Anal. Chem. 2008, 80, 1141-1145
Utilizing a CdTe Quantum Dots-Enzyme Hybrid System for the Determination of Both Phenolic Compounds and Hydrogen Peroxide Jipei Yuan, Weiwei Guo, and Erkang Wang*
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Graduate School of the Chinese Acadamy of Sciences, Changchun, Jilin 130022, China
In this paper, we attempt to construct a simple and sensitive detection method for both phenolic compounds and hydrogen peroxide, with the successful combination of the unique property of quantum dots and the specificity of enzymatic reactions. In the presence of H2O2 and horseradish peroxidase, phenolic compounds can quench quantum dots’ photoluminescence efficiently, and the extent of quenching is severalfold to more than 100-fold increase. Quinone intermediates produced from the enzymatic catalyzed oxidation of phenolic compounds were believed to play the main role in the photoluminescence quenching. Using a quantum dots-enzyme system, the detection limits for phenolic compounds and hydrogen peroxide were detected to be ∼10-7 mol L-1. The coupling of efficient quenching of quantum dot photoluminescence by quinone and the effective enzymatic reactions make this a simple and sensitive method for phenolic compound detection and great potential in the development of H2O2 biosensors for various analytes. Semiconductor nanoparticles (or quantum dots, QDs), due to their quantum confinement of electrons and the size-dependent light emitting with high efficiency under photoexcitation, have gained increasing interest in the fundamental studies of QDs for two decades. The unique tunable property of QDs makes them promising tools in biological and biomedical research, electronics, and light-emitting devices.1-3 QD-based chemical assay, especially a bioassay, has become one of the most exciting forefront fields in analytical chemistry. The size similarity of QDs and biomolecules paves the way to design biosensors with a combination of the unique property of QDs and the selective binding and catalytic functions of biomolecules.4-7 Many researchers have made efforts toward the development of QD-based optical sensors including * To whom correspondence should be addressed. Tel: +86-431-85262003. Fax: +86-431-85689711. E-mail:
[email protected]. (1) Green, M. Angew. Chem., Int. Ed. 2004, 43, 4129-4131. (2) Klostranec, J. M.; Chan, W. C. W. Adv. Mater. 2006, 18, 1953-1964. (3) Zhou, M.; Chang, S.; Grover, C. P. Opt. Express 2004, 12, 2925-2931. (4) Luo, X.; Morrin, A.; Killard, A. J.; Smyth, M. R. Electroanalysis 2006, 18, 319-326. (5) Shi, J.; Zhu, Y.; Zhang, X.; Baeyens, W. R. G.; Garcia-Campana, A. M. Trends Anal. Chem. 2004, 23, 351-360. (6) Willner, I.; Willner, B.; Katz, E. Bioelectrochemistry 2007, 70, 2-11. (7) Willner, I.; Baron, R.; Willner, B. Biosens. Bioelectron. 2007, 22, 18411852. 10.1021/ac0713048 CCC: $40.75 Published on Web 01/15/2008
© 2008 American Chemical Society
fluorescence resonance energy transfer-based sensors for detecting metal ions, small molecules, and biomolecules such as DNA and enzymes.8-16 As the photoluminescence (PL) property of QDs is strongly dependent upon the nature of the surface,17,18 the interactions between given chemical species and the surface of QDs would result in changes in the PL efficiency. It was reported that electron/hole acceptors absorbed on the QD surface exhibited efficient quenching effects on the QD PL intensity.19-23 This unique feature of QDs can be highly exploitable in the development of novel optical detection methods based on QDs for specific analytes.24 The utilization of QDs for the sensitive determination of analytes with importance is still in the initial stage and steadily increasing. Phenolic compounds are among the major classes of pollutants produced by industrial and agricultural activities. Many of these compounds and their derivatives are extremely harmful to humans through oral, dermal, or respiratory tracks and thus represent a serious environmental problem.25,26 To achieve sensitive and fast (8) Spanhel, L.; Haase, M.; Weller, H.; Henglein, A. J. Am. Chem. Soc. 1987, 109, 5649-5655. (9) Isarov, A. V.; Chrysochoos, J. Langmuir 1997, 13, 3142-3149. (10) Moore, D. E.; Patel, K. Langmuir 2001, 17, 2541-2544. (11) Chen, Y.; Rosenzweig, Z. Anal. Chem. 2002, 74, 5132-5138. (12) Huang, C-P.; Li, Y-K.; Chen, T-M. Biosens. Bioelectron. 2007, 22, 18351838. (13) Ji, X.; Zheng, J.; Xu, J.; Rastogi, V. K.; Cheng, T-C.; DeFrank, J. J.; Leblanc, R. M. J. Phys. Chem. B 2005, 109, 3793-3799. (14) Medintz, I. L.; Clapp, A. R.; Mattoussi, H.; Goldman, E. R.; Fisher, B.; Mauro, J. M. Nat. Mater. 2003, 2, 630-638. (15) Oh, E.; Hong, M-Y.; Lee, D.; Nam, S-H.; Yoon, H. C.; Kim, H-S. J. Am. Chem. Soc. 2005, 127, 3270-3271. (16) Shi, L.; Rosenzweig, N.; Rosenzweig, Z. Anal. Chem. 2007, 79, 208-214. (17) Brus, L. E. J. Chem. Phys. 1983, 79, 5566-5571. (18) Wang, Z. L. Characterization of Nanophase Materials, 1st ed.; Wiley-VCH Verlag GmbH: Weinheim, 2000. (19) Hasselbarth, A.; Eychmueller, A.; Weller, H. Chem. Phys. Lett. 1993, 203, 271-276. (20) Hagfeldt, A.; Gratzel, M. Chem. Rev. 1995, 95, 49-68. (21) Burda, C.; Green, T. C.; Link, S.; El-Sayed, M. A. J. Phys. Chem. B 1999, 103, 1783-1788. (22) Landes, C.; Burda, C.; Braun, M.; El-Sayed, M. A. J. Phys. Chem. B 2001, 105, 2981-2986. (23) Suchetti, C. A.; Lema, R. H.; Hamity, M. J. Photochem. Photobiol. A 2005, 169, 1-8. (24) Costa-Fernandez, J. M.; Pereiro, R.; Sanz-Medel, A. Trends Anal. Chem. 2006, 25, 207-218. (25) Abdullah, J.; Ahmad, M.; Heng, L. Y.; Karuppiah, N.; Sidek, H. Talanta 2006, 70, 527-532. (26) Gianfreda, L.; Iamarino, G.; Scelza, R.; Rao, M. A. Biocatal. Biotransform. 2006, 24, 177-187.
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sensorswithelectrochemiluminescence/electrochemicaldetection,32-34 the detection limit of this method was similar but the linear range was much wider. Also, the physiological buffer with neutral pH value used in this system is more biocompatible ensuring the enzymes keep their best activity.
Figure 1. Chemical structures of phenolic compounds investigated.
determination of phenol pollutants is still an extensively researched area. The peroxidase/H2O2 system gained intensive attention in the areas of both the sensing and biological removal of phenolic pollutants.26 Several kinds of peroxidase can catalyze the oxidation of phenolic pollutants to produce quinone intermediates and then nontoxic oligomers.27,28 The principle that quinone intermediates (electron acceptors) produced from the peroxidasecatalyzed oxidation of phenolic compounds27 can quench QD PL intensity efficiently12 is the basis to develop a QD-based detection method for phenolic compounds in this paper. In this paper, with a combination of the unique PL property of QDs that is highly dependent on the surface and the selective catalytic function of an enzyme, detection protocols for both phenolic compounds and hydrogen peroxide were developed with horseradish peroxidase (HRP, EC 1.11.1.7) chosen as a model enzyme. Quenching effects of phenolic compounds, with their structure shown in Figure 1, on QD PL under peroxidase-catalyzed oxidation were investigated. More efficient PL quenchers of QD PL (quinone or quinone intermediates) were enzymatic catalytically transformed from phenolic compounds with quenching effects elevated to severalfold or more than 100-fold. Relative low detection limits at 10-7 mol L-1 levels for phenolic compounds can be obtained. The detection limits for phenolic compounds are lower than the U.S. Environmental Protection Agency estimated wastewater discharge limit of 0.5 mg L-1 (at 10-6 mol L-1 level) and low enough to detect the common levels of phenolic pollutant in wastewater.29,30 Taking advantages of the facile water-soluble CdTe QDs preparation approach developed by our group previously,31 and the fast and efficient enzymatic reaction, the determination system was simple and the detection time was greatly shortened as intensive quenching effects can be observed once the analytes are added. With the features of facility, sensitivity, and wide quantitative concentration range (3 orders of magnitude), this promising method permitted the rapid screening of a family of pollutants. H2O2-sensitive biosensor has been used widely in the detection of analytes that can be oxidated with the catalysis of oxidase to produce H2O2. The sensitive detection of H2O2 is of great importance in the development of competent detection methods for such analytes. In the presence of H2Q/HRP, the QD PL intensity can be quenched with the addition of H2O2; based on which, there is great potential in the development of enzymeQD-based H2O2 biosensors. Compared with the QD-based H2O2 (27) Husain, Q.; Jan, U. J. Sci. Ind. Res. India 2000, 59, 286-293. (28) Aitken, M. D.; Massey, I. J.; Chen, T. P.; Heck, P. E. Water Res. 1994, 28, 1897-1889. (29) Sal, C. S. A.; Boaventura, R. A. R. Biochem. Eng. J. 2001, 9, 211-213. (30) Giti, E.; Mehdi, H.; Nasser, G. Int. Biodeterior. Biodegrad. 2005, 56, 231235. (31) Bao, H. F.; Wang, E. K.; Dong, S. J. Small 2006, 2, 476-480.
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EXPERIMENT SECTION Chemicals. All the starting materials of CdTe QDs synthesis were obtained from commercial suppliers and were used without further purification. Mercaptosuccinic acid was obtained from Aldrich Chemical (Milwaukee, WI). 3-Hydroxytyramine hydrochloride (dopamine hydrochloride) was purchased from Fluka (Milwaukee, WI). HRP was obtained from Roche. All the solutions were prepared with water purified by a Milli-Q system (Millipore, Bedford, MA). Other chemicals are at least analytical grade. Stock solutions of 0.1 mol L-1 hydroquinone, phenol, and dopamine were freshly prepared daily. Instrumentation. UV/vis absorption spectra were recorded on a Cary 500 UV-vis-near-IR Varian spectrophotometer. Fluorescence experiments were recorded on a Perkin-Elmer LS 55 luminescence spectrometer. Synthesis of Water-Soluble CdTe Quantum Dots. CdTe QDs stabilized by mercaptosuccinic acid were synthesized according to a previously reported method.31 Briefly, 4 mL of 0.04 mol L-1 cadmium chloride was diluted to 50 mL in a one-necked flask, and trisodium citrate dihydrate (0.2 g), mercaptosuccinic acid (0.1 g), Na2TeO3 (0.01 mol L-1, 2 mL), and NaBH4 (0.075 g) were added with stirring. The mixture was reacted at 90 °C under open-air conditions for a certain period of time. The obtained QDs were precipitated with ethanol, and the precipitates were separated by centrifugation and were redissolved in 20 mmol L-1 Tris-HCl buffer solution. Fluorescence Experiments. Working solutions were prepared by diluting aliquots of stock solutions and QDs with TrisHCl buffer. Under the excitation wavelength of 400 nm, the fluorescence spectra of QDs were recorded. The slot widths of the excitation and emission were both 10.0 nm. RESULTS AND DISCUSSION Quenching of QD PL by 1,4-Benzoquinone. In this work, CdTe QDs colloid was dispersed in a 20 mmol L-1 Tris-HCl buffer solution (pH 7.4), which comprises 50 mmol L-1 trisodium citrate dihydrate, in order to stabilize QDs. The size and the concentration in stock solution of QDs was calculated to be 3.27 nm and 4.9 µmol L-1, respectively, based on the QD UV-vis absorbance spectrum according to previous literature.35 It has been reported that electron acceptors absorbed on the surface of QDs can quench the exciton emission of QDs.19-21 The Burda group has investigated the bleach recovery of CdSe nanoparticles with 1,4-benzoquine (BQ), chosen as a classic electron acceptor, and their results revealed that quinone shuttled the electron from the conduction band to the valence band of the excited QD, resulting in the quenching of QD PL intensity.21 The quenching effect of BQ on QD fluorescence intensity was investigated in the present work. BQ was added into QD colloid (32) Zou, G.; Ju, H. Anal. Chem. 2004, 76, 6871-6876. (33) Xu, Y.; Liang, J.; Hu, C.; Wang, F.; Hu, S.; He, Z. J. Biol. Inorg. Chem. 2007, 12, 421-427. (34) Jie, G.; Liu, B.; Miao, J.; Zhu, J. Talanta 2007, 71, 1476-1480. (35) Yu, W. W.; Qu, L.; Guo, W.; Peng, X. Chem. Mater. 2003, 15, 2854-2860.
Figure 2. (A) PL spectra representing QDs (a) and the kinetic study of quenching effects of 0.5 mmol L-1 BQ on QD PL with the incubation time of 0, 2.5, 5.0, 7.5, 10.0, and 12.5 min (b-g). (B) Relationship between PL intensity and the incubation time.
Figure 4. Quenching effects of H2Q in the absence/presence of H2O2/HRP. (A) PL spectra of QDs (a), QDs with the addition of 1 mmol L-1 H2Q (b), 1 mmol L-1 H2Q and 1 mmol L-1 H2O2 (c), and 1 mmol L-1 H2Q, 1 mmol L-1 H2O2, and 5 µg mL-1 HRP in 20 mmol L-1 Tris-HCl buffer (pH 7.4) with the incubation time of 5 min (d). (B) Kinetic study of the quenching of PL intensity by 1 mmol L-1 H2Q and 1 mmol L-1 H2O2 in the absence/presence of 5 µg mL-1 HRP.
quinone (H2Q) with the HRP-catalyzed oxidation by H2O2 was investigated, as H2Q can be transformed to benzoquinone through the HRP catalytic oxidation by H2O2 shown in the following equation.36 HRP
H2O + H2O2 y\z BQ + 2H2O
Figure 3. (A) PL spectra representing the quenching of BQ with the concentrations of 0, 1.0 × 10-6, 1.0 × 10-5, 5.0 × 10-5, 1.0 × 10-4, 2.0 × 10-4, 3.0 × 10-4, 4.0 × 10-4, 6.0 × 10-4, 8.0 × 10-4, 1.0 × 10-3, and 1.5 × 10-3 mol L-1 (a-l). (B) Linear relationship between P0/P and concentration of BQ.
and the mixture underwent a fluorescence experiment immediately. Figure 2 shows the quenching effects on QD PL in the presence of 0.5 mmol L-1 BQ with different incubation times. BQ presents efficient quenching on QD PL intensity as the PL intensity decreased from 702 to ∼120 when BQ was added. The quenching effect strengthened with incubation time and PL intensity decreased to ∼10 after ∼6 min. Figure 3 illustrates the PL spectra (Figure 3A) as well as P0/P (Figure 3B, P0 and P refer to the PL intensity in the absence/presence of quencher) of CdTe QDs in the presence of different concentrations of BQ, respectively. An efficient quenching effect by BQ was obtained as 1.0 mmol L-1. BQ can produce a quenching extent of ∼99.1%, and also when BQ concentration was 1.0 × 10-8 mol L-1, a quenching extent of 1.0% can be observed. A linear relationship between P0/P and the concentration of BQ was obtained within a 4 orders magnitude linear range from 1.0 × 10-7 to 1.5 × 10-3 mol L-1 as shown in Figure 3B. Intensive quenching of QD PL intensity by BQ and the wide linear range makes it possible to develop a competent detection method for phenolic compounds. QD PL Quenching by Hydroquinone in the Presence of HRP/H2O2. As a model system, the quenching effect of hydro-
(1)
H2Q exhibits no intensive quenching of QD PL intensity as shown in Figure 4. When mixed with H2O2, an inhibition extent of ∼20% was observed, as a spot of H2Q was oxidated by H2O2 to BQ, which was a more intensive PL quencher. When 2.5 µg mL-1 HRP enzyme was added into the QD-H2Q-H2O2 system, a sharp drop (with a quenching extent of 98%) of PL intensity of QDs was detected immediately. And we can also observe from Figure 4B that when the QDs mixed with H2O2 and H2Q, PL intensity decreased linearly with the incubation time; while in the presence of HRP, a sharp decrease of PL intensity was observed. This indicates that, with the catalysis of HRP, the oxidation of H2Q by H2O2 becomes more efficient, BQ with high concentration can be produced in a short time, and the quenching effects have a ∼9-fold elevation as shown in Table 1. A previous study has reported that enough H2O2 could destroy the lattice structure of CdTe NCs completely in alkaline solution.37 In order to testify that the intensive quenching effect was produced from the addition of H2O2, the influence of H2O2 on PL intensity of QDs in Tris-HCl buffer with a neutral pH value was studied. Results showed that H2O2 at low concentrations (
