Electrochemical, Photoelectrochemical, and Piezoelectric Analysis of

the presence of O2, to the respective catechol derivatives, e.g.,. L-DOPA or dopamine, that are further oxidized by the enzyme to the respective quino...
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Anal. Chem. 2008, 80, 2811-2816

Electrochemical, Photoelectrochemical, and Piezoelectric Analysis of Tyrosinase Activity by Functionalized Nanoparticles Huseyin Bekir Yildiz, Ronit Freeman, Ron Gill, and Itamar Willner*

Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

The electrochemical and photoelectrochemical detection of tyrosinase (TR) activity (an indicative marker for melanoma cancer cells) is reported, using Pt nanoparticles (NPs) or CdS NPs as electrocatalytic labels or photoelectrochemical reporter units. The Pt NPs or CdS NPs are modified with a tyrosine methyl ester, (1), capping layer. Oxidation of the capping layer by TR/O2 yields the respective L-DOPA and dopaquinone products. The reduction of the resulting mixture of products with citric acid yields the L-DOPA derivative,(3), as a single product. The association of the (3)-functionalized Pt NPs or CdS NPs to a boronic acid monolayer-modified electrode enables the electrochemical transduction of TR activity by the Pt-NPs-electrocatalyzed reduction of H2O2 or the photoelectrochemical transduction of TR activity by the generation of photocurrents in the presence of triethanolamine as a sacrificial electron donor. The detection limits for analyzing TR corresponds to 1 U and 0.1 U by the electrochemical and photoelectrochemical methods, respectively. The association of the Pt NPs or CdS NPs to the functionalized monolayer electrode is followed by quartz crystal microbalance measurements. Tyrosinase is a copper-containing protein that catalyzes the oxidation of phenol derivatives, such as tyrosine or tyramine, in the presence of O2, to the respective catechol derivatives, e.g., L-DOPA or dopamine, that are further oxidized by the enzyme to the respective quinone products, eq 1. Elevated amounts of

tyrosinase were detected in melanoma cancer cells, and the enzyme is considered as an indicative marker for this type of malignant cells.1 Also, it was reported that the loss of dopamine * To whom correspondence should be addressed. E-mail: willnea@ vms.huji.ac.il. Phone: 972-2-6585272. Fax: 972-2-6527715. (1) Angeletti, C.; Khomitch, V.; Halaban, R.; Rimm, D. L. Diagn. Cytopathol. 2004, 31, 33-37. 10.1021/ac702401v CCC: $40.75 Published on Web 03/07/2008

© 2008 American Chemical Society

in neurons might cause diseases such as Parkinson disease.2 Similarly, dopamine is a central neurotransmitter, and its sensitive detection, particularly with integrated miniaturized devices, could be valuable for the invasive monitoring of neural response. Indeed, several recent reports addressed different optical and electrochemical methods to detect tyrosinase activity. The luminescence of semiconductor quantum dots modified by a tyrosine derivative was quenched by the tyrosinase-mediated oxidation of the tyrosine residues to the respective quinone units, thus providing an optical assay for the enzyme.3 Also, the tyrosinaseinduced generation of Au nanoparticles upon the oxidation of L-tyrosine in the presence of a Au(III) salt was used for the optical detection of tyrosinase activity.4 The biocatalytic oxidation of a tyramine monolayer-modified Au electrode by tyrosinase to the respective dopamine monolayer, followed by the association of ferrocene boronic acid, acting as a redox label, to the resulting catechol monolayer, was used to develop an electrochemical sensor that follows tyrosinase activities.5 Similarly, the tyrosinasestimulated oxidation, and consequently the accompanying potential changes of the tyramine- or dopamine-functionalized gate surfaces of field-effect transistor devices, was used to electronically monitor tyrosinase and its activity.6 Metallic nanoparticles or semiconductor quantum dots are often used as catalytic7 or optical8 labels for biosensing events. For example, the catalytic deposition of metals or metal nanoparticles and the generation of conducting paths between electrode gaps were used to detect DNA hybridization and antigen-antibody complexes.9 Also, the catalyzed deposition of Ag0 shells on Au NP labels was used for the amplified electrochemical detection of DNA.10 Similarly, the utility of catalytic NPs for bioanalysis by introducing an electrocatalytic nanoparticle-modified antibody that is sensitive to oxygen reduc(2) Olanow, C.W. Neurology 1990, 40, 32-37. (3) Gill, R.; Freeman, R.; Xu, J. P.; Willner, I.; Winograd, S.; Shweky, I.; Banin, U. J. Am. Chem. Soc. 2006, 128, 15376-15377. (4) Baron, R.; Zayats, M.; Willner, I. Anal. Chem. 2005, 77, 1566-1571. (5) Li, D.; Gill, R.; Freeman, R.; Willner, I. Chem. Commun. 2006, 5027-5029. (6) Freeman, R.; Elbaz, J.; Gill, R.; Zayats, M.; Willner, I. Chem. Eur. J. 2007, 13, 7288-7293. (7) (a) Daniel, M. C.; Astruc, D. Chem. Rev. 2004, 104, 293-346. (b) Baron, R.; Willner, B.; Willner, I. Chem. Commun. 2007, 323-332. (8) (a) Medintz, I. L.; Clapp, A. R.; Mattoussi, H.; Goldman, E. R.; Fisher, H.; Mauro, J. M. Nat. Mater. 2003, 2, 630-638. (b) Liz-Marzan, L. M.; Philipse, A. P. J. Phys. Chem. 1995, 99, 15120-15128. (9) Park, S. J.; Taton, E. W.; Mirkin, C. A. Science 2002, 295, 1503-1506. (10) Wang, J.; Polsky, R.; Xu, D. Langmuir 2001, 17, 5739-5741.

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tion was further examined.11 Semiconductor NPs are often used as fluorescent labels for biorecognition events.12 For example, different sized semiconductor NPs were used for the multiplexed analysis of different antigens.13 Fluorescent quantum dots (QDs) were used for the detection of single-nucleotide polymorphism in human oncogene p53 and for the multiallele detection of hepatitis C virus in microarray configurations.14 In the present study, we use L-tyrosine methyl ester-functionalized Pt nanoparticles (NPs) as catalytic probes for analyzing tyrosinase (TR) activity by electrochemical means. Similarly, we demonstrate the use of L-tyrosine methyl ester-modified CdS NPs for the photoelectrochemical analysis of tyrosinase activity. The results are further supported by using the functionalized Pt NPs or CdS NPs as “weight labels” that monitor the activity of tyrosinase on a piezoelectric quartz crystal microbalance.15 EXPERIMENTAL SECTION Materials and Reagents. Ultrapure water from NANOpure Diamond (Barnstead Int., Dubuque, U.S.A.) source was used throughout the experiments. All chemicals were purchased from Sigma-Aldrich and used as received without further purification. Preparation of L-Tyrosine Methyl Ester-Modified Pt Nanoparticles. Platinum particles were prepared by heating 100 mL of a 1 mM PtCl6- solution to reflux, followed by the addition of 10 mL of a 38.8 mM aqueous sodium citrate solution. After 10 min of boiling, the solution turned from clear to black colored, after which heating was then turned off and the solution was stirred for additional 10 min. Finally the solution was allowed to cool to room temperature, filtered through a 0.2 µm cellulose acetate filter (Schleicher and Schuell, Keene, NH), and rinsed two times through a 100 000 MW cutoff Centricon tube (Millipore Inc., Billerica, MA) with water. The Pt NPs were then mixed with an aqueous solution of 1 mM 3-mercaptopropionic acid. The mixture was placed on a shaker for 12 h and subsequently cleaned by repeated centrifugation and precipitation of the NPs at 5000 rpm for 5 min, followed by the resuspension of the NPs in HEPES buffer solution pH ) 7.4. The 3-mercaptopropionic acid-modified Pt NPs were mixed with l000-fold excess of tyrosine methyl ester (1), in a 10 mM HEPES buffer pH ) 7.4 that included 10 mM 1-ethyl-3-[3dimethylaminopropyl] carbodiimide hydrochloride (EDC). The mixture was placed on a shaker for 2 h, and the resulting amidated-L-tyrosine methyl ester, (2)-capped Pt NPs were cleaned by repeated centrifugation at 5000 rpm for 5 min and subsequent resuspension in a 10 mM HEPES buffer pH ) 7.4. Preparation of Amidated L-DOPA Methyl Ester (3) and Dopaquinone-Capped Pt NPs. For the analysis of tyrosinase, the (2)-functionalized Pt NPs were treated with different concentrations of tyrosinase. All experiments were performed in 10 mM (11) Polsky, R.; Harper, J. C.; Wheeler, D. R.; Dirk, S. M.; Rawlings, J. A.; Brozik, S. M. Chem. Commun. 2007, 2741-2743. (12) Bruchez, M., Jr.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013-2016. (13) Goldman, E. R.; Clapp, A. R.; Anderson, G. P.; Uyeda, H. T.; Mauro, J. M.; Medintz, I. L.; Mattoussi, H. Anal. Chem. 2004, 76, 684-688. (14) Gerion, D.; Chen, F.; Kannan, B.; Fu, A.; Parak, W. J.; Chen, D. J.; Majumdar, A.; Alivisatos, A. P. Anal. Chem. 2003, 75, 4766-4772. (15) Tombelli, S.; Mascini, M.; Turner, A. P. F. Biosens. Bioelectron. 2002, 17, 929-936.

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phosphate buffer pH ) 6.3. In the presence of tyrosinase and O2 (air), the tyrosine residues are converted to a mixture of (3) and the respective dopaquinone derivative. In order to ensure that all of capping of the NPs is in the (3) state, the mixture was treated with 1 mM citric acid for 10 min. Preparation of Boronic Acid Monolayer-Functionalized Au Wires. Au electrodes, 0.5 mm radius, were placed in a 10 mM aqueous solution of 3-mercaptopropionic acid for 3 h, rinsed with water, and dried under argon. The mercaptopropionic acidfunctionalized Au wires were treated with a 5 mM 3-aminophenyl boronic acid (4) solution (in 10 mM HEPES buffer pH ) 7.4) for 2.5 h. The resulting electrodes were washed with a HEPES buffer solution and subsequently dried under argon. Preparation of CdS Nanoparticles. A dioctyl sulfosuccinate sodium salt (AOT)/n-heptane water-in-oil microemulsion was prepared by the solubilization of 3.5 mL of water in 100 mL of n-heptane in the presence of 7.0 g of AOT as surfactant. The mixture was stirred until a clear phase was obtained. The resulting mixture was separated into 60 mL and 40 mL of reverse-micelle subvolumes. Aqueous solutions of Cd(ClO4)2 (240 µL, 1.35 M) and Na2S (160 µL, 1.33 M) were added to the 60 mL and 40 mL subvolumes, respectively, and the two solutions were combined and stirred for 1 h to yield the CdS NPs. For the preparation of 3-mercaptopropionic acid-capped CdS NPs, a mixture consisting of an aqueous solution of 2-mercaptoethane sulfonic acid sodium salt (330 µL, 0.32 M) and 3-mercaptopropionic acid (66 µL, 0.32 M) was added to the resulting micellar solution, and the mixture was stirred for 14 h under argon. Pyridine, 20 mL, was added to the system, and the resulting precipitate was centrifuged and washed with n-heptane, petrol ether, butanol, and methanol. Functionalization of Amidated L-Tyrosine Methyl Ester (2) and L-DOPA-Methyl Ester, (3). CdS NPs capped with 3-mercaptopropionic acid were mixed with l000-fold excess of tyrosine methyl ester (1) in a 10 mM HEPES buffer pH ) 7.4 that included 10 mM EDC. The mixture was stirred for 2 h, and the resulting modified CdS NPs were centrifuged at 5000 rpm for 5 min. For the preparation of (3)-modified CdS NPs, the same procedure as described for the preparation of Pt NPs with (3) was followed. Preparation of Boronic Acid-Functionalized Gold Slides for Photoelectrochemical Measurements. For the photoelectrochemical experiments, gold slides (Au-coated glass microarray slides were purchased from Nalge Nunc International, Rochester, U.S.A.)., cut to the size of 9 mm × 25 mm, were used. The Aucoated slides were modified with the boronic acid monolayer by the similar procedure described for the Au wires. Microgravimetric Quartz Crystal Microbalance (QCM) Experiments. Microgravimetric quartz crystal microbalance (QCM) measurements were performed with a home-built instrument linked to a frequency analyzer (Fluke) using Au/quartz crystals (AT-cut 10 MHz). The geometrical area of the Au electrode was 0.2 ( 0.05 cm2, roughness factor 3.5. Prior to each measurement the modified QCM crystals were dried under a flow of argon, and the crystal frequencies were determined under air. Experimental Setup. All electrochemical experiments were carried out using an Autolab electrochemical system (ECO Chemie, The Netherlands) driven by the GPES software. Cyclic voltammograms were recorded using a saturated calomel electrode as a reference and a carbon counter electrode.

Scheme 1. Electrochemical Analysis of Tyrosinase Activity by a Boronic Acid Monolayer Functionalized Electrode and Tyrosine Methyl Ester-Labeled Pt NPs

Photoelectrochemical experiments were performed using a home-built photoelectrochemical system that included a 300 W Xe lamp (Oriel, model 6258), a monochromator (Oriel, model 74000, 2 nm resolution), and a chopper (Oriel, model 76994). The electrical output from the cell was sampled by a lock-in amplifier (Stanford Research model SR 830 DSP). The shutter chopping frequency was controlled by a Stanford Research pulse/delay generator model DE 535. The CdS-NP-functionalized electrode was employed as the working electrode. A graphite electrode was used as the counter electrode. The photogenerated current was measured between the working and the counter electrode. The electrolyte solution consisted of 0.02 M triethanolamine in 0.01 M phosphate buffer, pH ) 7.3. RESULTS AND DISCUSSION Pt NPs have been recently used as catalytic labels for the amplified electrochemical and chemiluminescent detection of DNA or aptamer-protein complexes by the Pt-NPs-electrocatalyzed reduction of H2O2.16,17 The application of Pt NPs as a catalytic label for the analysis of tyrosinase is depicted in Scheme 1. Pt NPs prepared by citrate reduction were functionalized with mercaptopropionic acid, and the substrate of the enzyme, Ltyrosine methyl ester (1), was covalently linked to the capped Pt NPs to yield (2)-modified Pt NPs. In the presence of tyrosinase and O2, the tyrosine residues are converted to a mixture of the amidated L-DOPA-methyl ester (3)-functionalized NPs and the respective dopaquinone derivative. In order to retain the reaction product of TR in the L-DOPA state (3), citric acid, which reduces any generated quinone to the L-DOPA derivative (3), was added to the system. The resulting L-DOPA-functionalized Pt NPs were then interacted with a boronic acid (4) monolayer-functionalized Au electrode. The latter electrode was prepared by the covalent linkage of 3-aminophenyl boronic acid to a mercaptopropionic acid monolayer-modified electrode. The resulting L-DOPA boronate complex formed on the electrode support included the electro(16) Polsky, R.; Gill, R.; Kaganovsky, L.; Willner, I. Anal. Chem. 2006, 78, 22682271.

catalytic Pt NPs labels, and this electrocatalyzed the reduction of H2O2. As the content of (3)-functionalized Pt NPs is controlled by the activity of TR, the coverage of the electrode by the Pt NPs, and consequently the amperometric response of the system, reflects the activity of TR. Figure 1A, curves c-e, shows the cyclic voltammograms corresponding to the Pt-NP-mediated reduction of H2O2 upon treatment of the (2)-functionalized particles with TR, 10 U, for different time intervals, according to Scheme 1. As the reaction times are prolonged the electrocatalytic cathodic currents are enhanced, consistent with the formation of increased amounts of the catechol product, the (3)-functionalized NPs that bind to the electrode surface. Figure 1A, curve b, depicts the response of the electrode treated with the (2)-modified Pt NPs in the absence of TR. The low-value electrocatalytic current originates from nonspecific binding of the Pt NP to the electrode surface. In further control experiments, the (2)-modified Pt NPs were treated with TR for 15 min in the absence of O2. Only the low-value catalytic current characteristic to the nonspecifically adsorbed NPs was observed. Also, the cyclic voltammogram of the (2)-functionalized Pt NPs treated with TR/O2 and interacted with the (4)-functionalized monolayer that was recorded in the absence of H2O2 did not show any electrocatalytic current. All these control experiments indicate that TR and O2 are indeed essential to generate the catalytic (3)-Pt-NPs-modified electrode and that the Pt NPs, indeed, electrocatalyze the reduction of H2O2. Figure 1B shows the cyclic voltammograms observed upon analyzing different concentrations of tyrosinase through the oxidation of the (2)-functionalized Pt NPs to the catechol-(3)modified Pt NPs. In these experiments the NPs are interacted for a fixed time interval corresponding to 20 min with different concentrations of TR and a constant O2 concentration. As the concentration of TR increases, the electrocatalytic reduction of H2O2 is enhanced, consistent with the formation of elevated amounts of the (3)-functionalized Pt NPs and, thus, increased surface coverage of the electrode with the catalyst, as the concentration of TR is higher. Figure 1B, inset, shows the (17) Gill, R.; Polsky, R.; Willner, I. Small 2006, 2, 1037-1041.

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Table 1. Time-Dependent Mass Changes and Respective Surface Coverage of the Pt NPs on the Piezoelectric Crystal upon Oxidation of the (2)-Functionalized Pt NPs by 10 U of TR oxidation time (min)

∆m (g)

surface coverage of NPs (particles‚cm-2)

5 10 15

79.5 × 10-10 97.1 × 10-10 114.8 × 10-10

2.6 × 1010 3.2 × 1010 3.8 × 1010

Table 2. QCM Analysis of Variable Amounts of TR through the Association of (2)-Functionalized Pt NPs to the Piezoelectric Crystal [tyrosinase] (U)

∆m (g)

surface coverage of NPs (particles‚cm-2)

2.5 5 10

132.4 × 10-10 150.1 × 10-10 176.6 × 10-10

4.4 × 1010 5.1 × 1010 5.8 × 1010

to electrodes was used to amplify sensing events.22,23 The frequency changes, ∆f, of the quartz crystal are directly proportional to the mass changes, ∆m, occurring on the crystal, according to the Sauerbrey relation (eq 2). The surface coverage was estimated using this equation, where f0 is the resonance frequency of the quartz crystal, A is the piezoelectrically active area, Fq is the density of the quartz, and µq is the shear modulus for AT-cut quartz. Figure 1. (A) Cyclic voltammograms corresponding to (a) the (4)functionalized monolayer electrode, (b) the (4)-functionalized electrode in the presence of the (2)-functionalized Pt NPs in the absence of tyrosinase, and (c-e) the (4)-modified electrode treated with the (2)functionalized Pt NPs that were reacted in the presence of 10 U of tyrosinase and O2 for time intervals corresponding to 5, 15, and 20 min prior to the interaction with the modified electrode. (B) Cyclic voltammograms corresponding to (a) the (4)-functionalized monolayer electrode and (b-f) the analysis of different concentrations of tyrosinase by the primary reaction of the (2)-functionalized Pt NPs in the presence of variable concentrations of tyrosinase for a fixed time interval of 20 min and the subsequent interaction of the resulting NPs with the (4)-modified electrode: (b) 0, (c) 2.5, (d) 5, (e) 7.5, and (f) 10 U. Inset: Derived calibration curve. Currents were recorded at -0.6 V vs SCE. All experiments were performed in 10 mM phosphate buffer, pH ) 6.3, under air, using 1 mg‚mL-1 Pt NPs and 9 mM H2O2.

resulting calibration curve that depicts the cathodic current values (at E ) -0.6 V vs SCE) at different amounts of TR. The detection limit for analyzing TR is ca. 1 U (for a comparison of the present electrocatalytic detection procedure to other methods vide infra). Further support that the resulting (3)-functionalized Pt NPs, indeed, bind to the (4)-functionalized monolayer-modified surface was obtained from QCM measurements on (4)-functionalized Au/quartz piezoelectric crystals.18 The association of NPs to the QCM crystals was extensively used to characterize the surface coverage of NPs in monolayer or multilayer configurations on metallic surfaces,19-21 and the association of functionalized NPs (18) Buttry, D. A.; Ward, M. D. Chem. Rev. 1992, 92, 1355-1379. (19) Gabai, R.; Sallacan, N.; Chegel, V.; Bourenko, T.; Katz, E.; Willner, I. J. Phys. Chem. B 2001, 105, 8196-8202.

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∆f ) -R∆m

[R ) -2 (f0)2/A(µqFq)1/2]

(2)

The binding of the (3)-functionalized Pt NPs, generated by the TR/O2 system, to the (4)-modified Au/quartz crystal can then be followed by the frequency changes of the crystal. The system does not only allow us to quantitatively characterize the surface coverage of the NPs on the electrode, but it enables us to apply the QCM as an additional electronic readout method of the TR activity. In Table 1, the mass changes, and the respective Pt NP coverage, observed upon analyzing the time-dependent oxidation of the (2)-functionalized Pt NPs by 10 U of TR are given. As the time interval for the oxidation of the tyrosine methyl ester, functionalized Pt NPs is prolonged, the surface coverage of the NPs on the surfaces increases. Upon reacting the Pt NPs for 15 min with TR, the surface coverage was found to be 3.8 × 1010 particles‚cm-2. (This value corresponds to ca. 20% of a randomly densely packed monolayer of 3.0 nm sized Pt NPs on the surface.) Table 2 shows the mass changes, and the respective coverage of the Pt NPs on the Au surface observed upon the analysis of different concentrations of TR using a fixed time interval of 20 min, according to Scheme 1. As the concentration of TR increases, the surface coverage of the Pt NP linked to the surface is higher. (20) Weizmann, Y.; Patolsky, F.; Willner, I. Analyst 2001, 126, 1502-1504. (21) Sheeney-Haj-Ichia, L.; Pogorelova, S.; Gofer, Y.; Willner, I. Adv. Funct. Mater. 2004, 14, 416-424. (22) Patolsky, F.; Ranjit, K. T.; Lichtenstein, A.; Willner, I. Chem. Commun. 2000, 1025-1026. (23) Li, D.; Yan, Y.; Wieckowska, A.; Willner, I. Chem. Commun. 2007, 35443546.

Scheme 2. Photoelectrochemical Analysis of Tyrosinase by a Boronic Acid Monolayer-Functionalized Electrode and Tyramine-Labeled CdS NPs

A further approach to analyze the activity of tyrosinase involved the photoelectrochemical transduction of the biocatalytic process by following the photocurrents generated by CdS NPs acting as labels for the TR-stimulated oxidation process. Photoelectrochemistry has been employed as a transduction means of biorecognition events, and the activities of enzymes24 or DNA hybridization25 were analyzed by the generation of photocurrents. Also, functionalized CdS NPs were linked to modified electrodes by supramolecular interactions, and the resulting photocurrents were used to follow the formation of the NP assemblies on surfaces.26,27 Scheme 2 depicts the photoelectrochemical analysis of TR activity by CdS semiconductor NPs. CdS NPs were prepared in a water-in-oil microemulsion system followed by the capping of the resulting NPs with mercaptopropionic acid and the subsequent covalent linking of L-tyrosine methyl ester (1) to the NPs. The (2)functionalized CdS NPs were interacted with TR/O2, the product mixture was reduced with citric acid, and the resulting (3)modified CdS NPs were linked to the boronic acid monolayerfunctionalized Au electrode. The photoexcitation of the CdS NPs linked to the electrode yields the electron-hole pair. In the presence of the sacrificial electron donor, triethanolamine, TEOA, the valence-band holes oxidize TEOA, while the conduction band electrons are injected into the electrode, thus, yielding the photocurrent. Figure 2A shows the photocurrent action spectra observed upon the reaction of the (2)-modified CdS NPs with 10 U of TR and O2 for different time intervals and the subsequent association of the resulting (3)-functionalized CdS NP to the boronic acid monolayer-functionalized electrode. As the time interval for the oxidation of the (2)-functionalized CdS particles is longer, the photocurrent is intensified. This is consistent with (24) Pardo-Yissar, V.; Katz, E.; Wasserman, J.; Willner, I. J. Am. Chem. Soc. 2003, 125, 622-623. (25) Willner, I.; Patolsky, F.; Wasserman, J. Angew. Chem., Int. Ed. 2001, 40, 1861-1864. (26) Freeman, R.; Gill, R.; Beissenhirtz, M.; Willner I. Photochem. Photobiol. 2007, 6, 416-422.

the fact that the content of the (3)-modified CdS particles is higher as the reaction time is prolonged, and thus, the surface coverage of the CdS NPs on the electrode surface is higher. These conclusions are further supported by complementary QCM measurements. The surface coverage of the (3)-functionalized NPs on the boronic acid monolayer electrode was found to be 2.6 × 1011, 3.4 × 1011, and 4.2 × 1011 particles‚cm-2 after the reacting the (2)-modified CdS NPs with 10 U of TR and O2 for 5, 10, and 15 min, respectively. Control experiments revealed that no photocurrents were observed upon exclusion of TR from the (2)functionalized CdS NPs or upon the exclusion of O2 from the NPs system that included TR. Also, no photocurrent was observed upon reacting the (2)-modified CdS NPs in the presence of TR/O2 and the binding of the NPs to the electrode, but upon exclusion of the sacrificial electron donor, TEOA. This latter result implies that the scavenging of the valence-band holes by the solution solubilized electron donor is essential to yield the photocurrent, by eliminating the rapid electron-hole recombination process. The mechanism for the formation of the photocurrent is depicted in Scheme 2. Figure 2B depicts the photocurrent action spectra observed upon analyzing the activities of different concentrations of TR through the oxidation of the (2)-functionalized CdS NPs. In these experiments, the (2)-modified CdS NPs were interacted for a fixed time interval of 20 min with different concentrations of TR, and the resulting catechol-functionalized CdS NPs were, subsequently, linked to the electrode surface. As the concentration of TR is higher, the photocurrent intensities increase as a result of higher content of the biocatalytically generated (3)-CdS-NPs. The derived calibration curve, Figure 2B, inset, implies that the photoelectrochemical method enables the detection of tyrosinase with a sensitivity that corresponds to 0.1 of U. It should be noted that two of the methods for analyzing tyrosinase expressed the enzyme activity in terms of the trans(27) Xu, J.; Weizmann, Y.; Krikhely, N.; Baron, R.; Willner, I. Small 2006, 2, 1178-1182.

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Table 3. Analysis of TR by Different Sensor Configurations methods

a

Figure 2. (A) Photocurrent action spectra corresponding to (a) the (4)-functionalized electrode treated with the (2)-modified CdS NPs in the absence of tyrosinase and (b-d) the (2)-modified CdS NPs reacted with 10 U of tyrosinase and O2 for time intervals corresponding to 2, 5, and 10 min, respectively, and then interacted with the (4)-functionalized monolayer electrode. (B) Photocurrent action spectra corresponding to (a) the (4)-functionalized electrode exposed to the (2)-functionalized CdS NPs in the absence of tyrosinase and (b-d) the photocurrents of the (4)-modified electrode, interacted with the (2)-functionalized CdS NPs that were exposed to (b) 0.5, (c) 1.0, and (d) 10 U of tyrosinase and O2 for a time interval of 20 min. Inset: Calibration curve corresponding to the photocurrent values at λ ) 380 nm, resulting by the (4)-modified electrode treated with the (2)functionalized CdS NPs that were exposed to variable amounts of tyrosinase. All experiments were performed in a 10 mM phosphate buffer solution, pH ) 7.4, in the presence of 20 mM triethanolamine.

duced voltammetric responses or the photocurrents generated by the respective enzyme units present in the systems. Thus, the activity of the enzyme in any analyzed sample may be deduced from the value of the respective transduced stimuli and the accompanying calibration curves. Furthermore, one should note that the absolute turnover rates of tyrosinase on its substrate, which is associated with the NPs, is not known due to the unknown coverage of the substrate on the NPs. The QCM results reveal, however, that the number of CdS NPs that associate with the surface is ca. 10-fold higher as compared to the number of Pt NPs that bind to the surface after reaction with tyrosinase for the

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detection limit of tyrosinase (U)

Electrochemical Methods ferrocene-tethered peptidesa ISFET devicesb Pt nanoparticles (present study)

0.04 0.25 1

Optical Methods semiconductor quantum dotsc CdS nanoparticles (present study)

0.2 0.1

Ref 5. b Ref 6. c Ref 3.

same time interval. These results suggest that the biocatalytic turnover of tyrosinase on the substrate associated with the CdS NPs is ca. 10-fold higher than on the Pt NPs, consistent with the observed detection limits of the two assays. In conclusion, the present study has introduced different methods to analyze the activity of tyrosinase through the biocatalyzed oxidation of the tyrosine methyl ester-functionalized Pt or CdS NPs. The oxidation of the (2)-modified Pt NPs and the subsequent coordination of the resulting catechol-(3)-functionalized Pt NPs to the boronic acid monolayer functionalized electrode enabled the amplified analysis of TR activities by the electrocatalyzed reduction of H2O2 by the Pt NP labels. Similarly, we introduced the use of the (2)-modified CdS NPs for the photoelectrochemical analysis of TR through the association of the enzyme-generated (3)-functionalized CdS NPs to the boronic acid monolayer-functionalized electrode. Illumination of the CdS-NPsfunctionalized electrode generated photocurrents in the presence of TEOA as a sacrificial electron donor. The QCM method provided a complementary tool to characterize the association of the Pt NPs and CdS NPs to the boronic acid monolayerfunctionalized Au electrode. Besides the quantitative characterization of the surface coverage of the NPs on the piezoelectric crystal, the frequency change, and the accompanying mass change on the crystal, yielded an additional readout signal for the activity of TR. Table 3 compares the detection limits of the two analytical methods discussed in the present study to other electrochemical or optical procedures to detect TR. The electrochemical method using the Pt-functionalized NPs is the least sensitive method among the three electronic sensors for analyzing TR, yet reveals the most simple and inexpensive configuration. The photoelectrochemical detection of TR activity reveals high sensitivity as compared to the electrochemical sensors. ACKNOWLEDGMENT Parts of this research are supported by the NanoSci-ERA (NanoLICHT project) and the Converging Technologies Research Fund (Israel Science Foundation). Received for review November 22, 2007. Accepted January 18, 2008. AC702401V