Efficient Fluorescence Turn-On Probe for Zirconium via a Target

Letter pubs.acs.org/ac

Efficient Fluorescence Turn-On Probe for Zirconium via a TargetTriggered DNA Molecular Beacon Strategy Hong-Min Meng,† Ting Fu,† Xiao-Bing Zhang,*,† Nan-Nan Wang,† Weihong Tan,*,†,‡ Guo-Li Shen,† and Ru-Qin Yu† †

State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China ‡ Department of Chemistry and Shands Cancer Center, UF Genetics Institute and McKnight Brain Institute, University of Florida, Gainesville, Florida 32611, United States S Supporting Information *

ABSTRACT: It is well-known that Zr4+ could selectively bind with two phosphate-functionalized molecules through a coordinate covalent interaction to form a sandwich-structured complex (−PO32−−Zr4+−PO32−−). In this paper, we for the first time converted such interaction into fluorescence sensing systems for Zr4+ via a target-triggered DNA molecular beacon strategy. In the new designed sensing system, two phosphorylated and pyrene-labeled oligonucleotides were chosen as both recognition and reporter units, which will be linked by target Zr4+ to form a hairpin structure and bring the two labeled pyrene molecules into close proximity, resulting in a “turn-on” excimer fluorescence signal. Moreover, γ-cyclodextrin was introduced to afford an amplified fluorescence signal and, therefore, provided an improved sensitivity for the target Zr4+. This allows detection of Zr4+ with high sensitivity (limit of detection, LOD = 200 nM) and excellent selectivity. The proposed sensing system has also been used for detection of Zr4+ in river water samples with satisfactory result.

Z

fluorescent method has attracted much attention and been applied widely for the detection of various analytes. In the past decade, considerable attention has been focused on the design of fluorescent probes for a number of metal ions based on small-molecule fluorophores,7,8 or functional nucleic acids.9,10 However, up-to-date, few fluorescence probes are developed for Zr4+. Molecular beacons, a class of novel oligonucleotide-based fluorescence probes, have been widely applied in DNA/RNA and some protein assays11 since their first development in 1996 by Tyagi and Kramer.12 Molecular beacons are single-stranded oligonucleotides with a stem-loop structure in the absence of a target, which brings the quencher and fluorophore into close proximity, whereby fluorescence is quenched effectively. The melting temperature of intermolecular hybrid between target molecule and the loop sequence is designed to be higher than that of the stem helix, which construct the basis for MB-based target detection. Due to its high quenching efficiency, MBbased sensing systems usually show low background fluorescence and large signal-to-background ratio and therefore high sensitivity to targets. Recently, such hairpin-structured

irconium possesses unique physical and chemical properties such as strong resistance to heat and corrosion, high mechanical strength, and high transparency to thermal neutrons. It is widely applied in various industrial processes such as manufacturing of water-cooled nuclear reactors, photographic flashbulbs, surgical instruments, space vehicle parts, ceramics, serving as a hardening agent in alloys, and tanning of leather.1 Moreover, Zr4+ complex was reported to show high catalytic activity toward phosphodiester hydrolysis of DNA under mild conditions.2 Therefore, the development of methods for determination of trace Zr4+ in various samples is of considerable significance and has become an attractive subject in modern industry. Several methods for the detection of zirconium at trace quantity level in various samples have been proposed. They include glow discharge mass spectrometry (GD-MS),3 reversed-phase liquid chromatography (RP-LC),4 spectrophotometry,5 and electrochemical methods.6 However, most of these methods necessitate the use of sophisticated and relatively costly apparatus and require complicated pretreatment procedures,3,4 not suitable for online or in-field monitoring, and some of them show poor sensitivity toward Zr4+ with detection limit located at ppm level.5 Due to its simple, low-cost, high sensitivity, fast analysis with spatial resolution and nonsample-destructing, and easy to perform online or infield detection with the application of fiber optics, the © 2012 American Chemical Society

Received: January 2, 2012 Accepted: February 12, 2012 Published: February 12, 2012 2124

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the detection temperature (25 °C). Thus, in the absence of Zr4+, free P 1 and P 2 cannot hybridize with each other, and the labeled pyrenes could not be close to each other to induce the excimer fluorescent emission. However, in the presence of Zr4+, the P 1 and P 2 can both bind with Zr4+ to form a sandwichstructured complex through the strong −PO32−−Zr4+−PO32−−, which can be regarded as a whole single-strand DNA. The Tm of the stem helix was determined to be 31.5 °C under the experimental condition (see Supporting Information and Figure S1), and the complex could then form a hairpin structure under the detection temperature (25 °C), which brings the two labeled pyrene molecules into close proximity, resulting in strong excimer fluorescent emission of pyrene. Probes with shorter stem length will give a low melting temperature for the Zr4+ complex, which might provide a poor excimer fluorescent response with a low sensitivity toward Zr4+. Probes with longer stem length, however, might result in a high melting temperature for the free P 1 and P 2, which might show a high background excimer fluorescent signal in the absence of target Zr4+. In addition, γ cyclodextrin (γ-CD) was introduced to afford an amplified fluorescence signal and therefore provided an improved sensitivity for the target Zr4+, as it was reported to be able to modulate the space proximity of the two pyrenes labeled on both ends of stem-containing oligonucleotide probe through cyclodextrin/pyrene inclusion interaction.16 To investigate if γ-CD can also act on oligonucleotides, the experimental melting curve data for the sensing system in the absence and presence of γ-CD are also collected (see Supporting Information, Figure S2). Experimental results show that γ-CD does not affect the Tm of the stem helix of the sensing system, indicating it amplies the signal by just acting on the two labeled pyrene molecules. To verify the signal amplification function of γ-CD, targetinduced excimer fluorescence enhancements of the sensing system in the absence and presence of β-CD or γ-CD (8 mM) were recorded, respectively. The P 1 and P 2 (500 nM) were first incubated for 30 min at 4 °C (storing in common refrigerator). After the addition of Zr4+ (80 μM), the mixture was incubated for another 20 min at 25 °C, if necessary, β-CD or γ-CD was then added and incubated for another 10 min, after which the fluorescent spectra were recorded. As shown in Figure 1, although the introduction of γ-CD resulted in a

probe has been converted to construct fluorescence probes for non-nucleic acid targets including various metal ions. It is wellknown that Zr4+ could selectively bind with two phosphatefunctionalized molecules through a coordinate covalent interaction between the phosphate groups and the inorganic ions.13 It can be envisioned that such unique strong −PO32−− Zr4+−PO32−− interaction could be converted into sensing systems for Zr4+ using various signal transduction mechanisms, just like other metal−DNA interactions,14 such as the widely applied T−Hg2+−T interaction for design of Hg2+ sensing systems.15 However, to our best knowledge, no such probe was reported for Zr4+. In the context of our long-term interests in searching for fluorescent probes for metal ions,8b,d,9b,10c we try to use the −PO32−−Zr4+−PO32−− interaction to design a fluorescence probe for turn-on detection of Zr4+ in aqueous samples. Herein, we report the development of such a kind of fluorescence Zr4+ probe via a target-triggered DNA molecular beacon strategy (Scheme 1). Scheme 1. Schematics of Molecular Beacon-Based Sensing System for Amplification Detection of Zr4+ Using γ-CD as a Signal Amplifier

Different from classic MB probes, which show the targetinduced destructing of the hairpin structure,12 our proposed Zr4+ probe exhibits target-triggered formation of the hairpin structure. Therefore, we cannot use the classic MB design with fluorophore and quencher pairs as signal reporter units,12 which will give a “turn-off” fluorescent response to Zr4+. Fluorescence “turn-off” probes usually show lower sensitivity than “turn-on” probes and may report false positive results caused by other quenchers in practical samples, which are undesirable for practically analytical applications. To solve this problem, we choose pyrene as the signal unit, as it could afford a “turn-on” excimer fluorescence response to the target-triggered formation of the hairpin structure.16 Most studies have indicated that a 15−25 bases loop together with a 5−7 mer stem will provide an appropriate balance for the formation of a hairpin structure.17 In our new proposed MB-based sensing system, two phosphorylated and pyrene-labeled oligonucleotides P 1 and P 2 (contained 16 and 15 bases, respectively) were chosen as both recognition and reporter units, which were designed to complement each other with 6 base pairs at the pyrene-labeled terminals and therefore could form a DNA molecular beacon structure with 19 bases looped together with a 6 mer stem, upon the introduction of target Zr4+. The melting temperature (Tm) for the hybrid of free P 1 with P 2 is determined to be 6.5 °C under the experimental condition(see Supporting Information and Figure S1), which is designed to be much lower than

Figure 1. Fluorescence responses of the sensing system under different conditions: (a) P 1 (500 nM) + P 2 (500 nM) + Zr4+ (80 μM) + γ-CD (8 mM), (b) P 1 + P 2 + Zr4+, (c) P 1 + P 2 + γ-CD, and (d) P 1 + P 2.

slightly increased background excimer fluorescence (Figure 1, curve c) compared with that in the absence of γ-CD (Figure 1, curve d), fortunately, it also resulted in a much larger signal to 2125

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P 2 (estimated to be 6 °C) is much lower than the detection temperature (rt), and pyrence excimer could not be formed, which afforded a very low excimer background fluorescence for the sensing system. However, upon the addition of Zr4+, the monomeric fluorescence emissions of pyrene decreased, and a new broad emission with peak at 474 nm was observed, which belongs to the characteristic fluorescence peak of pyrene excimer, indicating that the formation of the hairpin structure of the complex of Zr4+ with P 1 and P 2 had brought the two labeled pyrene molecules into close proximity. A dramatic increase in pyrene excimer fluorescence intensity was observed as the Zr4+ concentration increased. With the concentration of Zr4+ larger than 100 μM, a 14-fold excimer fluorescence enhancement was observed. The large excimer fluorescence enhancement together with the low background excimer fluorescence of free probes benefited for a high sensitivity for target Zr4+. Figure 2b depicts the relationship between the excimer fluorescence intensity and the different concentrations of Zr4+. It shows a dynamic range from 500 nM to 100 μM with a low detection limit of 200 nM for Zr4+ (based on 3δ/slope). In contrast, control experiments with Zr4+ at various concentrations in the absence of γ-CD were also carried out (see Supporting Information, Figure S5). The emission intensity of pyrene excimer fluorescence at 474 nm was enhanced moderately with the increase of Zr4+, and a dynamic range only from 5 to 100 μM was observed. These results clearly demonstrate that the γ-CD does really play an important role in the signal amplification. Besides sensitivity, selectivity is another important parameter to evaluate the performance of a new developed fluorescent probe. Particularly, for a fluorescence probe with potential application in practical complex samples, a highly selective response to the target over other potentially competing species is a necessity. Therefore, the selectivity and competition experiments were extended to various metal ions. The selectivity was first tested with excitation fixed at 344 nm and recorded the fluorescence intensity change at 474 nm triggered by the competing metal ions. As shown in Figure 3 (the black

background ratio (SBR), assuring a higher sensitivity for detection of Zr4+. By introducing γ-CD amplification, we observed a SBR of 1348% for 80 μM of Zr4+. In contrast, in the absence of γ-CD, only a SBR of 420% was observed. However, the introduction of β-CD did not induce significant signal amplification for Zr 4+ (data not shown). The signal amplification function of γ-CD for the sensing system seems to be the result of housing a part of the dimer of pyrene within the γ-CD cavity (internal diameter of 8.5 Å18) to bring the two pyrene molecules more nearby to trigger remarkable excimer fluorescence enhancement upon the addition of target. In order to achieve the system’s best sensing performance, the concentrations for P 1, P 2, and γ-CD and ionic strength of the buffer solution were optimized. Experimental results showed that a concentration of 500 nM for both P 1 and P 2, 50 mM of NaCl, and 8 mM of γ-CD could provide maximum SBR for the sensing system (see Supporting Information, Figures S3 and S4), which were chosen as optimized conditions for further investigation. The proposed amplified sensing system is highly sensitive to Zr4+. Figure 2a shows the fluorescence-emission spectra of the

Figure 2. Sensitivity of the amplified fluorescence sensing system for Zr4+. (a) Fluorescence emission spectra in the presence of different concentrations of Zr4+. (b) The relationship of the excimer fluorescence enhancement with the target concentration. Inset shows the responses of sensing system to Zr4+ at low concentration.

Figure 3. Fluorescence response of the sensing system to 80 μM of Zr4+ or other metal ions (the black bar portion) and to the mixture of 80 μM of Zr4+ with other metal ions (the gray bar portion). The concentrations for other metal ions are as follows: 10 mM for K+, Mg2+, and Ca2+ and 800 μM for others.

sensing system upon the addition of Zr4+ at different concentrations. In the absence of Zr4+, free P 1 and P 2 are separated from each other, only the typical monomeric fluorescence emission of pyrene with peaks at 378 and 391 nm were observed. In such a case, the Tm for the hybrid of free P 1 with

bar portion), little fluorescence intensity ratio changes were observed with all other metal ions, indicating that our proposed 2126

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probe exhibits high selectivity to Zr4+ over other competing metal ions. To test practical applicability of our fluorescent probe for Zr4+, competition experiments were also carried out. An excess amount of above-mentioned metal ions (10 mM for K+, Mg2+, and Ca2+ and 800 μM for others) are added to 80 μM of Zr4+ in buffered aqueous solution, and the fluorescence intensity change at 474 nm of the probe was detected and then compared with that of buffer solution containing only 80 μM of Zr4+. Results are shown in Figure 3 (the gray bar portion). Our probe showed almost unchanged responses to Zr4+ before and after addition of other competing metal ions. These experimental results show that the response of the probe to Zr4+ is unaffected by the presence of the other possible contaminating metal ions, even existing in a concentration 10 times higher than that of Zr4+. All these results indicate that our proposed probe is highly selective and has great potential for environmental and industrial applications. To verify if Zr4+ can indeed bind with P 1 and P 2 to form a sandwich-structured complex through the strong −PO32−− Zr4+−PO32−−, gel electrophoresis experiments were then carried out under different conditions. The free P 1 and P 2 cannot be ligated together upon incubation at 4 °C for 2 h in the absence of Zr4+ (Figure 4, lane 3). However, there was a

Table 1. Recovery Study of Spiked Zr4+ in River Waters with Proposed Probe sample

Zr4+ spiked (μM)

Zr4+ recovered meana ± SDb

recovery (%)

river water 1 river water 2 river water 3

10.0 20.0 50.0

9.6 ± 0.3 19.8 ± 0.5 51.2 ± 2.4

96 99 102.4

a

Mean of three determinations. bSD: standard deviation.

samples show good recovery values. To further investigate the sensitivity of the fluorescence probe in the practical system, the Zr4+ titration experiments in the river water samples were also performed. Experimental results showed that the titration curve in the river water samples was similar to that in the buffer solution (see Supporting Information, Figure S6), with a dynamic concentration range from 1 to 100 μM for Zr4+. All these results indicated that the proposed sensing system was applicable for practical Zr4+ detection in real samples with other potentially competing species coexisting. In summary, the coordinate covalent interaction between Zr4+ and two phosphate-functionalized molecules was converted into a novel fluorescence sensing system for zirconium by employing a target-triggered DNA molecular beacon strategy. By choosing two labeled pyrene molecules as signal reporter, the proposed sensing system shows a “turn-on” excimer fluorescence response to target Zr4+. Moreover, by employing γ-CD as a signal amplifier, it affords an improved sensitivity for Zr4+ with a detection limit of 200 nM. Most importantly, the fluorescence responses of the sensing system are remarkably specific for Zr4+ in the presence of other heavy and transition metal ions, which meet the selective requirements for environmental and industrial monitoring application. This novel sensing system is simple in design and can be easily carried out by simple mixing and incubation. It has also been successfully applied for practical Zr4+ detection in river water samples, further demonstrating its value in the practical applications.

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Figure 4. Gel electrophoresis of the sensing system under different conditions: lane 1, 10 μM P 1 only; lane 2, 10 μM P 2 only; lane 3, 10 μM P 1 incubated with P 2 at 4 °C for 1.5 h; lane 4, 10 μM P 1 and P 2 incubated with 800 μM Zr4+ at 4 °C for 1.5 h; lane 5, DNA marker, respectively.

ASSOCIATED CONTENT

S Supporting Information *

Apparatus and experimental procedures; supplementary spectral data. This material is available free of charge via the Internet at http://pubs.acs.org.

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clearly observed complexation product of P 1 and P 2 in the presence of Zr4+ (Figure 4, lane 4). These results demonstrated that Zr4+ can bind with P 1 and P 2 to form a sandwichstructured longer single-strand DNA. The practical application of the designed fluorescent probe was then evaluated by determination of recovery of spiked Zr4+ in river water samples. The river water samples were obtained from Xiang River (Changsha, China). All the samples collected were simply filtered and showed that no Zr4+ was present. In order to reduce the pH influence in the detection, 1 mL of the river water samples was added to 1 mL of Tris−HCl buffered water containing P 1 and P 2 (500 nM, final concentration) to keep the pH value at 7.4, and then, its fluorescence intensity change was detected before and after being spiked with concentrated standard Zr4+ solution. The analytical results are shown in Table 1. All the measurements were performed three times. One observed that the results obtained in real water

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (X.-B.Z.); [email protected] (W.H.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS H.M.M. and T.F. contributed equally to this work. This work was supported by the National Key Scientific Program of China (2011CB911001 and 2011CB911003), the National Natural Science Foundation of China (Grant 20975034, 21177036), the National Key Natural Science Foundation of China (No. 21135001), and Hunan Provincial Natural Science Foundation of China (Grant 11JJ1002). 2127

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Letter

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