Intracellular MicroRNA Imaging with MoS2-Supported Nonenzymatic

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Biological and Medical Applications of Materials and Interfaces 2

Intracellular MicroRNA Imaging with MoS-Supported Nonenzymatic Catassembly of DNA Hairpins Dan Zhu, Jiaxuan Huang, Bang Lu, Yu Zhu, Yaqi Wei, Qi Zhang, Xixi Guo, Lihui Yuwen, Shao Su, Jie Chao, and Lianhui Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 22 May 2019 Downloaded from http://pubs.acs.org on May 22, 2019

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ACS Applied Materials & Interfaces

Intracellular

MicroRNA

Imaging

with

MoS2-Supported

Nonenzymatic

Catassembly of DNA Hairpins Dan Zhu,ǁ Jiaxuan Huang,ǁ Bang Lu, Yu Zhu, Yaqi Wei, Qi Zhang, Xixi Guo, Lihui Yuwen, Shao Su, Jie Chao,* Lianhui Wang* Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key Laboratory for Biosensors, Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center

for

Advanced

Materials

(SICAM),

Nanjing

University

of

Posts

and

Telecommunications, 9 Wenyuan Road, Nanjing 210023, China

Abstract Amplification strategies for low-level microRNA detection in living cells is pivotal for gene diagnosis and many cellular bioprocesses. In this work, we develop an amplification strategy for microRNA-21 (miRNA-21) imaging in living cells with MoS2-supported catassembly of DNA hairpins. MoS2 nanosheet with low cytotoxicity serves as the nanocarrier and excellent fluorescence quencher, which can transfer fluorescent metastable hairpin DNA into the cells easily with a nondestructive manner and significantly reduce background signals. The three branched catalyzed hairpin assembly (TB-CHA) probes contain three types of designed DNA molecular beacons with modification of Cy3 in the terminal. In the presence of miRNA-21, catalyzed hairpin assembly (CHA) reaction would be triggered and a “Y” shaped three branched duplex nanostructure would be formed, which would release from the surface of MoS2 nanosheet due to the reduced affinity between DNA duplex and MoS2 nanosheet. The multisite fluorescence modification and the circular reaction of TB-CHA probes allowed a significant fluorescence recovery in live-cell microenvironment. Ultrasensitive detection of miRNA-21 is achieved with a detection limit of 75.6 aM, which is ~five orders of magnitude lower than that of the simple strand displacementbased strategy (detection limit: 8.5 pM). The method offers great opportunities for ultrasensitive live-cell detection of miRNAs and helps deeper understanding of the

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physiological functions of miRNAs in cancer research and life processes. Keywords: MoS2 nanosheet, nonenzymatic catassembly, three branched-catalyzed hairpin assembly (TB-CHA), signal amplification, microRNA detection 1. Introduction MicroRNAs (miRNAs) are a kind of short endogenous non-coding RNAs, which play important roles in physiological progresses through imperfect pairing with target mRNAs and post-transcriptional gene regulation.1-4 A growing evidences have proved that the occurrence and development of cancers is closely related to the expression of particular kinds of miRNAs.5-7 Screening for miRNA expression in living cells could provide evidence for early diagnosis of tumors or other diseases.8, 9 Up to now, various analytical approaches have been reported for the quantification determination of cancer-related miRNAs, such as real-time polymerase chain reaction (RT-PCR), microarrays or northern blot, et al.10-13 Despite progress have been made, most methods need to extract RNA from cell lysate and are destructive to cells, which limit the application of live-cell detection. Developing strategies for nondestructive specific gene detection with high sensitivity and selectivity is urgently demanded.14-19 Considering of the low abundance, small size and high homology of miRNA, there exist plenty of challenges for intracellular detection. For instance, the relatively low readout signal from the single step of in situ hybridization limits the sensitivity of miRNA detection.20 Moreover, by reason of the complexity of the intracellular environment, false positive signal might be caused by nuclease degradation or nonspecific binding. To improve the sensitivity in intracellular analysis, methods based on enzyme-catalyzed cyclic reactions and isothermal amplification were introduced into living cells.21-27 For instance, Xiang et al. reported a target-triggered recycling amplification assay for amplified intracellular miRNA imaging by introducing a DNA fuel into living cells through liposome transfection.28 Lou et al. transfected Exonuclease III into cancer cells to obtain recycling amplified signals for miRNA imaging.29 In 2017,


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Tan et al. employed DNA nanostructures as nanocarriers to bring several oligonucleotides into living cells to conduct an autonomous DNA circuit without the assistance of transfection agents.30 Although numerous improvements have been made, there still exist some shortcomings. Firstly, essential enzyme or strands during cycling reactions should be externally introduced by transfection or other methods due to their poor cell permeability. Secondly, the transfected sequences for amplification cannot maintain stability in the intracellular environment without protection. Thirdly, multistep incubations are required when multiple strands need to be introduced via nanocarriers, which are tedious and time-consuming. Therefore, developing appropriate systems to improve the transportation efficiency could be the breakthrough for amplified intracellular detection. In recent years, nanomaterials-based strategies for specific target analysis have attracted widespread attention due to the excellent optical, electrical and structural properties of nanomaterials.31-37 Zero-dimentional (0D) to three-dimentional (3D) nanomaterials have been widely applied in the intracellular detection.38, 39 Among them, two-dimensional (2D) nanomaterials has large surface area, which enables one-step assembly of large amounts of probes on the nanosheet.40-45 To meet the above challenges, we herein utilize a MoS2-supported catassembly of DNA hairpins as the signal amplifier to acheive low-level microRNA-21 (miRNA-21) detection in living cells. MoS2 nanosheet is a type of novel 2D transition metal dichalcogenides which have attracted extensive attention due to their unique physical and chemical properties.46-50 In comparison with other two-dimensional materials, such as graphene oxides, halogenated graphenes and WSe2, MoS2 nanosheet possesses much less cytotoxicity, which is suitable for intracellular analysis.51,

52

As the nanocarrier of

metastable hairpin probes, MoS2 nanosheet could transfer into cells with a nondestructive manner to allow the recycling reaction taken place in the intracellular environment.20,

52-54

It is notable that the whole procedure for transportation and

detection was accomplished in one-step incubation without multiple transfections. MoS2 nanosheets also serves as excellent fluorescence quenchers to significantly



reduce background signals.45, 48 The large specific area of nanosheets enriches large amounts of three-branched catalyzed hairpin assembly (TB-CHA) probes with fluorophore modified in three branched terminal to achieve significant fluorescence enhancement. Therefore, low-level miRNA detection in living cells could be realized to discriminate cancer cells. The MoS2-based TB-CHA probe reaches an ultralow detection limit of miRNA-21 at 75.6 aM, which is ~five orders of magnitude lower than that of the system without TB-CHA. 2. Experimental 2.1. Materials and chemicals Oligonucleotides and reagents used in the experiments were demonstrated in the supporting information in details. 2.2. Preparation of MoS2-based TB-CHA probe MoS2 nanosheet were synthesized using an ultrasonic stripping method according to the steps in our previous work.55 Before being mixed, hairpin DNAs were retained at 95 °C for 10 min and followed by cooling to 25 °C slowly to form DNA beacons. The adsorption beween MoS2 nanosheet and hairpin DNAs was carried out in 200 μL phosphate buffer (2.3 mM NaH2PO4, 7.7 mM Na2HPO4 and 100 mM NaCl, pH 7.4) containing 25 nM hairpin DNA 1 (H1), 100 nM hairpin DNA 2 (H2), 25 nM hairpin DNA 3 (H3) and 4 U RNase inhibitor. 10 μg/mL MoS2 suspension was subsequently added and incubated for 20 min at room temperature to sufficiently absorb hairpin probes. The prepared MoS2-based TB-CHA probe was kept at 4 °C in the dark for further use. As the control, MoS2-based H1 probe was prepared by incubating 150 nM H1 with 10 μg/mL MoS2 in 200 μL phosphate buffer. 2.3. Polyacrylamide gel electrophoresis


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The products of amplification procedures were characterized using 8% polyacrylamide gel electrophoresis. Then, the electrophoresis was run in TBE buffer (88 mM Tris-acetic acid, 2 mM EDTA, pH 8.0) at 80 V for 120 min. After staining by GelRed, the gel was imaged using GBOX-F3-E (Gene Company Limited, the U.S.A). 2.4. Detection of miRNA-21 in vitro The prepared MoS2-based TB-CHA probe (10 μg/mL) and the MoS2-based H1 probe (10 μg/mL) were allowed to incubate with various concentrations of target miRNA-21 in phosphate buffer (2.3 mM NaH2PO4, 7.7 mM Na2HPO4 and 100 mM NaCl, pH 7.4) at 37 °C for 1 h. The fluorescence experiments was performed on a RF5301PC fluorescence spectrophotometer (Shimadzu, Tokyo, Japan) and the fluorescence spectra of Cy3 were recorded between 550 and 650 nm with 540 nm excitation. 2.5. MTT assay The cytotoxicity of the probe was evaluated by a MTT assay. Several groups of MCF-7 cells (1 × 105 cells, total volume of 200 μL per well) were incubated in the 96well plates at 37 °C for 24 h in a 5% CO2/95% air incubator. After removing the former medium, cells were allowed to incubate with different concentrations of MoS2 nanosheet (0, 2.5, 5, 10, 20 μg/mL) or MoS2-based TB-CHA probe (0, 2.5, 5, 10, 20 μg/mL) for 24 h, respectively. After that, 100 μL 0.5 mg/mL MTT solution was added for another 4 h incubation. After removing the remaining MTT solution, the formed formazan crystal was dissolved by adding DMSO (150 μL). Finally, the absorbance at 490 nm was recorded by a microplate reader（EL×808, BioTek, the U.S.A.） 2.6. Confocal fluorescence imaging All cells were incubated in the 96-well plates at 37 °C for 24 h in a 5% CO2/95% air incubator (1 × 105 cells per well of final density). After washing with 1 × PBS for 3 times, fresh DMEM medium containing 10 μg/mL MoS2-based TB-CHA probe or 10



μg/mL MoS2-based H1 probe was added and incubated for 8 h. After removing the incubation solutions, cells were washed with 1 × PBS buffer for 3 times and the confocol images were monitored under a FV1000 laser scanning confocal microscope (Olympus Optical Co., Ltd., Japan) with 559 nm excitation. 2.7. Flow cytometric assay After cells were treated with MoS2-based TB-CHA probe for 8 h, cells were trypsinized and suspended in a fresh medium. The fluorescence intensities were analyzed using a BD FACSCanto flow cytometry (Merck Millipore, Darmstadt, Germany) in real time. The fluorescence intensity of Cy3 was obtained with 561 nm extinction. 3. Results and Discussion 3.1. Principle of the assay As illustrated in Figure 1, three well designed free hairpin DNA probes (H1, H2 and H3) with the 5’ terminal modifying the fluorophore (Cy3) are attached to the surface of MoS2 nanosheet to fabricate the MoS2-based three-branched catalyzed hairpin assembly (TB-CHA) probe through the van der Waals force between MoS2 nanosheet and overhanging single-strand part of hairpin DNA probes. After sufficient adsorption, the fluorescence of Cy3 is efficiently quenched by the MoS2 due to fluorescence energy transfer effect.28 The neck and overhanging part of H1 is partly complementary to target miRNA-21. Therefore, the presence of miRNA-21 can trigger the hybridization between miRNA-21 and H1 specifically through the overhanging part of H1. Then, the hairpin nanostructure of H1 is opened up to produce a sticky end which is partly complementary to H2. Then, the opening up of H2 exposing a new sticky end to initiate the hybridization between H2 and H3. Finally, miRNA-21 is replaced due to more bases complementary between H3 and H1. After the processes of strand displacement and CHA reaction, a “Y” shaped three branched duplex nanostructure is formed. Due to the weak affinity between duplex DNA and MoS2 nanosheet, the


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nanostructures are released from the nanosheet and fluorescence signals are recovered. The released miRNA-21 will trigger the next circles of hybridization repeatedly. In this case, a single miRNA-21 can cause the production of large amounts of “Y” shaped three branched duplex nanostructures, which will produce a remarkable fluorescence enhancement. Besides, MoS2 also serves as an ideal carrier transferring metastable hairpin probes into living cells for intracellular imaging. Thus, signal amplified detection of miRNA with ultrahigh sensitivity in living cells is expected to be achieved.

Figure 1. Illustration of MoS2-based three-branched catalyzed hairpin assembly (TB-CHA) probe for the cycling signal amplified detection and imaging of low-level miRNA-21 expression in living cells. 3.2. Feasibility study of miRNA-21 detection The structure formation and feasibility of CHA reaction were firstly determined by 8% polyacrylamide gel electrophoresis (PAGE) analysis. From the band 7 in Figure 2A, the cross-hybridization between H1, H2 and H3 is almost blocked without the presence of target DNA-21. After incubated with 150 nM miRNA-21, the interaction between H1, H2 and H3 is initiated and a distinct band can be observed in band 6. The



relatively lower mobility of the main band in band 6 compared to that in band 2, 4 and 5 suggested that the H1/H2/H3 complex is generated with a larger molecular weight. Transmission electron microscopy (TEM) of MoS2 nanosheet before and after DNA hairpins assembly (Figure S1) shows that the nanosheet has a good dispersion in solution with a wrinkled and thin-layered nanostructure, suggesting MoS2 nanosheet is suitable for further in-solution applications. To test the quenching ability of MoS2 and the response for miRNA-21 of the MoS2-based TB-CHA probe, fluorescence measurements were then performed (Figure 2B). Curve a shows the fluorescence spectrum of the free hairpin probes under the excitation at 540 nm. After incubating with MoS2 nanosheets, the fluorescence is decreased ~86% because that the Cy3 labeled hairpin probes are brought into close proximity with MoS2 nanosheet (curve b in Figure 2B), indicating that MoS2 can serve as an efficient quencher. Then, the presence of miRNA initiates the cycling reaction of TB-CHA. Large amounts of “Y” shaped duplex probes are generated and released to restore the fluorescence (curve c in Figure 2B). The result indicates that the “turn-on” mode works well for miRNA-21 responding. To verify the amplification ability of TBCHA, the MoS2-based H1 probe was then tested by incubating the same total amount hairpin probes (H1) with MoS2 nanosheets. Curve e (Figure 2B) without TB-CHA reaction suggests that the fluorescence recovery is only ~35% of that in curve b with TB-CHA, confirming that TB-CHA can enhance the fluorescence signal for miRNA21 detection. Curves e and f in Figure 2B further verifies that the fluorescence recovery of CHA is initiated by the hybridization of H1 and miRNA-21 specifically.


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Figure 2. (A) Polyacrylamide gel electrophoresis (PAGE) image of the DNA nanostructures using 8% agarose gel. Lane 1: 20 bp marker; Lane 2: H1 probe; Lane 3: DNA-21; lane 4: H1 + DNA-21; Lane 5: H1 + H2 + DNA-21; Lane 6: H1 + H2 + H3 + DNA-21; Lane 7: H1 + H2 + H3. Concentrations: H1, H2, H3, 1 μM；DNA-21： 150 nM. (B) Fluorescence spectra of the free H1 + H2 + H3 (curve a), MoS2 + H1 + H2 + H3 (curve b), MoS2 + H1 + H2 + H3 + miRNA-21 (curve c), MoS2 + H1 (curve d), MoS2 + H1 + miRNA-21 (curve e), MoS2 + H2 + miRNA-21 (curve f). Concentrations for system a-c: H1, H3: 25 nM；H2: 100 nM; MoS2: 10 μg/mL; DNA-21: 100 nM. Concentrations for system d-e: H1:150 nM; MoS2: 10 μg/mL; DNA-21: 100 nM. Concentrations for system f: H2:150 nM; MoS2: 10 μg/mL; DNA-21: 100 nM. λex: 540 nm. 3.3. MoS2-based TB-CHA probe for amplified miRNA-21 detection in vitro Having confirmed the feasibility of the assay, the concentration ratio of hairpin DNA probes was optimized because that might affect the efficiency of the CHA-based reaction.56 With a constant total concentration of hairpin probes, the concentration ratio of H1:H2:H3 was adjusted from 1:1:1 to 1:5:1 in the existence and absence of miRNA21. The result in Figure 3A suggests that the fluorescence signal is greatly enhanced when the proportion of H2 was increased and the signal to noise ratio (S/N) was obtained the maximum when H1:H2:H3 was 1:4:1. Then, the concentration of MoS2 nanosheet is investigated because high concentration of MoS2 nanosheet would lead to low background signals while excessive nanosheet might absorb target gene in a nonspecific manner and reduce signals. The result represented in Figure 3B confirms that larger amounts of MoS2 would quench the fluorescence of Cy3 and the highest S/N was achieved at the MoS2 concentration of 10 μg/mL. Hence, 10 μg/mL was chosen for further experiments. Kinetics studies suggest that the fluorescence of hairpin probes is significantly quenched by MoS2 within 20 min (Figure 3C). After incubating with 100 nM target miRNA-21, the fluorescence is recovered to the maximum within 60 min



(Figure 3D). The rapid quenching and responding kinetics suggests that the biosensor can be fabricated and performed with high efficiency.

Figure 3. (A) Optimal experiments for the concentration ratios of H1:H2:H3. The total concentrations of DNA hairpins in each system are 150 nM. Concentration of MoS2: 10 μg/mL. (B) Optimal experiments for the concentrations of MoS2. Concentrations: H1: 25 nM; H2: 100 nM; H3: 25 nM. The yellow curves respect the ratio of signal to noise. (C) Fluorescence change of H1, H2 and H3 after being incubated with buffer (black curve) and MoS2 (red curve) for different time. Concentrations: H1: 25 nM; H2: 100 nM; H3: 25 nM; MoS2: 10 μg/mL. (D) Fluorescence change of probe after being incubated with 100 nM miRNA-21 for different time. Concentrations: H1: 25 nM; H2: 100 nM; H3: 25 nM; MoS2: 10 μg/mL; miRNA-21: 100 nM. λex: 540 nm; λem: 565 nm. To demonstrate the performance of the MoS2-based TB-CHA probe for the amplified detection of miRNA-21 in solution, the fluorescence response for miRNA21 with and without TB-CHA were compared (strategies were shown in the schemes


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of Figure 4A and 4B). Two batches of probes (MoS2-based TB-CHA probe and MoS2based H1 probe) were allowed to incubate with various concentrations of miRNA-21 (0-100 nM), respectively. The result suggests that the increase fluorescence values with TB-CHA (MoS2-based TB-CHA probe) are much higher than that without TB-CHA (MoS2-based H1 probes). From the calibration curves inset in Figure 4C and 4D, the system with TB-CHA reveals a wider detection range of 100 fM-1 nM, while the detection range of the system without TB-CHA is only 10 pM-100 nM. The limit of detection (LOD) of system with TB-CHA is calculated to be 75.6 aM (S/N=3), which is ~five orders of magnitude lower than that of the system without TB-CHA (LOD: 8.5 pM), confirming that the TB-CHA can obviously enhance the detection sensitivity for miRNA. The high sensitivity is attributed to the circular generation of fluorescent three branched H1/H2/H3 complex initiated by miRNA-21 (scheme in Figure 4A), while the fluorescence recovery of system without TB-CHA is limited due to the single-step strand displacement (scheme in Figure 4B). We also compared the detection performance of this strategy with other recent enzyme-free amplification based systems and listed in Table S2, which suggests that TB-CHA-based system is more sensitive than most recent enzyme-free systems. The selectivity experiments further test the responding of MoS2-based TB-CHA probe and MoS2-based H1 probe towards the target miRNA-21, single-base mismatched target (miRNA-21-SM), non-specific targets (miRNA-27a) and non-complementary random miRNA (NC). The result in Figure S2A reveals that the fluorescence intensity of target miRNA-21 is ~3 folds higher than that of miRNA-21-SM and ~4.3 folds higher than that of non-specific targets (miRNA-27a and NC). As a comparison, the fluorescence intensity of target miRNA-21 is only ~1.4 folds higher than that of miRNA-21-SM and ~1.8 folds higher than that of non-specific targets (miRNA-27a and NC) in the system without TB-CHA (Figure S2B). The result suggests that the MoS2-based TB-CHA probe retains high specificity for sequences in miRNA detection owing to the excellent ability of molecular beacons to distinguish base mismatches.57, 58



Figure 4. (A, B) Scheme and fluorescence spectra of systems (A) with and (B) without MoS2-based TB-CHA probe responding for various concentrations (0, 100 aM, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM and 100 nM) of miRNA21. (C, D) Concentration curves of systems (C) with and (D) without MoS2-based TBCHA probe. Insets are the calibration curves for each system. For system with TB-CHA, the calibration equation was y = 9.36 lg cmiRNA-21 (aM) + 17.26 (R2 = 0.9830); for system without TB-CHA, the calibration equation was y = 10.20 lg cmiRNA-21 (aM) − 37.51 (R2 = 0.9746). λex: 540 nm; λem: 565 nm. 3.4. Intracellular imaging of miRNA-21 Before the intracellular imaging, the cytotoxicity of the MoS2-based TB-CHA probe and bare MoS2 was investigated by a MTT assay in MCF-7 cell (human breast cancer cell line) as a model. Different concentrations of MoS2 and MoS2-based TBCHA probe from 0 to 20 μg/mL were added to MCF-7 cells for 24 h incubation at


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37 °C. According to the absorbance intensities of formed product at 490 nm, MCF-7 cells maintain high activity (> 90%) after sufficient uptake of both probes (Figure S3). The result suggests that the probe appears no obvious toxicity and side-effect to living cells, confirming that the MoS2-based TB-CHA probe is suitable for intracellular diagnosis. To test the stability of nanoprobe in complex physiological environment, fluorescence analysis was performed under nuclease degradation and 50% cell lysate degradation. After the fabrication of MoS2-based TB-CHA, the probe was diluted in 2 U/L Enzyme deoxyribonuclease (DNase I) or 50% cell lysate. As shown in Figure S4, the fluorescence intensities of the probe treated with DNase I (Figure S4A) or 50% cell lysate (Figure S4B) was not obviously changed after 12 h incubation, suggesting that the nanoprobe has no obvious degradation. After adding 100 nM target miRNA-21, the fluorescence was significantly recovered. The results suggests the probe have high stability in complex physiological environment for long-time detection. We selected MCF-7 cells as the positive cells and MCF-10A cells (normal human breast cell line) as the negative cells. After being incubated with MoS2-based TB-CHA probe for 8 h, obvious fluorescence signals of Cy3 can be observed in the group of MCF-7 cells, while faint fluorescence is observed in the MCF-10A cells (Figure 5). Statistical data of fluorescence intensity suggest that the MCF-7 treated with MoS2based TB-CHA probe is ~10 folds higher than that of MCF-10A cells. The relative miRNA-21 levels in these two cells are further confirmed by qRT-PCR method (Figure S5A). The result indicates that the probe can discriminate cancerous cells from normal ones efficiently. To study the incubation kinetics of probe and cells, MCF-7 cells were allowed to incubate with MoS2-based TB-CHA probe for different time (0 to 12 h). The fluorescence of cells were imaged with a confocal microscope and quantified by flow cytometry. The result in Figure S6 suggests that fluorescence signals in MCH-7 cells are increased with time. After 8 h incubation, bright fluorescence signals can be observed. Therefore, 8 h was selected to be suitable for the incubation between cells



and probes. Further, the ability of MoS2-based TB-CHA probe for intracellular signal amplification was then investigated. Two batches of MCF-7 cells were allowed to incubate with the same concentrations of MoS2-based TB-CHA probe and MoS2-based H1 probe for 8 h, respectively. The confocal imaging in Figure 5 suggests that the fluorescence intensity of MCF-7 cells incubating with MoS2-based TB-CHA probe is ~3.2 folds higher than that of cells incubating with MoS2-based H1 probe, indicating that the signal amplification of MoS2-based TB-CHA probe could work efficiently in the intracellular environment.


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Figure 5. (A) Confocal microscopic images of MCF-7 cells after being incubated with MoS2-based TB-CHA probe (the first column); MCF-10A cells after being incubated with MoS2-based TB-CHA probe (the second column) and MCF-7 cells after being incubated with MoS2-based H1 probe (the third column). The first row was Cy3 fluorescence recorded at 559 nm excitation. The scale bar is 50 μm. (B) Relative fluorescence intensity of MCF-7 cells and MCF-10A cells after being incubated with



MoS2-based TB-CHA probe. (C) Relative fluorescence intensity of MCF-7 cells after being incubated with MoS2-based TB-CHA probe (with TB-CHA) and MoS2-based H1 probe (without TB-CHA). To further investigate the universality of the MoS2-based TB-CHA probe for miRNA-21 detection in different kinds of cancer cells, the probe was applied with other two types of cancer cells, HeLa cells (human cervical carcinoma cell line) and HepG2 cells (human hepatocellular carcinoma cell line). Bright green fluorescence signals for miRNA are observed under the confocal microscopy after 8 h incubation (Figure 6), which has a similar phenomenon in MCF-7 cells. The qRT-PCR results in Figure S5A further confirm that the miRNA-21 levels are relatively high in both cell lines. The fluorescence signals produced by MoS2-based TB-CHA probe are in consistence with the expression levels of miRNA-21. To find a correlation relationship between the relative intracellular fluorescence and the amount of miRNA-21, the absolute amount of miRNA-21 was calculated by setting a synthetic miRNA-21 at a known concentration as the standard. By counting the cell numbers respectively by cell-count boards, the absolute amount of miRNA-21 determined by RT-PCR is (2.17 ± 0.15) × 104 copies/cell in MCF-7 cells, (1.96 ± 0.12) × 104 copies/cell in HeLa cells, (1.81 ± 0.21) × 104 copies/cell in HepG2 cells, (3.08 ± 1.12) × 103 copies/cell in MCF-10A cells, respectively. The result was also consistent with the previous work.59 As shown in Figure S5B, a correlation relationship between the relative intracellular fluorescence and the amount of miRNA-21 can be obtained to provide a reference for predicting the miRNA levels in unknown cells (F (relative fluorescence intensity) = 1.05 c (× 103 copies/cell) – 0.070, R2 = 0.9952). The above result indicated that the MoS2-based TBCHA probe was adapt to the detection and imaging of miRNA in various living cells.


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Figure 6. Confocal microscopic images of HeLa cells (the first row) and HepG-2 cells (the second row) incubated with MoS2-based TB-CHA probe. The first column is Cy3 fluorescence recorded at 559 nm excitation. The scale bar is 50 μm. 4. Conclusions In conclusion, we have represented a nonenzymatic TB-CHA probe based on MoS2 nanosheet for signal amplified live-cell detection and imaging of specific miRNAs with several advantages. First, one-step transportation and detection based on MoS2 nanosheet is fulfilled with good biocompatibility and low cytotoxicity, which allows the nondestructive imaging of miRNA-21 and distinction of cancerous cells from normal ones. Second, ultrasensitive miRNA-21 detection (with a detection limit of 75.6 aM) is realized, which is attributed to the multi-site fluorescence modification and circular production of three branched fluorescence signal probes. Third, high selectivity is achieved by the MoS2-based TB-CHA probe due to the excellent ability of molecular beacons to distinguish base mismatches. Moreover, the nanoprobe can be fabricated easily by one-step incubation within 20 min. Since protein expression has a close relationship with the cell types, aptamers for specific protein recognition on the surface of cells can be encoded into the nanoprobe to further expand the specificity of the nanoprobe for recognizing specific cell types. We anticipate this research can be



extended to a variety of signal amplified intracellular detection systems, which may have great potential in biological diagnosis and pathological studies. ASSOCIATED CONTENT Supporting Information “Materials and chemicals”, stability test, quantificational RT-PCR (qRT-PCR) procedues, supporting Table S1-S2 and supporting Figure S1-S6 is available free of charge on the ACS website. DNA and sequences; TEM images of MoS2 nanosheet before and after DNA assembly; Selectivity study of MoS2-based TB-CHA probe and MoS2-based H1 probe; Cytotoxicity experiment of MoS2-based TB-CHA probe; miRNA-21 level in cells determined by RT-PCR; Kinetics of miRNA-21 imaging in MCF-7 cells. AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected] Author Contributions ǁThese

authors contributed equally (D.Z. and J.H.).

ACKNOWLEDGEMENTS This work was financially supported by the National Key Research and Development Program of China (2017YFA0205302), the National Natural Science Foundation of China (21605087, 61671250, 61771253), the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R37), the Key Research and


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Development Program of Jiangsu (BE2018732), the Natural Science Key Fund for Colleges and Universities in Jiangsu Province (17KJA430011), the National Postdoctoral Program for Innovative Talents (BX201700123), and the China Postdoctoral Science Foundation funded project (2018M630586). CONFLICT OF INTEREST The authors declare no conflict of interest. REFERENCES (1) Bartel, D. MicroRNAs: Target Recognition and Regulatory Functions. Cell. 2009, 136, 215-233. (2) Fan, Q. ; Yang, L.; Zhang, X.; Peng, X.; Wei, S.; Su, D.; Zhai, Z.; Hua, X.; Li, H. The Emerging Role of Exosome-Derived Non-Coding RNAs in Cancer Biology. Cancer Lett. 2018, 414, 107-115. (3) He, L.; Hannon, G.; Micrornas. Small RNAs with A Big Role in Gene Regulation. Nat. Rev. Genet. 2004, 5, 522-531. (4) Winter, J.; Jung, S.; Keller, S.; Gregory, R.; Diederichs, S. Many Roads to Maturity: microRNA Biogenesis Pathways and Their Regulation. Nat. Cell. Biol. 2009, 11, 228234. (5) Rupaimoole, R.; Slack, F. MicroRNA Therapeutics: Towards A New Era for the Management of Cancer and Other Diseases. Nat. Rev. Drug. Discov. 2017, 16, 203221. (6) Calin, G.; Croce, C. MicroRNA Signatures in Human Cancers. Nat. Rev. Cancer 2006, 6, 857-866. (7) Croce, C. Causes and Consequences of MicroRNA Dysregulation in Cancer. Nat. Rev. Genet. 2009, 10, 704-714. (8) Makarova, J., Shkurnikov, M., Wicklein, D., Lange, T., Samatov, T., Turchinovich, A., Tonevitsky, A. Intracellular and Extracellular MicroRNA: An Update on Localization and Biological Role. Prog. Histochem. Cytochem. 2016, 51, 33-49.



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Intracellular MicroRNA Imaging with MoS2-Supported Nonenzymatic

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