still distinguishable [see (b) and (c) containing 25 and 50 mU/mL, respectively, in Figure 1B]. This is understandable, since the hydrolysis of acetylthiocholine into thiocholine would become faster if more AChE was present in the solution, as to be discussed below. It should be noted that only addition of AChE caused no aggregation of the Au-NPs (data not shown here). The absorption spectral variation observed for the ensemble solution of Au-NPs and acetylthiocholine after addition of AChE is due to the aggregation of Au-NPs as illustrated in Scheme 1. This is directly confirmed by TEM analysis. In the absence of AChE, Au-NPs were well dispersed; however, after addition of AChE, Au-NPs aggregated as shown in Figure 2. Since the hydrolysis of acetylthiocholine into thiocholine is facilitated by addition of AChE, it is expected that more thiocholine will be formed when the mixture solution is incubated for a long period of time. Accordingly, the aggregation degree of Au-NPs would increase, and thus, large absorption spectral variation would occur. This is indeed in agreement with the experimental observation as discussed above. Also, when more AChE was added to the solution, the hydrolysis reaction would become faster and more thiocholine would be generated, leading to remarkable absorption spectral change (see photo in Figure 1B). A control experiment with acetylcholine was performed under similar conditions; no absorption spectral variation due to the cross-linking of Au-NPs was observed even after the reaction mixture was incubated for 15 min as shown in Figure S1 of the Supporting Information. This control experiment provides further support for the assumption that the cross-linking of Au-NPs in the presence of

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Figure 1. Colorimetric assay for AChE by using Au-NPs. (A) Absorption spectrum of Au-NP solution containing acetylthiocholine and those after acetylthiocholine was hydrolyzed in the presence of AChE (0.6 mU/mL) for different periods at 25 °C; the solution under investigation was composed of 0.9 mL of Au-NPs (2.4 nM), acetylthiocholine (10 µM), AChE (0.6 mU), and 0.1 mL of phosphate buffered saline (PBS, 10 mM, pH ) 8.0). (B) Color change for the Au-NP solutions containing acetylthiocholine with and without AChE addition; the solutions contained 0.9 mL of Au-NPs (6.0 nM), acetylthiocholine (10 µM), 0.1 mL of PBS (10 mM, pH ) 8.0), and different amounts of AChE (a, 0 mU/mL; b, 25 mU/mL; c, 50 mU/mL; d, 100 mU/mL). Photos were taken after incubation at 25 °C for 5.0 min.

Figure 2. TEM images of Au-NPs (2.4 nM) containing (A) acetylthiocholine (10 µM) and (B) acetylthiocholine (10 µM), AChE (0.6 mU) after incubation for 20 min.

Figure 3. Variation of A650/A520 versus the reaction time for the Au-NP solutions containing acetylthiocholine (10 µM) with different concentrations of AChE (0, 0.6, 1.0, 1.5, and 2.0 mU/mL).

acetylthicholine and AChE is due to the formation of thiocholine generated by hydrolysis of acetylthicholine catalyzed by AChE. However, choline generated by the hydrolysis of acetylcholine catalyzed by AChE cannot lead to the cross-linking of Au-NPs. The AChE activity assay with Au-NPs was further performed by incubating the ensemble solution containing acetylthiocholine (10 µM) and different concentrations of AChE (0, 0.6, 1, 1.5, and 2 mU/mL) for different periods of time. The absorption spectrum of the ensemble solution in each case was measured after every 1 min. Figure 3 shows the plots of A650/A520 versus the reaction time (0-15 min) for different concentrations of AChE. As anticipated, A650/A520 increased with the reaction time. The increase of A650/A520 was obviously more remarkable for the ensemble solution containing a high concentration of AChE. A nearly linear relation between the AChE concentration and A650/ A520 resulted as shown in Figure S2 of the Supporting Information. At a high concentration of AChE, the hydrolysis reaction became fast, and as a result the hydrolysis reaction could be finished within a short time. For instance, when the concentration of AChE reached 2.0 mU/mL, no further absorption variation was detected after the ensemble was incubated for 6.0 min.

Figure 4. (A) Colorimetric assay for AChE inhibitor screening by using Au-NPs. The solutions contained 0.9 mL of Au-NPs (6.0 nM), acetylthiocholine (10 µM), AChE (30 mU), 0.1 mL of PBS (10 mM, pH ) 8.0), and different concentrations of tacrine (a and b, 0 nM; c, 50 nM; d, 100 nM; e, 200 nM; f, 400 nM). Sample (a) contained no AChE and was used for comparison. Photos were taken after incubation at 25 °C for 20 min. (B) Chemical structure of tacrine.

This convenient colorimetric assay for AChE activity with Au-NPs enables us to further explore its application to AChE inhibitor screening. Tacrine (9-amino-1,2,3,4-tetrahydro-acridine, Figure 4), a well-known inhibitor for acetylcholinesterase,14 was selected as an example to demonstrate the application of AuNPs in AChE inhibitor screening. The color of the ensemble solution of Au-NPs was changed from red to gray after introducing AChE to the solution as discussed above. However, the color variation became slow after addition of tacrine to the ensemble as shown in Figure 4. When more tacrine was added, the color of the ensemble kept closer to red under the same conditions. For instance, the color of the ensemble was nearly the same as that of the initial Au-NPs (red) when the concentration of tacrine reached 400 nM (30 mU/mL AChE; see Figure 4A, samples a and f). These results can be accountable, since tacrine can inhibit the activity of AChE and thus the hydrolysis of acetylthiocholine (14) Harel, M.; Schalk, I.; Ehret-Sabatier, L.; Bouet, F.; Goeldner, M.; Hirth, C.; Axelsen, P. H.; Silman, I.; Sussman, J. L. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 9031–9035.

Continuous Colorimetric Assay for AChE

Figure 5. Variation of A650/A520 versus the reaction time for the Au-NP ensemble containing acetylthiocholine (10 µM), AChE (1.5 mU), and different concentrations of tacrine (0, 4.0, 10, 20, and 40 nM).

into thiocholine will become slow. As a result, less thiocholine will be formed after addition of tacrine, leading to less aggregation of Au-NPs and thus less absorption variation (and less color change). Therefore, it is not only possible to perform a colorimetric assay for AChE activity with the ensemble of Au-NPs and acetylthiocholine conveniently, but also possible to screen the inhibitors of AChE with the ensemble by the naked-eye. In order to quantify the inhibition efficiency of tacrine toward AChE, absorption spectra of the Au-NP solutions containing acetylthiocholine (10 µM) and AChE (1.5 mU) in the absence and presence of different concentrations of tacrine (0, 4.0, 10, 20, 40, 100, and 150 nM) were measured after the solutions were incubated for different periods of time. Obviously, variation of A650/A520 versus the reaction time became smoother after addition of tacrine compared to that in the absence of tacrine as displayed in Figure 5. For instance, when the concentration of tacrine in the ensemble reached 40 nM, A650/A520 kept nearly unchanged even if the ensemble was incubated for 6 min. Such absorption spectral variation is consistent with the color change observed for the ensemble solution as detailed above. On the basis of the plot of A650/A520 (measured after incubation for 10 min) versus

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the concentration of tacrine (see Figure S3 of the Supporting Information), the corresponding IC50 was estimated to be 6.4 nM.15 In summary, a new continuous colorimetric method for AChE activity assay and inhibitor screening was successfully established with Au-NPs and acetylthiocholine. This new assay approach was based on the following facts: (1) the aggregation of Au-NPs results in the red-shift of the plasmon absorption due to interparticle plasmon interactions and (2) AChE can catalyze the hydrolysis of acetylthiocholine into thiocholine which can induce the aggregation of Au-NPs. The unique feature of this assay method is that the AChE activity can be analyzed by the nakedeye and AChE with a concentration as low as 0.6 mU/mL can be assayed. The results also clearly demonstrate the usefulness of this convenient assay method for screening inhibitors of AChE. Given its simplicity and easy operation, this method may extend to high-throughput screening of AChE inhibitors and relevant drug discovery. Acknowledgment. The present research was financially supported by NSFC, the State Basic Program and Chinese Academy of Sciences. Supporting Information Available: Absorption spectra and the plot of A650/A520 versus reaction time for the solution of Au-NPs containing acetylcholine and AChE; nearly linear relation between AChE concentration and A650/A520; plot of A650/A520 versus the concentration of tacrine. This material is available free of charge via the Internet at http://pubs.acs.org. LA803870V (15) The IC50 was determined with 1.5 mU/mL of AChE, and it was different from the IC50 values reported before (see refs 3 and 7a). However, it is understandable, since the IC50 values usually increase by increasing the enzyme concentration.