Coreactant Enhanced Anodic Electrochemiluminescence of CdTe

decade water-soluble and biocompatible QDs have been prepared4–6 and used as ... by using H2O2,15–17 S2O8 ... work proposed an energy-transfer mec...
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Anal. Chem. 2008, 80, 5377–5382

Coreactant Enhanced Anodic Electrochemiluminescence of CdTe Quantum Dots at Low Potential for Sensitive Biosensing Amplified by Enzymatic Cycle Xuan Liu and Huangxian Ju* Key Laboratory of Analytical Chemistry for Life Science (Ministry of Education of China), Department of Chemistry, Nanjing University, Nanjing 210093, P. R. China This work used sulfite as a coreactant to enhance the anodic electrochemiluminescence (ECL) of mercaptopropionic acid modified CdTe quantum dots (QDs). This strategy proposed the first coreactant anodic ECL of QDs and led to a sensitive ECL emission of QDs in aqueous solution at relatively low potential. In the presence of dissolved oxygen, the stable ECL emission resulted from the excited QDs. Thus, an ECL detection method was proposed at +0.90 V (vs Ag/AgCl) based on the quenching of excited QDs by the analyte. Using tyrosine as a model compound, whose electrooxidized product could quench the excited QDs and thus the ECL emission, an analytical method for detection of tyrosine in a wide concentration range was developed. Furthermore, by combining an enzymatic cycle of trace tyrosinase to produce the oxidized product with an energy-transfer process, an extremely sensitive method for ECL detection of tyrosine with a subpicomolar limit of detection was developed. The sulfite-enhanced anodic ECL emission provided an alternative for traditional ECL light emitters and a new methodology for extremely sensitive ECL detection of mono- and dihydroxybenzenes at relatively low anodic potential. This strategy could be easily realized and opened new avenues for the applications of QDs in ECL biosensing. Quantum dots (QDs) have attracted considerable attention due to their unique electric, magnetic and optical properties.1–3 In past decade water-soluble and biocompatible QDs have been prepared4–6 and used as photoluminescence (PL) probes for cellular imaging,7,8 * To whom correspondence should be addressed. E-mail: [email protected]. Tel. and Fax: +86-25-83593593. (1) Yin, Y. D.; Alivisatos, A. P. Nature 2005, 437, 664–670. (2) Alivisatos, A. P. Science 1996, 271, 933–937. (3) Burda, C.; Chen, X. B.; Narayanan, R.; El-Sayed, M. A. Chem. Rev. 2005, 105, 1025–1102. (4) Chan, W. C. W.; Nie, S. M. Science 1998, 281, 2016–2018. (5) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013–2016. (6) Liu, G. D.; Wang, J.; Kim, J.; Jan, M. R. Anal. Chem. 2004, 76, 7126–7130. (7) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat. Mater. 2005, 4, 435–446. (8) Zheng, Y. G.; Gao, S. J.; Ying, J. Y. Adv. Mater. 2007, 19, 376–380. 10.1021/ac8003715 CCC: $40.75  2008 American Chemical Society Published on Web 06/04/2008

immunoassays9 and enzyme-based bioanalysis.10,11 Due to the controllable merits of electrochemical methods, electrochemiluminescence (ECL) of II-VI QDs has been extensively studied in both organic12–14 and aqueous media.15–19 All these ECL emissions observed in aqueous medium are produced in a cathodic process by using H2O2,15–17 S2O82- 18 and O219 as coreactants. They have been applied in constructing immunosensor for low-density lipoprotein18 and enzyme-based ECL biosensor for glucose.19 In view of the importance of anodic ECL in bioanalysis, our previous work proposed an energy-transfer mechanism for anodic ECL of mercaptopropionic acid (MPA)-modified CdTe QDs at an indium tin oxide (ITO) electrode to detect catechol derivatives.20 The ECL analytical technique has many advantages over PL techniques, such as low cost, wide range of analytes, and high sensitivity. A series of coreactant ECL analytical methods have been developed for clinical diagnostics, environmental assays such as food and water testing, and biowarfare agent detection.21 The coreactant ECL technique includes two main systems, i.e., ruthenium complex with amine-containing compounds as coreactants22–24 and a luminol-H2O2 system.25 The overwhelming majority of commercially available tests are based on the anodic ECL of Ru(bpy)32+-tripropylamine system.21 However, the high oxidation potential needed for producing the anodic ECL emission of Ru(bpy)32+/3+ systems often suffers undesirable anodic reac(9) Lingerfelt, B. M.; Mattoussi, H.; Goldman, E. R.; Mauro, J. M.; Anderson, G. P. Anal. Chem. 2003, 75, 4043–4049. (10) Shi, L. F.; Rosenzweig, N.; Rosenzweig, Z. Anal. Chem. 2007, 79, 208– 214. (11) Gill, R.; Freeman, R.; Xu, J. P.; Willner, I.; Winograd, S.; Shweky, I.; Banin, U. J. Am. Chem. Soc. 2006, 128, 15376–15377. (12) Myung, N.; Ding, Z. F.; Bard, A. J. Nano Lett. 2002, 2, 1315–1319. (13) Myung, N.; Bae, Y.; Bard, A. J. Nano Lett. 2003, 3, 1053–1055. (14) Bae, Y. J.; Myung, N.; Bard, A. J. Nano Lett. 2004, 4, 1153–1161. (15) Zou, G. Z.; Ju, H. X. Anal. Chem. 2004, 76, 6871–6876. (16) Ding, S. N.; Xu, J. J.; Chen, H. Y. Chem. Commun. 2006, 3631–3633. (17) Jiang, H.; Ju, H. X. Anal. Chem. 2007, 79, 6690–6696. (18) Jie, G. F.; Liu, B.; Pan, H. C.; Zhu, J. J.; Chen, H. Y. Anal. Chem. 2007, 79, 5574–5581. (19) Jiang, H.; Ju, H. X. Chem. Commun. 2007, 404–406. (20) Liu, X.; Jiang, H.; Lei, J. P.; Ju, H. X. Anal. Chem. 2007, 79, 8055–8060. (21) Richter, M. M. Chem. Rev. 2004, 104, 3003–3036. (22) Yuan, J. P.; Li, T.; Yin, X. B.; Guo, L.; Jiang, X. Z.; Jin, W. R.; Yang, X. R.; Wang, E. K. Anal. Chem. 2006, 78, 2934–2938. (23) Zhan, W.; Bard, A. J. Anal. Chem. 2007, 79, 459–463. (24) Li, J. G.; Yan, Q. Y.; Ju, H. X. Anal. Chem. 2006, 78, 2694–2699. (25) Wilson, R.; Clavering, C.; Hutchinson, A. Anal. Chem. 2003, 75, 4244– 4249.

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tions, which limit the analytical application26 and prevent the further development of ECL analysis. Although great effort has focused on reducing the anodic ECL potential,26,27 little progress has been made for the analytical application. Thus, it is necessary to develop new anodic ECL systems at relatively low redox potential for improving the sensitivity and selectivity of ECL analysis and extending ECL to different application fields.21 This work observed an enhanced anodic ECL of MPA-modified CdTe QDs at relatively low anodic potential by using sulfite as a coreactant. Although the anodic ECL of CdTe QDs has been reported on an ITO working electrode in air-saturated solution,20 the relatively high anodic potential (+1.17 V vs Ag/AgCl) and weak alkaline condition (pH 9.3) limit the application of this system in bioanalysis. In neutral medium, the ECL is so weak that the quenching effect is undetectable for analytical performance. Thus, we herein further studied the effect of coreactants on the anodic ECL emission of CdTe QDs in aqueous solution. In the presence of dissolved oxygen sulfite could greatly enhance the weak anodic ECL emission of CdTe QDs at a paraffin-impregnated graphite electrode (PIGE), which occurred at relatively low anodic potential (+0.89 V vs Ag/AgCl) and showed a strong ECL emission in pH 7.5 buffer solution. To our best knowledge, it is the first report of an enhancer for anodic ECL of QDs. The proposed sulfite-coreactant ECL emission resulted from the excited QDs. Thus, a novel strategy for detecting the quenchers of excited QDs or their precursors was designed. Using tyrosine as a model compound, its electrooxidative product could quench the excited QDs by an energy-transfer process. The sulfiteenhanced ECL emission was sensitive enough for biosensing of tyrosine down to 46 nM in neutral medium. In order to further amplify the quenching signal and improve the sensitivity for many application purposes, because the amounts of many biologically important analytes available for analysis are extremely limited,28,29 this work further designed an enzymatic cycle to produce the oxidized product using tyrosinase30 and developed an extremely sensitive method for ECL detection of tyrosine with a subpicomolar limit of detection. This result made QDs a potential alternative for traditional ECL emitters as QDs are also reproducible through ECL process.31 Moreover, the size-dependent properties of QDs2 would provide the emitter promising behaviors for ECL application. This work opened new avenues for applying QDs in highly sensitive detection. EXPERIMENTAL SECTION Reagents. MPA, superoxide dismutase (SOD, EC 1.15.1.1, from bovine erythrocytes, 4200 units mg-1 solid), tyrosinase mushroom (EC 1.14.18.1, 5370 units mg-1 solid) were purchased from Sigma Chemical Co. (St. Louis, MO). Cadmium chloride (CdCl2 · 2.5H2O) was purchased from Alfa Aesar China Ltd. (26) Li, F.; Zu, Y. B. Anal. Chem. 2004, 76, 1768–1772. (27) Cao, W. D.; Ferrance, J. P.; Demas, J.; Landers, J. P. J. Am. Chem. Soc. 2006, 128, 7572–7578. (28) Ao, L. M.; Gao, F.; Pan, B. F.; He, R.; Cui, D. X. Anal. Chem. 2006, 78, 1104–1106. (29) Bao, P.; Frutos, A. G.; Greef, C.; Lahiri, J.; Muller, U.; Peterson, T. C.; Warden, L.; Xie, X. Y. Anal. Chem. 2002, 74, 1792–1797. (30) Power, O. P.; Ritchle, I. M. Anal. Chem. 1982, 54, 1985–1987. (31) Ding, Z.; Quinn, B. M.; Haram, S. K.; Pell, L. E.; Korgel, B. A.; Bard, A. J. Science 2002, 296, 1293–1297.

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Figure 1. UV-vis absorption spectra of MPA-modified CdTe QDs solutions prepared with refluxed times of 60, 80, 100, 140, 200, 300, 420, and 600 min.

Tellurium powder and sodium borohydride were obtained from Sinopharm Chemical Reagent Co. Ltd. Tyrosine was purchased from Sunshine, and sodium sulfite was from Merck’s Reagenzien. All the reagents were of analytical grade and used as received. The 0.1 M pH 7.5 phosphate buffer solution containing 0.1 M KNO3 (PBS) was used throughout the work. Doubly distilled water was used throughout. The MPA-modified CdTe QDs were prepared according to the previous report and confirmed by Fourier transform infrared (FT-IR) spectra.20 Apparatus. The electrochemical and ECL measurements were carried out on a MPI-A multifunctional electrochemical and chemiluminescent analytical system (Xi’an Remex Analytical Instrument Ltd. Co.) at room temperature with a configuration consisting of a PIGE as working electrode, a platinum counter electrode, and an Ag/AgCl (saturated KCl solution) reference electrode. PIGE (6.0-mm diameter) was homemade.15 During measurements, anodic potential supplied by the MPI-A electrochemical analyzer was applied to the PIGE working electrode by a cyclic voltammetric (CV) technique with a potential range from 0 to +1.10 V. At the same time, the ECL emission was recorded by the MPI-A multifunctional chemiluminescence analyzer. The emission intensity was detected at +0.90 V. The emission window was placed in front of the photomultiplier tube (detection range from 300 to 650 nm) biased at -1000 V. The ECL spectra were obtained by collecting the ECL data at +0.90 V during the cyclic potential sweep with seven pieces of filters at 510, 535, 550, 565, 580, 600, and 650 nm. Their thickness was 2 mm and transparent efficiency was ∼88%. PL experiments were performed on a Jasco FP 820 fluorometer (Jasco Co.). The UV-vis absorption spectra were obtained on Shimadzu UV-3600 UV-vis-NIR photospectrometer (Shimadzu Co.). FT-IR spectra were recorded on Nicolet 400 FT-IR spectrometer (Madison, WI).

RESULTS AND DISCUSSION Characterization and ECL Behaviors of As Prepared MPAModified CdTe QDs. Figure 1 shows the UV-vis spectra of eight as-prepared QDs samples obtained in a batch. The absorption wavelength of these samples increased from 498 to 607 nm with the increasing refluxed time from 1 to 10 h. The diameter of QD particles and concentration of QDs solution could be estimated

Figure 2. ECL (A) and CV (B) curves of 0.2 µM MPA-modified CdTe QDs (black), 1.0 mM Na2SO3 (blue), 0.2 µM QDs + 0.4 mM Na2SO3 (red), and 0.2 µM QDs + 1.0 mM Na2SO3 (green) in air-saturated pH 7.5 PBS at 100 mV s-1.

from the first adsorption peak in the UV-vis spectrum and several empirical equations below:32 for diameter: d ) 9.8127 × 10-7λ3 - 1.7147 × 10-3λ 2 + 1.0064λ - 194.84 (nm) for concentration: c ) A/(10043d 2.12 L) Here, d is the size of a given nanocrystal sample, λ (nm) and A are the wavelength and absorbance of the first excitonic absorption peak of the corresponding sample, respectively, c is the molar concentration (mol L-1) of the QD solution, and L is the path length (cm) of the radiation beam used for recording the absorption spectrum. In this work, L was fixed at 0.5 cm. The diameters of QD particles and concentrations of QDs solutions shown in Figure 1 were 2.3, 2.5, 2.8, 3.0, 3.2, 3.4, 3.5, and 3.7 nm, and 7.2, 5.1, 3.5, 2.9, 3.5, 2.0, 1.9, and 1.5 µmol L-1, respectively. In air-saturated pH 7.5 PBS, the cyclic voltammogram of 0.2 µmol L-1 QDs at PIGE showed an irreversible anodic peak at +0.826 V, which was coincident with the previous report.33 However the oxidation process was different from the reported mechanism,33 in which the binding of thiol molecules used as stabilizing agents to a gold electrode surface led to destruction of the shell-core structure of QDs, and the rest of the CdTe core was then oxidized to produce Cd2+ and TeO32-. Here the shell-core structure of QDs could be preserved at PIGE. Thus, the oxidation of thiol-capped CdTe NCs produced holes injected QDs, i.e., CdTe(h+)/CdSR, due to the protection of stabilizing agents (MPA), which led to an ECL emission with an onset potential of +0.75 V (Figure 2, black curve). The ECL emission was so weak that it could not be used for analytical purposes. However, after 0.4 mM Na2SO3 was added into this solution, the emission peak from QDs at +0.89 V was enhanced by 18 times, accompanied with the increasing of CV current peak (Figure 2, red curve). A control experiment showed that although sulfite could also be oxidized at potentials more than +0.4 V, it did not (32) Yu, W. W.; Qu, L. H.; Guo, W. Z.; Peng, X. G. Chem. Mater. 2003, 15, 2854–2860. (33) Poznyak, S. K.; Osipovich, N. P.; Shavel, A.; Talapin, D. V.; Gao, M. Y.; Eychmu ¨ ller, A.; Gaponik, N. J. Phys. Chem. B 2005, 109, 1094–1100.

Figure 3. ECL intensity of eight 0.2 µM QD samples with different sizes prepared in a batch in pH 7.5 PBS without (A) and with (B) the presence of 1.0 mM Na2SO3. ECL intensity data were collected at +0.90 V.

Figure 4. Dependence of ECL intensity of 0.2 µM QDs on sodium sulfite concentration. The ECL data were collected at +0.90 V.

show any ECL response (Figure 2, blue curve). Thus, the increasing ECL emission resulted from the sensitized effect of sulfite. At the size of 3.4 nm, the QDs could produce the maximum ECL intensity in the absence and presence of Na2SO3 (Figure 3). This was due to the dependence of ECL intensity on both the QD band gap for electron injection and the electron-transfer rate. With increasing QD size, the QD band gap decreased,32 leading to faster electron injection to the surface states of QDs and greater ECL intensity. However, when the nanoparticles were larger than 3.4 nm, the ECL intensity decreased quickly due to the decreasing surface-to-volume ratio of QDs, which slowed the electron-transfer rate between QDs and the working electrode.17 The compromise of these factors led to a maximum ECL intensity at 3.4 nm. Thus, the solution of 3.4-nm QDs was used for following experiments. Optimization of Sodium Sulfite Concentration. The enhanced ECL emission was related to the concentration of Na2SO3. High concentration of Na2SO3, e.g., 1.0 mM, would weaken the enhancing action (Figure 2, green curve), which was attributed to the decrease of dissolved O2 concentration due to the presence of Na2SO3 (seen below). The effective enhancement occurred at the sulfite concentration of 0.4 mM (Figure 4). The stability of the ECL emission also depended on sulfite concentration (Figure 5). High sulfite concentration led to a relatively low consuming rate of sulfite by dissolved oxygen. Thus, Analytical Chemistry, Vol. 80, No. 14, July 15, 2008

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Figure 5. Enhanced ECL intensity of 0.2 µM QD solution in the presence of 1.0 and 0.4 mM (inset) sodium sulfite vs time of exposing the solution to air.

the ECL intensity was more stable at higher sulfite concentration. At a sulfite concentration of 0.4 mM, the ECL intensity decreased by 11 and 16% after the solution was kept in air for 85 and 96 min, respectively (inset, Figure 5). At high SO32- concentration, the decrease was slowed. When the SO32- concentration was 1.0 mM, only a 3.5% decrease was observed after being kept in air for 165 min, indicating better stability (Figure 5). Considering the stability and the need of dissolved oxygen for producing the ECL emission, 1.0 mM sodium sulfite was used for following experiments and biosensing, at which the ECL intensity increased for 12 times (Figure 2A, green curve). Upon consecutive cyclic potential scanning, the emission intensity could maintain at a constant value. Thus, the detection solution contained 0.2 µmol L-1 3.4 nm QDs and 1.0 mM Na2SO3. Mechanism for the Enhanced Anodic ECL of CdTe QDs. When the concentration of coreactant was more than 0.4 mM, the enhancing action weakened (Figure 4). As described previously,20 dissolved oxygen played an importance role during the ECL emission procedure. To confirm this fact, the detection solution was bubbled with highly pure N2 for 30 min to remove the dissolved O2. In air-free detection solution, no ECL emission was observable, even in the presence of Na2SO3 (Figure 6A, curve 1). When the air-free detection solution was then exposed to air, which led to gradually increasing dissolved oxygen, the ECL intensity recovered gradually with on increasing the exposure time (Figure 6A, curve 2), indicating that the ECL emission was dependent on the dissolved O2. This experiment was stopped at the time of 45 min, which was insufficient for forming air-saturated solution, leading to the weakened enhancing action. In air-saturated pH 7.5 PBS, upon addition of SOD, an efficient scavenger of superoxide anion, into the detection solution, the ECL intensity decreased to a large extent, and the decrease increased with increasing SOD concentration (Figure 6B), indicating that superoxide anion was a key species in the ECL emission. Thus, dissolved oxygen participated in the ECL reaction in the species of •O2-. The emission peak of ECL spectrum of 3.4-nm QDs occurred at 589.8 nm, the same position as that of the PL spectrum recorded with the same detection solution (excited at 360 nm) (Figure 7), which was far from the emission range of SO2* light emitter from 300 to 450 nm.34 In addition, two other samples with QD diameters of 2.8 and 2.3 nm showed emission peaks at 549.0 and 536.4 nm, which were also close to the PL (34) Sun, H. W.; Li, L. Q.; Chen, X. Y. Anal. Chim. Acta 2006, 576, 192–199.

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Figure 6. Dependence of ECL intensity enhanced by 1.0 mM Na2SO3 on dissolved O2 (A) and SOD (B) concentration in pH 7.5 PBS containing 0.2 µM QDs collected at +0.90 V. (1) Detection solution was bubbled with highly pure N2 for 30 min; (2) deoxygenated detection solution was exposed to air for different times.

Figure 7. PL (solid line) and ECL (dashed line) spectra of 0.2 µM 3.4-nm QDs in pH 7.5 PBS containing 1.0 mM Na2SO3. The excitation wavelength of PL spectrum is 360 nm, and the ECL data were collected at +0.90 V.

peak positions (not shown), indicating the ECL light emitter was the excited QDs (CdTe/CdSR* or QDs*).19 Furthermore, the UV-vis absorption of QDs solution did not overlap with the emission range of SO2*; thus, the excited QDs were not produced by the energy transfer of SO2*.35 According to those reported previously,12,19,20 the excited QDs could be produced from the combination of hole (QDs (h+)) and electron (QDs (e-)) injected QDs. When QDs*, which were formed by the collision of oxidized QDs (h+) and the reduced QDs (e-), returned to the groundstate accompanied with photon irradiation, the ECL emission occurred. In this potential sweep range 0-+1.10 V, QDs (h+) were generated directly by the electric oxidation of QDs, and QDs (e-) could be formed by the interaction between QDs and the superoxide anion, which injected an electron into the 1Se quantum-confined orbital of MPA-modified CdTe QDs.36 In the proposed system, the coreactant sulfite could accelerate the (35) Clapp, A. R.; Medintz, I. L.; Mauro, J. M.; Fisher, B.; Bawendi, M. G.; Mattoussi, H. J. Am. Chem. Soc. 2004, 126, 301–310. (36) Poznyak, S. K.; Talapin, D. V.; Shevchenko, E. V.; Weller, H. Nano Lett. 2004, 4, 693–698.

formation of superoxide anion,36 thus enhancing the ECL emission. The whole ECL process could be expressed as follows: CdTe ⁄ CdSR - e- f CdTe(h+) ⁄ CdSR

(1)

SO32- - e- f •SO3-

(2)

2OH- + •SO3- + 2O 2 f •O2- + SO42- + H2O

(3)

CdTe ⁄ CdSR + •O2- f CdTe(e-1se ) ⁄ CdSR + O2

(4)

CdTe(h+) ⁄ CdSR + CdTe(e-1se) ⁄ CdSR f CdTe ⁄ CdSR* (5) Simultaneously, the injection of electrons from superoxide anion into the holes injected QDs might also exist: CdTe(h+) ⁄ CdSR + •O2- f CdTe ⁄ CdSR * + O2

(6)

Then CdTe ⁄ CdSR/ f CdTe ⁄ CdSR + hv(589.8 nm)

(7)

Analytical Application of the Anodic ECL of QDs. After tyrosine was added into pH 7.5 PBS containing 0.2 µmol L-1 QDs and 1.0 mM Na2SO3, the anodic ECL emission decreased, indicating a quenching effect. At the electrode surface, tyrosine could be oxidized to produce an o-quinone product.39 The o-quinone compound could interact with the excited QDs and act as a quencher to quench the QDs* by an energy-transfer process.20 Thus, the decrease in ECL intensity could be attributed to the interaction between the electrooxidized product of tyrosine with the excited QDs. With the increasing concentration of tyrosine from 46 nM to 1.4 mM, the ECL emission decreased linearly (R ) 0.991) (inset in Figure 8A), leading to a method for detection of tyrosine. At the concentration of 46 nM, the ECL intensity decreased by 9.4%, which was enough for accurate detection of tyrosine. Enzyme-Sensitized ECL Detection. In order to further increase the detection sensitivity, tyrosinase was used to catalyze the oxidation of tyrosine by dissolved O2 to produce o-quinone (Scheme 1).30 In air-saturated detection solution containing 8.5 U mL-1 tyrosinase, upon successive addition of tyrosine, the ECL intensity at +0.90 V greatly decreased after the solution was set for 2 min. The quenching efficiency was much higher than that in absence of enzyme (Figure 8A). The enzymatically amplified quenching process resulted in a great decrease of the ECL emission even at very low concentration of tyrosine (Figure 8B). Furthermore, at a definite concentration of analyte, with increasing tyrosinase concentration, the decrease of ECL intensity increased due to the increasing enzymatic reaction rate and tended to a constant value (not shown). In this work, 8.5 U mL-1 tyrosinase was used for analytical purpose. (37) Geng, H.; Meng, Z. Q. Spectrochim. Acta, Part A 2006, 64, 87–92. (38) Liang, Y. D.; Gao, W.; Song, J. F. Bioorg. Med. Chem. Lett. 2006, 16, 5328– 5333. (39) Tsai, H.; Weber, S. G. Anal. Chem. 1992, 64, 2897–2903.

Figure 8. Calibration plots for tyrosine detection in the presence and absence (inset) of 8.5 U mL-1 tyrosinase in air-saturated pH 7.5 PBS containing 0.2 µM QDs and 1.0 mM Na2SO3 (A) and ECL curves of 0, 0.1, 1.0, and 100 pM tyrosine in the presence of 8.5 U mL-1 tyrosinase (from high to low) at 100 mV s-1 (B).

Scheme 1. Procedure of Highly Sensitivity Detection of Tyrosine Based on the Enhanced Anodic ECL and Catalytic Oxidation of Tyrosinase in Air-Saturated PBS

Under optimal conditions, the tyrosine quenching amplified by the enzymatic cycle showed a linear range up to 0.2 nM (R ) 0.997) (Figure 8A), with a quenching degree of 17% at 0.1 pM (Figure 8B, red curve), indicating an extremely high sensitivity for detection of tyrosine. The relative standard deviations for five measurements of 0.1 and 100 pM tyrosine were 0.6 and 2.6%, respectively, indicating acceptable reproducibility. Interference Investigation. Although tyrosinase can also catalyze the oxidation of adrenaline, dopamine, and noradrenaline, tyrosine is the only amino acid that can be a substrate for tyrosinase; thus, the enzyme-sensitized method could be specific for solutions containing hydrolyzed proteins or amino acid mixtures.40 The tolerable capacities of four potential interferents, uric acid, ascorbic acid, L-phenylalanine, and L-tryptophan, for the tyrosine detection were examined by measuring the quenching degree of the ECL intensity by the interferents in air-saturated pH 7.5 PBS containing 8.5 U mL-1 tyrosinase, 0.1 nM tyrosine, 1.0 mM Na2SO3, and 0.2 µM QDs. The results were listed in Table 1. The four interferents could be tolerated at least 1000 times, even 10 000 or 20 000 times concentration of tyrosine, indicating that this method had an excellent specificity for highly sensitive detection of tyrosine. Mono- and dihydroxybenzenes could be oxidized to o-benzoquinone in the presence of oxygen and tyrosinase.30,40 Thus, they would interfere with the determination of tyrosine. This problem could be dissolved by coupling with a (40) Rivas, G. A.; Solis, V. M. Anal. Chem. 1991, 63, 2762–2765.

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Table 1. Tolerable Concentration of Interferents in Air-Saturated pH 7.5 PBS Containing 8.5 U mL-1 Tyrosinase, 0.1 nM Tyrosine, 1.0 mM Na2SO3, and 0.2 µM QDs interferent concentration (µM) quenched degree (%)

uric ascorbic acid acid 1 6.5

2 5.2

L-phenylalanine

L-tryptophan

1 9.7

0.1 8.4

separation system. Furthermore, this appearance suggested a potential application of this method in highly sensitive detection of all these substrates. CONCLUSIONS We report a sulfite-enhanced anodic ECL of MPA-modified CdTe QDs in air-saturated aqueous solution. Sulfite acts as a coreactant to sensitize the ECL signal at a relatively low potential of +0.89 V. The enhanced ECL emission is stable and has been proved to result from the excited QDs (CdTe/CdSR*). The electrooxidized product of tyrosine can quench the ECL emission, leading to an analytical method for detection of tyrosine. Further-

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more, with the help of trace tyrosinase, the quenching of ECL emission by tyrosine can be amplified in the presence of dissolved oxygen, producing an extremely sensitive method for anodic ECL detection of tyrosine down to subpicomolar concentrations. This method shows good reproducibility and selectivity. This work provides an alternative for traditional ECL light emitters and a new methodology for extremely sensitive ECL detection of monoand dihydroxybenzene derivatives at relatively low anodic potential. Thus, it opens new avenues for the analytical applications of QDs in ECL biosensing. ACKNOWLEDGMENT We gratefully acknowledge the support of the National Science Fund for Creative Research Groups (20521503), the Key Program (20535010), and Major Research Plan (90713015) from the National Natural Science Foundation of China.

Received for review February 22, 2008. Accepted April 25, 2008. AC8003715