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() Images showing MDA-MB-231 cells after incubation with AuNPs functionalized with the substrate strand but not the DNAzyme strand (negative control). () Images showing MDA-MB-231 cells after incubation with the DNAzyme motor system but not subsequently treated with the cofactor Mn (negative control). () Images showing the target MDA-MB-231 cancer cells after incubation with a mutant DNAzyme motor system and the subsequent treatment with the cofactor Mn (negative control). () Images showing the target MDA-MB-231 cancer cells after incubation with the DNAzyme motor system and the subsequent treatment with the cofactor Mn. Fluorescence images of the target cells are the result of intracellular operation of the DNAzyme motor. () Images showing the negative control MCF-10a cells after the same treatment as for the MDA-MB-231 cells in . () Images showing the negative control MCF-7 cells after the same treatment as for the MDA-MB-231 cells in . All images were collected at the end of 60 min operation time (i.e., 60 min after the addition of the cofactor Mn). Fluorescence imaging of living cells was performed using an Olympus IX-81 microscope equipped with a Yokagawa CSU × 1 spinning disk confocal scan-head and a Hamamatsu EMCCD camera with × 40/1.3 Oil and × 20/0.85 Oil objective lenses. A pumped diode laser at 491 nm was used for the excitation of FAM. Magnification was chosen to allow a final pixel size of 447 nm with × 20 lens and 223 nm with × 40 lens. The length of the scale bar is 17 μm.

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Inside the cells, the specific miR-10b miRNA hybridizes to the locking strand of the motor system through the strand displacement reaction, exposing the sequestered Arm 2 of the DNAzyme motor and freeing the DNAzyme to interact with a substrate strand. Further treatment of the cells with 5 mM MnCl 2 initiated the DNAzyme motor to walk along the AuNP autonomously and processively. Each walking step restores the fluorescence of one FAM molecule. After 60 min operation, the fluorescence is detectable from the MDA-MB-231 cancer cells ( Kay Jewelers Diamond Enhancer Ring 1 ct tw Roundcut 14K Yellow Gold byas7Ao
), suggesting that the intracellular operation of the DNAzyme motor has been accomplished. These cells show various fluorescence intensities, suggesting that the intracellular miR-10b levels are different among these cells. When cells were not treated with Mn 2+ solution, no fluorescence was observed ( Fine Jewelry Lab Created Blue Sapphire Sterling Silver Necklace 4fpne5w
), confirming that the operation of the DNAzyme motor requires both the target miRNA and the cofactor. This control experiment also suggests that the substrate strand on the AuNPs is stable and is not released without the active operation of the DNAzyme motor. We also tested the mutant DNAzyme motor incubated with the target MDA-MB-231 cells. As expected, no fluorescence was observed ( Novica Amethyst dangle earrings Moon Garden Handmade Sterling Silver and Amethyst Dangle Earrings cz4D2xt
), proving the high stability of the substrate strand on AuNPs in the absence of an active DNAzyme motor. Another control, using AuNPs conjugated with the substrate strands but not with the DNAzyme, showed no fluorescence ( Novica Silver dangle earrings Potato Blossom Handcrafted Floral Fine Silver Dangle Earrings CMyJbH
), further proving that substrate strands on the AuNP are stable within the cells. We also introduced the motor system into two control cells deficient in miR-10b, MCF-10a and MCF-7 cells . After 60 min incubation, little fluorescence is observed from these two cells ( Ti2 Titanium Triple Four Petal Flower Pendant Yellow Dy0r9
), proving the specificity of the DNAzyme motor for the specific miRNA target.

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