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Target-Responsive DNAzyme Cross-Linked Hydrogel for Visual Quantitative Detection of Lead Yishun Huang, Yanli Ma, Yahong Chen, Xuemeng Wu, Luting Fang, Zhi Zhu,* and Chaoyong James Yang* The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, Innovation Center of Chemistry for Energy Materials, State Key Laboratory of Physical Chemistry of Solid Surfaces, the Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, Fujian, People’s Republic of China S Supporting Information *

ABSTRACT: Because of the severe health risks associated with lead pollution, rapid, sensitive, and portable detection of low levels of Pb2+ in biological and environmental samples is of great importance. In this work, a Pb2+-responsive hydrogel was prepared using a DNAzyme and its substrate as cross-linker for rapid, sensitive, portable, and quantitative detection of Pb2+. Gold nanoparticles (AuNPs) were first encapsulated in the hydrogel as an indicator for colorimetric analysis. In the absence of lead, the DNAzyme is inactive, and the substrate cross-linker maintains the hydrogel in the gel form. In contrast, the presence of lead activates the DNAzyme to cleave the substrate, decreasing the cross-linking density of the hydrogel and resulting in dissolution of the hydrogel and release of AuNPs for visual detection. As low as 10 nM Pb2+ can be detected by the naked eye. Furthermore, to realize quantitative visual detection, a volumetric bar-chart chip (V-chip) was used for quantitative readout of the hydrogel system by replacing AuNPs with gold−platinum core−shell nanoparticles (Au@PtNPs). The Au@PtNPs released from the hydrogel upon target activation can efficiently catalyze the decomposition of H2O2 to generate a large volume of O2. The gas pressure moves an ink bar in the V-chip for portable visual quantitative detection of lead with a detection limit less than 5 nM. The device was able to detect lead in digested blood with excellent accuracy. The method developed can be used for portable lead quantitation in many applications. Furthermore, the method can be further extended to portable visual quantitative detection of a variety of targets by replacing the lead-responsive DNAzyme with other DNAzymes.

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reaching the defined detection limits,15 they require sophisticated instrumentation and complicated sample preparation processes. In recent decades, various sensors for Pb2+ have been reported, including Pb2+-dependent DNAzyme-based sensors.16,17 The DNAzyme has the RNA nuclease activity to cleave the ribonucleotide in the substrate and is highly specific for Pb2+ as its cofactor. The DNAzyme has been utilized in various designs to achieve sensitive detection of lead ion via different readout methods, including fluorescence,17−19 electrochemical signal,20,21 and chemiluminescence.22,23 However, these methods are still limited by the need for expensive and complicated instrumentation and skilled operators. To achieve portable detection, Liu and Lu designed DNAzyme−gold nanoparticles for a colorimetric method which can detect as low as 100 nM lead in leaded paint with tunable dynamic range.24 Li’s group also demonstrated a gold nanoparticle-based colorimetric method.25 Further, Mazumdar et al. developed a

s a heavy metal, lead is severely toxic at high doses, possibly due to its interference with various enzyme systems. Lead ion can bind to thiol groups of enzymes or displace other essential metal ions, thus affecting a wide range of biological systems.1 Lead pollution severely endangers human health, especially in children, by irreversible brain damage, mental retardation, and development issues, leading to attention deficit and learning disabilities.2 Lead exposure can occur through a variety of sources, including air, bare soil, home remedies, drinking water, toy jewelry, lead-based paints, and others.1,3,4 According to the United States Environmental Protection Agency (EPA), the level of lead in the blood is considered toxic when its concentration is higher than 0.1 mg/ L (483 nM). In drinking water systems, the maximum allowable level of lead is 0.015 mg/L (72.4 nM). Therefore, as a public health concern, the development of ultrasensitive assays for rapid and accurate detection of lead is very important for water quality control, clinical toxicology, and industrial monitoring. Although traditional methods, such as atomic absorption spectrometry (AAS),5−7 inductively coupled plasma mass spectrometry (ICPMS),8−12 and inductive coupled plasma atomic emission spectrometry (ICP-AES),13,14 are capable of © XXXX American Chemical Society

Received: September 22, 2014 Accepted: October 23, 2014

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dipstick test with a detection limit of 0.5 μM which is ideal for lead detection in household paints.26 However, because the enzymatic activity was decreased when the DNAzyme was bound to the surface of the nanoparticles,27 the limit of detection was too high for detection of Pb2+ in field work or in remote areas. Stimuli-responsive smart hydrogels have attracted particular attention in the development of biosensor devices with the advantages of simplicity, sensitivity, cost-effectiveness, and portability, as well as ease of storage. In particular, DNA hydrogels with synthetic polymers as backbone and functional DNA as cross-linker have been widely used for portable detection of various targets,28,29 such as small molecules,30,31 ions,32−35 and even proteins.36 Previously, we have constructed hydrogels responsive to ATP, cocaine,30,31 and copper ion (Cu2+)33 by utilizing aptamer and DNAzyme as cross-linker. The hydrogel systems displayed excellent portability with multiple convenient readout features.30−32 In this work, a lead ion responsive hydrogel utilizing the substrate of a Pb2+-dependent DNAzyme as cross-linker was designed and synthesized for rapid, sensitive, portable, and quantitative detection of Pb2+. The presence of Pb2+ activates the DNAzyme and induces the cleavage of the substrate sequence to destabilize the hydrogel, thus releasing the indicator AuNPs for colorimetric detection. To further realize quantitative visual detection, a volumetric bar-chart chip (Vchip), in which indicator AuNPs were replaced by gold− platinum core−shell nanoparticles (Au@PtNPs), was used as quantitative readout for the hydrogel system. The released Au@PtNPs from the hydrogel can efficiently catalyze the decomposition of H2O2 to generate a large volume of O2, leading to movement of an ink bar in the V-chip for portable, visual, and quantitative detection of lead with a detection limit less than 5 nM. Our method demonstrates that metal ions can be visually quantified using a metal-responsive DNAzyme crosslinked hydrogel as the signal recognition element and a V-chip as the visual and quantitative readout.

in the DNA synthesizer to obtain acrydite−DNA. 1H NMR (400 MHz, CDCl3): 5.85 (s, 1H), 5.67 (s, 1H), 5.30 (s, 1H), 3.80−3.75 (m, 2H), 3.70−3.50 (m, 4H), 3.35−3.25 (m, 2H), 2.65 (t, 2H), 1.95 (m, 3H), 1.68−1.52 (m, 4H) 1.47−1.30 (m, 4H) 1.22−1.15 (m, 12H). 13C NMR (400 MHz, CDCl3): 168.4, 140.2, 119.1, 117.7, 63.5, 58.3, 42.9, 39.5, 31.0, 29.5, 26.6, 25.6, 24.6, 20.3, 18.7. Synthesis of Oligonucleotides. All oligonucleotides used in the work were synthesized on an ABI 394 DNA synthesizer according to the standard DNA synthesis protocol. DNA sequences used for this work are shown in Supporting Information Table S1. Strands A1 and strand A2 were modified at the 5′-end using the acrydite phosphoramidite. After DNA synthesis and modification, the product was cleaved from the solid support, deprotected with ammonia−water treatment, and purified with an LC3000 semipreparative high-performance liquid chromatography (HPLC) system using a reversed-phase C18 column (Chuang Xin Tong Heng, Beijing, China). A solution of 0.1 M triethylamine acetate (pH 7.0) was used as HPLC buffer A, and HPLC-grade acetonitrile was used as HPLC buffer B. After detritylation by 80% (v/v) acetate acid, the DNA product was desalted with a 3k NMWL (nominal molecular weight limit) ultracentrifugal filter three times, quantified by UV−vis spectrometer, and stored at −20 °C for future use. Substrate strand containing adenosine triphosphate (rA) was purified according to the protocols of Glen Research with the following changes. After the above-mentioned detritylation, another 2′-O-triisopropylsilyloxymethyl group was removed by incubating in 100 μL of DMSO plus 125 μL of triethylamine trihydrofluoride for 2.5 h at 65 °C. Then, 25 μL of 3 M sodium acetate and 1.5 mL of N-butyl alcohol were added and the mixture was held at −20 °C for at least 30 min to precipitate DNA. The DNA pellet was dissolved by RNasefree water and desalted by 3k filter three times and stored at −20 °C.37 Preparation of Polyacrylamide−DNA Conjugates. Solutions of 1 mM concentration of strands A1 and A2 were prepared separately in centrifuge tubes containing 4% acrylamide. After vacuum desiccation for 10 min to remove air at 37 °C, 1.4% (v/v) of freshly prepared initiator (0.05 g of APS dissolved in 0.5 mL of ultrapure water) and accelerator (25 μL TEMED dissolved in 0.5 mL of ultrapure water) solution were added to the solutions immediately. The centrifuge tubes were placed in the vacuum desiccator again for 15 min at 37 °C to allow polymerization to take place, yielding polyacrylamide− DNA conjugates pA1 and pA2. A 100k NMWL ultracentrifugal filter was used for the elimination of unpolymerized strands. Preparation of DNAzyme Hydrogel. To avoid the aggregation of AuNPs and Au@PtNPs in the hydrogel at high salt concentrations, the nanoparticle surfaces were coated with polymers. Specifically, 2.5 nM 13 nm AuNPs were treated with 1 μM thiol-polyethylene glycol (MW 5000) and then centrifuged and redispersed three times using RNase-free water and concentrated to 150 nM. The 2.5 nM 16 nm Au@PtNPs were treated with 100 μM DNAzyme strand at 85 °C for 2 h before use to remove protecting reagents on the nanoparticles which may influence the subsequent DNAzyme catalytic reaction and meanwhile protect nanoparticles with DNA. To generate hydrogel, pA1 and pA2 were mixed with substrate and DNAzyme strands at a molar ratio of 100 μM (pA1)/100 μM (pA2)/50 μM (substrate)/100 μM (DNAzyme strand) with addition of AuNPs or Au@PtNPs at a final concentration of 30 or 4.6 nM in reaction buffer. The mixture was shaken

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EXPERIMENTAL SECTION Materials. The reagents for DNA synthesis were purchased from Glen Research (Sterling, VA, U.S.A.). Lead(II) acetate, acetate acid, sodium chloride, ammonium persulfate (APS), tetramethylethylenediamine (TEMED), and acrylamide were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Other reagents were purchased from Sinopharm Chemical Reagents (Shanghai, China). Synthesis of Acrydite Phosphoramidite. A mixture of 6amino-1-hexanol (1 g, 8.53 mmol) and triethylamine (2.36 mL, 17 mmol) was precooled to 0 °C. Methacryloyl chloride (2.67 g, 2.55 mmol) was added dropwise, and afterward stirred at 0 °C for 2 h. After evaporating all solvent, the reaction product was redissolved in 10 mL of ethanol and 4 mL of 15% sodium hydroxide. The solvent was evaporated, and 6-hydroxyhexyl methacrylamide was chromatographed on a column of silica gel G using ethyl acetate. To a solution containing 6-hydroxyhexyl methacrylamide (0.50 g, 2.70 mmol) in anhydrous CH2Cl2 (10 mL), N,N′-diisopropylethylamine (DIPEA) (0.98 g, 7.50 mmol) was added slowly at 0 °C, followed by dropwise addition of 2-cyanoethyl diisopropyl chlorophosphoramidite (0.87 mL, 3.25 mmol). The reaction mixture was stirred at 0 °C for 2 h. After removing the solvent, the residue was purified by column chromatography (ethyl acetate/hexane/triethylamine 40:60:3) and dried to a colorless oil. The final product was used B

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Figure 1. Working principle of DNAzyme cross-linked hydrogel for visual detection of lead ions. In the presence of Pb2+, the DNAzyme cleaves the cross-linking substrate strand and releases the encapsulated AuNPs to the supernatant, which can be monitored by the naked eye.

vigorously three times at high temperature (60 °C) to guarantee the homogeneity of the hydrogel, and then allowed to slowly cool to room temperature to produce DNAzyme cross-linked hydrogel with AuNPs or Au@PtNPs trapped inside. The reaction buffer is 10 mM Tris−acetate (pH 8.0, containing 300 mM NaCl). Colorimetric Detection of Pb2+. Aliquots of 50 μL of different concentrations of Pb2+ were added to 10 μL of AuNPencapsulated hydrogel. The reaction was allowed to take place at 25 °C with gentle shaking at 150 rpm for 2.5 h. Naked eye observation of color change can be recorded by camera for determination of concentration of Pb2+. Alternatively, UV−vis absorbance of the supernatant can be measured for more precise results. Hydrogel Integrated with V-chip for Quantitative Detection of Pb 2+ . Aliquots of 50 μL of different concentrations of Pb2+ were added to 10 μL of Au@PtNPencapsulated hydrogel. The reaction was allowed to take place at 25 °C with gentle shaking at 150 rpm for 2.5 h. The supernatant was then transferred to V-chip, where the released Au@PtNPs was mixed with H2O2. Catalytic decomposition of H2O2 generate a large amount of O2, pushing the ink bar to move in the V-chip. The travel distance ink bar was then used for quantitative detection of the concentration of Pb2+. Safety Warning. Experiments involving heavy-metal ions should be performed with protective gloves. The waste solutions should be reclaimed.

collapses. Meanwhile, the DNAzyme strand and the substrate strand also dissociate to release Pb2+, which can bind to another preformed DNAzyme/substrate complex and facilitate the cleavage of another substrate strand. If gold nanoparticles (AuNPs) are added prior to the formation of hydrogel, they will be trapped inside the three-dimensional (3-D) network of the hydrogel. When target Pb2+ is introduced to dissolve the gel, the AuNPs will be released and change the supernatant from colorless to red. Therefore, the degree of color change can be used to indicate the concentration of Pb2+. Feasibility of DNAzyme Cross-Linked Hydrogel for Pb2+ Detection. To validate the feasibility of the DNAzyme cross-linked hydrogel for lead ion detection, the DNAzyme cross-linked hydrogel was prepared with AuNPs trapped inside. The sequence of DNAzyme and substrate was initially adopted from Lan et al.38 However, gel electrophoresis experiment results (Supporting Information Figure S1) suggested that nonspecific cleavage may occur without Pb2+ when high concentration (10 μM) of substrate and DNAzyme strand are used. Therefore, we optimized the sequences by removing one base (Enz-1), two bases (Enz-2), and three bases (Enz-3) from the DNAzyme strand on the 5′ end. The optimized Enz-3 was found to have high stability in the absence of Pb2+ and high cleaving efficiency in the presence of Pb2+ and was therefore used in the subsequent experiments. Then, the concentrations of DNAzyme and cross-linker strand were also optimized to be 100 and 50 μM, respectively (Supporting Information Figure S2). As shown in Figure 2A, at these concentrations, the hydrogel was well-formed and sufficiently stable to keep the AuNPs inside. Upon the addition of 1 μM Pb2+, the hydrogel dissociated to release AuNPs into the supernatant, showing an obvious change from colorless to red for the supernatant solution. The kinetics of AuNP release were observed by monitoring the absorbance of AuNPs in the supernatant at 520

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RESULTS AND DISCUSSION Working Principle of Lead Ion Responsive DNAzyme Cross-linked Hydrogel. The working principle of the lead ion responsive DNAzyme cross-linked hydrogel is schematically shown in Figure 1. Two short DNA strands (A1 and A2) are copolymerized with acrylamide to form linear DNA− polyacrylamide conjugates, pA1 and pA2, respectively. The cross-linker strand is the substrate sequence of the lead DNAzyme with its two ends extended to be complementary to A1 and A2, respectively. To prepare a lead ion responsive hydrogel, linear DNA−polyacrylamide conjugates pA1 and pA2 are mixed with the cross-linker and DNAzyme so that the substrate binds with the DNAzyme to form a stem−loop structure and hybridizes with pA1 and pA2 to cross-link the polymer chain and eventually form the DNA hydrogel. Because the DNAzyme requires lead ion as its cofactor, in the absence of lead ion, the substrate strand is stably hybridized with A1, DNAzyme, and A2, leading to a stable hydrogel. In contrast, in the presence of lead ion, binding of Pb2+ with DNAzyme activates its enzymatic activity, leading to the cleavage of the cross-linker strand at the middle ribonucleotide site. As a result, the substrate strand is split into two parts and the hydrogel

Figure 2. Feasibility of DNAzyme cross-linked hydrogel for rapid visual detection of lead ions. Colorimetric comparison of the response of the prepared hydrogel after the addition of 1 μM Pb2+ and no Pb2+ (A) and their corresponding release kinetics obtained by monitoring the absorbance of AuNPs at 520 nm (B). C

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Figure 3. Sensitivity of DNAzyme cross-linked hydrogel for Pb2+ detection. Different concentrations of Pb2+ in the samples induced the decomposition of DNAzyme cross-linked hydrogel to different degrees, as observed by the naked eye (A) and corresponding absorbance of AuNPs at 520 nm (B). The linear range is observed between 0 and 100 nM.

nm. (Figure 2B). The AuNPs were rapidly released, reaching a plateau after 2 h. In contrast, there was no obvious absorbance change when there was no target added. These experiments clearly suggest that the DNA hydrogel were successfully synthesized and can rapidly respond to lead ions. Sensitive and Specific Colorimetric Detection of Pb2+. The performance of the AuNP-encapsulated hydrogel for sensitive colorimetric detection of Pb2+ was evaluated by treating the hydrogel with different concentrations of Pb2+. As shown in the naked-eye observation in Figure 3A, increasing concentrations of Pb2+ resulted in more cleavage reactions and a decrease in cross-linker density, thereby enhancing the dissociation of the hydrogel and subsequent release of AuNPs to the supernatant. The color change of the sample with 10 nM Pb2+ could be clearly distinguished from the background by direct observation. UV−vis spectroscopy (Figure 3B) shows the relationship between the absorbance of AuNPs at 520 nm and the concentration of Pb2+. The linear range was obtained between 0 and 100 nM. As the concentration of Pb2+ increased further, the cross-linkers were depleted and the absorbance reached a plateau. The detection limit was calculated to be 3.4 nM based on the 3σ rule. This value is lower than the requirement of the EPA’s drinking water regulations for lead. Therefore, our colorimetric method is suitable for sensitive and visual detection of Pb2+. Supporting Information Figure S3 shows the release kinetics of AuNPs in the presence of different concentrations of Pb2+. For high concentrations of Pb2+, the release rate of AuNPs showed an initial delay, followed by a rapid rise and a gentle plateau at the end. For low concentrations (0−100 nM) of Pb2+, the release rate was still rising after 2.5 h of reaction, and these concentrations were in the linear range for analysis. The detection of Pb2+ by the DNAzyme cross-linked hydrogel is highly selective due to the high selectivity of the DNAzyme sequence. In the presence of high concentrations of other cations, including Cu2+, Fe3+, Mg2+, Ca2+, Cd2+, Zn2+, Mn2+, Ni2+, and Co2+ (1 mM, 1000 times that of Pb2+), no observable absorbance changes were observed visually or with a UV−vis spectrometer, as shown in Figure 4A and 4B, respectively. In contrast, 1 μM Pb2+ can completely dissociate the hydrogel and give significant color and absorbance changes. The high selectivity of the Pb2+ DNAzyme hydrogel successfully eliminates the interference of other cations and demonstrates the high fidelity of this method in specific Pb2+ detection. To further evaluate the specificity of our method in a complex sample, seawater, which was proven to have negligible Pb2+ concentration by ICPMS, was spiked with 30, 100, and 300 nM Pb2+. As shown in Figure 4C, seawater with the 30 nM Pb2+ spike was distinct from the blank sample. With increasing

Figure 4. Specificity of DNAzyme hydrogel. (A) Response of hydrogel to 1 mM cations and 1 μM Pb2+. (B) The corresponding absorbance at 520 nm of the supernatant. (C) Responses of DNAzyme hydrogel to different concentrations of lead ion in seawater.

concentration of Pb2+, the hydrogel gradually dissociated to give a more obvious color change. Therefore, despite the complex matrix of the seawater, as low as 30 nM Pb2+ could be detected by our method, demonstrating the sensitivity and specificity of our hydrogel method for Pb2+ detection in complex samples. DNAzyme Cross-Linked Hydrogel with Microfluidic Chip for Portable and Visual Quantitative Detection of Pb2+. Colorimetric detection is sometimes limited when the color contrast is not high enough for detection of low concentrations of Pb2+. Moreover, colorimetric detection can provide only qualitative or semiquantitative results, while in certain cases a quantitative result is needed. Recently, we have integrated aptamer cross-linked hydrogel as a molecular recognition element with volumetric bar-chart chip (V-chip) as a distance-based visual and quantitative readout apparatus for sensitive detection of cocaine.39−42 In this work we further demonstrated that combining the DNAzyme hydrogel as the target recognition element and a V-chip as the quantitative readout device, lead ion can be portably, visually, and quantitatively detected. The working principle of the target-responsive DNAzyme cross-linked hydrogel with volumetric bar-chart chip readout for visual quantitative detection is shown in Figure 5A. First, Au core/Pt shell nanoparticles (Au@PtNPs) were entrapped in the hydrogel instead of AuNPs, as we have previously demonstrated that Au@PtNPs can accelerate the decomposition of H2O2 to produce O2 gas.39 In the presence of lead ion, the hydrogel dissociates to release the encapsulated Au@PtNPs into the supernatant solution, which is then loaded into the Vchip. The V-chip consists of two glass slides with different D

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Figure 5. Sensitive and portable detection of lead with volumetric bar-chart chip as visual readout device. (A) The working principle of the targetresponsive DNAzyme cross-linked hydrogel with volumetric bar-chart chip readout method for visual quantitative detection. (B) Different travel distance of the ink bar with different concentration of Pb2+ in buffer and digested blood sample. (C) The linear response of ink bar distance to Pb2+ concentration.

that the accuracy our method is as good as that of the standard ICPMS method for real sample analysis.

disconnected channel patterns etched (shown in Supporting Information Figure S4). The disconnected channel patterns of these two glass slides are designed in such a way that, after being assembled, by adjusting the relative position of these two glass slides, their otherwise disconnected channels can join together to form horizontally or vertically complementarily connected long channels. For example, as shown in Figure 5A, at the sample loading position, all vertical channels are disconnected while the horizontal channels are connected, allowing the loading of samples containing Au@PtNPs in the bottom lane, H2O2 in the middle lane, and ink in the top lane. After sample loading, the top layer slides up to the reading position for mixing, reaction, and reading, where the horizontal channels are disconnected yet the vertical channels are connected. At the reading position, the hydrogel supernatant is brought to mix with H2O2. When there are Au@PtNPs present in the supernatant, O2 is generated and the ink in the top row of wells is pushed into the small bars and moves up the channel. The distance that each ink bar chart moves within a specified time depends on the number of catalyst Au@PtNPs, which, in turn, is proportional to the lead concentration. Thus, the lead concentration can be visually and quantitatively detected without the need for an external electrical device and power source. As shown in Figure 5B, with increasing concentrations of Pb2+, the migration distance of the ink bar increased. The linear relationship between the distances and the concentration of Pb2+ was obtained from 0 to 150 nM (Figure 5C), and the detection limit was calculated to be 2.6 nM according to the 3σ rule. The linear regression equation was obtained as y = 0.14x + 0.07, with a correlation coefficient of 0.996. The DNAzyme−cross-linked hydrogel−V-chip system was further tested with a digested blood sample. The concentration of Pb2+ in blood sample was determined to be 60 ± 2 nM in a 10-fold dilution of the original blood sample. This result agrees well with the ICPMS result of 61.8 ± 14.4 nM, demonstrating

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CONCLUSIONS In conclusion, a lead ion responsive hydrogel was prepared using DNAzyme and its substrate as cross-linker for rapid, sensitive, visual, portable, and quantitative detection of Pb2+. The presence of Pb2+ activates the DNAzyme and induces cleavage of the substrate strand to dissolve the hydrogel, thus releasing the indicator AuNPs for colorimetric detection. As low as 10 nM Pb2+ can be visually detected with high specificity. To realize quantitative and visual detection, a volumetric barchart chip was used for quantitative readout of the hydrogel response by replacing AuNPs with Au@PtNPs. The Au@ PtNPs released from the hydrogel in the presence of Pb2+ can efficiently catalyze the decomposition of H2O2 to generate a large volume of O2 to move an ink bar in the chip for portable, visual, and quantitative detection of Pb2+ with a detection limit of 2.6 nM. The result of the hydrogel−V-chip method for Pb2+ detection in a digested blood sample agreed well with the ICPMS result, establishing the accuracy and reliability of our method. By replacing the lead-responsive DNAzyme with other DNAzymes, the method can potentially be further extended to portable visual quantitative detection of a variety of targets.

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ASSOCIATED CONTENT

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 Authors

*Phone: +86 592-218-7601. Fax: +86 592-218-9959. E-mail: [email protected]. *Phone: +86 592-218-7601. Fax: +86 592-218-9959. E-mail: [email protected]. E

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Notes

(30) Yan, L.; Zhu, Z.; Zou, Y.; Huang, Y.; Liu, D.; Jia, S.; Xu, D.; Wu, M.; Zhou, Y.; Zhou, S.; Yang, C. J. J. Am. Chem. Soc. 2013, 135, 3748− 3751. (31) Zhu, Z.; Wu, C.; Liu, H.; Zou, Y.; Zhang, X.; Kang, H.; Yang, C. J.; Tan, W. Angew. Chem., Int. Ed. 2010, 49, 1052−1056. (32) Dave, N.; Chan, M. Y.; Huang, P.-J. J.; Smith, B. D.; Liu, J. J. Am. Chem. Soc. 2010, 132, 12668−12673. (33) Lin, H.; Zou, Y.; Huang, Y.; Chen, J.; Zhang, W. Y.; Zhuang, Z.; Jenkins, G.; Yang, C. J. Chem. Commun. 2011, 47, 9312−9314. (34) Guo, W.; Qi, X.-J.; Orbach, R.; Lu, C.-H.; Freage, L.; MironiHarpaz, I.; Seliktar, D.; Yang, H.-H.; Willner, I. Chem. Commun. 2014, 50, 4065−4068. (35) Guo, W.; Orbach, R.; Mironi-Harpaz, I.; Seliktar, D.; Willner, I. Small 2013, 9, 3748−3752. (36) Zhang, L.; Lei, J.; Liu, L.; Li, C.; Ju, H. Anal. Chem. 2013, 85, 11077−11082. (37) Glen Research. Procedure for the synthesis, deprotection and isolation of RNA using TOM-protected monomers. http://www. glenresearch.com/Technical/TB_RNA_TOM_Deprotection.pdf. (38) Lan, T.; Furuya, K.; Lu, Y. Chem. Commun. 2010, 46, 3896− 3898. (39) Zhu, Z.; Guan, Z.; Jia, S.; Lei, Z.; Lin, S.; Zhang, H.; Ma, Y.; Tian, Z.-Q.; Yang, C. J. Angew. Chem., Int. Ed. [Online early access]. DOI: 10.1002/anie.201405995. Published Online: Aug 11, 2014. http://onlinelibrary.wiley.com/doi/10.1002/anie.201405995/abstract. (40) Song, Y.; Zhang, Y.; Bernard, P. E.; Reuben, J. M.; Ueno, N. T.; Arlinghaus, R. B.; Zu, Y.; Qin, L. Nat. Commun. 2012, 3, 1283. (41) Song, Y.; Wang, Y.; Qin, L. J. Am. Chem. Soc. 2013, 135, 16785− 16788. (42) Song, Y.; Xia, X.; Wu, X.; Wang, P.; Qin, L. Angew. Chem., Int. Ed. [Online early access]. DOI: 10.1002/anie.201404349. Published Online: July 17, 2014. http://onlinelibrary.wiley.com/doi/10.1002/ anie.201404349/abstract.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Basic Research Program of China (2010CB732402, 2013CB933703), the National Science Foundation of China (91313302, 21205100, 21275122, 21075104, 21422506), National Instrumentation Program (2011YQ03012412), National Found for Fostering Talents of Basic Science (NFFTBS, J1310024), the Natural Science Foundation of Fujian Province (2013J01061), and the National Science Foundation for Distinguished Young Scholars of China (21325522) for their financial support.

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dx.doi.org/10.1021/ac503540q | Anal. Chem. XXXX, XXX, XXX−XXX