A Novel Fluorescence Switch for MicroRNA Imaging in Living Cells

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Biological and Medical Applications of Materials and Interfaces

A Novel Fluorescence Switch for MicroRNA Imaging in Living Cells based on DNAzyme Amplification Strategy. Peixin Li, Min Wei, Fen Zhang, Juan Su, Wei Wei, Yuanjian Zhang, and Songqin Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b15330 • Publication Date (Web): 26 Nov 2018 Downloaded from http://pubs.acs.org on November 27, 2018

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ACS Applied Materials & Interfaces

A Novel Fluorescence Switch for MicroRNA Imaging in Living Cells based on DNAzyme Amplification Strategy.

Peixin Lia, Min Weib, Fen Zhanga, juan Sua, Wei Weia*, Yuanjian Zhanga, Songqin Liua

aJiangsu

Engineering Laboratory of Smart Carbon-Rich Materials and Device, Jiangsu

Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 211189, China bCollege

of Food Science and Technology, Henan University of Technology,

Zhengzhou, 450001, China

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ABSTRACT MicroRNAs (miRNAs) play important roles in the regulation of target gene expression and cell development. Therefore, developing of accurate and visual detection methods for miRNAs is important for early diagnosis of cancer. In this study, we established a visual detection method for miRNA 155 based on DNAzyme amplification strategy in living cells. MnO2 nanosheets were employed to deliver Locked DNAzyme and Substrate DNA into cells. AuNPs-Probe were taken up by cells autonomously. Then, MnO2 nanosheets were reduced to Mn2+ by glutathione (GSH) in cells and DNA modules were released. MiRNA 155 took away Locker DNA by strand displacement reaction to activate the DNAzyme. Then the DNAzyme cleaved substrate DNA and released single-stranded DNA named Key DNA. Key DNA opened the hairpin DNA that modified on gold nanoparticles (AuNPs) and turn on the fluorescence of cy5. One target miRNA led to plenty of released Key DNA when lots of substrate DNA were added. Thus, the visual detection of miRNA 155 in living cells would be initiated. Under confocal laser microscopy, the fluorescence was obviously observed in tumor cells but not in normal cells. The method has a linear range from 0.1 nM to 10 nM and a low detection limit of 44 pM in vitro detection.

Keywords: microRNA, DNAzyme, AuNPs, DNA walker, Signal amplification, MnO2, Fluorescence imaging

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1. INTRODUCTION MicroRNAs (miRNAs), which contain about 22 nucleotides, are a class of non-coding small molecule RNAs. They participate in regulation of ontogeny, cell proliferation, differentiation and apoptosis by means of incomplete hybridization with the target gene and inhibition of target gene translation.1-3 MiRNAs are also closely related to the pathogenesis, metastasis, drug resistance and other pathological processes of tumor.4-6 Such as miRNA 155, a typical multifunctional miRNA, which is located in the third exon of human BIC (B cell integration cluster) gene non-coding transcriptional area, is a potential oncogene or cancer suppressor gene.7 More and more researches showed that miRNA 155 played a role as oncogene in lots of types of cancer.8-10 Therefore, the development of reliable methods for miRNA detection is significant for early diagnostics of cancer. In the last two decades, a variety of techniques are developed for miRNA detection. For example, electronic detection,11-16 surface plasmon resonance (SPR),17 surface plasmon resonance enhanced light scattering (SP-LS),18 fluorescent assays,19-26 upconversion luminescence (UCL)27 and fluorescence anisotropy (FA).28 Recently, metal nanoparticle especially gold nanoparticles (AuNPs) based methods have been used for miRNA imaging in single cell.29-31 AuNPs are easy to preparation and widely used in biochemistry due to its stable chemical property. DNAzymes are DNA sequences that have high catalytic efficiency and structure identification activities.32 The discovery of DNAzymes extended the concept of enzyme. There are two constituent parts of DNAzymes: the binding site and the catalytic site. The binding site makes DNAzymes hybridize with substrate and the catalytic site cleaves substrate with the help of cofactors. Different types of DNAzyme need variety classes of cofactor, such as Mg2+, Pb2+, Zn2+, Mn2+, etc.33-36 DNAzymes have many advantages compared with traditional protein enzymes. They are low cost, stable at both room temperature and higher temperatures, easy to synthesize and label. They play important roles in different reaction systems with various catalytic functions by changing binding sequences. Therefore, DNAzymes have a wide application prospect and have gradually become the focus tools for 3



researchers. There are many applications of DNAzymes in miRNA detection. Zhu37 et al. designed a multicomponent nucleic acid enzymes (MNAzyme) nanodevices for multiple miRNAs imaging and drug delivery. The MNAzyme motifs were employed to lock drugs inside the nanodevices. In particular, MNAzyme motifs probes responded to multiple miRNAs by fabricating a logic gate. Wang et al.38 developed a hairpin DNAzyme for miRNA 141 detection based on AuNP. Fluorophores labeled hairpin DNA which contained both sequences of DNAzyme and substrate was modified on AuNPs, and an active secondary structure formed to enhance the fluorescence in the presence of miRNA 141. They also developed a AuNP loaded split-DNAzyme probe 39. Target miRNAs hybridized with split DNAzymes to build complete DNAzymes so that fluorophore-labeled substrate strands were cleaved and their fluorescence were enhanced. Yang40 et al. constructed a DNAzyme based nanomachine that used DNA walker as signal amplification strategy. Herein, we report a DNAzyme-based amplification strategy for the detection of miRNA 155 both in vitro and in living cells. As shown in Scheme 1A, a complementary strand of miRNA 155 named Locker DNA was used to hybridize with the DNAzyme to plug its active site. In the presence of miRNA 155, the DNAzyme would be activated because of the hybridization between the Locker and miRNA 155 due to strand displacement reaction. Then, the active DNAzyme combined with a hairpin DNA named Substrate which had the cleavage site of the DNAzyme. The Substrate would be cleaved to two single DNA strands by the active DNAzyme. One of them is partly complementary to FL DNA named Key. Subsequently, AuNPs probes which were modified with a lot of cy5 labelled hairpin DNA named FL DNA were added into the solution. The fluorescence of cy5 modified on AuNPs was quenched. Once upon the Key was released, they would hybridize with the FL DNA and turn on the fluorescence of cy5. Plenty of the Substrates were added so that lots of the Key was released to turn on the fluorescence of the AuNPs-probe. The fluorescence was greatly enhanced due to the above amplification process. Therefore, DNAzyme-based amplification strategy offered this method high detection sensitivity. Compared with previously reported DNAzyme, where additional cofactors were 4


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needs and most of them were toxic to cells, here, Mn2+ was reduced from MnO2 nanosheets by glutathione (GSH) in cells. It avoided the additional addition of cofactors. On the other hand, MTT experiments proved that Mn2+ at the used concentration was harmless to cells. The principle of detection miRNA 155 in living cells is showed in Scheme 1B. MnO2 nanosheets were employed as a transport to deliver DNA modules including the Locker, the DNAzyme and the Substrate into cells owing to its strong absorptivity for single stranded DNA (ssDNA). MnO2 nanosheets and the AuNPs-Probe could penetrate across the cell membrane efficiently. The glutathione (GSH) in cells reduced MnO2 nanosheets to Mn2+ so that the DNA modules would be released to initiate the detection of miRNA 155. The high level of miRNA 155 in tumor cells would turn on the fluorescence of the AuNPs-probe. By contrast, the fluorescence of the AuNPs-Probe would not be turned on in normal cells because of their low level of miRNA 155. In summary, the method in this work has achieved the detection and imaging of miRNA 155 in living cells.

2. MATERIALS AND METHODS 2.1. Chemicals and Materials. All reagents were analytical grade and from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Ultrapure water (18.2 MΩ cm, Barnstead, Thermo Scientific) was used throughout the experiments. Sequences of all oligonucleotides are listed in Table S1. 2.2. Apparatus. Fluorescence and UV-vis spectra were detected by fluorescence spectrometer (Fluoromax-4) from Horiba Jobin Yvon, Japan, and UV-vis spectrophotometer (Shimadzu UV-2450, Kyoto, Japan). Transmission electron microscopy was from Hitachi, Japan (JEM-2010). Zeta potential and DLS measurements were taken using a laser particle analyzer (NanoBrook Omni, Brookhaven, USA). 2.3. Preparation of Probe. Citrate-stabilized AuNPs were prepared with traditional method.41 Detailed procedures were described in supporting information. Prepared AuNPs was functionalized with FL DNA. 42 The SH- modified on FL DNA 5



was reduced by TCEP·HCl followed by mixed with 1 mL AuNPs. Then, 30 μL of 1 M NaCl was added to the mixture stepwise and shaked rapidly for stabilizing the obtained probe, followed by stirring at room temperature. All experiments were operated in the dark. After 24 h, the prepared DNA-AuNPs were purified through three rounds of centrifugation (13 000 rpm, 15 min) and resuspended in Tris-HCl buffer (25 mM Tris, 200 mM NaCl, pH 8.0). 2.4. Synthesis of MnO2/DNA Composite. Synthesis of MnO2 nanosheet was based on literature reported procedures.

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Detailed procedures and information are

placed in supporting information. 10 μL of 4 μM DNAzyme and Locker were incubation at 37 °C for 2 h. Subsequently, 20 μL of 10 μM Substrate and 0.8 mg/mL MnO2 nanosheets were added into the above solution. Then, the mixed solution were stirred at room temperature for 20 min. 2.5. Fluorescence Experiments. 10 μL of 1 μM DNAzyme and Locker were added into Tris-HCl buffer for 2 h incubation at 37 °C. 250 nM Substrate, 500 μM Mg2+ and increasing concentrations of miRNA 155 from 0 to 150 nM were added and incubated at 37 °C for another 2 h. Then, 50 μL of 4 nM AuNPs-Probe were added into above solution. After incubation for 2 h, their fluorescence spectra were measured. 2.6. Cell Culture. HepG2 cells were cultured in DMEM medium and LO2 cells were cultured in RPMI-1640 medium. 10% fetal bovine serum and 100 U/mL of 1% antibiotics penicillin/streptomycin were supplemented to both of the medium. Then, they were incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. 2.7. Confocal Fluorescence Imaging. Cell lines were seeded on confocal dish at 37 °C overnight. Then, 500 μL serum-free culture media containing 4 nM AuNPs-Probe and 20 μg/mL of MnO2/DNA were added for 4 h incubation. Three times wash of cells was necessary. Olympus FV3000 laser scanning confocal microscopy and 60× oil-immersion objective were used. 3. RESULTS AND DISCUSSION 3.1. Characterization of Materials. The uniform distribution and less aggregation of 20 nm AuNPs were proved by transmission electron microscopy 6


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(TEM) images (Figure 1A) and dynamic light scattering (DLS) results (Figure 1B). After AuNPs were functionalized with the FL DNA, the UV-vis spectrum showed the characteristic peak of DNA between 250 and 300 nm (Figure 1C). The Zeta potential also showed the probe had a more negative charge compared with the AuNPs because of the large amount of negative charge on the FL DNA (Figure 1D). The MnO2 nanosheets also had negative charged potential. The TEM image of MnO2 nanosheets showed their sheet structure (Figure 1E). Figure 1F showed the UV-vis absorption peak of MnO2 nanosheets was at 360 nm. 3.2. Feasibility of the Proposed Method. In order to test the feasibility of the strategy for miRNA 155 detection, the fluorescence intensity of the AuNPs-Probe and the mixture of the AuNPs-Probe with different reagents in this reaction system were recorded (Figure 2). Fluorescence of cy5 was quenched by AuNPs efficiently when there was only the AuNPs-Probe in Tris-HCl buffer (curve a). When the Substrates were added into the AuNPs-Probe solution, the fluorescence had a weak enhancement (curve b). In the presence of only DNAzyme, the fluorescence of above solution (AuNPs-Probe / Substrate) did not change because DNAzyme cannot work without Mn2+, (curve c). When Mn2+ was added, the DNAzyme was activated to cleave the Substrate and the Key would be released to recover the fluorescence of the AuNPs-Probe, as a result, the fluorescence intensity increased extremely (curve d). In the presence of Locker, the binding site of DNAzyme with the Substrate was plugged and the fluorescence of the AuNPs-Probe was quenched (curve e). After added the target, miRNA 155 hybridized with locker by strand displacement reaction and the fluorescence recovered again (curve f). The cleavage result of the DNAzyme was proved by gel electrophoresis experiments (Fig. S2, supporting information). 3.3. Optimize the Detection Conditions. In order to obtain the best performance, the effects of pH and concentrations of the Locker, the Substrate and Mn2+ were investigated. Figure S1A showed the plugging efficiency of the Locker. It was found that the fluorescence intensity decreased with the increment of the Locker concentration. In the presence of 50 nM Locker, the fluorescence intensity of the AuNPs-Probe decreased at utmost. When the concentration of the Locker was greater 7



than 50 nM, the fluorescence intensity didn’t change obviously. On the other hand, the high concentration of the Locker would reduce the sensitivity because miRNA 155 would hybridize with the excessive Locker and the DNAzyme would not be activated. Accordingly, 50 nM of Locker was chosen. As shown in Figure S1B, the fluorescence intensity enhanced with the increase of the Substrate. More Key was released when the concentration of the Substrate increased, so, more FL DNA labelled on AuNPs was opened. In this way, the fluorescence signal was amplified and achieved peak value when 250 nM Substrate was added. So, 250 nM Substrate was chosen for the following experiments. Figure S1C clearly indicated the influence of pH. The fluorescence intensity was rise and then fall with the increased of pH. The highest fluorescence intensity was obtained at pH=8.0. Therefore, the reaction condition was chosen to be pH 8.0. Finally, we investigated the effect of the concentration of Mn2+ (Figure S1D). As a cofactor, DNAzyme had no activity without Mn2+. The DNAzyme exhibited high cleavage efficiency in the presence of 500 μM Mn2+. Hence, operation of the DNAzyme was feasible in living cells because 500 μM Mn2+ don’t harm cells.44 3.4 Performance of the Proposed Biosensor. Under optimal conditions, quantitative detection performance of the proposed method for miRNA 155 was investigated (Figure 3A). Fluorescence increased gradually with the increasing concentrations of miRNA 155. The functional relation between miRNA 155 concentrations and fluorescence intensities is showed in Figure 3B. There was a wide linearity range of the detection method that was 0.1 nM to 10 nM. According to the rule of S/N = 3, a detection limit of 44 pM was obtained. On the basis of the above performance, it would be concluded that the method offered a high sensitivity and a wide linearity range for the detection of miRNA 155, which is mainly due to the DNAzyme-based signal amplification. To verify the specificity of miRNA 155 detection, the influences of four different types of miRNA including miRNA 21, miRNA 141, miRNA 182 and miRNA 197, which have high similar sequence, were tested under the same conditions. In Figure 4, strong fluorescence was obtained when miRNA 155 was used and almost no 8


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fluorescence recovery was observed for other miRNAs at equal concentration. These experiments proved that the DNAzyme based strategy had high selectivity for miRNA detection, which has good prospect be applied in complex cellular environment. To testify the analytical performance in actual samples, six human plasma samples were diluted in a 1:10 ratio with Tris-HCl buffer and then added various concentrations of miRNA 155. Results are showed in Table 1. Recoveries were in the range of 90 % ~ 109 %, which indicated the proposed detection method had high accuracy in complex systems. 3.5. MiRNA 155 Imaging in Living Cells. HepG2 cells and LO2 cells were used as target and control cells for miRNA 155 imaging. We have explored the concentration of AuNPs-Probe and DNA modules in cell experiment. The fluorescence in HepG2 cells was not evident by laser scanning confocal microscope(LSCM) when 1 nM AuNPs-Probe, 50 nM DNAzyme, 50 nM Locker and 250 nM Substrate were used as that optimized in vitro experiments. With the concentration of AuNPs-Probe and DNA modules increasing, the fluorescence increased effectively in HepG2 cells. Significant different fluorescences between LO2 cells and HepG2 cells were observed under LSCM when 4 nM AuNPs-Probe, 100 nM DNAzyme, 100 nM Locker and 500 nM Substrate were added. Higher concentration of Substrats, AuNPs-Probe, DNAzyme and Lockers were used in cell imaging because all of them cannot be taken up by cells completely. Incubating time of AuNPs-Probe and DNA modules with cells were also studied carefully. The AuNPs-Probe and MnO2/DNA composite could not enter into cells and reaction completely when the incubating time was less than 4 h. On the other hand, cell activity decreased when the incubating time was more than 6 h. The experimental results showed that 4 h was the best incubating time. All cells were incubated with AuNPs-Probe and MnO2/DNA composite for 4 h. After washing cells three times with PBS (pH=7.40) to remove the AuNPs-Probe that had not enter cells, they were imaged with the confocal laser scanning microscopy. Figure 5 showed that strong red fluorescence emitted by cy5 were found in HepG2 cells and almost no fluorescence was observed in LO2 cells. The results revealed that 9



the probes and MnO2/DNA composite entered cells successfully and the strategy for miRNA 155 detection worked well. It was evident that this fluorescence switch for microRNA imaging in living cells based on DNAzyme amplification strategy is successful. Cytotoxicity assay were done to prove that 500 μM of Mn2+ used in the experiments had almost no toxicity to cells. (Fiugre S3, Supporting information.) 4. CONCLUSIONS In conclusion, we have developed a novel method for detection of miRNA 155 in living cells based on DNAzyme amplification strategy. In the presence of miRNA 155, DNAzyme would be activated and cleave the Substrate to release the Key. Then the Key would open the FL DNA modified on the AuNPs and turn on the fluorescence. The introduction of DNAzyme-based signal amplification strategy makes the method highly sensitive with a low detection limit of 44 pM and a wide linearity range of 0.1 nM to 10 nM. By using the method in multiple miRNA mixture or real samples, we demonstrated its high selectivity and accuracy. In living cells, the fluorescence was turned on in tumor cells but not in normal cells. These advantages enabled this method to detect miRNA 155 rapidly and effectively. More importantly, this method may contribute to tumor cells tracking and clinical definite in early-stage cancer.

ASSOCIATED CONTENT Supporting Information Optimization of experimental conditions for miRNA 155 detection; preparation of AuNPs; gel electrophoresis; cytotoxicity assay; sequences of oligonucleotides used for this work.

AUTHOR INFORMATION 10


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Corresponding author: Tel.: 86-25-52090613 Fax: 86-25-52090618 E-mail address: [email protected] (W. Wei)

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ACKNOWLEDGMENTS We gratefully appreciate the support from National Natural Science Foundation of China (21775019, 21475020, 21635004 and 81730087), Fundamental Research Funds for the Central Universities and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (Grant Nos. 2242018K3DN04), The Open Project of The Key Laboratory of Modern Toxicology of Ministry of Education, Nanjing Medical University (NMUAMT201804). REFERENCES (1) Bartel, D. P. MicroRNAs: Genomics, Biogenesis, Mechanism, and Function. Cell 2004, 116, 281-297. (2) Ambros, V. The Functions of Animal MicroRNAs. Nature 2004, 431, 350−355. (3) Mallory, A. C.; Vaucheret, H. Functions of MicroRNAs and Related Small RNAs in Plants. Nat. Genet. 2006, 38, S31-36. (4) Calin, G. A.; Croce, C. M. MicroRNA Signatures in Human Cancers. Nat. Rev. Cancer 2006, 6, 857-866. (5) Tricoli, J. V.; Jacobson, J. W. MicroRNA: Potential for Cancer Detection, Diagnosis, and Prognosis. Cancer Res. 2007, 67, 4553-4555. (6) Ruan, K.; Fang, X. G.; Ouyang, G. L. MicroRNAs: Novel Regulators in the Hallmarks of Human Cancer. Cancer Lett. 2009, 285, 116-126. (7) Eis, P. S.; Tam, W; Sun, L. P.; Chadburn, A.; Li, Z. D.; Gomez, M. F.; Lund, E.; Dahlberg, J. E. Accumulation of MiR-155 and BIC RNA in Human B Cell Lymphomas. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 3627-3632. (8) Iorio, M. V.; Ferracin, M.; Liu, C. G.; Veronese, A.; Spizzo, R.; Sabbioni, S.; Magri, E.; Pedriali, M.; Fabbri, M.; Campiglio, M.; Ménard, S.; Palazzo, J. P.; Rosenberg, A.; Musiani, P.; Volinia, S.; Nenci, I.; Calin, G. A.; Querzoli, P.; Negrini, M.; Croce, C. M. MicroRNA Gene Expression Deregulation in Human Breast Cancer. Cancer Res. 2005, 65, 7065-7070.

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Figure captions Scheme 1. Schematic Illustration for the Detection of miRNA 155 in vitro (A) and in tumor cells (B). Figure 1. (A) TEM image of AuNPs. (B) DLS characterization of the prepared AuNPs. (C) UV-vis spectra of (a) AuNPs and (b) the AuNPs-Probe. (D) Zeta potentials of AuNPs, the AuNPs-probe in H2O and MnO2 nanosheets in H2O. (E) TEM image of MnO2 nanosheets. (F) UV-vis spectra of MnO2 nanosheets. Figure 2. The fluorescence emission spectra under different conditions: (a) AuNPs-Probe, (b) AuNPs-Probe / Substrate, (c) AuNPs-Probe / Substrate / DNAzyme, (d) AuNPs-Probe / Substrate / DNAzyme / Mn2+, (e) AuNPs-Probe / Substrate / DNAzyme / Mn2+ / Locker, (f) AuNPs-Probe / Substrate / DNAzyme / Mn2+ / Locker / miRNA (50 nM). The concentrations of the AuNPs-Probe, Substrate, DNAzyme, Mn2+ and Locker were 1 nM, 250 nM, 50 nM, 500 μM, 50 nM, respectively. Figure 3. (A) Fluorescence spectra of the method with the increasing concentrations of miRNA 155 from 0 to 150 nM. (B) The fluorescence intensity at 663 nm with different concentrations of miRNA 155. Inset shows calibration curves for miRNA. Figure 4. Specificity of the method over several miRNA targets. The concentrations of different miRNAs were all 150 nM. Figure 5. Confocal fluorescence imaging of intracellular miRNA 155 in HepG2 cells and L02 cells. The excitation wavelength was 640 nm, and the images were collected in the range of 650-750 nm. Table 1. Recovery results of miRNA added in human plasma samples.

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Scheme 1.

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Figure 1. 80

B

A

Intensity

60 40 20 0

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Figure 2. 400

Fluorescence(a.u.)

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a b c d e f

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A

Flourescence(a.u.)

Figure 3. Fluorescence(a.u.)

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Figure 4. 400

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miRNA155 miRNA 21 miRNA141 miRNA182 miRNA197

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Figure 5.

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Table 1. Recovery results of miRNA added in human plasma samples sample no. added (nM) found (nM) recovery (%) S1 0.5 0.478 95.6 S2 1 1.09 109 S3 3 3.21 107.1 S4 5 4.74 94.8 S5 7 7.22 103.2 S6 10 10.16 101.6

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Table of Content:

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A Novel Fluorescence Switch for MicroRNA Imaging in Living Cells

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