Inhibition of the Acetycholine Esterase-Stimulated Growth of Au

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NANO LETTERS

Inhibition of the Acetycholine Esterase-Stimulated Growth of Au Nanoparticles: Nanotechnology-Based Sensing of Nerve Gases

2005 Vol. 5, No. 4 649-653

Valeri Pavlov, Yi Xiao, and Itamar Willner* Institute of Chemistry, The Hebrew UniVersity of Jerusalem, Jerusalem, 91904 Israel Received January 11, 2005; Revised Manuscript Received February 23, 2005

ABSTRACT The acetylcholine esterase, AChE, mediated hydrolysis of acetylthiocholine (1) yields a reducing agent thiocholine (2) that stimulates the catalytic enlargement of Au NP seeds in the presence of AuCl4-. The reductive enlargement of the Au NPs is controlled by the concentration of the substrate (1) and by the activity of the enzyme. The catalytic growth of the Au NPs is inhibited by 1,5-bis(4-allyldimethylammoniumphenyl)pentane-3-one dibromide (3) or by diethyl p-nitrophenyl phosphate (paraoxon; 4), thus enabling a colorimetric test for AChE inhibitors. The colorimetric assay was also developed on glass supports.

The use of metal and semiconductor nanoparticles (NPs) as active components for the optical and electrical sensing of biorecognition processes is rapidly developing.1 The interparticle plasmon interactions of Au NP-functionalized nucleic acids were employed to analyze DNA and to detect single base mismatches in nucleic acids.2 Also, nucleic acidfunctionalized semiconductor NPs were used as labels for the electrochemical,3 optical,4 or photoelectrochemical5 detection of DNA. Recently, the coupling of enzymes to the biocatalytic growth of Au NPs was employed for the analysis of NAD(P)H cofactors and for following NAD+-dependent biocatalyzed reactions.6 Also, the biocatalytic growth of Au NPs in the presence of glucose/glucose oxidase7 or by the tyrosine/O2/tyrosinase8 system was employed to sense glucose or to follow the tyrosinase activity, respectively. Here we report on the acetylcholine esterase-stimulated growth of Au NPs. We demonstrate that the enzyme inhibitor blocks the growth of the Au NPs. As a result, the system provides a novel nanotechnology-based sensing method for nerve gases. Acetylcholine is a central neurotransmitter, and its hydrolysis by acetylcholine esterase, AChE, is a key process for the regulation of the neural response system.9 The inhibition of AChE, e.g., by nerve gases, leads to the perturbation of the nerve conduction system and to the rapid paralysis of vital functions of the living systems.10 Different methods to sense the activity of AChE and to follow its inhibition were developed.11 The most common method involves the activation of an enzyme cascade where AChE * Corresponding author. Tel. 972-2-6585272; Fax 972-2-6527715; E-mail: [email protected]. 10.1021/nl050054c CCC: $30.25 Published on Web 03/09/2005

© 2005 American Chemical Society

hydrolyses acetycholine to choline, the subsequent oxidation of choline to betaine by choline oxidase with the concomitant formation of H2O2, followed by the horseradish peroxidasemediated colorimetric12 or electrochemical13 detection of the H2O2. Recently, CdS-NPs were coupled to AChE, and the resulting photocurrent was used to follow the enzyme activity and its inhibition.14 The present system consists of the enzyme AChE, HAuCl4, 1.1 × 10-3 M, gold NP seeds, 2-3 nm, 3.6 × 10-8 M (particles prepared by NaBH4 reduction and capped with citrate),15 and the enzyme substrate acetylthiocholine, 1. Figure 1A shows the absorbance spectra of the resulting Au NPs in the presence of a fixed concentration of 1, 1.4 × 10-4 M, and variable concentrations of the enzyme. As the concentrations of the enzyme increase, the plasmon absorbance bands are intensified, turn broader, and are blue shifted. Figure 1B shows the absorbance spectra of the Au NPs formed in the presence of a fixed concentration of enzyme, 0.13 units/mL, and variable concentrations of the substrate. As the concentrations of 1 are elevated, the plasmon absorbance bands of the NPs increase in their intensities, become broader, and are blue shifted. Control experiments reveal that all of the components are essential to stimulate the spectral changes of the NP solution. Substitution of acetylthiocholine with acetylcholine does not lead to any spectral change that indicates the formation of Au NPs. TEM analyses reveal that as the content of the enzyme increases or the concentration of 1 is elevated, larger particles are formed, reaching a diameter of 300 to 500 nm, and concomitantly, very small particles 8 to 30 nm are produced in the system. The large particles consist of a dense Au core

Scheme 1. Detection of Acetylcholine Esterase Activity by Growing Au Nanoparticles

Figure 1. (A) Absorbance spectra of the Au NPs systems formed in the presence of different concentrations of AChE: (a) 0 units/ mL; (b) 0.026 units/mL; (c) 0.054 units/mL; (d) 0.077 units/mL; (e) 0.11 units/mL; (f) 0.13 units/mL; (g) 0.15 units/mL; (h) 0.21 units/mL. In all experiments the system includes HAuCl4, 1.1 × 10-3 M, Au NP seeds, 3.6 × 10-8 M, [1] ) 1.4 × 10-4 M. (B) Absorbance spectra of the Au NPs formed in the presence of AChE, 0.13 units/mL, HAuCl4, 1.1 × 10-3 M, Au NP seeds, 3.6 × 10-8 M, and variable concentrations of 1: (a) 0 M; (b) 2.4 × 10-5 M; (c) 4.8 × 10-5 M; (d) 9.5 × 10-5 M; (e) 1.4 × 10-4 M; (f) 1.9 × 10-4 M; (g) 2.4 × 10-4 M; (h) 3.8 × 10-4 M. Spectra were recorded after 5 min of particle growth.

bands. These small NPs may be generated by the detachment of the thin nanoclusters associated with the surface of the large particles. Further support that the thiocholine acts as the reducing agent for the catalytic enlargement of the Au NP seeds was obtained by treatment of the Au NP seeds with variable concentrations of thiocholine, 2, in the absence of the enzyme. Figure 3 shows the absorbance spectra of the Au NPs formed in the presence of different concentrations of 2 in the presence of AuCl4- and the Au NP seeds. As the concentrations of 2 are elevated, the absorbance spectra of the Au NPs are intensified in analogy to the spectra observed with the enzyme and 1 as described for the biocatalytic process.

Figure 2. TEM images of: (A) Au NP mixture formed upon the treatment of the Au NP seeds with AChE and 1, 1.4 × 10-4 M, for 5 min. Large “flower-like” Au NPs with a diameter of 300 nm to 500 nm are observed, together with very small, 8-30 nm, Au NPs. The left inset shows an enlarged image of the large Au NPs. The right inset shows an aggregate consisting of small Au NPs. (B) The image of the Au NPs formed upon enlargement of the Au NP seeds with AChE and acetylthiocholine, 1.4 × 10-4 M, in the presence of the inhibitor 3, 2.4 × 10-5 M, for 5 min. The left inset shows a typical aggregate of Au NPs with a diameter of 5 nm to 20 nm.

surrounded by deposited irregular and thin nanoclusters, (Figure 2A). The nanoclustering and roughness of the large particles is enhanced as the contents of enzyme or substrate increase. These observations explain nicely the spectral changes observed in the present NP systems. As the concentrations of the enzyme (or substrate, 1) increase, the hydrolysis of acetylthiocholine to thiocholine is enhanced. The latter product acts as the reducing agent for the reduction of AuCl4- and the deposition of gold on the Au NP seeds. As the amount of reductant increases, larger Au particles with increased nanoclustering on the particle surface are formed, giving rise to intensified absorbance bands (Scheme 1). Nonetheless, as the concentrations of the enzyme (or substrate) increase, the content of the small Au NPs (8-30 nm) is substantially higher, and these small NPs lead to the blue shifted, yet more broadly intensified, plasmon 650

Figure 3. Absorbance spectra of the Au NPs formed in the presence of HAuCl4, 1.1 × 10-3 M, Au NP seeds, 3.6 × 10-8 M, and variable concentrations of thiocholine, 2: (a) 0 M; (b) 2.4 × 10-5 M; (c) 4.8 × 10-5 M; (d) 9.5 × 10-5 M; (e) 1.4 × 10-4 M; (f) 1.9 × 10-4 M; (g) 2.4 × 10-4 M; (h) 3.8 × 10-4 M. Spectra were recorded after 5 min of particle growth.

From a practical point of view it is important to follow the inhibition of the activity of AChE. Toward this goal, we have examined the inhibition of AChE by 1,5-bis(4-allyldimethylammoniumphenyl)pentane-3-one dibromide (3), a common AChE inhibitor that mimics the functions of nerve gases,16 by following the inhibited growth of Au NPs. Figure 4A shows the absorbance spectra of the Au-NPs formed Nano Lett., Vol. 5, No. 4, 2005

Figure 4. (A) Absorbance spectra of the Au NPs formed in the presence of different concentrations of the inhibitor, 3: (a) 0 M; (b) 2.4 × 10-6 M; (c) 4.8 × 10-6 M; (d) 9.5 × 10-6 M; (e) 2.4 × 10-5 M. All systems include AChE, 0.13 units/mL, HAuCl4, 1.1 × 10-3 M, Au NP seeds, 3.6 × 10-8 M, [1] ) 1.4 × 10-3 M. (B) Lineweaver-Burk plots corresponding to the inhibition of the Au NP growth by the respective concentrations of 3. Spectra were recorded after a fixed time-interval of 5 min.

by AChE, 0.13 units/mL, and 1, 1.4 × 10-4 M, and Au NP seeds, 3.6 × 10-8 M, in the presence of different concentrations of the inhibitor. Clearly, as the concentration of the inhibitor increases the enlargement of the Au NPs is blocked, as reflected by the lower intensities of the absorbance bands, due to the lower yield of the hydrolysis product. Figure 4B shows the Lineweaver-Burk plots corresponding to the kinetic analysis of the inhibition process, characterized by following the absorbance of the NPs. From the derived plots we conclude that a competitive inhibition process prevails in the system, KI ) 0.069 µM and KM ) 0.14 mM. The derived value of KI is in excellent agreement with the reported value.17 Further support that the inhibitor 3 retards the growth of the particles is obtained from TEM measurements (Figure 2B). In contrast to the large particles formed in the absence of the inhibitor, only small Au NPs, 5 nm to 20 nm, are formed in the presence of 3, 2.4 × 10-5 M, for 5 min. This result implies that the inhibitor blocks the growth of the Au NPs. To generalize the concept of following the inhibition of acetylcholine esterase by nerve gas analogues that retard the biocatalytic growth of Au NPs, we examined the effect of diethyl p-nitrophenyl phosphate (paraoxon-ethyl; 4) on the activity of AChE. Paraoxon, 4, is widely examined as a model system for organophosphate inhibitors of AChE (and thus, for organophosphate nerve gases).18,19 In contrast to the previously studied inhibitor, 3, that reveals competitive inhibition of the AChE binding site, paraoxon inhibits AChE by an irreversible inhibition path, eq 1. According to the well-established mechanism, the organophosphate inhibitor (CX) leads to the phosphorylation of the active site with the concomitant release of the leaving group X (X ) p-nitrophenol for paraoxon, 4).

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Figure 5. (A) Absorbance spectra of the Au NPs formed in the presence of AChE and acetylthiocholine, 1, in the presence of different concentrations of the inhibitor, 4: (a) 0 M; (b) 1.0 × 10-8 M; (c) 5.0 × 10-8 M; (d) 1.0 × 10-7 M; (e) 2.0 × 10-7 M; (f) 4 × 10-7 M. All systems include AChE, 0.13 units/mL, HAuCl4, 1.1 × 10-3 M, Au NP seeds, 3.6 × 10-8 M, and acetylthiocholine, 1, 1.4 × 10-3 M. In all experiments AChE was incubated with the respective concentration of the inhibitor for a time-interval of 20 min, and the absorbance spectra of the enlarged Au NPs was recorded after a 5 min of biocatalytic growth. (B) Plots of logarithm of rates of Au NP formation in the presence of different concentrations of 4: (a) 1.0 × 10-8 M; (b) 5.0 × 10-8 M; (c) 1.0 × 10-7 M; (d) 2.0 × 10-7 M; (e) 4 × 10-7 M. In all systems AChE, 0.13 units/mL, HAuCl4, 1.1 × 10-3 M, Au NP seeds, 3.6 × 10-8 M, and acetylthiocholine, 1, 1.4 × 10-3 M.

The overall rate constant for the inhibition of the enzyme is given by eq 2, where [E] is the concentration of the noninhibited enzyme, [Eo] is the initial concentration of the enzyme, and [I] is the concentration of the inhibitor. ln [E] ) ln [Eo] - ki [I]t

(2)

Figure 5A shows the absorbance spectra of the AChEgenerated Au NPs in the presence of 1 and variable concentrations of the inhibitor 4, using a fixed reaction time interval of 5 min. As the concentration of the inhibitor increases, the absorbance of the Au NPs decreases, implying that the biocatalytic growth of the Au NP is inhibited. Figure 5B shows the analysis of the kinetics of inhibition of AChE by 4. The absorbance of the Au NPs reflects the activity of AChE. Accordingly, Figure 5B depicts the time-dependent absorbance changes of the Au NP in the presence of variable concentrations of the inhibitor 4 (cf. eq 2). From the respective slopes, and knowing the concentration of the inhibitor 4, the value of ki ) 3.3 × 105 M-1 min-1 was extracted. This value is in good agreement with the reported value (2.9 × 105 M-1 min-1).19 For any future homeland security use of this NPs-based sensor system, it would be advantageous to construct a surface-immobilized assay. Accordingly, glass plates were functionalized with an aminopropylsiloxane film and the Au NP seeds were bound to the surface.20 Figure 6 shows the absorbance spectra of the Au NPs deposited on the surface in the presence of AChE, 0.13 units/mL, and 1, 1.4 × 10-4 M, in the absence of the inhibitor, curve (a), and in the presence of different concentrations of the inhibitor 3, curves (b-e). In the absence of the inhibitor, the surface exhibits a 651

Figure 6. Absorbance spectra corresponding to the inhibition of the Au NPs growth on glass supports recorded in the presence of AChE, 0.13 units/mL, HAuCl4, 1.1 × 10-3 M, [1] ) 1.4 × 10-4 M, and different concentrations of 3: (a) 0 M; (b) 5.9 × 10-7 M; (c) 1.2 × 10-6 M; (d) 2.4 × 10-6 M; (e) 5.9 × 10-6 M. Inset: Images of glass slides formed in the presence of the respective concentrations of the inhibitor, 3, (left no inhibitor, right [3] ) 2.4 × 10-6 M).

blue color with a maximum absorbance at λ ) 570 nm. As the concentrations of the inhibitor increase, the plasmon absorbance bands of the NPs decrease in their intensities and are blue shifted. Interestingly, the absorbance maximum of the surface-confined enlarged Au NPs is red shifted by ca. 12 nm as compared to the absorbance band in solution. These results are explained by the fact that the enlargement of the surface-confined Au NPs is followed by a rinsing step that washes off all the small nanoclusters, giving rise to the blue shift in solution. As the concentration of the inhibitor increases, the surface-associated particles are smaller, leading to blue-shifted absorbance bands of lower intensities. Figure 6, inset, shows the color images of the glass plates formed upon reaction of AChE/1 with different concentrations of the inhibitor 3. The system without the inhibitor yields a blue-colored surface while the added inhibitor generates an almost nondetectable pale pink glass support. Thus, a surface engineered sensor that includes an active sensing domain and a control domain may provide an effective optical strip for the detection of nerve gases. Experimental Section. Materials. Acetylcholine esterase (from electric eel) and other chemicals were obtained from Sigma-Aldrich and were used as supplied. Au NP seeds (2-3 nm) stabilized with citrate were prepared according to the literature.15 The concentration of the Au NPs was determined by following the absorbance spectra, λ ) 510 nm, and by using the extinction coefficient 6.5 × 105 cm-1 M-1. Acetylcholine Esterase Assay. 80 µL of 1.6 mM acetylthiocholine chloride in 0.1 M Tris buffer (pH 8.0) and 4 µL of acetylcholine esterase solution in 0.1 M Tris buffer were incubated at 35 °C for 15 min. Next, 800 µL of 1.25 mM HAuCl4 and 30 µL of 2-3 nm aqueous gold NP seeds were added to give the final concentrations of acetylthiocholine chloride, 1.4 × 10-4 M, HAuCl4 1.1 × 10-3 M, gold NP seeds, 3.6 × 10-8 M, and after 5 min the absorbance spectrum of the resulting solution was measured. 652

Determination of the Inhibition Constant of 1,5-Bis(4allyldimethylammonium-phenyl)pentan-3-one dibromide (3). 80 µL of variable concentrations of acetylthiocholine chloride in 0.1 M Tris buffer (pH 8.0) that included variable concentrations of 1,5-bis(4-allyldimethylammonium-phenyl)pentan-3-one dibromide, 3, and 4 µL of acetylcholine esterase solution in 0.1 M Tris buffer (0.13 units/mL) were incubated at 35 °C for 15 min. Afterward, 800 µL of 1.25 mM HAuCl4 and 30 µL of 1.1 × 10-6 M of 2-3 nm aqueous gold NP seeds were added to the enzyme mixture. The absorbance spectra of the formed Au-NPs were recorded after 5 min. Inhibition of Au NP Growth on Glass Slides by 1,5-Bis(4-allyldimethylammonium-phenyl)pentan-3-one dibromide (3). 45 µL of 1.6 mM acetylthiocholine chloride in 0.1 M Tris buffer (pH 8.0) containing variable concentrations of 2 and 16 µL of acetylcholine esterase solution in 0.1 M Tris buffer (0.13 units/mL) were incubated at 35 °C for 15 min. Afterward, the enzyme solution was mixed with 3.2 mL of an aqueous solution of HAuCl4, 1.25 mM. The Au NPfunctionalized glass slides were immersed in the resulting solution for 5 min. The slides were then washed with water and their absorbance was followed in water. Acknowledgment. This research is supported by the German-Israel Program (DIP). References (1) (a) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128-4158. (b) Katz, E.; Shipway, A. N.; Willner, I. In Nanoparticles-From Theory to Applications; Schmid, G., Ed.; Wiley-VCH: Weinheim, 2003; Chapter 6, pp 368-421. (c) Katz, E.; Willner, I. Angew. Chem. Int. Ed. 2004, 43, 6042-6108. (2) (a) Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 1959-1964. (b) Storhoff, J. J.; Mirkin, C. A. Chem. ReV. 1999, 99, 1849-1862. (3) (a) Wang, J.; Liu, G.; Polsky, R.; Merkoci, A. Electrochem. Commun. 2002, 4, 722-726. (b) Wang, J.; Liu, G.; Merkoci, A. J. Am. Chem. Soc. 2003, 125, 3214-3215. (c) Wang, J.; Xu, D. K.; Kawde, A. N.; Polsky, R. Anal. Chem. 2001, 73, 5576-5581. (4) (a) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013-2016. (b) Chan, W. C. W.; Nie, S. Science 1998, 281, 2016-2018. (5) Willner, I.; Patolsky, F.; Wasserman, J. Angew. Chem., Int. Ed. 2001, 40, 1861-1864. (6) Xiao, Y.; Pavlov, V.; Levine, S.; Niazov, T.; Markovitch, G.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 4519-4522. (7) Zayats, M.; Baron, R.; Popov, I.; Willner, I. Nano Lett. 2005, 5, 2125. (8) Baron, R.; Zayats, M.; Willner, I. Anal. Chem. 2005, 77, 15661571. (9) (a) Wessler, I.; Kirkpatrick, C. J.; Racke, K. Clinic. Exp. Pharmacol. Physiol. 1999, 26, 198-205. (b) Perry, E.; Walker, M.; Grace, J.; Perry, R. Trends Neurosci. 1999, 22, 273-280. (10) (a) Pauluhn, J.; Machemer, L.; Kimmerle, G. Toxicology 1987, 46, 177-190. (b) Clement, J. G. Fundam. Appl. Toxicol. 1983, 3, 533535. (11) (a) Wilson, I. B. In Methods in Enzymology; Colowick, S. P.; Kaplan N. O., Eds.; Academic Press: New York, 1955. (b) Alfonta, L.; Katz, E.; Willner, I. Anal. Chem. 2000, 72, 927-935. (c) Liu, W.; Tsou, C. L. Biochim. Biophys. Acta 1986, 870, 185-190. (12) Zhou, M.; Zhang, C.; Haugland, R. P. Proc. SPIE-Int. Soc. Opt. Eng. 2000, 3926, 166-171. (13) Riklin, A.; Willner, I. Anal. Chem. 1995, 67, 4118-4126. (14) Pardo-Yissar, V.; Katz, E.; Wasserman, J.; Willner, I. J. Am. Chem. Soc. 2003, 125, 622-623. (15) Busbee, B. D.; Obare, S. O.; Murphy, C. J. AdV. Mater. 2003, 15, 414-416.

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