Concatemeric dsDNA-Templated Copper Nanoparticles Strategy with

Jun 24, 2014 - DNA-directed assembly of copper nanoblocks with inbuilt fluorescent and electrochemical properties: Application in simultaneous amplifi...
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Concatemeric dsDNA-Templated Copper Nanoparticles Strategy with Improved Sensitivity and Stability Based on Rolling Circle Replication and Its Application in MicroRNA Detection Fengzhou Xu,† Hui Shi,† Xiaoxiao He, Kemin Wang,* Dinggeng He, Qiuping Guo, Zhihe Qing, Lv’an Yan, Xiaosheng Ye, Duo Li, and Jinlu Tang State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Institute of Biology, Hunan University, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Changsha 410082, China S Supporting Information *

ABSTRACT: DNA-templated copper nanoparticles (CuNPs) have emerged as promising fluorescent probes for biochemical assays, but the reported monomeric CuNPs remain problematic because of weak fluorescence and poor stability. To solve this problem, a novel concatemeric dsDNA-templated CuNPs (dsDNA-CuNPs) strategy was proposed by introducing the rolling circle replication (RCR) technique into CuNPs synthesis. In this strategy, a short oligonucleotide primer could trigger RCR and be further converted to a long concatemeric dsDNA scaffold through hybridization. After the addition of copper ions and ascorbate, concatemeric dsDNA-CuNPs could effectively form and emit intense fluorescence in the range of 500−650 nm under a 340 nm excitation. In comparison with monomeric dsDNA-CuNPs, the sensitivity of concatemeric dsDNA-CuNPs was greatly improved with ∼10 000 folds amplification. And their fluorescence signal was detected to reserve ∼60% at 2.5 h after formation, revealing ∼2 times enhanced stability. On the basis of these advantages, microRNA let-7d was selected as the model target to testify this strategy as a versatile assay platform. By directly using let-7d as the primer in RCR, the simple, low-cost, and selective microRNA detection was successfully achieved with a good linearity between 10 and 400 pM and a detection limit of 10 pM. The concatemeric dsDNA-CuNPs strategy might be widely adapted to various analytes that can directly or indirectly induce RCR. s promising alternatives to common fluorophores like organic dyes, few-atom metal nanoparticles (NPs) with strong and robust fluorescence emission have recently attracted considerable research interest.1−4 In particular, DNA-templated fluorescent metal NPs show great potential as fluorescent probes for biochemical applications due to their advantages of ultrafine size, low toxicity, good biocompatibility, outstanding photophysical properties, as well as facile integration with nucleic acid−based target-recognition abilities and signal amplification mechanisms.5−8 For example, cytosine-rich DNA-templated silver nanoclusters have been widely studied for detection of various targets, such as metal ions,9−11 proteins,12 microRNA,13,14 single-nucleotide mutation,15 cancer cells,16−18 and so on. Compared with other existing fluorescent metal NPs, DNAtemplated copper NPs (CuNPs), including random doublestranded DNA-templated CuNPs (dsDNA-CuNPs) reported by Mokhir et al.19 and poly(thymine)-templated CuNPs (poly T-CuNPs) developed by our group,20,21 are a type of newly emerged functional biochemical probe. Its formation might be resulted from the high-affinity clustering of Cu0, which is formed through chemical reactions between Cu2+ and the reducing agent in solution, on DNA scaffolds.19 Due to the

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© 2014 American Chemical Society

strong quantum-confinement effect, CuNPs can be excited with a maximum absorption wavelength of 340 nm and exhibit an overall fluorescence emission in the range of 500−650 nm (dsDNA-CuNPs: max λem = 570 nm; poly T-CuNPs: max λem = 615 nm). The MegaStokes shifting is quite favorable for the elimination of interference from background signals of complex biological systems. Furthermore, the synthesis of CuNPs is highly efficient, simple, and rapid (about 5 min at room temperature), thus laying a substantial foundation for their widespread applications in biosensing.22−25 Besides, dsDNACuNPs, which are formed through accumulation of Cu0 in the major groove of dsDNA scaffolds,19 also hold unique advantages of excellent discrimination between dsDNA and single-stranded DNA (ssDNA) as well as no need for specific sequence design. These properties can be facilely utilized to contrive versatile nucleic acid-related detection strategies. For example, Mokhir et al. realized DNA assay by using dsDNA products of target DNA hybridized with complemented DNA Received: March 15, 2014 Accepted: June 24, 2014 Published: June 24, 2014 6976

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to synthesize CuNPs.19 Dong et al. further extended the application of dsDNA-CuNPs to ATP and cocaine sensing through the integration of DNA aptamer-target binding mechanisms,26 and Yu’s group developed several enzyme activity detection methods by utilizing the enzyme-induced degradation of dsDNA scaffolds for CuNPs formation.27,28 Despite the above studies, exploration of DNA-templated CuNPs in biochemical application is still at its early stage. In particular, design of CuNPs-based detection strategies is confined to the simple adoption of monomeric CuNPs formed with monomeric DNA templates for signal generation. Because a distinct fluorescence signal of monomeric CuNPs could be detected only in the presence of a relatively high concentration of DNA scaffolds (at least dozens of nanomoles),19,26 the highly sensitive bioassay might fail. Moreover, the fluorescence emission of monomeric CuNPs could be stable for just ∼20 min and thereafter decrease quickly, which might greatly impede its applications in real-time and long-time monitoring research. Therefore, it should be a very interesting and significant study to overcome these tangible defects of DNAtemplated CuNPs so as to facilitate its further applications in more research fields. To our knowledge, however, this has not been reported yet. Rolling circle replication (RCR) is a simple and powerful isothermal DNA replication technique, which can convert dNTPs into a concatemeric ssDNA molecule comprising thousands of tandem periodic copies of the circular template through a short oligonucleotide primer-initiated enzymatic process.29 In combination with various signal readout strategies like DNA-staining dyes,30 fluorescent DNA probes,31 DNAmodified gold NPs,32 etc., RCR could generally achieve a signal amplification of about 1000−10 000-fold.33−35 Herein, aiming at the improvement of fluorescent CuNPs’ sensitivity and stability, we first introduce a DNA amplification mechanism into CuNPs synthesis and propose a novel RCR-mediated concatemeric CuNPs strategy by adopting dsDNA-CuNPs as the model (Figure 1). In this strategy, a circular DNA template is designed with two functional regions (R and H). Region R (blue) is a recognition region, a part of which is complementary to the primer. Region H (green) is a hybridization region, and its dsDNA form is designated to template the formation of CuNPs. In the presence of phi29 polymerase and dNTPs, a

primer can trigger the RCR process with a continual replication of the circular template. As a result, a short oligonucleotide primer is extended to a long concatemeric ssDNA with periodically repeated complementary parts of regions R and H (named as R′ and H′). Through ensuing hybridization of the H′ region with complementary DNA H, a long concatemeric dsDNA scaffold comprising two distinct alternating regions of R′ ssDNA (blue) and H/H′ dsDNA (green) can be obtained to synthesize concatemeric dsDNA-CuNPs. The remarkable increase of thousands of dsDNA scaffold units is expected to endow concatemeric dsDNA-CuNPs with a substantial improvement of fluorescent properties, thus providing an opportunity to develop novel CuNPs-based detection methods for various targets that can directly or indirectly initiate RCR. Indeed, after a systematic investigation in comparison with monomeric dsDNA-CuNPs, concatemeric dsDNA-CuNPs have been found to hold markedly enhanced sensitivity and stability. Thereupon, microRNA, which plays an important role in transcriptional and post-transcriptional regulation of gene expression and is considered as an essential biomarker in cancer research,36,37 has been selected as the model target. By directly using the target as the primer for RCR, a simple, low-cost, sensitive, and selective microRNA assay has been successfully achieved with the concatemeric dsDNA-CuNPs strategy.

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EXPERIMENTAL SECTION Materials and Reagents. All the DNA or RNA molecules reported in this article were custom-designed and then synthesized by Takara biotechnology (Dalian) Co., Ltd. or Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). Sequences of the oligos are listed in Table S-1. DNA polymerase (phi29) was obtained from New England Biolabs (Beijing), Ltd. 3-(N-morpholino) propanesulfonic acid (MOPS) was purchased from Dingguo Biotech (Beijing, China). Copper sulfate (CuSO4·5H2O) and sodium Disoascorbate (sodium ascorbate) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other chemicals were of the highest grade available. All stock and buffer solutions were prepared using ultrapure water (18.2 MΩ·cm from Millpore purification system). In RNA-related experiments, all the reagents and tools were dealt with using 1‰ DEPC to achieve RNase inactivation. Rolling Circle Replication. For feasibility investigation, 0.5 nM primer (DNA1 or let-7d) and a 0.5 nM circular template were first mixed in MOPS buffer (10 mM MOPS, 0.15 M NaCl, 10 mM MgCl2, pH 7.50, 25 °C). Then, the mixture was heated to 95 °C for 5 min and slowly cooled down to room temperature in about 4 h to ensure a relatively complete hybridization of the primer with the circular template. After the addition of 500 μM dNTPs and 100 U/mL phi29, the mixture was allowed to react at 30 °C for 12 h in a Mastercycler pro PCR instrument (Eppendorf, Germany). Finally, RCR was terminated by a thermal treatment at 65 °C for 10 min, and the resulted products were stored at 4 °C. In sensitivity investigation experiments, targets with different concentrations were annealed with a 1 nM circular template, and other operations were as fore-mentioned. Formation of dsDNA-CuNPs. To prepare concatemeric dsDNA-CuNPs, the RCR product was first hybridized with 1000 nM complementary DNA to form concatemeric dsDNA scaffolds. Then, 200 μM Cu2+ and 4 mM sodium ascorbate were added and allowed to react for about 10 min at room temperature. The resulted concatemeric dsDNA-CuNPs were

Figure 1. Schematic showing the principle of the RCR-mediated concatemeric dsDNA-CuNPs strategy. 6977

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Figure 2. Feasibility of the concatemeric dsDNA-CuNPs strategy. (A) Fluorescence spectra of CuNPs which were synthesized using the hybridized complex of 0 or 0.5 nM DNA1-induced RCR product with 1000 nM DNA2. (Insets: fluorescent photos of the corresponding CuNPs samples under the UV lamp excitation; right, 0 nM DNA1-RCR; left, 0.5 nM DNA1-RCR.) (B) 1% agarose electrophoresis analysis of 0 (lane 2) or 0.5 nM (lane 3) DNA1-induced RCR product (lane 1 is DNA marker). HRTEM images of (C) monomeric dsDNA-CuNPs formed with 500 nM DNA2/DNA3 dsDNA and (D) concatemeric dsDNA-CuNPs formed with the hybridized complex of 1 nM DNA1-induced RCR product with 1000 nM DNA2. (Insets: HRTEM images with higher amplification showing the obvious crystal lattice structure of CuNPs.)

CuNPs strategy, the RCR system was designed including a 19mer primer DNA1, a 65-mer circular template embedded with a 32-mer DNA2 as region H, phi29 polymerase, and dNTPs in MOPS buffer. The RCR product ought to be a long concatemeric ssDNA containing 32-mer DNA3 as region H′. Upon the addition of DNA2, a long concatemeric dsDNA scaffold containing repeated DNA2/DNA3 hybridization regions could form and then template the synthesis of fluorescent CuNPs in the presence of Cu2+ and ascorbate. As displayed in Figure 2A, concatemeric dsDNA-CuNPs, which were prepared using the hybridized complex of 0.5 nM DNA1-induced RCR product with 1000 nM DNA2, exhibited an intense fluorescence emission in the range of 500−650 nm (max λem = 570 nm) under 340 nm excitation. Whereas under the same condition, the RCR sample without DNA1 did not produce obvious signal at 570 nm, revealing the failed formation of CuNPs as a result of lacking RCR products. Also, under UV light irradiation, fluorescent photos of the above two samples clearly presented the efficacious synthesis of RCR-mediated concatemeric dsDNA-CuNPs with a strong red fluorescence (insets in Figure 2A). The RCR product was further verified by conducting 1% agarose gel electrophoresis (Figure 2B), in which 0.5 nM DNA1-induced RCR product showed a bright band near the sample hole because it was too long to move forward in the agarose gel (lane 3). Subsequently, the morphology of concatemeric dsDNA-CuNPs was studied

immediately characterized or used for following experiments. For monomeric dsDNA-CuNPs, the synthesis method was the same, but monomeric dsDNA scaffolds were used. Characterization of dsDNA-CuNPs. The morphology of CuNPs was studied using the high resolution transmission electron microscopy (HRTEM). Samples were prepared by spin coating 10 μL of as-prepared CuNPs onto carbon-coated copper grid substrates, which were then dried naturally overnight and measured by Tecnai G 2 20ST TEM. Fluorescence spectra of CuNPs were measured on a F-4500 fluorescence spectrophotometer (Hitachi, Japan) with an excitation wavelength of 340 nm and an emission wavelength range of 500−650 nm (max λem = 570 nm). For stability investigation, fluorescence intensities of CuNPs at 570 nm were detected every 10 min since Cu2+ and sodium ascorbate were added into the DNA scaffolds solution. Agarose Electrophoresis Analysis. Generally, nucleic acid samples were analyzed by 1% agarose gel in 1 × TBE buffer (89 mM Tris-boric acid, 2.0 mM EDTA, pH 8.3) followed by electrophoresis for 1 h at 100 V. After 1 × SYBR Gold staining for 30 min, the gel was imaged on a Tanon2500R gel imaging system (Shanghai, China).

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RESULTS AND DISCUSSION Feasibility of the Concatemeric dsDNA-CuNPs Strategy. To investigate feasibility of the concatemeric dsDNA6978

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Figure 3. Sensitivity investigation of the concatemeric dsDNA-CuNPs strategy with DNA1 as the model target. (A) Fluorescence spectra of concatemeric dsDNA-CuNPs that were formed using the hybridized complex of 1000 nM DNA2 with the RCR product induced by different concentrations of DNA1. (B) The corresponding calibration curve of the concatemeric dsDNA-CuNPs strategy for the DNA1 assay. The inset shows the linear response at low concentrations of DNA1. (Error bars represent standard deviations from three repeated experiments.)

Figure 4. Stability investigation of concatemeric dsDNA-CuNPs in comparison with monomeric dsDNA-CuNPs. (A) Time-dependent fluorescence intensity variation of CuNPs which were synthesized using different concentrations of DNA2/DNA3 dsDNA scaffolds or 1 nM DNA1-induced concatemeric dsDNA scaffold. (B) The normalized fluorescence intensities result in A. (C) 1% agarose electrophoresis analysis of DNA scaffolds after incubation with Cu2+ and ascorbate for different times. (Lane 1) DNA marker. (Lane 2) Concatemeric dsDNA scaffolds of 1 nM DNA1induced RCR product hybridized with 1000 nM DNA2. (Lanes 3−7) Concatemeric dsDNA scaffolds with the addition of Cu2+ and ascorbate were incubated for 0, 0.5, 1, 2, and 4 h, respectively. (Lane 8) Monomeric dsDNA scaffolds of 500 nM DNA2/DNA3. (Lanes 9−13) Monomeric dsDNA scaffolds with the addition of Cu2+ and ascorbate were incubated for 0, 0.5, 1, 2, and 4 h, respectively. (Lane 14) Monomeric dsDNA scaffold with the addition of ascorbate was incubated for 4 h. (Lane 15) Monomeric dsDNA scaffold with the addition of Cu2+ was incubated for 4 h.

scaffold. The results strongly support that RCR could be successfully introduced to produce concatemeric dsDNACuNPs with intense fluorescence emission, thus laying a foundation to develop detection methods for targets that can trigger RCR. Sensitivity Investigation of the Concatemeric dsDNACuNPs Strategy. Before the sensitivity investigation, prepara-

using HRTEM, in comparison with monomeric dsDNACuNPs formed with monomeric DNA2/DNA3 dsDNA scaffolds. Figure 2C indicated that monomeric dsDNACuNPs were scattered with a size of about 3−4 nm. In contrast, concatemeric dsDNA-CuNPs were obviously agglomerative (Figure 2D), showing an accumulation or overlap of several CuNPs in one RCR-induced concatemeric dsDNA 6979

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ascorbate for different times. Results in Figure 4C showed that with the incubation time increased, degradation of monomeric dsDNA was gradually exacerbated (lanes 8−13). After 2 h, DNA bands were difficult to see, indicating little intact dsDNA survived (lanes 12 and 13). This perfectly corresponds to the fluorescence decay of monomeric dsDNACuNPs and verifies that its instability might be resulted from template degradation. Also, intact bands of monomeric dsDNA with the addition of either ascorbate (lane 14) or Cu2+ (lane 15) revealed that degradation only occurred when Cu2+ and ascorbate coexisted, indirectly proving that ·OH might be a cause of DNA damage. As for concatemeric dsDNA, a much slower degradation rate was observed, although a similar tendency was indicated. The concatemeric dsDNA of 1 nM DNA1-induced RCR product hybridized with DNA2 was immediately degraded into smaller fragments (∼500 bp) after the addition of Cu2+ and ascorbate (lane 3). With time increased, degraded fragments were shorter and shorter. After 1 h, DNA bands were stable at a location identical with monomeric dsDNA and distinctly visible until 4 h. It was deduced that because ssDNA is more prone to be damaged by · OH,40 the RCR-induced abundant ssDNA regions might consume ·OH and reduce dsDNA degradation, thus producing fragments with a similar size to monomeric dsDNA. Since these dsDNA fragments could also template the formation of CuNPs, a greatly improved stability was achieved in the concatemeric dsDNA-CuNPs strategy. MicroRNA Detection with the Concatemeric dsDNACuNPs Strategy. To demonstrate the concatemeric dsDNACuNPs strategy as a versatile assay platform, studies were conducted by adopting microRNA (miRNA) as the analyte. miRNA is a class of small noncoding ssRNA with an average of 22 nucleotides, which can modulate gene expression through interaction with translation.41 As a typical cancer-associated biomarker,36,37,42 the sequence-dependent detection of miRNA has recently been explored for cancer diagnostic research. In this work, let-7d, a member of let-7 human miRNA family targeting caspase-3 mRNA,43 was used as the model to be detected with the concatemeric dsDNA-CuNPs strategy. In the detection principle, target let-7d was directly used as the primer for RCR, and the circular template was designed to embed a region R with 22-mer sequence complementary to let7d and a region H of 32-mer DNA3. In the presence of circular a template, phi29 and dNTPs, let-7d could trigger RCR and be extended to a long concatemeric nucleic acid molecule, which could hybridize with DNA3 to produce a long concatemeric dsDNA scaffold for the formation of concatemeric dsDNACuNPs with strong fluorescence emission (Figure S-4). Contrarily, without let-7d, almost no obvious signal at 570 nm could be detected. It was indicated that the presence of let7d could be successfully sensed by this concatemeric dsDNACuNPs assay platform. Then, let-7d with different concentrations (0−1000 pM) was used to induce RCR, and the resulted concatemeric dsDNA-CuNPs were measured on a fluorescence spectrophotometer. With the increase of let-7d concentration, the bands of RCR products in agarose gel were brighter and brighter (Figure S-5A). Correspondingly, fluorescence signals of concatemeric dsDNA-CuNPs were detected to be gradually enhanced (Figure 5A). A good linearity was obtained from 10 pM to 400 pM (R2 = 0.9983) with the lowest concentration of 10 pM detected (Figure 5B). We next investigated specificity of the concatemeric dsDNACuNPs strategy for the let-7d assay by using two miRNA

tion conditions of concatemeric dsDNA-CuNPs, including concentrations of DNA2, Cu2+, and ascorbate, were optimized (Figure S-1). In the following experiments, 1000 nM DNA2, 200 μM Cu2+, and 4 mM ascorbate were routinely used to synthesize concatemeric dsDNA-CuNPs. To inspect sensitivity of the concatemeric dsDNA-CuNPs strategy in a comprehensible way, DNA1, which was adopted as the primer in RCR, was identified as the target. The RCR products produced in the presence of DNA1 with different concentrations (0−1000 pM) were used to hybridize with DNA2 and then prepare concatemeric dsDNA-CuNPs. As shown in Figure 3, with an increase of DNA1 concentration, the fluorescence signal of concatemeric dsDNA-CuNPs was measured to be gradually enhanced. There was a good linear correlation between fluorescence intensity and DNA1 concentration in the range of 0.5−100 pM (R2 = 0.9907), with the lowest concentration of 500 fM detected. Compared with the monomeric dsDNACuNPs strategy (Figure S-2, LOD ≈ 5 nM), the sensitivity was greatly improved with ∼10 000-fold amplification in the concatemeric dsDNA-CuNPs strategy. It was indicated that the introduction of RCR could convert a short oligonucleotide into an extremely long DNA with a remarkable increase of thousands of dsDNA scaffold units, thus endowing the concatemeric dsDNA-CuNPs strategy with great promise for highly sensitive biochemical sensing. Stability Investigation of the Concatemeric dsDNACuNPs Strategy. A stable and long-lasting fluorescence emission is critically desired by CuNPs to facilitate real-time and long-time monitoring applications. However, conventionally adopted monomeric CuNPs are generally unstable and exhibit a continually decaying fluorescence in a short period after formation. Figure 4A and B illustrate the recorded variation of fluorescence intensities of CuNPs versus time. It was shown that with time prolonged, fluorescence signals of monomeric CuNPs formed with DNA2/DNA3 dsDNA first sharply increased to the highest value with a short stability in ∼20 min and thereafter quickly decreased. Especially when dsDNA concentration was low (100 nM), a weak fluorescence and a poor stability were simultaneously exposed. Although as the concentration of monomeric CuNPs increased, stability was slightly enhanced, the fluorescence signal still presented a sharp decline of 79% and 68% at 2.5 h for 500 nM and 1000 nM, respectively. In contrast, concatemeric dsDNA-CuNPs synthesized using the hybridized complex of 1 nM DNA1-induced RCR product with 1000 nM DNA2 displayed a much slower fluorescence decaying rate. At 2.5 h post-formation, the fluorescence signal of concatemeric dsDNA-CuNPs still reserved ∼60% of the highest value. The stability was ∼3 times the 500 nM monomeric CuNPs holding a comparative original fluorescence intensity, and even much better than 1000 nM monomeric CuNPs holding a much higher original fluorescence intensity (Figure S-3). It was revealed that the concatemeric dsDNA-CuNPs strategy could substantially ameliorate the stability of CuNPs, thus endowing it a longlasting signal function with effective fluorescence emission for future applications. We hypothesized that instability of CuNPs might be due to the effect of ·OH, which can be generated by Cu2+ and ascorbate (a Fenton-like reaction),38 and abstract an H atom from the DNA deoxyribose phosphate backbone,39 thus leading to template damage and fluorescence decay of CuNPs. To confirm this conjecture, agarose electrophoresis of dsDNA scaffolds was performed after incubation with Cu2+ and 6980

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Correspondingly, as shown in Figure 5C, the value of (FmiRNA − F0)/F0 in 1 nM let-7d-induced concatemeric dsDNA-CuNPs was ∼2.4 times and ∼3.8 times of that in let-7c and let-7i samples, respectively. Moreover, there was nearly no obvious fluorescent signal detected for the miR-21 control. It was demonstrated that let-7d could be easily differentiated from other let-7 family members and noncognate miRNA, suggesting good specificity of the concatemeric dsDNA-CuNPs assay platform. It is also worth mentioning that we first studied the feasibility of CuNPs synthesis using double-stranded RNA/RNA or RNA/DNA (Figure S-6). Results showed that dsRNA (let-7d/ c-let-7d RNA) seemed unable to template the formation of CuNPs, and only a faint fluorescence was detected even at a very high concentration of 2000 nM. By replacing c-let-7d RNA with c-let-7d DNA, double-stranded RNA/DNA-templated CuNPs displayed a slight fluorescence enhancement, but compared with dsDNA-CuNPs holding the same length of 22 bp, the signal was much weaker. It was clarified that the nucleic acid type could significantly affect the formation of CuNPs. It might be infeasible to directly detect miRNA by using double-stranded RNA/RNA or RNA/DNA to synthesize CuNPs. In contrast, through transformation of the miRNA target into DNA scaffolds, the concatemeric CuNPs strategy effectively solved the problem and achieved a simple, low-cost, sensitive, and selective miRNA assay.

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CONCLUSION In this paper, we presented the first attempt to overcome drawbacks of conventional monomeric dsDNA-CuNPs in sensitivity and stability. By introducing the RCR mechanism, a novel concatemeric dsDNA-CuNPs strategy was developed, in which a short oligonucleotide primer could be converted to a long concatemeric dsDNA scaffold to template the formation of CuNPs. The remarkable increase of thousands of dsDNA scaffold units endowed concatemeric dsDNA-CuNPs substantial improvement of fluorescent properties, including ∼10 000fold enhanced sensitivity and a ∼2 times elevated stability in comparison with monomeric dsDNA-CuNPs. These advantages might greatly facilitate the application of CuNPs in highly sensitive biochemical sensing as well as real-time and long-time monitoring. As proof of concept, a simple, low-cost, sensitive, and selective miRNA detection was performed using let-7d as the model target. By directly adopting let-7d as the primer in RCR, good linearity between 10 and 400 pM and a detection limit of 10 pM was obtained for let-7d assay with the concatemeric dsDNA-CuNPs strategy. Actually, this strategy might be applicable to various targets that can directly or indirectly induce RCR. Especially by integrating nucleic acid aptamers, its application might be further widened. Moreover, because of similar properties, this strategy might also be efficacious for the sensitivity and stability improvement of poly T-CuNPs, suggesting great potential in biochemical sensing research.

Figure 5. miRNA detection with the concatemeric dsDNA-CuNPs strategy. (A) Fluorescence spectra of concatemeric dsDNA-CuNPs formed using the hybridized complex of 1000 nM DNA3 with the RCR product induced by different concentrations of let-7d. (B) The corresponding calibration curve of the concatemeric dsDNA-CuNPs strategy for let-7d assay. The inset shows the linear response at low concentrations of let-7d. (Error bars represent standard deviations from three repeated experiments.) (C) Assay specificity investigation of the concatemeric dsDNA-CuNPs strategy to detect different miRNA sequences with the same concentration of 1 nM, including the let-7d target and three control miRNAs. (FmiRNA was the fluorescence intensity of CuNPs formed using the hybridized complex of 1000 nM DNA3 with the RCR product induced by different miRNA; F0 was the background signal produced by the RCR system without any primer in the presence of Cu2+ and ascorbate.)

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molecules in the same family of let-7 (let-7c and let-7i with three and six nucleotides different from let-7d, respectively) and one noncognate miRNA (miR-21) as controls. Agarose electrophoresis analysis of RCR products induced with different RNA sequences revealed that almost no RCR product was imaged for the noncognate miR-21 (Figure S-5B). As for let-7c and let-7i, the bands of RCR products were slightly brighter than the miR-21 control but much darker than that of let-7d.

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S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: 86-731-88821566. E-mail: [email protected]. 6981

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Author Contributions

(27) Hu, R.; Liu, Y. R.; Kong, R. M.; Donovan, M. J.; Zhang, X. B.; Tan, W. H.; Shen, G. L.; Yu, R. Q. Biosens. Bioelectron. 2013, 42, 31− 35. (28) Zhang, L. L.; Zhao, J. J.; Zhang, H.; Jiang, J. H.; Yu, R. Q. Biosens. Bioelectron. 2013, 44, 6−9. (29) Zhao, W. A.; Ali, M. M.; Brook, M. A.; Li, Y. F. Angew. Chem., Int. Ed. 2008, 47, 6330−6337. (30) Cheng, Y. Q.; Zhang, X.; Li, Z. P.; Jiao, X. X.; Wang, Y. C.; Zhang, Y. L. Angew. Chem., Int. Ed. 2009, 48, 3268−3272. (31) Larsson, C.; Koch, J.; Nygren, A.; Janssen, G.; Raap, A. K.; Landegren, U.; Nilsson, M. Nat. Methods 2004, 1, 227−232. (32) Hu, J.; Zhang, C. Y. Anal. Chem. 2010, 82, 8991−8997. (33) Nilsson, M.; Malmgren, H.; Samiotaki, M.; Kwiatkowski, M.; Chowdhary, B. P.; Landegren, U. Science 1994, 265, 2085−2088. (34) Fire, A.; Xu, S. Q. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 4641− 4645. (35) Liu, D. Y.; Daubendiek, S. L.; Zillman, M. A.; Ryan, K.; Kool, E. T. J. Am. Chem. Soc. 1996, 118, 1587−1594. (36) Calin, G. A.; Croce, C. M. Nat. Rev. Cancer 2006, 6, 857−866. (37) Lu, J.; Getz1, G.; Miska, E. A.; Alvarez-Saavedra, E.; Lamb, J.; Peck, D.; Sweet-Cordero, A.; Ebert, B. L.; Mak, R. H.; Ferrando, A. A.; Downing, J. R.; Jacks, T.; Horvitz, H. R.; Golub, T. R. Nature 2005, 435, 834−838. (38) Stadtman, E. R. Am. J. Clin. Nutr. 1991, 54, 1125S−1128S. (39) Balasubramanian, B.; Pogozelski, W. K.; Tullius, T. D. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 9738−9743. (40) Liu, M.; Zhang, Q.; Zhao, H. M.; Chen, S.; Yu, H. T.; Zhang, Y. B.; Quan, X. Chem. Commun. 2011, 47, 4084−4086. (41) Friedman, R. C.; Farh, K. K. H.; Burge, C. B.; Bartel, D. P. Genome Res. 2009, 19, 92−105. (42) Dalmay, T.; Edwards, D. R. Oncogene 2006, 25, 6170−6175. (43) Brueckner, B.; Stresemann, C.; Kuner, R.; Mund, C.; Musch, T.; Meister, M.; Sultmann, H.; Lyko, F. Cancer Res. 2007, 67, 1419−1423.

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These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grants 21175039, 21190044, 21221003, 21322509, 21305035, and 21305038), Hunan Province Science and Technology Project of China (2013FJ4042), and Hunan Provincial Graduate Research and Innovation Projects (CX2013B139).

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REFERENCES

(1) Zheng, J.; Petty, J. T.; Dickson, R. M. J. Am. Chem. Soc. 2003, 125, 7780−7781. (2) Xie, J. P.; Zheng, Y. G.; Ying, J. Y. J. Am. Chem. Soc. 2009, 131, 888−889. (3) Yuan, X.; Luo, Z. T.; Zhang, Q. B.; Zhang, X. H.; Zheng, Y. G.; Lee, J. Y.; Xie, J. P. ACS Nano 2011, 5, 8800−8808. (4) Tanaka, S. I.; Miyazaki, J.; Tiwari, D. K.; Jin, T.; Inouye, Y. Angew. Chem., Int. Ed. 2011, 50, 431−435. (5) Ma, N.; Sargent, H. E.; Kelley, O. S. Nat. Nanotechnol. 2009, 4, 121−125. (6) Petty, J. T.; Zheng, J.; Hud, N. V.; Dickson, R. M. J. Am. Chem. Soc. 2004, 126, 5207−5212. (7) Wang, Z. D.; Zhang, J. Q.; Ekman, J. M.; Kenis, P. J. A.; Lu, Y. Nano Lett. 2010, 10, 1886−1891. (8) Ritchie, C. M.; Johnsen, K. R.; Kiser, J. R.; Antoku, Y.; Dickson, R. M.; Petty, J. T. J. Phys. Chem. C 2007, 111, 175−181. (9) Guo, W. W.; Yuan, J. P.; Wang, E. K. Chem. Commun. 2009, 23, 3395−3397. (10) Su, Y. T.; Lan, G. Y.; Chen, W. Y.; Chang, H. T. Anal. Chem. 2010, 82, 8566−8572. (11) Lan, G. Y.; Huang, C. C.; Chang, H. T. Chem. Commun. 2010, 46, 1257−1259. (12) Sharma, J.; Yeh, H. C.; Yoo, H.; Werner, J. H.; Martinez, J. S. Chem. Commun. 2011, 47, 2294−2296. (13) Liu, Y. Q.; Zhang, M.; Yin, B. C.; Ye, B. C. Anal. Chem. 2012, 84, 5165−5169. (14) Yang, S. W.; Vosch, T. Anal. Chem. 2011, 83, 6935−6939. (15) Guo, W. W.; Yuan, J. P.; Dong, Q. Z.; Wang, E. K. J. Am. Chem. Soc. 2010, 132, 932−934. (16) Yu, J. H.; Choi, S.; Richards, C. I.; Antoku, Y.; Dickson, R. M. Photochem. Photobiol. 2008, 84, 1435−1439. (17) Yu, J. H.; Choi, S.; Dickson, R. M. Angew. Chem., Int. Ed. 2009, 48, 318−320. (18) Sun, Z. P.; Wang, Y. L.; Wei, Y. T.; Liu, R.; Zhu, H. R.; Cui, Y. Y.; Zhao, Y. L.; Gao, X. Y. Chem. Commun. 2011, 47, 11960−11962. (19) Rotaru, A.; Dutta, S.; Jentzsch, E.; Gothelf, K.; Mokhir, A. Angew. Chem., Int. Ed. 2010, 49, 5665−5667. (20) Qing, Z. H.; He, X. X.; He, D. G.; Wang, K. M.; Xu, F. Z.; Qing, T. P.; Yang, X. Angew. Chem., Int. Ed. 2013, 52, 9719−9722. (21) Qing, Z. H.; He, X. X.; Qing, T. P.; Wang, K. M.; Shi, H.; He, D. G.; Zou, Z.; Yan, L. A.; Xu, F. Z.; Ye, X. S.; Mao, Z. G. Anal. Chem. 2013, 85, 12138−12143. (22) Chen, J. H.; Liu, J.; Fang, Z. Y.; Zeng, L. W. Chem. Commun. 2012, 48, 1057−1059. (23) Zhang, L. L.; Zhao, J. J.; Duan, M.; Zhang, H.; Jiang, J. H.; Yu, R. Q. Anal. Chem. 2013, 85, 3797−3801. (24) Jia, X. F.; Li, J.; Han, L.; Ren, J. T.; Yang, X.; Wang, E. K. ACS Nano 2012, 6, 3311−3317. (25) Liu, G. Y.; Shao, Y.; Peng, J.; Dai, W.; Liu, L. L.; Xu, S. J.; Wu, F.; Wu, X. H. Nanotechnology 2013, 24, 345502. (26) Zhou, Z. X.; Du, Y.; Dong, S. J. Anal. Chem. 2011, 83, 5122− 5127. 6982

dx.doi.org/10.1021/ac500955r | Anal. Chem. 2014, 86, 6976−6982