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• W3088003301 abstract "Open AccessCCS ChemistryRESEARCH ARTICLE1 Aug 2021Smart Engineering of a Self-Powered and Integrated Nanocomposite for Intracellular MicroRNA Imaging Mei-Rong Cui, Xiang-Ling Li, Hong-Yuan Chen and Jing-Juan Xu Mei-Rong Cui State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023 Google Scholar More articles by this author , Xiang-Ling Li *Corresponding author: E-mail Address: [email protected]n E-mail Address: [email protected] State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023 State Key Laboratory of Materials-Oriented Chemical Engineering, College of Life Science and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816 Google Scholar More articles by this author , Hong-Yuan Chen State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023 Google Scholar More articles by this author and Jing-Juan Xu *Corresponding author: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.020.202000419 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail Over the past 2 years, many DNA motors have been synthesized and run in living cells, but there are still challenges in designing integrated DNA motors self-powered to enable autonomous intracellular walking without auxiliary additives. Herein, we report a smart strategy based on a DNA motor–MnO2 nanocomposite, which successfully meets these requirements of intracellular analysis and enables sensitive imaging of specific microRNAs (miRNAs) in living cells. Once the motor system enters the cells, MnO2 nanosheets are reduced by intracellular glutathione (GSH), which not only releases the DNA motors that can be activated by the intracellular target miRNA via binding-induced DNA assembly, but also produces cofactors, Mn2+, that can be used as fuels for autonomous and progressive walking. In addition, the false-positive signal generated by GSH on the DNA motor destruction can be greatly reduced due to the consumption of GSH during this process. This strategy not only combines the advantages of previous dynamic nanomachines based on Au nanoparticles but also has merits of higher integration, lower background fluorescence, and self-powered performance, which provide an efficient avenue for visualizing various biomolecules in living cells. Download figure Download PowerPoint Introduction MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate the post-transcriptional process.1,2 They not only participate in the biological processes of cell development, differentiation, apoptosis, and proliferation, but are also related to many pathological processes, such as tumor formation, metastasis, and resistance to drug therapy.3–7 Specifically, miRNAs have become a valuable tumor marker and drug target, highlighting its important roles in the diagnosis and treatment of cancer.8–12 Therefore, imaging miRNA in living cells is desperately needed for the identification and diagnosis of cancer and efficient drug treatment. However, there are still challenges in viewing miRNA in living cells due to the low abundance and complicated physiological environment.13 Since nanoflares were first been proposed by the Mirkin group14 in 2007, nucleic acid–Au nanoparticle (AuNP) nanoprobes have attracted more attention and have been successfully applied in ultrasensitive imaging of miRNA in living cells.15–20 Especially over the past 3 years, inspired by highly complicated and efficient molecular motors presented in living systems, researchers have attempted to design artificial molecular motor nanocomposites (such as robots, springs, and walkers) with excellent biostability for imaging miRNA in living cells and studying physiological processes.21–29 For instance, the DNA motor and DNAzyme motor reported by the Le group30,31 have provided good examples of DNA nanocomposites operating inside cells. However, the operation of the DNA motor requires the participation of specific endonuclease, which limits potential intracellular applications, and the cofactor Mn2+ needs to be additionally supplied for the walking of DNAzyme motors due to the insufficient Mn2+ level in native cells. To overcome these limitations, the Ye group32 has constructed an endogenous adenosine 5′-triphosphate (ATP)-powered DNA motor for imaging intracellular miRNA. This strategy provided a new idea for DNA motor system design for efficient miRNA imaging in living cells without auxiliary additives. Still, considering that the consumption of endogenous molecules may have adverse effects on cell viability and limit wider intracellular molecules imaging, it is necessary and valuable to design a more integrated and self-powered DNA motor to image biomolecules in living cells. Herein, we report a DNA motor–MnO2 nanocomposite that can meet these requirements. As shown in Scheme 1, a DNA motor–MnO2 nanocomposite is composed of walking legs (W), MnO2 nanosheets, and AuNPs assembled with substrate strands (S) and affinity strands (A) (DNA-AuNP). The S labeled with Cy5 and A tether to the AuNP surface by Au–S bonds, in which the fluorescence emission of Cy5 is quenched via nanomaterial surface energy transfer. The target miRNA-21 (miR-21, T) recognition sequences are designed to embed in A and W. DNA-AuNP and the walking leg (W) are assembled on MnO2 nanosheets via strong adsorption between DNA single strands (ssDNA) and MnO2 nanosheets. After the DNA motor–MnO2 nanocomposite enters the cells by endocytosis, the MnO2 nanosheets are reduced to Mn2+ by intracellular glutathione (GSH) and then the DNA-AuNP and W strands are released. Subsequently, the DNA motor initiates the following self-powered catalytic reaction when encountering the target miRNA: the target miRNA hybridizes with A and W and connects them, the DNAzyme portion at one end of the W strand then hybridizes to the adjacent S, which is a cleavage site of the DNAzyme and forms Mn2+-specific DNAzyme. In the presence of Mn2+, S cleaves into two parts and then the Cy5-labeled part is released from AuNP and fluorescence is recovered. Meanwhile, the DNAzyme portion is released from the other part of S and hybridizes with another adjacent S to trigger autonomous and continuous fluorescence single output. This proposed method has merits of higher integration, lower background fluorescence, and self-powered performance, which provide efficient avenues to visualize miRNAs or other small molecules in living cells. Scheme 1 | Schematic illustration of the DNA motor–MnO2 nanocomposite for intracellular miRNA imaging. Download figure Download PowerPoint Experimental Section Reagents and apparatuses All HPLC (high performance liquid chromatography)-purified oligonucleotides in this paper were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The sequences of the involved oligonucleotides are listed in Supporting Information Table S1. Further details of other reagents and apparatus used in this paper can be found in the Supporting Information. Preparation of DNA-AuNP First, AuNPs (diameter of ∼18 nm) were prepared according to previously reported procedures.33 After that, S (100 μL, 1 μM) and A (10 μL, 1 μM) were added to the solution of AuNPs (435 μL, 2.4 nM) and stirred overnight. Then, SH-PEG solution (0.1 mL, 10 μM) was added to the mixture and incubated for 12 h in the dark. In order to stabilize DNA-AuNP, NaCl solution (4.3 μL, 2 M) was added 8 times to reach a final concentration of 0.1 M. After each addition of NaCl, the mixture was incubated at room temperature for another 6 h. Then, the solution was centrifuged (13,000 rpm, 20 min) and resuspended in 10 mM Tris–HCl (pH 7.4) solution. Finally, this product was stored at 4 °C prior to use. Preparation of the DNA motor–MnO2 nanocomposite The synthesis of MnO2 was performed according to previous reports.34,35 The physisorption of DNA motor on MnO2 nanosheet was carried out by mixing the solution of MnO2 nanosheet (120 μg/mL), DNA-AuNP (4 nM), and W (200 nM) for 60 min followed by the addition of 50 µL of Tris–HCl buffer (pH = 7.4). The concentration of nanocomposite was defined by AuNP concentration. Optimization of the performance of the proposed nanocomposite in vitro To investigate the influence of the ratio of DNA-AuNP to W, a solution of MnO2 nanosheet (120 μg/mL), DNA-AuNP (4 nM, in which the ratio of S∶A is 7∶1) was mixed with different concentrations of W to form different DNA motor–MnO2 nanocomposites, maintaining the concentration ratios of DNA-AuNP to W at 1∶30, 1∶40, 1∶50, 1∶60, and 1∶70. About 1 mM GSH and 200 pM miR-21 were added to the Tris–HCl buffer (pH 7.4) with 1 nM of prepared DNA motor–MnO2 nanocomposite and incubated for another 3 h. After that, the fluorescent intensity of the product was measured at an excitation wavelength of 633 nm, and then the corresponding curves were obtained. To investigate the influence of the ratio of S to A, a solution of MnO2 nanosheet (120 μg/mL) and W (200 nM) was mixed with different concentrations of DNA-AuNP (in which the ratio of S:A ranges from 20∶1 to 10∶4) to form different DNA motor–MnO2 nanocomposites, and then aforementioned fluorescence experiment was carried out. Determination of ssDNA strand amount on each AuNP A fluorescence-based method was used to determine the quantity of ssDNA on each AuNP. DNA-AuNP was obtained by the substrate stand (S), which was labeled with Cy5 tether to the AuNP surface by Au–S bonds. About 12 mM mercaptoethanol was mixed with the prepared 0.5 nM DNA (S)-AuNP solution to displace S via a thiol exchange reaction and reacted for 12 h at room temperature. Subsequently, the AuNPs were removed by centrifugation. By comparing the fluorescence intensity of the supernatant and a standard calibration curve acquired by testing several known concentration gradients of S, the total amount of ssDNA on each AuNP could be determined. Stability investigation of the DNA motor–MnO2 nanocomposite To compare the stability of DNA-AuNP and the DNA motor–MnO2 nanocomposite in the presence of GSH, DNA-AuNP and the DNA motor–MnO2 nanocomposite were diluted with Tris–HCl buffer (25 mM, pH 7.4) to a concentration of 1 nM and then incubated with 1 mM GSH at 37 °C for different times up to 12 h. At time points of 0, 2, 4, 6, 8, 10, and 12 h, the sample (200 μL) was centrifuged (16,000 rpm, 20 min, 4 °C) to remove AuNPs, and the fluorescence of the supernatant was measured (λex = 633 nm and λem = 650–750 nm). All experiments were repeated at least three times. In vitro response of DNA motor–MnO2 nanocomposite to target miRNA About 1 mM GSH and different concentrations of miR-21 ranging from 0 to 200 pM were added to the Tris–HCl buffer (pH 7.4) with 1 nM prepared DNA motor–MnO2 nanocomposite and incubated for another 3 h. After that, the fluorescent intensity of the product was measured at an excitation wavelength of 633 nm, and then the corresponding curves were obtained. The specificity of this strategy was investigated with miR-141, miR-155, miR-182, and miR-197 under the same conditions. Cell culture This work uses four kinds of cell lines. HeLa cells (human cervical cancer cells), HEK-293 cells (human embryonic kidney cells) were grown in Dulbecco’s modification of Eagle’s medium (DMEM) (10% fetal bovine serum) in a 5% CO2, 37 °C incubator. MCF-7 cells (human breast cancer cells) were cultured in RMPI 1640 medium (10% fetal bovine serum), and MCF-10A cells (human normal mammary epithelial cells) were grown in DMEM/F12 (1∶1) medium supplemented with horse serum (5%), insulin (10 μg/mL), epidermal growth factor (20 ng/mL), cholera toxin (100 ng/mL), and hydrocortisone (0.5 μg/mL). Fluorescence confocal microscopy imaging Four types of cells were seeded on 35 mm confocal dishes and remained in an incubator at 37 °C. After 24 h incubation, these cells were washed with 1× phosphate-buffered saline (PBS) and incubated in the corresponding medium containing 1 nM DNA motor–MnO2 nanocomposite for 3 h. Before imaging by confocal microscopy, these cells were cleaned by PBS three times to remove excess DNA motor–MnO2 nanocomposite. For the specificity tests, cells were pretreated with transfection of miR-21 or anti-miR-21 by Lipofectamine 2000. The excitation wavelength was 633 nm, and the images were collected in the range of 650–700 nm. Evaluation of cytotoxicity To estimate the cytotoxicity of the DNA motor–MnO2 nanocomposite, MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) experiments were conducted in MCF-7 cells. MCF-7 cells were seeded on 96-well plates and remained in an incubator at 37 °C. After 24 h incubation, these cells were washed with 1× PBS and incubated in fresh medium with 1 nM DNA-AuNP or DNA motor–MnO2 nanocomposite for 3, 6, 9, and 18 h, respectively. Then, these cells were cleaned with PBS three times to remove excess and then incubated with 200 μL MTT solution (0.5 mg/mL in PBS) for 4 h. After that, MTT solution was removed and dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. Finally, the absorption of MTT at 490 nm was collected, which represented cell viability. Quantification of miR-21 by RT-PCR The relative expression levels of miR-21 in MCF-7 cells, HEK-293 cells, HeLa cells, and MCF-10A were conducted by qRT-PCR (reverse transcription-polymerase chain reaction) analysis.36 The primers used for gene expression analyses in this paper were designed and synthesized by Sangon and their sequences are listed in Supporting Information Table S2. Total RNA was isolated by Trizol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocols. The first-strand cDNAs were reverse-transcribed and synthesized from 0.5 µg total cellular RNA with random hexamers. Briefly, the RT-PCR cycles were conducted to 40 cycles for amplification of miR-21. The fluorescent dye used in RT-PCR reactions was SYBR Green PCR Master Mix. Following the PCR cycling program, it was set for one cycle of predenaturation at 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, then the melting curve from 55 to 95 °C was read every 0.2 °C and held for 1 s between reads. All qRT-PCR reactions were performed in triplicate. The relative expression of miRNA was calculated using the 2−ΔCT method, in which ΔCT = CTmiRNA – CTU6. The experiment was repeated three times. Results and Discussion Characterization of the DNA motor–MnO2 nanocomposite The DNA motor–MnO2 nanocomposite consists of three components: DNA-AuNP, MnO2 nanosheets, and W. As the transmission electron microscopy (TEM) images and the atomic force microscopy (AFM) image show in Figures 1a and 1b and Supporting Information Figure S1, DNA-AuNP had a spherical structure and was ∼20 nm in diameter, the single-layer MnO2 nanosheets (<150 nm) exhibited 2D and ultrathin planes with occasional wrinkles. The TEM image of DNA motor–MnO2 nanocomposite in Figure 1c and the corresponding elemental mappings of Au, Mn, O in the rectangular part of Figure 1c demonstrated that DNA-AuNP and single-layer MnO2 nanosheets were successfully assembled (Figure 1d). The synthesis of the DNA motor–MnO2 nanocomposite was also estimated by the UV–vis absorption spectroscopy and Zeta potential analysis. As the UV–vis spectra show in Supporting Information Figure S2, the characteristic DNA peak at 260 nm of DNA-AuNP suggested S and A tethered to the surface of AuNP and the obvious slope changes in the range of 300–500 nm occurred when DNA-AuNP combined with MnO2 nanocomposite. Even when incubating the MnO2 and DNA motor–MnO2 nanocomposites in weakly acidic conditions, no significant changes in the characteristic absorption curves were observed, revealing that the nanocomposite showed excellent stability. The Zeta potential analysis suggested that the DNA motor–MnO2 nanocomposite had a more negative potential than AuNP and DNA-AuNP ( Supporting Information Figure S3). These all clearly indicate the successful formation of the DNA motor–MnO2 nanocomposite. Figure 1 | (a–c) TEM image of DNA-AuNP, MnO2 nanosheets, and the DNA motor–MnO2 nanocomposite. (d) Corresponding elemental mappings of Au, Mn, and O in the rectangular part of (c). Download figure Download PowerPoint Optimization and performance evaluation of the DNA motor–MnO2 nanocomposite To obtain better performance of the DNA motor–MnO2 nanocomposite, we optimized the W sequence by testing the performance of five nanocomposites containing different W sequences (W1, W2, W3, W4, and W5). These W strands vary by different lengths of arms 1 and 2 of DNAzyme (detailed sequences are listed in Supporting Information Table S1). Length optimization is important because the relevant works have revealed that the length of arm 1 is crucial for inducing false-positive signals due to the spontaneous hybridization between W and S, and the length of arm 2 is associated with the catalytic rate of DNAzyme.37 To compare the performance of DNA motor–MnO2 nanocomposites, we determined their background fluorescence resulting from the target-independent substrate cleavage produced by spontaneous hybridization between W and S. As the result shows in Figure 2a, the background fluorescence intensities of these five DNA motor–MnO2 nanocomposites were enhanced with time, and DNA motor–MnO2 nanocomposite built with W3-W5 had a lower background, which suggested that the background fluorescence was mainly impacted by the length of arm 2, and arm 2 with seven bases had lower background levels. The length of arm 1 was important to the catalytic rate of DNAzyme as hybridization between arm 1 and the substrate remains as an intramolecular association after cleavage. We evaluated the response of these DNA motor–MnO2 nanocomposites to 200 pM miR-21. As shown in Supporting Information Figure S4a, the DNA motor–MnO2 nanocomposites built with W2 and W4 had the strongest fluorescence response after reacting with miR-21 for 2 h. This was because arm 1 with five bases had faster cleavage rates with the substrate than arm 1 with six bases, and arm 1 with three or four bases was too short to ensure efficient association with the substrate. As described earlier, combining with the background fluorescence intensity of these five DNA motor–MnO2 nanocomposites, we concluded that W4 had the highest signal-to-background ratio ( Supporting Information Figure S4b), and it was used in the rest of the experiments as the optimal sequence. Figure 2 | (a) Background fluorescence changes from target-independent substrate cleavage by DNA motor–MnO2 nanocomposite built with different W. (b) Background fluorescence changes of DNA-AuNP and the DNA motor–MnO2 nanocomposite in the presence of 1 mM GSH. (c) Fluorescence profile of the DNA motor–MnO2 nanocomposite responds to different concentration of miR-21 (0, 1, 10, 20, 50, 100, 150, and 200 pM) in the presence of 1 mM GSH in vitro at 37 °C, excited at 633 nm. (d) Calibration curves for the fluorescence intensity versus corresponding miR-21 concentrations (1, 10, 20, 50, 100, 150, and 200 pM). Error bars were estimated from three replicate measurements. Download figure Download PowerPoint Second, the ratios of A and S, DNA-AuNP, and W have also been optimized, and as the results show in Supporting Information Figure S5, the DNA motor–MnO2 nanocomposite with a ratio of S∶A=10∶1 and DNA-AuNP∶W=1∶50 had the highest fluorescent response to 200 pM miR-21, so these ratios were used in the subsequent experiments. After that, the amount of ssDNA on each AuNP and the reaction time were determined. As the results show in Supporting Information Figure S6, the amount of ssDNA on single AuNP was calculated to be ∼108 molecules. According to the amount of ssDNA on each AuNP and the optimized molar ratio (S∶A), the amount of S and A on each AuNP was estimated to be ∼98 and 10 strands, respectively. While investigating reaction time, the fluorescence signal gradually increased with increasing reaction time and reached the platform at 3 h at a higher concentration of the target ( Supporting Information Figure S7), which was utilized as the reaction time in the following experiments in vitro. Furthermore, the stability of the DNA motor–MnO2 nanocomposite was estimated by the control experiment. It is known that the false signal produced by GSH breaking the Au–S bonds of DNA-AuNP is a major limitation of the motor walking mechanism.38 However, this DNA motor–MnO2 nanocomposite can overcome this obstacle, and it was illustrated by comparing changes in the background fluorescence intensities of the DNA-AuNP and the DNA motor–MnO2 nanocomposite solutions in the presence of 1 mM GSH, respectively. As shown in Figure 2b, in the presence of 1 mM GSH, the background fluorescence intensity of DNA-AuNP significantly increased with time, while the signal of the DNA motor–MnO2 nanocomposite was almost unchanged. This was because MnO2, a member of this DNA motor–MnO2 nanocomposite, can convert active GSH to inactive glutathione disulfide (GSSG), inhibiting the false-positive signal generated by GSH destroying the Au–S bonds of DNA-AuNP. Meanwhile, previous research has reported that the presence of nanomaterials, including MnO2 nanosheets and AuNPs, successfully increased the biostability of the DNA motor–MnO2 nanocomposite, because the increased steric hindrance prevents the nuclease access.39–41 To investigate the biostability of the DNA motor–MnO2 nanocomposite, we performed a fluorescence spectrum experiment. As the results show in Supporting Information Figure S8, there was almost no obvious fluorescence enhancement after the DNA motor–MnO2 nanocomposite was incubated with the deoxyribonuclease I (DNase I, 6 U/mL, a concentration much higher than in living cells), which suggested that the DNA motor–MnO2 nanocomposite showed excellent stability and can successfully avoid the nonspecific degradation of nucleases. Under optimized conditions, the feasibility of the DNA motor–MnO2 nanocomposite to detect miR-21 was verified. As shown in Supporting Information Figure S9, only in the presence of the DNA motor-MnO2 nanocomposite, target miR-21, and GSH, the self-powered catalytic reaction could be initiated and a significant fluorescent signal could be generated. This also illustrated the stability of the DNA motor–MnO2 nanocomposite. Then, we investigated the sensitivity of this strategy by observing in vitro fluorescence response to different concentrations of miR-21 (ranging from 0 to 200 pM). As shown in Figure 2c, the fluorescence intensity of the mixture solution increased with increasing concentration of miR-21, which showed good linearity in the range of 1–200 pM with a relationship of Y = 16.84X (pM) + 491.13 (R2 = 0.9902), where Y is the fluorescence intensity and X is the concentration of miR-21 (Figure 2d), and a detection limit of 0.1 pM on the basis of 3σ per slope, which was obviously lower than the previous method.42 Furthermore, the specificity of the DNA motor–MnO2 nanocomposite was investigated by comparing the signals from miR-197, miR-155, miR-141, and miR-182. As the results show in Supporting Information Figure S10, relatively low fluorescence intensities were acquired in the presence of 200 pM miR-197, miR-155, miR-141, or miR-182, but a very high fluorescence intensity was obtained in the presence of miR-21. These results suggest that this strategy exhibited great selectivity for miR-21 over other miRNAs and is an excellent candidate for the detection of multiple low-abundance miRNAs. The intracellular miRNA imaging capability of the DNA motor–MnO2 nanocomposite Before estimating the performance of the DNA motor–MnO2 nanocomposite for imaging intracellular miR-21, MTT assay was conducted in advance to evaluate the cytotoxicity of our DNA motor–MnO2 nanocomposite. As the results show in Supporting Information Figure S11, more than 90% of the cells were variably even cultured with the nanocomposites for 18 h, suggesting that the DNA motor–MnO2 nanocomposite has little side effects on cell viability and could be used for intracellular imaging experiments. Then, MCF-7 cells were selected as the cell model to evaluate the intracellular miRNA imaging capability of our strategy, as the miR-21 is overexpressed in these types of cells.43 MCF-7 cells were incubated with the pure medium, medium with 1 nM DNA motor–MnO2 nanocomposite, and medium with 1 nM mutant DNA motor–MnO2 nanocomposite (in which the W sequence has three mismatch bases and the detailed sequence information corresponding to W6 in Supporting Information Table S1). As the confocal images and fluorescence intensity quantification analysis show in Figures 3a and 3b, there was an obvious fluorescent signal presented in the MCF-7 cells incubated in the medium with DNA motor–MnO2 nanocomposite (1); however, there was almost no fluorescent signal in the MCF-7 cells while they were incubated with mutant DNA motor–MnO2 nanocomposite (2), which has three mismatch bases in the W sequence) or the pure medium (3). These results suggest that the designed DNA motor–MnO2 nanocomposite could lead to a self-powered catalytic reaction after being initiated by target miR-21 and has high selectivity. Moreover, we assessed the content of elemental Mn in living cells by inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis and explored the cellular uptake efficiency of the DNA motor–MnO2 nanocomposite according to the reported method.44,45 Based on the test results, there was around 400 μM of Mn element in the living cells after the cells were incubated with a 1 nM DNA motor–MnO2 nanocomposite for 3 h. As the concentration of GSH in living cells was about 1.0–15 mM,46 almost all of the elemental Mn could be present as Mn2+. The content of Mn element in living cells is enough to support rapid and autonomous operation of DNA motors in living cells as the DNAzyme motor reliably operates in the presence of 250–2000 μM Mn2+.31 Furthermore, the cellular uptake efficiency via ICP-OES analysis was calculated to be 26% and the detailed calculated process is presented in the Supporting Information. Figure 3 | (a) Confocal microscopy images of MCF-7 cells treated with various conditions, from top to bottom: medium containing 1 nM DNA motor–MnO2 nanocomposite (1), medium containing 1 nM mutant DNA motor–MnO2 nanocomposite (2), and the pure medium (3). (b) Fluorescence intensity corresponding to the dotted lines in (a). Scale bars: 20 μm. Download figure Download PowerPoint We also investigated the imaging effect of our strategy for miR-21 in living cells at different incubation times. As the imaging results show in Figures 4a and 4b, there was a faint fluorescent signal observed after cells were incubated with 1 nM DNA motor–MnO2 nanocomposite for 1 h. When the incubated time increased, the fluorescence signals were significantly enhanced, and after 3 h incubation, the fluorescence signals were brilliant and no longer increased with increased incubation time. These results illustrate that 3 h incubation time was sufficient for the DNA motor–MnO2 nanocomposite entering the cells to react with miR-21 and lead to the subsequent self-powered catalytic reaction. Meanwhile, these cells maintained a good state after 3 h incubation, which is consistent with the MTT assay results. We also investigated the location of the nanocomposite in living cells by confocal cell imaging after treatment with nanocomposite and nuclear dye Hoechst 33342. As the results show in Figure 4c and 4d, there was almost no overlap between the two channels of Hoechst 33342 and Cy5, which illustrated that miR-21 was mostly distributed in the cytoplasm. Then, the specificity of the DNA motor–MnO2 nanocomposite for miR-21 detection was estimated via fluorescence confocal microscopy imaging. As shown in Supporting Information Figure S12, compared with the control group (lane i), an obviously decreased trend of fluorescence signal was detected in the MCF-7 cells pretreated with anti-miR-21 transfection (lane ii). While transfect" @default.
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