A Cell-Anchored and Self-Calibrated DNA Nanoplatform for in Situ Imaging and Quantification of Endogenous MicroRNA in Live Cells: Introducing Two Controls to Normalize the Sensing Signals

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLES11 Mar 2022A Cell-Anchored and Self-Calibrated DNA Nanoplatform for in Situ Imaging and Quantification of Endogenous MicroRNA in Live Cells: Introducing Two Controls to Normalize the Sensing Signals Wenjuan Song, Zhi-Ling Song, Qian Li, Chengwen Shang, Qiqi Chao, Xingfu Liu, Rongmei Kong, Gao-Chao Fan and Xiliang Luo Wenjuan Song Key Laboratory of Optic-Electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 Google Scholar More articles by this author , Zhi-Ling Song *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Key Laboratory of Optic-Electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 Google Scholar More articles by this author , Qian Li Key Laboratory of Optic-Electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 Google Scholar More articles by this author , Chengwen Shang Key Laboratory of Optic-Electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 Google Scholar More articles by this author , Qiqi Chao Key Laboratory of Optic-Electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 Google Scholar More articles by this author , Xingfu Liu Key Laboratory of Optic-Electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 Google Scholar More articles by this author , Rongmei Kong School of Chemistry and Chemical Engineering, Qufu Normal University, Qufu, Shandong 273165 Google Scholar More articles by this author , Gao-Chao Fan Key Laboratory of Optic-Electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 Google Scholar More articles by this author and Xiliang Luo *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Key Laboratory of Optic-Electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.022.202101618 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Quantifying the microRNAs (miRNAs) levels in living cells, while essential for the study of fundamental biology and medical diagnostics, has barely been achieved due to insufficient probe delivery and unquantifiable signals. We report a cell-anchored and self-calibrated DNA nanoplatform, a cholesterol-headed DNA nanowire that is capable of efficiently delivering to various cells and simultaneously detecting two target miRNAs. One miRNA target can be utilized as an endogenous control against cell-to-cell variations. Moreover, the photocleavable linkers inserted in the nanostructures allow us to precisely regulate the probe structure and fluorescence signaling at the desired time and location in vivo. As a second control, the maximum fluorescence can be elicited by UV light, which further facilitates the normalization of the absolute fluorescence signal. With two introduced internal controls, the maximum fluorescence and endogenous control gene, this approach displays excellent stability and self-calibration performance, effectively avoiding the interference from operating conditions and cell-to-cell variations, such as the laser powers and intracellular probe concentrations. Importantly, this design is capable of unifying the output signal intensity between in vitro test and cell imaging, making the in vitro linear calibration curve appropriate for the quantification of miRNA expression in living cells. Download figure Download PowerPoint Introduction MicroRNAs (miRNAs) are involved in essential physiological processes, including cellular survival, proliferation, apoptosis, and differentiation.1–3 Aberrant miRNA expression is associated with the occurrence and progression of some diseases such as malignant tumors.4,5 Hence, monitoring intracellular miRNA levels is especially important in medical diagnostics. Alternative in vitro methodologies,6–8 such as quantitative real-time polymerase chain reaction (RT-PCR), northern blotting, and microarray screening, while achieving significant advances in detecting the expression levels of miRNAs in cell lysates, are unable to work in living cells. Fluorescence imaging approaches9–13 have been believed to be powerful in cellular biosensing due to in situ visualization, high resolution, and their noninvasive capability. However, quantifying miRNAs in living cells has been limited by insufficient probe delivery and unquantifiable signals. As for the delivery of nucleic acid probes, alternative strategies, including liposomes, inorganic nanomaterials, organic polymers, and proteins, have been proposed for endogenous miRNA analysis.14–17 While these methods have achieved the goal of delivering cargo into specific cells, there is still a need to improve the performance of the delivery system, so as to reduce costs, simplify the preparation process, improve the biocompatibility, and generalize the system to multiple cells. Given these findings, DNA self-assembly based on the hybridization chain reaction (HCR)18–20 is a good choice, which can be easily synthesized by roughly mixing DNA strands. Moreover, to achieve generalized detection, the DNA nanosystem also needs to be able to recognize multiple cells, whether tumor or normal. The hydrophobic DNA label, cholesterol, possesses a strong hydrophobic affinity with the phospholipid bilayer in the cell membrane, and it can bind to different cells without complex chemical modifications, additional genetic manipulation, or metabolic regulation.21–23 Therefore, integrating cholesterol with fluorescent DNA self-assembly will contribute to efficient probe delivery into a variety of cells. Additionally, for further quantitative miRNA analysis in living cells, the most commonly used fluorescent approaches have suffered from the challenges of unequal signal intensity between cell imaging and in vitro test, making the in vitro calibration curve unavailable for intracellular detection. The key factors contributing to these challenges include the inhomogeneity of probe transfection efficiency and the variation of operating parameters, such as the probe concentration and laser power. For the in vitro techniques, such as RT-PCR, a key part in relative quantification of miRNA levels involves the adoption of a control gene, including the endogenous and exogenous control genes.24,25 In particular, miR-16, a stable and highly expressed gene, has been generally utilized as a reference to calibrate the raw experimental data.24 The Miller group26 indicated that the Let-7a and miR-16 genes were suitable endogenous control genes for miRNA analysis in human breast cancer. The Zhang group27 also revealed that miR-16 was one of the most suitable miRNA endogenous control genes across all cell lines. Accordingly, researchers have sought to introduce these control strategies into their in vivo genetic analysis. The Mirkin group28 developed a multiplexed nanoflare labeled with Cy3 and Cy5 fluorophores for simultaneous detection of two mRNA targets (survivin-target and actin-control) inside cells. In other words, these fluorescence probes should carry two independent fluorescence signals, one for the first target, the miRNA of interest, and the other for the second target, the endogenous control gene. However, due to the two distinct excited lasers, the two absolute-fluorescence outputs tend to fluctuate inconsistently with operating parameters, leading to the target-independent cell-to-cell variations. Therefore, quantitative detection of miRNA in living cells remains a major challenge. Motivated by the control strategy26,27 and single laser-excited ratiometric strategy,29,30 we hypothesize that the absolute fluorescence intensity can be normalized by a control fluorescencethat is excited under the same laser parameter. In this regard, the maximum fluorescence of the identical fluorophore is a desirable candidate. The key step toward this goal is to obtain the maximum fluorescence, which requires precise regulation of the probe structure and fluorescence signaling in vivo at the desired time and location. Light tools with low cost, clean energy, and remote controllability have been widely used to manipulate molecular engineering, fluorescence signaling, gene expression, and tumor therapeutics.31–33 The photocleavable (PC) probe is a typical example, whose molecular structure is locked by a PC linker and then cut by light irradiation at the right time and place.31–33 With the advantages of high spatiotemporal resolution, this light-controllable approach allows us to decide when and where to edit the probe structure to trigger the maximum fluorescence and ultimately normalize the initial absolute fluorescence. However, the incorporation of light-controllable technique into the control gene strategies for intracellular quantification assay has not been reported thus far. Herein, we develop a cell-anchored and self-calibrated DNA nanoplatform for in situ imaging and quantification of miRNA in living cells. As shown in Scheme 1, this DNA nanoplatform, a large DNA fluorescence nanowire, is self-assembled by three functional domains (A, H1–P1, and H2–P2). The activator, A strand, induces HCR assembly of hairpin probes H1–P1 and H2–P2 to construct a cholesterol-headed HCR nanowire (HCRW). Cholesterol is known to anchor strongly to the cell membrane. Thus, a strand with the cholesterol label at the head also serves as a guidance for the effective delivery of HCRW nanoprobes to various cells. Two other functional regions are the signaling elements, hairpin probes H1–P1 and H2–P2, which are designed to signal the two targets, the control miRNA and the target miRNA of interest. Furthermore, the H1 and H2 sequences are inserted with the PC-linkers containing an o-nitrobenzyl group, facilitating further spatiotemporal regulation. Scheme 1 | Schematic illustration of the synthesis of HCRW nanoplatform and its application in quantification of endogenous miRNA in living cell. Download figure Download PowerPoint miR-21, a highly expressed tumor marker, is designated as a model target of interest. miR-16 is selected as an endogenous control gene because of its stable expression in all human breast cell lines. In the presence of miR-21 and miR-16, their complementary probe sequences P1 and P2 are released from the HCRW to distance the fluorophores from black hole quenchers (BHQ), leading to the enhancement of the corresponding Cy5 fluorescence (FCy5) and tetramethylrhodamine (TAMRA) fluorescence (FTAMRA). After target-triggered responses, normalization of their absolute signals is performed with reference to the second control, the maximum fluorescence. The UV light irradiation is added to release all BHQ quenchers, thereby triggering the fully recovered maximum fluorescence of Cy5 and TAMRA, namely FCy5-max and FTAMRA-max. Then, the fluorescence recovery efficiencies, called normalized intensities, are calculated by plugging the four absolute fluorescence signals of individual cells into eqs. 1 and 2. Referring to two controls, our designed HCRW adopts the normalized fluorescence ratio (NCy5/NTAMRA) as the final output signal for the quantitative detection of miR-21. Compared with the commonly used signaling modes, it displays excellent stability and self-calibration performance, effectively shielding the influences of various experimental conditions such as the laser power and probe concentration. In this way, the linear calibration curve obtained in vitro allows for the miRNA quantification in living cells. Accordingly, the mean concentrations of endogenous miR-21 in the MCF-7, MDA-MB-231, and MCF-10A cells have been calculated to be 7.09, 2.15, and 0.75 nM, respectively, presenting a higher expression of miR-21 in cancer cells than in normal cells. This approach provides a solution for in situ quantitative detection of miRNA in living cells, and it can also be extended to the analysis of other biomolecules. Experimental Methods Preparation of HCRW Before the synthesis of the nanoprobe, all the DNA sequences were solved in phosphate-buffered saline (PBS) solution. First, hairpin DNA sequences H1 and H2 were heated at 90 °C for 10 min and then slowly cooled to room temperature for the later experiment. Next, equivalent probe sequences P1 and P2 were incubated with H1 and H2 for 30 min at 37 °C, respectively, so as to form the monomers H1–P1 and H2–P2. Finally, a mixture of H1–P1, H2–P2, and A was incubated at 37 °C for 1 h to trigger the HCR self-assembly, and then the HCRW was synthesized. All nucleotide sequences used in this study were shown in Supporting Information Tables S1–S3. Verification of the fluorescence sensing capacity of HCRW HCRW nanoprobes (10 μL) were added to 90 μL PBS solution containing different concentrations of targets (miR-21 and miR-16). After incubation for 40 min at 37 °C, the fluorescence changes were monitored by an F-7000 fluorescence spectrophotometer (Hitach, Japan). The independent fluorescence signals of TAMRA and Cy5 were detected under the excitation wavelengths of 543 and 633 nm, respectively. All experiments were conducted at least three times. In vitro UV irradiation test The PC-linkers inserted in the HCRW nanostructures allowed us to precisely regulate the probe structure and fluorescence signaling at the right time and place. To investigate this, a nanostructure without PC-linkers, HCRW-noPC, was synthesized for comparision. The common UV light (365 nm, 1 W) was used to cleave the PC-linkers and trigger the maximum fluorescence signals. The HCRW and HCRW-noPC solutions were irradiated under UV light for different periods of time, ranging from 0 to 10 min, and then the dynamic fluorescence signals were monitored with the F-7000 fluorescence spectrophotometer. In this way, the optimization of irradiation time was also conducted. Stability and self-calibration test Laser power was used as a model experimental variate to explore the detection performance of the HCRW probe. Typically, 10 μL HCRW nanoprobes were added to 90 μL PBS solution containing fixed miR-16 (5 nM) and different concentrations of miR-21. After incubation for 40 min at 37 °C, the target-triggered fluorescence signals of TAMRA and Cy5 were detected under different laser powers and read as FCy5 and FTAMRA, respectively. Next, the mixed solution was irradiated under UV light for 6 min to elicit the maximum fluorescence signals, FCy5-max and FTAMRA-max, which were detected under different laser powers. Then, the normalized fluorescence intensities, NCy5 and NTAMRA, were calculated by plugging the data into eqs. 1 and 2. Finally, the normalized ratio, NCy5/NTAMRA, was used as the final output signal. A comparison among the absolute intensity FCy5, normalized intensity NCy5, absolute ratio FCy5/FTAMRA, and normalized ratio NCy5/NTAMRA was performed to demonstrate the changes of detection signal under different laser powers. The test under varying probe concentrations was similar to the above steps, except that the laser power was constant throughout the test, while the probe concentration was changed. All the experiments were conducted at least three times. Confirmation of cholesterol-guided intracellular delivery The cholesterol label plays an important role in effectively guiding the delivery of nanoprobe to various cells. A cholesterol-headed HCR flare system (Cho-HCRF) was labeled with Cy5 and TAMRA to light up the transfection trace along with its intracellular location. Meanwhile, an HCR flare system without cholesterol label, HCRF, was also synthesized for comparison. Before the addition of probes, MCF-7 cells were preinoculated in 96-well plates at 37 °C for 24 h and then washed with PBS solution three times. Next, 150 μL 250 nM Cho-HCRF or HCRF probes were added to each well and incubated at 37 °C for 50 min. During this time period, real-time confocal imaging was performed on the cells at 10, 20, 30, 40, and 50 min. Fluorescence images were captured under a 10× objective lens (Leica, Wetzlar, Germany). The laser with a wavelength of 543 nm/633 nm was adopted as the excitation source of Cy5 (red)/TAMRA (green) fluorescence, and the bandpass filter range was set at 570–610 nm/650–690 nm. Optimization of UV irradiation time of HCRW in living cells MCF-7 cells were preinoculated in 96-well plates at 37 °C for 24 h and then washed three times with PBS. Then, 150 μL 250 nM HCRW was added into each well and incubated at room temperature under dark conditions for 40 min. After washing three times with PBS buffer, the MCF-7 cells were exposed to UV irradiation for different times (0, 1, 5, 10, and 15 min). Then, the temporal changes of Cy5 and TAMRA fluorescence in the living cells were measured with confocal microscopy. Fluorescence imaging in live cells For the in situ imaging of living cells, the HCRW nanoprobes were incubated with MCF-7 MDA-MB-231 and MCF-10A cells for 40 min under dark conditions in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. Then the cell imaging was analyzed with confocal microscopy under the lasers of 543 and 633 nm, and the emissions of TAMRA and Cy5 were collected in the green and red channels, respectively. Next, the cells were exposed to UV irradiation for 10 min. Finally, the maximum fluorescence of Cy5 and TAMRA in the cells was measured with confocal microscopy. Results and Discussion Construction and characterization As shown in Scheme 1, the HCRW nanoplatform was synthesized starting from the HCR self-assembly of five DNA oligonucleotides (H1, H2, P1, P2, and A). miR-21 was designated as a target gene in this work because of its high relevance in tumor progression.34–37 miR-16 was chosen as another target and an endogenous control gene, which was stably and highly expressed across human breast cells and frequently used as a control in conventional biological methodologies. The probing sequence P1 was partially hybridized with PC-linker-embedded hairpin strand 1 (termed H1), and then the hairpin probe H1–P1 capable of binding miR-21 was prepared. Another hairpin probe H2–P2 for miR-16 was also formed in this way. Different dyes (Cy5 for miR-21 and TAMRA for miR-16) were labeled into the 5′ ends of the probe sequences of HCRW to make the independent determination of each target. The synthetic hairpin probes were then mixed with cholesterol-guided activator (termed A) to induce HCR self-assembly, thus constructing the large nanowire HCRW. A polyacrylamide gel electrophoresis (PAGE) assay was used to characterize the synthesis of HCRW. In Figure 1a, the gel migration rate slowed with the sequential addition of five DNA oligonucleotides, indicating successful synthesis of HCRW (Figure 1a, lanes 1–6). Moreover, the target recognition performance of P1 and P2 in this large nanowire remained good, as evidenced by an extra accelerated band in lane 7 compared with lane 6. Figure 1 | Characterization and verification of HCRW. (a) Native PAGE analysis. From lanes 0 to 8: lane 0: marker; lane 1: H1; lane 2: P1; lane 3: H1 + P1; lane 4: H1 + P1 + H2 + P2; lane 5: A; lane 6: lane 4 + A; lane 7: lane 6 + DNA21 + DNA16; lane 8: DNA21 + DNA16. Kinetic curve responses of HCRW platform for 20 nM miR-21 (b) and 5 nM miR-16 (c), where FCy5 represents the absolute intensity of Cy5 fluorescence and FTAMRA represents the absolute intensity of TAMRA fluorescence. (d) Original Cy5 fluorescence spectra of HCRW platform (50 nM) in response to different concentrations of miR-21 (0, 0.5, 2, 5, 10, 20, 30, 40, 50, and 100 nM). (e) Original TAMRA fluorescence spectra of HCRW platform (50 nM) in response to different concentrations of miR-16 (0, 0.5, 2, 5, 10, 20, 30, 40, 50, and 100 nM). Download figure Download PowerPoint Next, the fluorescence-sensing capacity of the HCRW was investigated. As designed in Supporting Information Figure S1a, in the initial HCRW nanostructure, the fluorescence of TAMRA and Cy5 was greatly quenched by the proximal BHQ quenchers. In the presence of miR-21 target, it hybridized with the P1 strand to form a completely complementary duplex, while Cy5 fluorophore was far away from the BHQ quencher, generating an enhanced Cy5 fluorescence. As shown in Figure 1b, the Cy5 fluorescence at 663 nm showed significant increase with the addition of miR-21. This effect was also observed in the fluorescence behavior of TAMRA at 582 nm after the addition of miR-16 (Figure 1c). In addition, HCRW demonstrated gradual enhancement in Cy5 fluorescence (FCy5) with increasing miR-21 concentration (Figure 1d and Supporting Information Figure S1). Moreover, a linear relationship (FCy5 = 112.2 + 45.7x) for target miR-21 in the range of 0.5–40 nM has been found, and the limit of detection was 127 pM (S/N = 3). FCy5 and x represent the Cy5 fluorescence intensity and miR-21 concentration, respectively. Meanwhile, the HCRW nanoprobe showed good specificity ( Supporting Information Figure S1) for miR-21 detection. Similarly, with miR-16 concentration rising, gradually increased P2 strands tended to separate from H1 strands, leading to enhanced TAMRA fluorescence (Figure 1e and Supporting Information Figure S2). Therefore, an obvious rise in the absolute intensity of TAMRA fluorescence (FTAMRA) was observed with increasing miR-16 concentration ( Supporting Information Figure S2). In addition, the specificity assay displayed that enhanced TAMRA fluorescence was triggered only by miR-16 ( Supporting Information Figure S2). To further verify that the probe recognition was sequence-specific and fluorescence detection was independent, the influence of miR-16 on the detection of miR-21 was tested ( Supporting Information Figure S3). The fluorescence response for miR-21 was hardly affected by miR-16, and the two corresponding calibration curves for target miR-21 with and without the addition of miR-16 almost coincided with each other ( Supporting Information Figures S3a–S3d). Together, all the results indicated that HCRW allowed for the response to miRNA-21 and miR-16 targets with two independent fluorescence signals in a sequence-specific manner in vitro. Stability and self-calibration capability In addition to the independent-sensing ability of HCRW, its stability and self-calibration capability were also important to the achievement of intracellular miRNA quantification. To address the challenge of unequal signals in different experimental conditions, this HCRW probe was used to conduct detection in two measurement steps (Figure 2a). First, under the trigger of miR-21 and miR-16 targets, the absolute fluorescence signals FCy5 and FTAMRA were obtained. The second step following this target-triggered response was the rapid activation of maximum fluorescence signals (FCy5-max and FTAMRA-max) under UV irradiation, which was confirmed by the fluorescence changes of HCRW ( Supporting Information Figure S4). After these fluorescence measurements, data conversion and calculation were performed. Accordingly, the two absolute fluorescence signals could be easily normalized by setting the maximum fluorescence signals to 1, i.e., the normalized signals were eqs 1 and 2. Next, the ratio of normalized fluorescence (eq. 3) acted as the final output signal for the miR-21 detection. We then investigated the optical stability of free fluorophores under UV irradiation ( Supporting Information Figure S5a) before further testing. The few fluorescence changes of Cy5 and TAMRA indicated that the imposed weak UV light hardly affected the original optical properties of the fluorophores ( Supporting Information Figures S5b–S5d). N Cy 5 = F Cy 5 / F Cy 5 − max (1) N TAMRA = F TAMRA / F TAMRA − max (2) N Cy 5 / N TAMRA = ( F Cy 5 F TAMRA − max ) / ( F Cy 5 − max F TAMRA ) (3) Figure 2 | Comparison of the response stabilities of traditional fluorescence methods and this method for the detection of target miR-21 under different laser powers. (a) Schematic illustration of HCRW for the detection of target miR-21 under different laser powers. (b) The fluorescence responses of HCRW platform (50 nM) for the 10 nM target miR-21 under different laser powers, when the miR-16 was fixed at 5 nM. (c) The maximum fluorescence spectra of HCRW platform after UV light cleavage. (d) The responses of the absolute fluorescence intensity FCy5 of HCRW platform for the 10 nM target miR-21 under different laser powers. (e) The corresponding ratios (NCy5/NTAMRA) of normalized fluorescence intensity NCy5 to NTAMRA under different laser powers. (f) The linear relationships of absolute fluorescence FCy5 versus the concentration of miR-21 under different laser powers. (g) The linear relationships of NCy5/NTAMRA versus the concentration of miR-21 under different laser powers. Download figure Download PowerPoint As a primary requirement, the probe should be able to calibrate the effects of different operating parameters, providing a stable and consistent detection signal to the same target. The laser power was used as a model operating variate to explore the detection behavior of the HCRW probe (Figure 2a). The fluorescence changes of both the Cy5 and TAMRA labels were studied in buffer solution. First, the concentrations of miR-21 and miR-16 in buffer solution were fixed at 10 and 5 nM, respectively. Next, the target miR-21 signal was read as the ratio of NCy5 to NTAMRA, namely NCy5/NTAMRA, while miR-16 was utilized as the first control and maximum fluorescence as the second control for normalization in fluorescence readings. For comparison, the traditional fluorescent methods based on other signaling output modes, such as absolute intensity FCy5, normalized intensity NCy5, and absolute ratio FCy5/FTAMRA, were also recorded. Figure 2 and Supporting Information Figure S6 display the signal changes of HCRW responding to the same targets under different laser powers. When the concentration of target miR-21 was fixed, a large variation was observed in target-triggered Cy5 fluorescence (Figure 2b), and the maximum Cy5 fluorescence also underwent the same change (Figure 2c and Supporting Information Figure S6). During this process, the TAMRA fluorescence signals were also monitored for the further calculation of output signals ( Supporting Information