Stabilization of ssRNA on Graphene Oxide Surface: An Effective Way

Jan 16, 2013 - As illustrated in Scheme 1, without any protection, an RNA probe [e.g., ... lysis buffer (150 mM NaCl, 1.0% Tritonx-100, 50 mM Tris-HCl...
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Stabilization of ssRNA on Graphene Oxide Surface: An Effective Way to Design Highly Robust RNA Probes Liang Cui, Zirong Chen, Zhi Zhu, Xiaoyan Lin, Xi Chen, and Chaoyong James Yang* State Key Laboratory of Physical Chemistry of Solid Surfaces, the Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Science, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, P. R. China S Supporting Information *

ABSTRACT: RNA probes constitute an important class of functional nucleic acids (FNAs). However, because of their notorious vulnerability to enzymatic degradation, extremely careful and special protocols must be followed when dealing with RNA probes. To fully use the large number of RNA FNAs available for bioanalysis and biomedicine, it is important to explore effective methods to protect RNA probes from enzymatic digestion. In this work, we systematically demonstrate that graphene oxide (GO) can effectively protect RNA probes from enzymatic digestion. Based on this finding, we propose an effective way to design robust RNA biosensors by simply mixing RNA probes with GO for analysis of nucleic acids, proteins, and small molecules. The entire assay is sensitive, selective, rapid, and more importantly, does not require any special protocols. The ability to protect ssRNA from enzymatic digestion by GO offers an exciting new way to stabilize ssRNA, which will not only provide new opportunities to utilize the large number of currently available, yet rarely explored, RNA FNAs for bioanalysis but also offer a new solution to protect important ssRNA molecules, such as microRNA and antisense ssRNA, for a great variety of biomedical applications.

O

the detection of a variety of analytes.14,19,20 It has also been found that DNA absorbed on GO surfaces can be effectively protected from nuclease digestion. For example, Lin’s group reported that ssDNA absorbed on GO surfaces can be effectively protected from enzymatic cleavage by DNase I.21 Zhang’s group and Li’s group found that in the presence of sufficient GO, dsDNA can also be adsorbed and exhibit enhanced resistance to several types of nucleases.22,23 The excellent protective property of GO against DNA nuclease digestion has, thus, further extended the applications of DNA probes both in vivo and in vitro.24−27 Furthermore, dsRNA has been immobilized on positively charged functionalized GO via electrostatic interaction with the aid of 1-pyrenemethylamine hydrochloride or polyethylenimine for siRNA delivery.28,29 The ssRNA has also been immobilized on GO via directed chemical conjugation30 for toxin sensing. It has been found that ssRNA chemically immobilized on GO can be effectively protected against nuclease digestion. Surprisingly, until now, the noncovalent binding ability of ssRNA to GO and the nuclease resistance property of the resulting ssRNA/GO complex has not been explored. In an attempt to develop an effective method to construct highly stable RNA probes for biosensing and bioapplications, in this work, the binding ability and stability of ssRNA on GO was

ver the last two decades, numerous functional nucleic acids (FNAs), including aptamers, riboswitches, ribozymes, and DNAzymes have been discovered.1−3 These FNAs have found wide applications in bioanalysis and biomedicine, including biomolecule sensing, biomarker discovery, drug screening, targeted delivery, gene regulation, and disease diagnosis.4−7 Specifically, a wide variety of FNA probes have been proposed for sensitive and selective detection of cells, nucleic acids, proteins, small molecules, and metal ions.5,6 However, most of the reported probes are based on DNA, while RNA probes are rarely explored. Taking aptamers as an example, although there have been a great number of RNA aptamer sequences discovered, only a few RNA aptamers, such as VEGF, thrombin, and theophylline, have been utilized for biosensing.8−10 The primary reason for this lack of popularity is the vulnerability of RNA probes to enzymatic degradation. Because extremely careful and special protocols must be followed when RNA probes are used, more stable DNA probes are preferred in bioassay development.11−13 To fully use the large number of RNA FNAs available for bioanalysis and biomedicine, it is essential that effective methods to protect RNA from enzymatic digestion be explored. In recent years, a novel inorganic nanomaterial, graphene, has become extremely popular in nanoelectronic and biological applications.14−16 It has been reported that graphene oxide (GO) can bind and quench dye-labeled single-stranded DNA (ssDNA) probes, while it has less affinity toward doublestranded DNA (dsDNA) or secondary and tertiary structured ssDNA.17,18 On the basis of this finding, GO has been used for © 2013 American Chemical Society

Received: October 31, 2012 Accepted: January 16, 2013 Published: January 16, 2013 2269

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Scheme 1. Protection of RNA Probe from Nuclease Digestiona

a

(A) Without the protection of GO, the RNA probes are vulnerable to nuclease digestion, thus resulting in false positive results. (B) In the presence of GO, protection of RNA probes from nuclease digestion avoids false positive signals, allowing effective and accurate detection of target molecules.

systematically investigated. Our results indicated that ssRNA can bind strongly to the GO surface and be effectively protected from enzymatic cleavage. On the basis of this finding, we propose an effective way to design robust RNA biosensors by simply mixing RNA probes with GO for sensitive and selective bioanalysis. As illustrated in Scheme 1, without any protection, an RNA probe [e.g., an RNA molecular beacon (rMB)] is easily subjected to nuclease destruction. As a result, a false positive signal will be generated even in the absence of a target molecule (Scheme 1A). In contrast, in the presence of GO, rMB will absorb on the surface of GO and be protected from nuclease digestion. Therefore, the fluorescence intensity increase can be observed only when the target is present (Scheme 1B). As a proof-of-concept, a robust RNA-based biosensing platform was developed for sensitive and selective detection of proteins, small molecules, and nucleic acids. The entire assay is sensitive, selective, rapid, and more importantly, does not require any special protocols. The ability to protect ssRNA from enzymatic digestion by GO offers an exciting new way to stabilize ssRNA, which will not only provide new opportunities to utilize the large number of currently available yet rarely explored RNA FNAs for bioanalysis but also provide a new solution to protect important ssRNA molecules, such as microRNA and antisense ssRNA, for a great variety of biomedical applications.

Table 1. Sequences Used in This Study name

sequence

rProbe cDNA smDNA VEGF−RNA aptamer theophylline−RNA aptamer

5′-FAM-CCU AGC UCU AAA UCA CUA UGG UCG CGC UAG G-Dabcyl-3′ 5′-FAM-UGA GGU AGU AGA UUG UAU AGU U-3′ 5′-AAC TAT ACA ATC TAC TAC CTC A-3′ 5′-AAC TAT ACA ACC TAC TAC CTC A-3′ 5′ -FAM-CCG GUA GUC GCA UGG CCC AUC GCG CCC GG-3′ 5′-FAM-GGC GAU ACC AGC CGA AAG GCC CUU GGC AGC GUC-3′

rMB

mM EDTA, and 1 mM PMSF) for 30 min and centrifugation at 2000 rpm for 5 min to collect the supernatant. Fluorescence Measurements. Fluorescence measurements were carried out on an RF-5301-PC fluorescence spectrophotometer (Shimadzu, Japan). For all the RNA probes in the time scan mode, excitation and emission wavelengths were set at 490 and 520 nm, respectively, with a 5 nm bandwidth. The emission spectra were obtained by exciting the samples at 490 nm and scanning the emission from 500 to 650 nm in steps of 1 nm. The GO-protected rMB experiments were conducted in 20 mM Tris-HCl (pH 8.0) buffer containing 5 mM MgCl2 and 50 mM NaCl. VEGF and Theophylline Detection. In the VEGF detection experiment, the target solution was added to 200 μL of PBS buffer solution (10.0 mM Na2HPO4, 137 mM NaCl, and 2.7 mM KCl, pH 7.4) containing an aptamer probe (100 nM) and GO (0.02 mg/mL), and the mixture was equilibrated at room temperature for 10 min prior to the measurement. For theophylline, the parameters were the same as those for VEGF, except that the buffer (20 mM Tris-HCl, 5 mM MgCl2, and 50 mM NaCl) was different. RNase H-assisted Signal Amplification Assay (RASA) for DNA Detection. The RASA detection in the presence of different concentrations of target DNA (0 to 200 nM) was carried out in 10 μL of the RNase H buffer (20 mM Tris-HCl, 20 mM KCl, and 10 mM MgCl2, pH 7.5) containing RNase H (3 units/μL), rMB (1 μM), and GO (0.2 mg/mL) at 37 °C for 2 h. After incubation, the sample was diluted to 200 μL in an RNase H buffer and analyzed via spectrofluorometer. Gel Electrophoresis for Monitoring Cryonase and Cell Lysate Digestion of rMB. Eight tubes of 10 μM RNA molecular beacons (rMBs) in 10 μL of Tris-HCl buffer were



EXPERIMENTAL SECTION Materials and Reagents. Cryonase and HPLC-purified RNA probes were purchased from Takara Biotechnology Company Ltd. (Dalian, China). Target DNA was synthesized on a PolyGen Column 12 DNA synthesizer, and all DNA synthesis reagents were purchased from Glen Research (Sterling, VA). All DNA/RNA sequences are listed in Table 1. Stains-All was purchased from Sigma-Aldrich (St. Louis, MO). Theophylline and caffeine were purchased from Xiya Reagent Company Ltd. (Chengdu, China). VEGF was obtained from R&D Systems (Minneapolis, MN). Thrombin was obtained from Haematologic Technologies, Inc. (Essex Junction, VT). GO was synthesized using a modified Hummers method.25,31,32 Cell lysate was obtained from breast cancer cell lines MDA-MB-231 by incubating the cells on ice with lysis buffer (150 mM NaCl, 1.0% Tritonx-100, 50 mM Tris-HCl, 1 2270

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Figure 1. (A) The kinetic response and (B) fluorescence emission spectra of 50 nM rProbe to sequential addition of 0.02 mg/mL GO and 5 × cDNA.

Figure 2. (A) Fluorescence response of rMB and rMB-GO to cryonase digestion. Inset: gel electrophoresis analysis of probe samples treated with cryonase. (B) Fluorescence spectra of rMB and rMB-GO incubated under normal conditions (20 mM Tris-HCl, 5 mM MgCl2, 50 mM NaCl, pH 7.6, room temperature) for 0−15 days.

a strong fluorescence intensity was observed from the solution containing FAM-labeled rProbe. Upon the addition of GO, an immediate decrease of the fluorescence intensity occurred. In less than 5 min, the fluorescence intensity dropped dramatically, yielding a 96.9% quenching efficiency. After 1 h of incubation, the quenching efficiency was found to be around 99.5% as shown in Figure 1B (red line). The result established that similar to ssDNA, ssRNA can also effectively absorb onto the surface of GO. When 5-fold excess cDNA was added, the fluorescence of the solution was instantly restored, with 89% restoration in less than 15 min. After 30 min incubation, more than 93% of the fluorescence was restored (Figure 1B, blue line), suggesting that the rProbe hybridized with cDNA to form a RNA/DNA duplex, resulting in desorption of the rProbe from the GO surface and subsequent restoration of fluorescence. Stability of GO-protected rMB. After demonstrating the adsorption and desorption process of RNA on the GO surface, the protection effect of GO on RNA against enzymatic cleavage was investigated. An RNA molecular beacon (rMB) was prepared, and the degradation reaction was monitored with a spectrofluorometer. Because the quencher on rMB can adequately quench the fluorescence of the fluorophore when

prepared. Four of the tubes were incubated with 0.2 mg/mL GO at room temperature for 30 min and the other four tubes without GO. To all of the tubes, 20 units of cryonase were added. The four tubes with GO-rMB were incubated at 37 °C for 0, 1, 2, and 4 h, while the other four tubes with free rMB were incubated at 37 °C for 0, 2, 10, and 30 min. Afterward, the samples were heated to 95 °C for 15 min to deactivate cryonase. A 20% denaturing polyacrylamide gel was prepared using 1 × TBE buffer (pH 8.3). The gel was run at 1 W power for about 1 h in 1 × TBE buffer, stained for 30 min with StainsAll solution (500 mL formamide, 100 mL 10 × TBE, 400 mL H2O, 200 mg Stains-All), and finally photographed with a digital camera.



RESULTS AND DISCUSSION

Adsorption and Desorption of RNA on GO Surface. To investigate the adsorption and desorption properties of RNA on the GO surface, the kinetic process of the RNA/GO interaction was studied by monitoring the fluorescence change of a dye-labeled ssRNA probe (rProbe) upon incubation with GO. A decrease in fluorescence intensity is observed if the rProbe binds to GO, due to the excellent quenching ability of GO against a variety of fluorophores.18 As shown in Figure 1A, 2271

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Figure 3. (A) Time course of GO-protected theophylline RNA aptamer fluorescence restoration at different target concentrations (0, 5, 10, 25, 50, 100, 500, and 1000 μM) and (B) the relationship between the fluorescence enhancement and target concentration.

the rMB is in a hairpin structure, the background fluorescence intensity of the rMB was similar regardless of the presence/ absence of GO (Figure 2A). However, with the addition of cryonase (a nonspecific nuclease that digests all types of nucleic acids), the fluorescence intensity of the rMB solution without GO increased instantaneously, reaching a maximum in less than 5 min. In contrast, no observable change in fluorescence intensity was recorded for rMB with GO even after incubation with cryonase for 1 h. Further gel electrophoresis experiments (Figure 2A inset) revealed that unprotected rMB was partially digested after incubation with cryonase for 2 min. After 10 min, the rMB band became invisible, indicating complete enzymatic hydrolysis of the rMB by cryonase. In contrast, there was no detectable hydrolysis of the rMB with the protection of GO even after 4 h. The stability of GO-protected rMB was also tested in cell lysate, as shown in Figure S1 of the Supporting Information. After 30 min of incubation in 30% cell lysate, most of the GO-protected rMB remained intact, while the free rMB decomposed completely. All these data established that rMB was effectively protected by GO from enzymatic digestion. One of the major challenges for the application of RNA probes is long-term storage due to the ubiquitous nature of RNase. Even at room temperature and in a normal buffer, RNA will be gradually hydrolyzed. In order to investigate the longterm stability of GO-protected RNA probes, 16 tubes of GOrMB were maintained at normal conditions (20 mM Tris-HCl, 5 mM MgCl2, 50 mM NaCl, pH 7.6, room temperature) for 0− 15 days. As a control, another 16 tubes of rMB solution without GO were treated under the same conditions. As shown in Figure 2B, at normal conditions without the protection of GO, the fluorescence intensity of the rMB gradually increased day by day, indicating that the RNA probe was unstable and was hydrolyzed with time. In contrast, there was almost no detectable change in fluorescence intensity of the rMB-GO sample even after 15 days of incubation. The results demonstrated that with the protection of GO, the RNA probes are stable and can be maintained at the normal conditions for an extended period of time. As a brief summary, cryonase digestion, cell lysate treatment, and long-term incubation experiments clearly suggested that GO can effectively protect RNA probes from enzymatic digestion. The protective property of different nanomaterials to DNA has widely been reported, and several mechanisms have been proposed,21,33−36 which could also be applied to explain the

protection property of GO on RNA. First, the change of local ion concentration induced by nanomaterials inhibits enzyme activity. This mechanism was proposed to explain the protective properties of silica nanoparticles and gold nanoparticles.34−36 Second, the interactions between DNA (RNA) and nanomaterials may cause the conformational change of the DNA (RNA) to be unrecognizable for enzyme binding pockets, thereby protecting DNA (RNA) from cleavage.23,33 Third, the most popular explanation is the steric hindrance effect that prevents nuclease from binding to the DNA (RNA) to initiate enzymatic digestion.21,22,35 On the basis of the above mechanisms, it is highly possible that when ssRNA absorbs onto the surface of GO, the resulting steric effect, changes in local ion concentration and/or probe conformation plays a major role in protecting the RNA probe from nuclease digestion. This ability to protect ssRNA from enzymatic digestion by GO offers an exciting new solution to stabilize ssRNA, which will allow the usage of the large number of currently available yet rarely explored RNA probes for bioanalytical and biomedical applications. GO-stabilized RNA Aptamer Probe for Theophylline Sensing. Taking advantage of the GO protection property, we proposed a simple and effective way to design robust RNA biosensors for sensitive and selective bioanalysis by simply mixing RNA probes with GO. As a proof-of-concept, we first designed a GO-protected anti-theophylline RNA aptasensor for the detection of theophylline.37 Theophylline is a methylxanthine drug used in therapy for respiratory diseases such as chronic obstructive pulmonary disease and asthma. Its RNA aptamer is well-characterized with remarkable specificity, and has been used in several biosensor engineering studies.10,38,39 However, due to the instability of RNA, extreme care,40 special protocols11 or sophisticated designs10 were required. Alternatively, by simply mixing dye-labeled ssRNA aptamer with GO, an anti-theophylline GO-aptasensor can be constructed with the ssRNA aptamer absorbed on the GO surface, and the fluorescence of the fluorophore can thus be effectively quenched. Only when binding with theophylline, will the aptamer desorb with the target from GO surface, resulting in an increase in fluorescence intensity. We measured the fluorescence intensity with different target concentrations using this method. As shown in Figure 3A, in the absence of theophylline, the anti-theophylline GO-aptasensor generated a very weak background signal, indicating the fluorescence of FAM-labeled 2272

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Figure 4. Response of (A) GO-protected VEGF RNA aptamer probe to different concentrations (0, 2.5, 5, 10, 25, 50, and 100 nM) of VEGF and (B, [VEGF] = 50 nM, [Thrombin] = 50 nM, [1X BSA] = 50 nM, [10X BSA] = 500 nM) control proteins.

Figure 5. RNase H-assisted signal amplification assay (RASA). (A) Working principle, (B) sensitivity, and (inset of B) selectivity of RASA for detection of DNA based on GO-protected RNA probes.

related macular degeneration.43 As shown in Figure 4A, in the absence of VEGF, the GO-protected FAM-labeled anti-VEGF RNA aptamer generated a very weak background signal, indicating the fluorescence of FAM-labeled aptamer was efficiently quenched by GO due to the excellent quenching property of GO. With the addition of 2.5 nM VEGF, fluorescence intensity slightly increased, suggesting the desorption of the probe from the GO surface by forming a VEGF/RNA aptamer complex. In the presence of different concentrations of VEGF, such as 5, 10, 25, 50, and 100 nM, the fluorescence intensity of the GO aptasensor increased with the increasing concentration of VEGF. The detection limit was estimated to be about 2 nM. Meanwhile, to investigate the response behaviors of RNA aptamer toward other proteins, the fluorescence intensity changes by two control proteins (Thrombin and BSA) were studied. In Figure 4B, no obvious fluorescence change was observed by adding the same concentration (50 nM) of BSA, VEGF, and even 10 × BSA (500 nM), indicating excellent selectivity of the GO-protected RNA platform. Moreover, since ssRNA adsorbed on the GO surface is effectively protected from enzymatic cleavage, it should be able to work in complex biological samples such as cell lysate or serum. To test this possibility, cell lysate was used as a model matrix for our assay. In 30% cell lysate, 10 nM VEGF can easily be detected (Figure S3 of the Supporting Information), demonstrating that the GO-aptasensor can

aptamer was efficiently quenched by GO. With the addition of 100 μM theophylline, fluorescence intensity significantly increased, suggesting the desorption of aptamer from GO surface by forming a theophylline/aptamer complex. In the presence of different concentrations of theophylline, such as 5, 10, 25, 50, and 100 μM, the fluorescence intensity of the GOaptasensor increased with the increasing concentration of theophylline. The relationship between fluorescence intensity change and theophylline concentration is plotted in Figure 3B. The fluorescence intensity change exhibits a good linear positive correlation with theophylline concentration ranging from 0 to 100 μM. The detection limit was calculated to be 2 μM, which was carried out in a short time (5 min) and is comparable to the existing theophylline aptasensors.10,39 Meanwhile, the GO-aptasensor was found to be highly selective toward theophylline. As shown in Figure S2 of the Supporting Information, caffeine, the structural derivatives of theophylline, did not generate significant signal output, although these two compounds differ in only a methyl group.41 GO-stabilized RNA Aptamer Probe for VEGF Detection. To verify the generality of our GO-aptasensor strategy, another RNA aptamer sequence, which can selectively recognize the 165-amino acid form of vascular endothelial growth factor (VEGF165),42 was used to develop a protein detection platform. VEGF165 has been used as a biomarker for rheumatoid arthritis and cancer and is also associated with age2273

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ssRNA molecules, such as microRNA and antisense ssRNA, for a great variety of biomedical applications.

effectively work in the complex biological samples. From small molecule to protein, these results demonstrate that GOprotected RNA that aptasensors are sensitive, selective, rapid, and more importantly, they do not require any extremely careful or special protocols. RNase H-Assisted Signal Amplification Assay (RASA) for DNA Detection. The susceptibility of RNA to enzymatic digestion, if utilized properly together with GO protection, could also become an advantage. By introducing GO for RNA protection and RNase H for RNA digestion, we also developed an RNase H-assisted signal amplification assay (RASA) for sensitive and selective detection of DNA, in which the degradation of RNA was precisely controlled by target DNA. RNase H degrades the RNA strand in a RNA−DNA hybrid, but it cannot degrade DNA strands or unhybridized RNA strands.44 The working principle of RASA is shown in Figure 5A. The rProbe is incubated with GO to allow the absorption of the rProbe on the GO surface. Because of the excellent quenching effect of GO to fluorophores, the rProbe will be quenched and fluoresce weakly. In the presence of target DNA, the DNA and RNA hybridize to form DNA/RNA duplex which is then desorbed from the GO surface. The rProbe will immediately become a substrate for RNase H digestion, generating free fluorophores and liberating the target DNA. The released target DNA then hybridizes with another rProbe, and the cycle starts anew. This cyclic reaction repeats again and again until all rProbes on the GO surface are consumed and all fluorophores fluoresce freely, resulting in significant fluorescence signal amplification. Thus, sensitive detection of DNA can be achieved. Figure 5B shows the fluorescence intensity observed with different concentrations of the target DNA by this method. The results indicate that fluorescence intensity increased as the concentration of the target DNA increased. The limit of detection was found to be less than 100 fM, which is superior to many existing signal amplification technologies, including Exo III-assisted signal amplification.45,46 More importantly, no special and tedious manipulation of the RNA probes is needed during the experiment. We also demonstrated that our method has excellent specificity to distinguish mismatched DNA sequences. As shown in the inset of Figure 5B, the completely matched target induced a 3 times higher signal change than single-base mismatched target DNA (smDNA). All these results demonstrated that a robust RASA was successfully established based on GO-protected RNA probes for the detection of target DNA with high sensitivity and selectivity.



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.



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (+86) 592-218-7601. Fax: (+86) 592-218-9959. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank the National Basic Research Program of China (Grant 2010CB732402), National Science Foundation of China (Grants 21205100, 21275122, 21075104), and Natural Science Foundation of Fujian Province for Distinguished Young Scholars (Grant 2010J06004) for their financial support.



REFERENCES

(1) Tuerk, C.; Gold, L. Science 1990, 249, 505−510. (2) Ellington, A. D.; Szostak, J. W. Nature 1990, 346, 818−822. (3) Breaker, R. R. Nat. Biotechnol. 1997, 15, 427−431. (4) Famulok, M.; Hartig, J. S.; Mayer, G. Chem. Rev. 2007, 107, 3715−3743. (5) Liu, J.; Cao, Z.; Lu, Y. Chem. Rev. 2009, 109, 1948−1998. (6) Li, D.; Song, S.; Fan, C. Acc. Chem. Res. 2010, 43, 631−641. (7) Fang, X. H.; Tan, W. H. Acc. Chem. Res. 2010, 43, 48−57. (8) Li, Y.; Lee, H. J.; Corn, R. M. Anal. Chem. 2007, 79, 1082−1088. (9) Ferapontova, E. E.; Olsen, E. M.; Gothelf, K. V. J. Am. Chem. Soc. 2008, 130, 4256−4258. (10) Lau, P. S.; Coombes, B. K.; Li, Y. Angew. Chem., Int. Ed. 2010, 49, 7938−7942. (11) Ferapontova, E. E.; Gothelf, K. V. Langmuir 2009, 25, 4279− 4283. (12) Lee, H. J.; Li, Y.; Wark, A. W.; Corn, R. M. Anal. Chem. 2005, 77, 5096−5100. (13) Famulok, M.; Mayer, G.; Blind, M. Acc. Chem. Res. 2000, 33, 591−599. (14) Liu, Y.; Dong, X.; Chen, P. Chem. Soc. Rev. 2012, 41, 2283− 2307. (15) Rao, C. N.; Sood, A. K.; Subrahmanyam, K. S.; Govindaraj, A. Angew. Chem., Int. Ed. 2009, 48, 7752−7777. (16) Loh, K. P.; Bao, Q.; Eda, G.; Chhowalla, M. Nat. Chem. 2010, 2, 1015−1024. (17) Lu, C. H.; Yang, H. H.; Zhu, C. L.; Chen, X.; Chen, G. N. Angew. Chem., Int. Ed. 2009, 48, 4785−4787. (18) He, S.; Song, B.; Li, D.; Zhu, C.; Qi, W.; Wen, Y.; Wang, L.; Song, S.; Fang, H.; Fan, C. Adv. Funct. Mater. 2010, 20, 453−459. (19) Merkoçi, A.; Morales-Narváez, M. Adv. Mater. (Weinheim, Ger.) 2012, 24, 3298−3308. (20) Wang, Y.; Li, Z.; Wang, J.; Li, J.; Lin, Y. Trends Biotechnol. 2011, 29, 205−212. (21) Tang, Z.; Wu, H.; Cort, J. R.; Buchko, G. W.; Zhang, Y.; Shao, Y.; Aksay, I. A.; Liu, J.; Lin, Y. Small 2010, 6, 1205−1209. (22) Lei, H.; Mi, L.; Zhou, X.; Chen, J.; Hu, J.; Guo, S.; Zhang, Y. Nanoscale 2011, 3, 3888−3892. (23) Tang, L.; Chang, H.; Liu, Y.; Li, J. Adv. Funct. Mater. 2012, 22, 3083−3088. (24) Wang, Y.; Li, Z.; Hu, D.; Lin, C. T.; Li, J.; Lin, Y. J. Am. Chem. Soc. 2010, 132, 9274−9276. (25) Lu, C. H.; Li, J.; Lin, M. H.; Wang, Y. W.; Yang, H. H.; Chen, X.; Chen, G. N. Angew. Chem., Int. Ed. 2010, 49, 8454−8457.



CONCLUSIONS In conclusion, we demonstrated that ssRNA have similar absorption and desorption properties toward GO as that of ssDNA, and more importantly, when adsorbed on the GO surface, ssRNA can be effectively protected from enzymatic cleavage. On the basis of this finding, we have developed a robust RNA aptasensor platform and an RNase H-assisted amplification platform for sensitive and selective detection of small molecules, proteins, and nucleic acids. With the protective ability of GO, no special or tedious manipulations of RNA probes are required, making our methods simple and robust. The ability to protect ssRNA from enzymatic digestion by GO offers an exciting new way to stabilize ssRNA, which will not only provide new opportunities to utilize the large number of currently available yet rarely explored RNA FNAs for bioanalysis but also provide a new solution to protect important 2274

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(26) Cui, L.; Lin, X.; Lin, N.; Song, Y.; Zhu, Z.; Chen, X.; Yang, C. J. Chem. Commun. (Cambridge, U.K.) 2012, 48, 194−196. (27) Lu, C. H.; Zhu, C. L.; Li, J.; Liu, J. J.; Chen, X.; Yang, H. H. Chem. Commun. (Cambridge, U.K.) 2010, 46, 3116−3118. (28) Yang, X.; Niu, G.; Cao, X.; Wen, Y.; Xiang, R.; Duan, H.; Chen, Y. J. Mater. Chem. 2012, 22, 6649−6654. (29) Zhang, L.; Lu, Z.; Zhao, Q.; Huang, J.; Shen, H.; Zhang, Z. Small 2011, 7, 460−464. (30) Hu, X.; Mu, L.; Wen, J.; Zhou, Q. J. Hazard. Mater. 2012, 213214, 387−392. (31) Hummers, W. S., Jr.; Offeman, R. E. J. Am. Chem. Soc. 1958, 80, 1339−1339. (32) Cote, L. J.; Kim, F.; Huang, J. J. Am. Chem. Soc. 2009, 131, 1043−1049. (33) Wu, Y.; Phillips, J. A.; Liu, H.; Yang, R.; Tan, W. ACS Nano 2008, 2, 2023−2028. (34) He, X. X.; Wang, K.; Tan, W.; Liu, B.; Lin, X.; He, C.; Li, D.; Huang, S.; Li, J. J. Am. Chem. Soc. 2003, 125, 7168−7169. (35) Seferos, D. S.; Prigodich, A. E.; Giljohann, D. A.; Patel, P. C.; Mirkin, C. A. Nano Lett. 2009, 9, 308−311. (36) Giljohann, D. A.; Seferos, D. S.; Daniel, W. L.; Massich, M. D.; Patel, P. C.; Mirkin, C. A. Angew. Chem., Int. Ed. 2010, 49, 3280−3294. (37) Zimmermann, G. R.; Jenison, R. D.; Wick, C. L.; Simorre, J. P.; Pardi, A. Nat. Struct. Biol. 1997, 4, 644−649. (38) Topp, S.; Gallivan, J. P. J. Am. Chem. Soc. 2007, 129, 6807− 6811. (39) Carrasquilla, C.; Lau, P. S.; Li, Y.; Brennan, J. D. J. Am. Chem. Soc. 2012, 134, 10998−10005. (40) Ferapontova, E. E.; Olsen, E. M.; Gothelf, K. V. J. Am. Chem. Soc. 2008, 130, 4256−4258. (41) Jenison, R. D.; Gill, S. C.; Pardi, A.; Polisky, B. Science 1994, 263, 1425−1429. (42) Jellinek, D.; Green, L. S.; Bell, C.; Janjic, N. Biochemistry 1994, 33, 10450−10456. (43) Li, Y.; Lee, H. J.; Corn, R. M. Anal. Chem. 2007, 79, 1082−1088. (44) Goodrich, T. T.; Lee, H. J.; Corn, R. M. J. Am. Chem. Soc. 2004, 126, 4086−4087. (45) Zuo, X.; Xia, F.; Xiao, Y.; Plaxco, K. W. J. Am. Chem. Soc. 2010, 132, 1816−1818. (46) Cui, L.; Ke, G.; Wang, C.; Yang, C. J. Analyst 2010, 135, 2069− 2073.

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