DNAzyme self-assembled gold nanoparticles for determination of metal ions using fluorescence anisotropy assay

Analytical Biochemistry 401 (2010) 47–52

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DNAzyme self-assembled gold nanoparticles for determination of metal ions using fluorescence anisotropy assay Bin-Cheng Yin 1, Peng Zuo 1, Hao Huo, Xinhua Zhong, Bang-Ce Ye * Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China

a r t i c l e

i n f o

Article history: Received 11 November 2009 Received in revised form 9 February 2010 Accepted 11 February 2010 Available online 14 February 2010 Keywords: DNAzyme Gold nanoparticles Copper Lead Fluorescence anisotropy

a b s t r a c t Gold nanoparticles can be exploited to facilitate a highly sensitive and selective metal ion detection based on fluorescence anisotropy assay with metal ion-dependent DNA-cleaving DNAzyme. This assay allows rapid and accurate determination of metal ions in aqueous medium at room temperature. The method has been demonstrated for determination of Cu2+ and Pb2+ ions. The detection sensitivity can be significantly improved to 1 nM by using a ‘‘nanoparticle enhancement” approach. Moreover, the assay was also tested in 384-well plates for high-throughput routine determination of toxic metal ions in environmental samples. The method showed distinct advantages over conventional methods in terms of its potential sensitivity, specificity, and ability for rapid response. Ó 2010 Elsevier Inc. All rights reserved.

Heavy metals are ubiquitous environmental contaminants, and their high toxicity makes their presence undesirable. In general, metal ions can be classified into two categories: essential and nonessential. Nonessential heavy metals, such as lead (Pb)2 and mercury (Hg), can cause a number of adverse health effects even at low-level exposure. Essential metals, such as copper (Cu) and zinc (Zn), are required to support biological activities. However, even these essential metals are toxic in excess [1]. A large number of heavy metals can cause disease or even death, constituting a serious threat to human health. Metal Cu2+ and Pb2+ ions also pose serious environmental problems and are potentially toxic for all living organisms like any other heavy metal. Due to their toxicity, the U.S. Environmental Protection Agency (EPA) has set the maximum allowable level (MAL) of copper and lead in drinking water at 1.3 ppm (20 lM) and 15 ppb (72 nM), respectively. Therefore, accurate determination of these metals at the trace level in environmental and biological samples has become increasingly important. To meet these objectives, a number of analysis methods for the detection of heavy metal ions have been developed over the past few years, including not only atomic absorption spectroscopy [2], vol* Corresponding author. Fax: +86 21 64252094. E-mail address: [email protected] (B.-C. Ye). 1 These authors contributed equally to this work. 2 Abbreviations used: Pb, lead; Hg, mercury; Cu, copper; Zn, zinc; EPA, Environmental Protection Agency; MAL, maximum allowable level; SPR, surface plasmon resonance; FA assay, fluorescence anisotropy assay; AuNP, gold nanoparticle; QCM, quartz crystal microbalance; PBS, phosphate-buffered saline; HEPES, 4-(2-hydroxyerhyl)piperazine-1 erhanesulfonic acid; DTT, dithiothreitol; rA, ribonucleotide adenosine; DEPC, diethyl pyrocarbonate. 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.02.014

tammetric detection [3], and inductively coupled plasma mass spectrometry [4] as classical detection methods but also some other strategies such as sensors [5], surface plasmon resonance (SPR) [6], peptide [7], and peptide-coated quantum dots [8]. Recent studies by the Lu group have delivered fluorescent sensors using DNAzyme for the detection of a number of metal ions. These studies demonstrated high sensitivity and selectivity. For examples, Liu and Lu [9] reported nanoparticle-based colorimetric sensors using DNAzyme-catalyzed ligation reaction for Cu2+ with a detection limit of approximate 5 lM. Furthermore, they reported fluorescent metal sensors using a Cu2+-dependent DNA-cleaving DNAzyme with a detection limit of 35 nM (2.3 ppb) [10]. DNAzyme-based sensors for other metal ions, such as Hg2+ [11,12], Pb2+ [13–17], Zn2+ [18,19], and UO22+ [20], have also been reported. Here we present a novel highly sensitive and selective fluorescence anisotropy assay (FA assay) for detecting Cu2+ and Pb2+ ions based on metal ion-dependent DNAzyme. The detection sensitivity of Cu2+ and Pb2+ ions can be significantly improved to approximately 1 nM (65 and 200 ppt, respectively) by using a ‘‘nanoparticle enhancement” approach that is much lower than the MAL of approximately 20 lM and 72 nM, respectively, in the United States. The strong enhancement of gold nanoparticles (AuNPs) has been studied in different biosensing systems to substantially improve the performance and sensitivity. The Willner group developed a novel method, based on the AuNP-catalyzed deposition of gold, for the amplified microgravimetric quartz crystal microbalance (QCM) detection of DNA [21] and thrombin [22] with excellent detection sensitivity. The group also developed the SPR

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DNAzyme AuNPs for determination of metal ions / B.-C. Yin et al. / Anal. Biochem. 401 (2010) 47–52

method [23] and two electrical methods (Faradaic impedance spectroscopy and ion-sensitive field effect transistor) [24] to detect cocaine using AuNPs as amplifying labels. Yan and coworkers [25] described electrochemical detection by the application of AuNPs as electron relays for the facilitation of the interfacial electron transfer on electrode. Recently, Dong and coworkers [26] reported that the detection sensitivity of single-walled carbon nanotube network field effect transistors for DNA can be significantly improved to approximately 100 fM by using a nanoparticle enhancement approach. Tokarev and coworkers [27] investigated localized plasmon resonance excited in AuNPs coupled with a responsive polymer gel. Driven by these researches, we demonstrate in the current article, for the first time, that the DNAzyme-based FA assay can be used for detection of Cu2+ and Pb2+ ions with excellent sensitivity and selectivity. Materials and methods Materials Hydrogen tetrachloroaurate(III) hydrate (HAuCl43H2O) was purchased from Strem Chemicals (Newburyport, MA, USA). The other chemicals were purchased from Sinopharm Chemcial Reagent (Shanghai, China). All chemicals used in this work were used directly without additional purification. The water was prepared with ultrapure water (18.2 MX/cm) from a Milli-Q water purification system (Millipore, Billerica, MA, USA). The nucleic acids Cu–Enz (50 -GGTAA GCCTGGGCCTCTTTCTTTTTAAGAAAGAAC-30 ), Cu–Sub (50 -SH-T20AG CTTCTTTCTAATACGGCTTACC-FAM-30 ), Pb–Enz (50 -ACAGACATCTCT TCTCCGAGCCGGTCGAAATAGTGAGT-30 ), and Pb–Sub (50 -FAM-ACT CACTATrAGGAAGAGATGTCTGTT20-SH-30 ) were synthesized using a standard procedure and purified by reverse-phase high-performance liquid chromatography (HPLC) from Songon (Shanghai, China). All oligonucleotides were quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Oligos were dissolved in Milli-Q water to a stock concentration of 250 lM before use. The stock solutions of Cu2+ and Pb2+ (500 mM) and other metal ion stock solutions (500 mM) were prepared in Milli-Q water, and we also used Milli-Q water for further dilution. Instrumentation Fluorescence anisotropy was measured by a Genios Pro fluorescence reader with an excitation wavelength at 485 nm and an emission wavelength at 535 nm (Tecan, Männedorf, Switzerland) using a black 96-well microplate (Fluotrac 200, Greiner Bio-One, Frickenhausen, Germany). Fluorescence anisotropy principle The FA assay is an intrinsically powerful technique for the rapid and homogeneous analysis of molecular interactions in biological/ chemical systems. Fluorescence anisotropy is sensitive to changes in the rotational motion of fluorescently labeled molecules. If a molecule is small, it will rotate faster and, hence, will have a smaller anisotropy. Conversely, the larger molecules will have larger anisotropy due to their slow rotation. The relationship among steady-state anisotropy r, fluorescence lifetime s, and rotational correlation time h using the Perrin equation [28]:

r¼

r0 ; 1 þ s=h

ð1Þ

where r0 is the fundamental anisotropy in the absence of rotational diffusion.

The rotational correlation time h is directly related to the volume V of the rotating molecular by:

h¼

gV RT

;

ð2Þ

where g is the viscosity of the solution, V is the molecular volume, T is the Kelvin temperature, and R is the gas constant. The molecular volume is related to the molecular weight by the following equation:

V ¼ mM;

ð3Þ

where v is the partial specific volume (ml/g), and M is the molecular weight of the molecule. DNA-functionalized AuNP preparation AuNPs of 13 nm were prepared by the citrate reduction of HAuCl4 according to a reported literature method [29]. The HAuCl4 solution was added to 3.5 ml of 1% trisodium citrate solution and was rapidly stirred and kept boiling for 20 min. Then the nanoparticle solution was cooled to room temperature and stored at 4 °C before use. AuNPs were functionalized [30,31] with oligonucleotides by adding Cu–Sub or Pb–Sub to aqueous nanoparticle solution (particle concentration 10 nM) to a final oligonucleotide concentration of 3.0 lM. After 16 h of incubation at room temperature, NaCl solution was added (final concentration of 0.1 M) by the stepwise addition of 1 M NaCl/100 mM phosphate-buffered saline (PBS, pH 7.4). The solution was allowed to ‘‘age” under these conditions for an additional 24 h. Excess reagent was then removed by centrifugation at 13,000 rpm and 4 °C for 30 min. Following removal of the supernatant, the red oily precipitate was washed twice with 10 mM PBS buffer (pH 7.4) by successive centrifugation and redispersion and then finally was suspended in stock solution (25 mM Tris–HCl buffer [pH 8.2] and 300 mM NaCl) and stored at 4 °C for further use. The surface coverage of thiol-modified oligonucleotide adsorbed onto AuNPs was determined by a fluorescence-based method [32] that employs dithiothreitol (DTT) to replace thiol/FAM-modified oligos from AuNP surfaces and then detect the released FAM-labeled oligos and was quantified by fluorescence spectroscopy. The surface-loading assay shows that there are approximately 100 DNA strands per as-prepared particle. Thus, the parameters for the AuNP system is set at a stoichiometry of approximately 100 DNA strands per particle. Fluorescence anisotropy measurement Before the experiment for metal ion detection, it is important to indicate that the Pb–Sub is a DNA/RNA chimera with ribonucleotide adenosine (rA) and is not stable and easily degradable like RNA; thus, great care should be taken to avoid inadvertently introducing RNases into the Pb–Sub solution during operation. To create and maintain an RNase-free environment, solutions (water and other solutions) were treated with 0.1% diethyl pyrocarbonate (DEPC). When preparing Tris buffers, water should be treated with DEPC first before dissolving Tris to make the appropriate buffer. The tips and 96-well plate are RNase free and do not require pretreatment to inactivate RNases. The Cu2+ sensor was prepared in 248 ll with a final Cu–Sub concentration of 20 nM (0.2 nM particle concentration, corresponding to 20 nM substrate Cu–Sub) and a Cu–Enz concentration of 100 nM in 1.5 M NaCl and 50 mM 4-(2hydroxyerhyl)piperazine-1 erhanesulfonic acid (HEPES) (pH 7.0). The Pb2+ sensor was prepared as the Cu2+ sensor in 248 ll with a final Pb–Sub concentration of 20 nM (0.2 nM particle concentration, corresponding to 20 nM substrate Pb–Sub) and a Pb–Enz concentration of 100 nM in 100 mM NaCl and 25 mM Tris acetate (pH 8.2). The mixture was warmed to 80 °C for 2 min and allowed

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to cool naturally to room temperature for 30 min. Then 1 ll of 12.5 mM ascorbate was added to the mixture with the final concentration of 50 lM. Ascorbate can significantly enhance the reaction rate and is also useful for suppressing fluorescence quenching by Cu2+ ion, Pb2+ ion, or other metal ions. The mixture was vortexed to mix all of the reagents and was placed into the well of a black 96-well microtiter plate to measure the initial fluorescence anisotropy. The temperature of a Genios Pro fluorescence reader was set at 25 °C (approximate room temperature). Subsequently, 1 ll of CuCl2 (PbCl2) solution or another metal ion solution or double distilled H2O with a different concentration was added, and the mixture was vortexed to mix all of the reagents (250 ll/well) and was incubated for an additional 15 min at room temperature before fluorescence anisotropy measurement. Each sample was measured four times. All experiments were repeated two times.

Results and discussion Principle of metal ion detection Fig. 1 outlines the principle of using the FA assay enhanced by AuNPs for the Cu2+ and Pb2+ ions detection. From the Perrin equation, the fluorescence anisotropy (r) is sensitive to changes in the rotational motion of fluorescently labeled molecules. The r value of a fluorophore is proportional to its rotational relaxation time, which in turn depends on its molecular volume (molecular weight). If a molecule is small, it will rotate faster and, hence, will have a smaller anisotropy value. Conversely, larger molecules will have larger anisotropy values due to their slow rotation. On the basis of Cu2+dependent DNA-cleaving DNAzyme, we first designed and prepared two DNA strands: the DNAzyme named as Cu–Enz and its substrate DNA strand named as Cu–Sub. Cu–Sub DNA labeled with 6-fluores-

49

cein-CE phosphoramidite (FAM) at the 30 end and thiol group at the 50 end (Cu–Sub, 50 -SH-T20AGCTTCTTTCTAATACGGCTTACC-FAM-30 ) was immobilized onto the surface of AuNPs (13 nm) via a thiol–Au interaction. The Cu–Enz and Cu–Subs on AuNPs form the complexes through two base-pairing regions; the 50 portion of the DNAzyme binds the substrate via Watson–Crick base pairs, and the 30 region does so through formation of a DNA triplex (Fig. 1D). The FAM fluorophore in the ‘‘big” AuNP-mediated Cu–Sub and Cu–Enz complexes displays the high anisotropy (Fig. 1A and B). In the presence of Cu2+ ion, the Cu–Enz as a recognition element specifically cleaves AuNPmediated Cu–Sub at the guanine base (marked in black). The AuNPmediated Cu–Sub and Cu–Enz complexes are separated due to a lack of thermal stability. The FAM-labeled cleaved products are released in the solution with rapid rotation and depolarize the light (Fig. 1C). Thus, the anisotropy of the system will significantly decrease correspondingly. In the absence of Cu2+ ion, the AuNP-mediated Cu–Sub and Cu–Enz associates would have stable high anisotropy. Therefore, via the free fluorescent cleavage products, this cleave event is translated to a measurable fluorescence anisotropy decrease proportional to the Cu2+ concentration. This design can also be easily expanded to Pb2+ ion detection by simply displacing the Cu–Sub and Cu–Enz for Pb–Sub and Pb–Enz. The structural model and sequences for bimolecular DNA complexes of Pb2+-dependent DNA-cleaving DNAzyme and its substrate are also presented in Fig. 1E.

Investigation of Cu2+-dependent DNA-cleaving DNAzyme specificity Here we provide additional evidence to verify the specificity of DNA-cleaving DNAzyme relying on the Cu2+ ion. We performed the control experiments to investigate whether the DNA molecules (Cu–Sub) undergo cleavage in the absence of the corresponding DNAzyme (Cu–Enz) and whether the Cu2+ ion is used as the only

Fig. 1. Schematic illustration of the strategy of Cu2+ and Pb2+ ions detection using Cu2+- or Pb2+-dependent DNA-cleaving DNAzyme fluorescence anisotropy via AuNP enhancement. (A) is shown the ‘‘big” AuNP-mediated Cu-Sub with high anisotropy. (B) is shown the formation of ‘‘big” AuNP-mediated Cu-Sub and Cu-Enz complexes with higher anisotropy after adding Cu-Enz. (C) is shown the FAM-labeled cleaved products from Cu-Sub with low anisotropy after adding Cu2+ ion and ascorbate. (D) and (E) are shown the structural models and sequences for bimolecular DNA complexes of DNAzyme and its substrate for Cu2+ and Pb2+ ions, respectively. The points of scission are indicated by a black arrow.

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cofactor. Three group experiments were carried out as follows: titrating Cu2+, Hg2+, or Pb2+ ion to the Cu–Sub-functionalized AuNP solution (Cu–Enz added); titrating Cu2+, Hg2+, or Pb2+ ion to the Cu–Sub-functionalized AuNP solution (no Cu–Enz added); and titrating Cu2+, Hg2+, or Pb2+ ion to the Cu–Sub-functionalized AuNP solution with fully complementary DNA (Cu–Sub–Comp, 50 -GGTAA GCCGTATTAGAAAGAAGCT-30 ). As shown in Fig. 2, there was little anisotropy change regardless of titrating Cu2+, Hg2+, or Pb2+ ion to the Cu–Sub-functionalized AuNP solution (Cu–Enz added). The results were the same when the tested metal ions were added to the solution containing Cu–Sub-functionalized AuNPs and the complementary DNA. In contrast, when Cu–Enz coexisted with Cu–Sub, there was a significant anisotropy change as Cu2+ ion was added. Sensitivity of Cu2+ ion detection As a proof-of-concept experiment, Cu–Enz- and Cu–Sub-functionalized AuNPs were dissolved in NaCl (1.5 M) and HEPES buffer (pH 7.0, 50 mM, 248 ll). The mixture was warmed to 80 °C for 2 min, cooled naturally to room temperature for 1 h to form the Cu–Enz and Cu–Sub complexes, and then transferred to the well of a black 96-well microtiter plate. An aqueous solution of ascorbate (12.5 mM, 1.0 ll) was added to the mixture to measure the initial anisotropy. Then the 1.0-ll aqueous solution of CuCl2 was added to the well, and the plate was vortexed to mix the solution and incubated at room temperature for 15 min before measurement. The release of Cu–Sub pieces cleaved by Cu–Enz in the system without AuNP modification at the Cu2+ concentrations of 10 and 100 nM resulted in anisotropy change corresponding to 0.002 and 0.019, respectively. The AuNP-mediated complexes led to a pronounced decrease in the anisotropy corresponding to 0.026 and 0.056, respectively. The results represent a remarkable AuNP enhancement function for fluorescence anisotropy in analyzing Cu2+ ion. The sensitivity of the FA assay was investigated by using various Cu2+ concentrations (0, 0.001, 0.01, 0.1, 1, and 10 lM) with a series of solutions containing Cu–Enz (100 nM) and Cu–Sub (20 nM). The anisotropy change was sensitive to Cu2+ ion in a concentrationdependent manner (Fig. 3A). This system shows good performance when the Cu2+ concentration lies between 1.0 nM and 10 lM. The Fig. 3A inset shows a good linear correlation (R = 0.9982) between the Dr value and the concentration of Cu2+ ion over the range of 0.001 to 10 lM. Thus, the current limit of detection for this method is approximately 1.0 nM (65 ppt) Cu2+ based on the three times of

Fig. 2. Fluorescence anisotropy changes on titrating Cu2+, Hg2+, and Pb2+ ions in three group experiments: (A) solution containing Cu–Sub-functionalized AuNPs and Cu–Enz; (B) solution containing only Cu–Sub-functionalized AuNPs; (C) solution containing Cu–Sub-functionalized AuNPs and the fully complementary DNA (Cu–Sub–Comp). The concentration of all tested metal ions was 500 nM.

signal-to-noise level of the blank sample, which is the most sensitive detection system for Cu2+ ion to the best of our knowledge. It should be noted that when the Cu2+ concentration ranged from 20 to 100 lM, the FAM emission was quenched approximately 10– 24%, whereas FAM quenching was less than 5% when the Cu2+ concentration was less than 10 lM. It is reasonable to believe that the high Cu2+ concentration (>20 lM) would decrease the system stability and degrade the system performance. Further experiments were carried out to explore the fluorescence quenching properties of AuNPs in this sensing system. The results demonstrated that the quenching properties of AuNPs had little effect on the FA assay of Cu2+ (<10 lM), in agreement with our previous work [33]. Therefore, this method is stable and reliable for detecting the Cu2+ ion in the range of 0.001 to 10 lM. Moreover, we studied the system with varying concentrations of Cu–Enz under the Cu2+ concentration of 500 nM. As shown in Fig. 3B, the anisotropy change was enhanced as Cu–Enz increased, where the minimum concentration of DNAzyme detectable was approximately 5 nM. Selectivity of Cu2+ ion detection The selectivity of the method was also examined by probing the response of the assay to other metal ions including Hg2+, Mn2+, Cd2+, Pb2+, Mg2+, Zn2+, Fe2+, Ca2+, K+, and Na+ ions at two concentrations: 10 and 500 nM (Fig. 4). The results demonstrate excellent selectivity over alkali, alkaline earth, and transition heavy metal ions with the exceptions of Hg2+ and Pb2+ ions. A small anisotropy decrease (Dr) was observed at the high Hg2+ and Pb2+ concentration (500 nM). No anisotropy decrease (Dr) was seen at the low Hg2+ and Pb2+ concentration (10 nM). The control experiments demonstrated that the decrease of anisotropy with Hg2+ and Pb2+ at 500 nM may result from the fluorophore interacting with the two metal ions, not from the cleavage of DNA (see Fig. 2). The specific detection of Cu2+ ion is clearly attributed to Cu2+-dependent DNAzyme cleaving action, resulting in the release of fluorescent cleavage products that lead to a decrease in anisotropy. Pb2+ ion detection using AuNP-enhanced FA assay The method can also be applied to Pb2+-dependent DNAzyme. Similarly, the nucleic acids Pb–Enz (50 -ACAGACATCTCTTCTCCGA GCCGGTCGAAATAGTGAGT-30 ) and Pb–Sub (50 -FAM-ACTCACTAT rAGGAAGAGATGTCTGTT20-SH-30 ) were employed for the design of the Pb2+ sensor. The release of Pb–Sub pieces cleaved by Pb–Enz in the system without AuNP modification at the Pb2+ concentrations of 10 and 100 nM resulted in anisotropy change corresponding to 0.005 and 0.021, respectively. The AuNP-mediated complexes led to a pronounced decrease in the anisotropy corresponding to 0.025 and 0.046, respectively. The results also represent a remarkable AuNP enhancement function for fluorescence anisotropy in analyzing the Pb2+ ion. Fig. 5A illustrates the decrease of fluorescence anisotropy (Dr) on the addition of different concentrations of Pb2+ ion, revealing that the Dr was strongly dependent on the concentration of Pb2+ ion. As the Pb2+ concentration increases, the corresponding Dr increases gradually. To evaluate the minimum concentration of Pb2+ ion in aqueous solution detectable by our method, we added Pb2+ to the mixture of AuNPs to obtain Pb2+ concentrations of 0, 0.001, 0.01, 0.1, 1, and 10 lM. An obvious change in Dr was observed when 1 nM Pb2+ was added to the mixture, leading to a limit of detection of 1 nM based on the three times of signal-to-noise level of the blank sample. The Fig. 5A inset shows a good linear correlation (R = 0.9977) between the Dr value and the concentration of Pb2+ ion over the range of 0.001 to 10 lM. The selectivity of the sensor was determined by challenging it with other metal ions, as shown in Fig. 5B. Another 10 divalent me-

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Fig. 3. Plots of fluorescence anisotropy changes as a function of Cu2+ concentration range from 1 nM to 10 lM (A) and as a function of Cu–Enz concentration range from 5 to 500 nM (B).

Fig. 4. Selectivity of Cu2+ assay method over other metal ions with two different concentrations: 500 nM and 10 nM.

Fig. 5. (A) Plot of fluorescence anisotropy changes as a function of Pb2+ concentration ranges from 1 nM to 10 lM. (B) Selectivity of Pb2+ assay method over other metal ions with two different concentrations: 500 nM and 10 nM.

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tal ions including Hg2+, Mn2+, Cd2+, Cu2+, Mg2+, Zn2+, Fe2+, Ca2+, K+, and Na+ ions were tested at two concentrations: 10 and 500 nM. The response to these ions is effectively indistinguishable from the response observed for the sample with added Pb2+ ion. In each case, Pb2+ gave the highest fluorescence anisotropy change, indicating the fastest cleavage. Thus, the Pb2+ sensor appears to be as specific as the Cu2+ sensor that we designed. Application To investigate whether this method was applicable to natural samples, we tested the spiked river samples with four different concentrations of Cu2+ or Pb2+ ion: 80 nM, 400 nM, 800 nM, and 1.6 lM. The interfering materials in the river samples did not influence the Cu2+ or Pb2+ detection with the described method. As expected, the method reveals the good recoveries of standard addition from 93.1% to 108.8%. It suggests that the DNAzymebased FA assay can be applied to facile detection of aqueous Cu2+ or Pb2+ ions in most cases. Conclusion We have demonstrated that AuNPs can enhance and improve the performance and sensitivity of the FA assay. As a proof-of-concept experiment, a novel and practical method relying on metal ion-dependent DNAzyme-based fluorescence anisotropy via AuNP enhancement was presented for detecting Cu2+ and Pb2+ ions in aqueous medium at room temperature. The FAM-labeled DNA substrates in the big AuNP-mediated complexes were cleaved and released. The AuNP enhancement function results in a significant decrease. This effect can be used for Cu2+ and Pb2+ detection as low as 1 nM. Based on the findings and results, the method opens up a new possibility for rapid and easy detection of toxic metal ions in environmental samples. Indeed, for a practical application purpose, preliminary experiments were performed on the real river water samples and the spiked river water samples. The results reveal good recoveries. Thus we believe that the method shows distinct advantages over conventional methods in terms of its potential sensitivity, specificity, and ability for rapid response. Acknowledgments This work was supported by grants of the National Science Foundation (NSF, 20627005, 20776039), SKLBE Fund (2060204), Shanghai Project (09JC1404100), NCET-07-0287, and Shuguang (06SG32). References [1] P.G. Georgopoulos, A. Roy, M.J. Yonone-Lioy, R.E. Opiekun, P.J. Lioy, Environmental copper: its dynamics and human exposure issues, Toxicol. Environ. Health B 4 (2001) 341–394. [2] M. Chan, S. Huang, Direct determination of cadmium and copper in seawater using a transversely heated graphite furnace atomic absorption spectrometer with Zeeman-effect background corrector, Talanta 51 (2000) 373–380. [3] M. Etienne, J. Bessiere, A. Walcarius, Voltammetric detection of copper(II) at a carbon paste electrode containing an organically modified silica, Sens. Actuat. B 76 (2001) 531–538. [4] J. Wu, E.A. Boyle, Low blank preconcentration technique for the determination of lead, copper, and cadmium in small-volume seawater samples by isotope dilution ICP–MS, Anal. Chem. 69 (1997) 2464–2470. [5] R.F.H. Viguier, A.N.J. Hulme, A sensitized europium complex generated by micromolar concentrations of copper(I): toward the detection of copper(I) in biology, Am. Chem. Soc. 128 (2006) 11370–11371. [6] S. Hong, T. Kang, J. Moon, S. Oh, J. Yi, Surface plasmon resonance analysis of aqueous copper ions with amino-terminated self-assembled monolayers, Colloids Surf. A 292 (2007) 264–270.

[7] Y. Zheng, K.M. Gattás-Asfura, V. Konla, R.M. Leblanc, A dansylated peptide for the selective detection of copper ions, Chem. Commun. 20 (2002) 2350–2351. [8] K.M. Gattás-Asfura, R.M. Leblanc, Peptide-coated CdS quantum dots for the optical detection of copper(II) and silver(I), Chem. Commun. 21 (2003) 2684– 2685. [9] J.W. Liu, Y. Lu, Colorimetric Cu2+ detection with a ligation DNAzyme and nanoparticles, Chem. Commun. 46 (2007) 4872–4874. [10] J.W. Liu, Y. Lu, A DNAzyme catalytic beacon sensor for paramagnetic Cu2+ ions in aqueous solution with high sensitivity and selectivity, J. Am. Chem. Soc. 129 (2007) 9838–9839. [11] M. Hollenstein, C. Hipolito, C. Lam, D. Dietrich, D. Perrin, A highly selective DNAzyme sensor for mercuric ions, Angew. Chem. Intl. Ed. 47 (2008) 4346– 4350. [12] J.W. Liu, Y. Lu, Rational design of turn-on allosteric DNAzyme catalytic beacons for aqueous mercury ions with ultrahigh sensitivity and selectivity, Angew. Chem. Intl. Ed. 46 (2007) 7587–7590. [13] H. Wang, Y. Kim, H. Liu, Z. Zhu, S. Bamrungsap, W. Tan, Engineering a unimolecular DNA-catalytic probe for single lead ion monitoring, J. Am. Chem. Soc. 131 (2009) 8221–8226. [14] T.J. Yim, J.W. Liu, Y. Lu, R.S. Kane, J.S. Dordick, Highly active and stable DNAzyme–carbon nanotube hybrids, J. Am. Chem. Soc. 127 (2005) 12200– 12201. [15] Y. Xiao, A.A. Rowe, K.W. Plaxco, Electrochemical detection of parts-per-billion lead via an electrode-bound DNAzyme assembly, J. Am. Chem. Soc. 129 (2007) 262–263. [16] L. Shen, Z. Chen, Y.H. Li, S.L. He, S.B. Xie, X.D. Xu, Z.W. Liang, X. Meng, Q. Li, Z.W. Zhu, M.X. Li, X.C. Le, Y.H. Shao, Electrochemical DNAzyme sensor for lead based on amplification of DNA–Au bio-bar codes, Anal. Chem. 80 (2008) 6323–6328. [17] J. Elbaz, B. Shlyahovsky, I. Willner, A DNAzyme cascade for the amplified detection of Pb2+ ions or L-histidine, Chem. Commun. 13 (2008) 1569– 1571. [18] S.W. Santoro, G.F. Joyce, K. Sakthivel, S. Gramatikova, C.F. Barbas, RNA cleavage by a DNA enzyme with extended chemical functionality, J. Am. Chem. Soc. 122 (2000) 2433–2439. [19] H. Kim, J. Liu, J. Li, N. Nagraj, M. Li, C.M.B. Pavot, Y. Lu, Metal-dependent global folding and activity of the 8–17 DNAzyme studied by fluorescence resonance energy transfer, J. Am. Chem. Soc. 129 (2007) 6896–6902. [20] J.W. Liu, A.K. Brown, X. Meng, D.M. Cropek, J.D. Istok, D.B. Watson, Y. Lu, A catalytic beacon sensor for uranium with parts-per-trillion sensitivity and millionfold selectivity, Proc. Natl. Acad. Sci. USA 104 (2007) 2056–2061. [21] V. Pavlov, Y. Xiao, B. Shlyahovsky, I. Willner, Aptamer-functionalized Au nanoparticles for the amplified optical detection of thrombin, J. Am. Chem. Soc. 126 (2004) 11768–11769. [22] Y. Weizmann, F. Patolsky, I. Willner, Amplified detection of DNA and analysis of single-base mismatches by the catalyzed deposition of gold on Aunanoparticles, Analyst 126 (2001) 1502–1504. [23] E. Golub, G. Pelossof, R. Freeman, H. Zhang, I. Willner, Electrochemical, photoelectrochemical, and surface plasmon resonance detection of cocaine using supramolecular aptamer complexes and metallic or semiconductor nanoparticles, Anal. Chem. 81 (2009) 9291–9298. [24] E. Sharon, R. Freeman, R. Tel-Vered, I. Willner, Impedimetric or ion-sensitive field-effect transistor (ISFET) aptasensors based on the self-assembly of Au nanoparticle-functionalized supramolecular aptamer nanostructures, Electroanalysis 21 (2009) 1291–1296. [25] Y.M. Yan, R. Tel-Vered, O. Yehezkeli, Z. Cheglakov, I. Willner, Biocatalytic growth of Au nanoparticles immobilized on glucose oxidase enhances the ferrocene-mediated bioelectrocatalytic oxidation of glucose, Adv. Mater. 20 (2008) 2365–2370. [26] X.C. Dong, C.M. Lau, A. Lohani, S.G. Mhaisalkar, J. Kasim, Z. Shen, X. Ho, J.A. Rogers, L.J. Li, Electrical detection of femtomolar DNA via gold-nanoparticle enhancement in carbon-nanotube-network field-effect transistors, Adv. Mater. 20 (2008) 2389–2393. [27] I. Tokarev, I. Tokareva, S. Minko, Gold-nanoparticle-enhanced plasmonic effects in a responsive polymer gel, Adv. Mater. 20 (2008) 2730–2734. [28] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, third ed., Springer, New York, 2006. [29] K.C. Grabar, R.G. Freeman, M.B. Hommer, M.J. Natan, Preparation and characterisation of Au colloid monolayers, Anal. Chem. 67 (1995) 735–743. [30] L.M. Demers, C.A. Mirkin, R.C. Mucic, R.A. Reynolds, R.L. Letsinger, R. Elghanian, G. Viswanadham, A fluorescence-based method for determining the surface coverage and hybridization efficiency of thiol-capped oligonucleotides bound to gold thin films and nanoparticles, Anal. Chem. 72 (2000) 5535–5541. [31] T.A. Taton, C.A. Mirkin, R.L. Letsinger, Scanometric DNA array detection with nanoparticle probes, Science 289 (2000) 1757–1760. [32] C.S. Thaxton, H.D. Hill, D.G. Georganopoulou, S.I. Stoeva, C.A. Mirkin, A biobarcode assay based upon dithiothreitol induced oligonucleotide release, Anal. Chem. 77 (2005) 8174–8178. [33] B.C. Ye, B.C. Yin, Highly sensitive detection of mercury(II) ions by fluorescence polarization enhanced by gold nanoparticles, Angew. Chem. Intl. Ed. 47 (2008) 8386–8389.