Fluorescence and SERS Imaging for the

Mar 30, 2017 - miR-21 and miR-203 were successfully detected in living MCF-7 cells, in agreement with in vitro results from the same batch of cell lys...
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Fluorescence and SERS Imaging for the Simultaneous Absolute Quantification of Multiple miRNAs in Living Cells Sujuan Ye,†,‡ Xiaoxiao Li,‡ Menglei Wang,‡ and Bo Tang*,† †

College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, P.R. China ‡ Key Laboratory of Sensor Analysis of Tumor Marker Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, P.R. China S Supporting Information *

ABSTRACT: The simultaneous imaging and quantification of multiple intracellular microRNAs (miRNAs) are particularly desirable for the early diagnosis of cancers. However, simultaneous direct imaging with absolute quantification of multiple intracellular RNAs remains a great challenge, particularly for miRNAs, which have significantly different expression levels in living cells. We designed dual-signal switchable (DSS) nanoprobes using the fluorescence-Raman signal switch. The intracellular uptake and dynamic behaviors of the probe are monitored by its fluorescence signal. Meanwhile, real-time quantitative detection of multiple miRNAs is made possible by measurements of the surface-enhanced Raman spectroscopy (SERS) ratios. Moreover, the signal 1:n ratio amplification mode only responds to lowabundance miRNA (asymmetric signal amplification mode) for simultaneous visualization and quantitative detection of significantly different levels of miRNAs in living cells. miR-21 and miR-203 were successfully detected in living MCF-7 cells, in agreement with in vitro results from the same batch of cell lysates. The reported dual-spectrum imaging method promises to offer a new strategy for the intracellular imaging and detection of various types of biomolecules.

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expression.13 Recently, the methods for in situ quantitation intracellular miRNA with a functionalized nanoprobe,14,15 or chiroplasmonic nanopyramids16 have been reported, and multiple miRNAs were detected by the carbon nitride nanosheet probe.17 DNA has recently emerged as a versatile material for nanoscale construction, resulting in a wide variety of welldefined and functional objects.18−20 A distinct advantage of DNA as a biological molecule is its ability to be used as a template in cellular processes. Indeed, several groups have reported the use of DNA probes for imaging of living cells.21−24 Tan et al. reported a DNA cascade amplifier probe to perform mRNA imaging inside live cells,21 and Devaraj et al. designed nucleic-acid-templated reactions to detect intracellular DNA and microRNA.22 Au nanoparticles (AuNPs), such as their optical and physical properties, have the excellent biocompatibility and the selfdelivery ability to enter cells without the use of transfection reagents.25 More importantly, AuNPs could adequately protect the DNA structure that was bound on the surface.25−27 Further, AuNP functional DNA probes have been already used for in

ariations in intracellular RNA levels provide valuable information regarding the early detection and medical diagnosis of cancer.1,2 MicroRNAs (miRNAs) are crucial gene regulatory factors in a number of biological processes.3,4 Alterations in the expression levels of tumor-related miRNAs are associated with tumor burden and malignant progression.5 Therefore, the intracellular monitoring and detection of miRNAs are particularly desirable for early cancer diagnosis. However, because of their characteristic small size, vulnerability to degradation, and sequence similarities, it is very difficult to quantitate intracellular miRNA levels in vivo. Currently, fluorescence microscopy-based molecular imaging techniques, because of their high selectivity, high resolution, and noninvasive capability, are the general strategies for visualizing gene expression inside cells.6−10 Fluorescence in situ hybridization (FISH) is a common method for visualizing gene expression inside cells and enables the detection of genes that are expressed at low levels.11 Li et al. also developed a toehold-initiated rolling circle amplification approach that allowed the visualization of individual miRNAs.12 However, the application of cell fixation or exotic enzyme tools has impeded its further utility inside live cells. The photocontrolled aptamer-based molecular beacon (MB) technique has also been used to quantitatively analyze intracellular mRNA levels, but its use is limited to a single type of mRNA with abundant © 2017 American Chemical Society

Received: February 24, 2017 Accepted: March 30, 2017 Published: March 30, 2017 5124

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Preparation of Cell Lysate A. The cells were collected in culture medium and centrifuged at 3000 rpm for 5 min, washed once with PBS buffer and twice with buffer solution, and then centrifuged at 3000 rpm for 5 min. The cell pellets were suspended in 600 μL of PBS buffer. Then, the cells were centrifuged at 12 000 rpm for 5 min at 4 °C. After the supernatant was removed, the cell precipitate was washed and centrifuged three times. The cell precipitate was finally dispersed into 30 μL of 0.01 M PBS buffer and stored at 4 °C. miRNAs Analysis in PBS Buffer and Cell Lysate A. The miRNAs analysis was carried out as follows: miR-21 and miR203 samples with different concentrations were added to the DSS-probe solution (the DSS probes were dispersed into 30 μL of 0.01 M PBS buffer or 30 μL cell lysate A). SERS measurements were conducted after incubation for 2 h. Ten spectra were obtained from different sites of each sample and averaged to represent the SERS results, and the experiments were carried out in triplicate. Error bars show the standard deviation of the three experiments. miRNAs Analysis in Cell Lysate B. The cell lysate B is prepared as follows: the different numbers of cells were disrupted with an ultrasonic cell disruptor and finally dispersed into 30 μL of 0.01 M PBS buffer. The miRNAs analysis in cell lysate B was carried out. A series of the cell lysate B were dispersed into 10 μL of the DSS probe solution. SERS measurements were conducted after incubated for 2 h. The experiment procedure was the same as above. Cellular Uptake of the DSS Probes. The cells were seeded at a density of 1 × 106 cells on a glass slide in a selfmade PDMS reaction tank. The DSS probe solution (15 μL) was added to the tank, and serum DMEM was added to the cells. The cells were incubated for the times indicated. SERS Analysis in Living Cells. The culture slide was fitted to a small incubator on the microscope stage to conduct the imaging and SERS measurements under cell culture conditions. SERS images were acquired on a Renishaw Invia Raman microscope equipped with a 633 nm HeNe laser using a streamline mode with a 50× objective lens. The laser power on the sample was 5 mW, and the accumulation time was 5 s. The diameter of the laser spot was 1 μm. The Raman microscope system has a motorized stage with a precision XYZ stage controller. The WiRE Raman Software Version 2.0 from Renishaw Ltd. was used to acquire and analyze the Raman data. Ten spectra were obtained from different cites of cytoplasm in each cell and averaged to represent the SERS results. Fluorescence Microscopy Analysis. Fluorescence imaging was performed with a Leica TCS SP5 inverted confocal microscope (Leica, Germany). The cellular images were acquired using a 40× objective. The excitation sources for the Rox probe and Cy3 probe were 514 nm, respectively, and a 540−640 nm band-pass filter was used for fluorescent signal detection.

situ intracellular imaging and discrimination between wild-type and mutant p53,26 in situ imaging and detection of intracellular telomerase,28−30 or RNA.31,32 However, given the limitations of the probes, it is difficult to achieve simultaneous imaging with adequate quantification of multiple intracellular miRNAs using these live-cell fluorescenceimaging techniques, particularly for miRNAs with significantly different levels. Because they contribute to the leveling effect of resolution, the signals of high-concentration analytes often overlap the signals of lower expression analytes.33,34 Therefore, this is a challenge that remains for the cell-imaging field. Surface-enhanced Raman spectroscopy (SERS) has significant advantages over fluorescence in terms of its abundant spectral information, resistance to peak overlapping, and low background signals in complex biological systems.35−37 These comprehensive advantages render SERS highly competitive in meeting the needs of multiplex quantification of molecules in living cells. However, to the best of our knowledge, SERS-based imaging and quantitative intracellular detection of a single type of miRNA has not yet been reported. To visualize and absolutely detect multiple miRNAs, three requirements must be satisfied in the molecular imaging field: the real-time monitoring of the dynamic uptake of the detection probes into cells and their intracellular behavior, the absolute quantitation of miRNA levels in complex systems, and the simultaneous detection of miRNAs with different concentration levels. To address these three requirements, we designed a fluorescence−SERS switching nanoprobe, termed the dualsignal switchable (DSS) probe. Fluorescence can provide a rapid and direct visualization for intracellular monitoring,6−10 and SERS can offered high sensitivity with narrow characteristics peaks for multiplex qualitative evaluation.35−37 The DSS probe possesses the complementary advantages of the fluorescence and SERS techniques. First, the dynamic uptake and intracellular behavior of the probe is monitored in real time by its fluorescence signal. Second, the essentially constant Raman peak is used as the internal reference for two types of quantitative ratiometric miRNA measurements. Third, for lowexpression miRNA detection, the target strand displacement reaction is introduced in the probe design to amplify the signal n times, termed 1:n asymmetric signal amplification, compared with high-expression miRNA.

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EXPERIMENTAL SECTION Preparation of the DSS Probe. The probe-functionalized AuNPs (DSS probes) were prepared as described below. To prepare the DSS-1 probe, the H1 and H2 hairpin DNAs were heated in a 90 °C water bath for 5 min and then allowed to cool to room temperature for 1 h before use. Briefly, 50 μL of 1 × 10−6 M (3-thiol modified) H1 DNA and 50 μL of 1 × 10−6 M H−C (3′-thiol and 5′-Cy3 modified DNA) were added to 1 mL of a freshly prepared AuNP solution and shaken gently overnight (approximately 12 h) at 37 °C. To prepare the DSS-2 probe, 50 μL of 1 × 10−6 M Rox-DNA (3′-thiol and 5′Rox modified DNA) was added to 1 mL of freshly prepared AuNPs (15 nm) and shaken gently overnight (approximately 12 h) at 37 °C. Subsequently, the excess reagents were removed by centrifugation at 10 000 rpm for 20 min. After the supernatant was removed, the red precipitate was washed and centrifuged three times. The resulting DSS probes were finally dispersed into 30 μL of 0.01 M phosphate buffer (PBS, pH 7.4).

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RESULTS AND DISCUSSION Principle of Imaging and Quantitation of Intracellular Multiple miRNAs Using the DSS Probe. For instance, in cancer cells, miR-203 expression levels are often low, while the amount of miR-21 is relatively high.38,39 Two types of DSS nanoprobes are designed for the simultaneous imaging and detection of miR-21 and miR-203. As shown in Figure 1, the Au nanoparticles (AuNPs) were functionalized by a gold−thiol bond with a dense monolayer of DNA functional strands, the probes take advantage of the unique properties of AuNPs, such 5125

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Figure 1. (A) Detection of miR-21 and miR-203 in living cells using DSS probes. (B) Mechanism for sensing the asymmetric signal amplification of the DSS probes for miR-21 and miR-203.

Figure 3. (A) SERS spectra for increasing concentrations of miR-21 and miR-203. (B,C) Variances of the I1586/I783 and I1499/I783 with the concentration of miR-21 and miR-203, respectively.

Figure 4. Specificity of the detection of miR-21 and miR-203 compared to miR-210, miR-214, and miR-49 at a concentration of 1.0 × 10−10 M for miR-21, miR-210, miR-214, and miR-49 and 1.0 × 10−12 M for miR-203.

Figure 2. (A−C) Fluorescence (Cy3-DSS probe, 543 nm excitation), bright-field, and TEM images of MCF-7 cells after a 4 h incubation with the DSS probes are shown from left to right. Scale bars = 100 μm. (D) SERS spectra and (E) variance of the Raman intensity with the diameter of the AuNPs.

as their fluorescence-quenching efficiency,40,41 SERS enhancing efficiency,42,43 protection of DNA25−27 and the self-delivery ability to enter cells without the use of transfection reagents.25 One DSS nanoprobe, termed DSS-1, consists of AuNPs, 5126

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Figure 6. (A,B) Comparison of the miRNA average contents in 50, 100, 150 living cells versus the lysate from 50, 100, 150 cells from the same batch, (A) miR-21, (B) miR-203.

on. For the highly expressed miR-21 target molecule, the H−C strand is gradually displaced from the dsDNA via binding with miR-21. The resulting single-stranded H1 then tends to form a hairpin structure through intramolecular hybridization. Such conformational changes result in a dual-signal switch, a decrease in the fluorescence of Cy3, and an increase in the SERS signal due to the close proximity of Cy3 to the AuNPs and subsequent resonance energy transfer. The SERS signal can be used to detect miR-21 in an equivalent reaction ratio (1:1) because of linear hybridization. The target-strand displacement amplification-dual-signal switch (TSDA-DSS) reaction is activated in the presence of the target miR-203 molecule. The miR-203-H2 catalytic duplex is formed, which opens the hairpin structure of H2; the complementary domain of H2 is exposed to DSS-2 and H2 and DSS-2 bind. Because the H2 and DSS-2 duplex is more stable than the hybrids between miR-203 and H2, a disassembly reaction occurs, initiating a branch migration that displaces miR-203 from H2.44,45 As a result, the fluorescence of DSS-2 is decreased, and the SERS signal is increased. The free miR-203 becomes available to trigger successive reaction cycles. In the TSDA, the low expression levels of miR-203 are able to yield multiple signal outputs (1:n), thus achieving sensitive detection of miRNA targets with different expression levels and avoiding signal interference and overlap with highly expressed miRNAs. Investigation of Cellular Uptake of DSS Probe. The process used to assemble the DSS probes was characterized using UV−visible spectroscopy (Figure S1, see Supporting

Figure 5. (A−E): Fluorescence images for (A) miR-21 and (B) miR203, SERS images for (C) miR-21 and (D) miR-203, and (E) bright field images in MCF-7 cells are shown from left to right. Average SERS intensity ratios for (F) one peak and (G) multiple peaks in a single MCF-7 cell incubated with the DSS probes for 1, 2, 4, and 6 h. (H) SERS spectra acquired from different positions within the culture, including the cytoplasm (red), cell nucleus (blue), and the surrounding environment (green). (I) Intensity ratio for increasing incubation time.

dsDNA strands (Cy3-modified DNA attached to AuNPs, H1; complementary strand, H−C), and a hairpin probe (H2); the other, termed DSS-2, is a AuNP-functionalized hairpin probe (Rox is attached to the probe at the positions indicated in Figure 1). The probes feature both “signal-on” and “signal-off” elements. As shown in Figure 1B, in the absence of miR-21 and miR-203, the probe is in a state of fluorescence signal-on and SERS signal-off. The fluorescence signal can be used to show cellular uptake and distribution of the probes, and the SERS signal cannot be detected because of the distance between the AuNPs and the dyes. In the presence of target miRNA molecules, an asymmetric signal amplification reaction is performed to trigger the dual-signal switching; the probe is switched to the state of fluorescence signal-off and SERS signal5127

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also investigated (1586 cm−1 for Cy3). As shown in Figure 2D,E, larger AuNP size was associated with higher SERS intensity. Considering the above two factors, the AuNPs with a diameter of approximately 25 nm could be considered as the optimal SERS substrate and carrier for the DSS-1 probe. However, for the AuNPs used as the carrier for DSS-2, the intrinsic SERS signal of the probe only increased the background signal, and thus a diameter of approximately 15 nm was selected. Performance Evaluation of miRNAs Detection in Vitro. Using optimized conditions (the details of the optimization experiments are provided in the SI, Figures S3, S4), the analytical performance of the DSS probes was investigated. Mixtures of different concentrations of miR-21 and miR-203 standards were simultaneously analyzed. Figure 3 shows the Raman signals of Cy3 and Rox in response to sample mixtures containing different concentrations of miR-21 and miR-203. As shown in the spectrum of the mixed probes in Figure 3A, characteristic peaks of both Cy3 (1193, 1391, 1465, and 1586 cm−1) and Rox (1344, 1499, and 1644 cm−1) are observed and enhanced with increasing concentrations of the target miRNA. Notably, the intensities of the 783 cm−1 band, which is ascribed to ring breathing and asymmetric O−P−O stretching of DNA,48,49 were similar to different target miRNA concentrations, indicating that it could be used as the internal peak. In addition, external stimulation had little impact on the internalizing ability of the band. Moreover, as shown in Figure 3B and C, the ratiometric I1586/I783 and I1499/I783 peak intensities display a linear relationship with the logarithmic concentrations of miR-21 and miR-203, respectively. To verify the validity of the calibration curves, we performed measurements of the miRNA concentrations in cell lysate A. In Figure 3B,C, the curves from the cell lysate were consistent with the standard curves in buffer. Thus, the calibration curves could be adopted for the detection of miR-21 and miR-203 levels in living cells. Specificity of the Method. To assess the specificity of the method for the simultaneous detection of miR-21 and miR-203, control experiments were performed by determining the signals for the same concentrations of miR-21, miR-203 and other candidate miRNAs (i.e., miR-200 and miR-141). The results are shown in Figure 4. Compared with the SERS signal obtained for miR-21 and miR-203, responses as low as those of the background were observed when the concentrations of other molecules were determined, indicating that these miRNAs do not significantly influence the simultaneous detection of miR21 and miR-203 because of the specificity of the asymmetric signal amplification reaction and the fingerprinting feature of SERS. These results indicate that the method provided good selectivity for its targets over the same level of nontarget molecules in physiological media. Dynamic Behavior of the Intracellular Probes. MCF-7 cells, in which miR-21 is overexpressed and miR-203 expression levels are often low, were incubated with the DSS probes to image the dynamic behavior of the intracellular probes and the behavioral response of the target miRNA.38,39 Figure 5A,B shows the MCF-7 cells uptaking the DSS probes; the fluorescence signal was used to monitor its distribution. After 1 h of incubation, the probes were sufficiently taken up by the cell, and the spatial distribution of the fluorescent signals of the DSS probe was detected both near the cell periphery and in the cytoplasm. In addition, the fluorescence intensity inside the cytoplasm did not exhibit a marked difference (Figure 5A,B).

Figure 7. Comparison of the SERS intensity ratios in each living cell versus the average intensity ratio from 100 cells, (A) miR-21, (B) miR203.

Information (SI)). The kinetics and feasibility of the target miRNA-triggered asymmetric signal amplification reaction in vitro were tested with the prepared DSS-1 and DSS-2 probes (Figure S2, see SI). As the carrier of the DSS probe, the size of the AuNPs is related to their cellular uptake and intracellular behavior. In our studies, we selected the DSS-1 probe (the H− C strand is substituted with H−C-1 strand that is not complementary to miR-21) for use in the MCF-7 cell line as a model system in which to monitor the uptake efficiency and dynamics of the DSS probes in living cells. AuNPs could be synthesized measuring 15−50 nm in diameter, as shown in the TEM images in Figure 2C(I-III). In Figure 2A(I−III), the intracellular fluorescence intensity of Cy3 demonstrates that cellular uptake was heavily dependent upon the size of the AuNPs. The maximum uptake by a cell occurred at a nanoparticle size of 15 nm. The intracellular fluorescence intensity of the 25 nm AuNPs was slightly less than those measuring 15 nm. At a particle size of approximately 50 nm, moderate intensity fluorescence was clearly observed at the edge of the cells, and intracellular intensity was weak. The fluorescence intensity was the lowest with the 50 nm particles. Although Chan et al. qualitatively showed that 50 nm AuNPs entered the cells more efficiently than the smaller nanoparticles,46 the DSS probe consists of AuNPs that are densely functionalized with oligonucleotides, thereby differing from the bare AuNPs. Meanwhile, it is well-known that SERS activity critically depends not only on the nature of the metal but also on the sizes of nanoparticles; the Raman enhanced effect of the 15 nm AuNPs is not significant.47 Therefore, the effect of different sizes of H1-modified AuNPs on SERS intensity was 5128

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toxicity experiments are important for intracellular probes. Thus, the cytotoxicity of the DSS probes was assessed. More than 90% of the cells remained viable compared with the control samples after the cells were incubated with the DSS probe for up to 48 h (Figure S7). Thus, the DSS probe showed almost no cytotoxicity or side effects in living cells, confirming that the nanoprobe could be applied to intracellular miRNA imaging and detection. These data showed that the asymmetric amplification reaction was efficient in living cells.

The redistribution of the DSS probes is relatively homogeneous, mainly because of the intracellular machinery capable of transporting AuNPs inside the cells. Meanwhile as shown in Figure 5A−D, a time-dependent decrease in fluorescence and an increase in SERS intensities were observed as a result of the structure switch in the DSS probes triggered by the intracellular miRNAs, indicating that the reaction occurred. The SERS intensity of the characteristic peak increased with time (Figure 5F,G). From 4 to 6 h, the SERS and fluorescence intensities remained relatively stable. When incubated for a longer time (6 h), the MCF-7 cells generated strong SERS signals, but only a slight increase in SERS intensity was observed when compared with that at 4 h. This result indicates that the optimized reaction time is 4 h. Figure 5H shows the SERS spectra from different cellular regions with different signal intensities. The target miRNA responses are mostly focused on the cytoplasm (red), whereas the SERS spectra from the nuclear region (blue) are similar to the background (green). Intracellular Stability of the DSS Probe. To further investigated the intracellular stability of the nanoprobe, the dynamic SERS behavior of the intracellular probes were observed. The results demonstrated the nanoprobe possesses high resistance of the degradation with intracellular nuclease (Figure 5I). Figure 5I revealed that the Raman intensity increased rapidly as the incubation time prolonged and reached a plateau after 6 h, in the following 7, 8, 9, and 10 h. The SERS intensities of the nanoprobes were not obviously changed, and the results demonstrated the nanoprobe possesses high resistance of the degradation with intracellular nuclease. Performance Evaluation of miRNA Levels Simultaneous Detection in Living Cells. The proposed probes could be used for the simultaneous quantification of miR-21 and miR-203, with intracellular concentrations of miRNAs quantified based on the calibration curve of solutions with known concentrations (Figure 3B,C). The peak ratiometric intensities, I1586/I783 and I1499/I783, were used to detect miR-21 and miR-203 in single cells, respectively. In a single MCF-7 cell, 2.1 × 10−10 M and 6.5 × 10−9 M concentrations of miR-203 and miR-21 were detected, respectively. Considering the average volume of MCF-7 cells,50,51 the average amounts of miR-203 and miR-21 were 350 and 8900 copies/cell from 100 cells, which is in good agreement with previously reported values.52,53 To assess the accuracy of this method, we further compared the miRNA contents in living cells with cell lysate B from the same batch. The comparison of average contents of different numbers of living cells versus the lysate from the same batch are shown in Figure 6A,B, and the results obtained from the SERS assay in living cells show an acceptable agreement with those obtained from the cell lysate. Meanwhile, we further compared SERS assay in living cell with quantitative real-time polymerase chain reaction (RTPCR) for miRNAs in the cell lysate. In Figure 6A,B, the results obtained by the SERS assay shows an acceptable agreement with those obtained by RT-PCR. The detailed description and data were added in the Supporting Information (see ESI, Figure S5). The comparison of SERS intensity ratio of each cell versus the average intensity ratio from 100 cells are shown in Figure 7A,B. As shown in Figure 7A,B, the content distributions of the various cells in the same batch are relatively homogeneous. Relative standard deviations (RSD) of 7.8% and 9.3% for miR21 and miR-203, respectively, were obtained from the measurements of 10 cells (Figure S6, see SI). In addition,

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CONCLUSION In summary, we have developed a self-delivering DSS probe for the simultaneous intracellular imaging and quantification of miRNAs with significantly different expression levels. Combined with the complementary advantages of the fluorescence and SERS techniques, the method achieved in situ tracking of the intracellular behavior of the probe with good performance and could quantitatively determine multiple miRNAs using SERS ratiometric detection. Moreover, the accurate detection of multiple miRNAs with significantly different expression levels in living cells was successfully achieved via an asymmetric amplification mode. This novel method could offer a powerful approach for the simultaneous imaging and detection of other biomolecules in living cells.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b00697. Experimental Section: Materials and Reagents Equipment, Synthesis of the Gold Nanoparticles, Cell Culture, qRT-PCR Procedure for miRNA Analysis; Results and Discussion: UV−visible Spectra of the DSS Probes, The Kinetic Study and the Feasibility of the Asymmetric Amplification Reaction, Optimization of the Reaction Temperature, pH and HCR Time, MTT Assay; Figures S1−S7, Table S1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86 531 86180017. ORCID

Bo Tang: 0000-0002-8712-7025 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the 973 Program (Grant 2013CB933800), the Natural Science Foundation of China (Grants 21227005, 21390411, 91313302, 21205065, and 21405087), the Postdoctoral Science Foundation of China (Grants 2012M521371, 2015M572074), the Shandong Provincial Natural Science Foundation (Grant ZR2014BM034), and the College and University Development Program (J15LC15).

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REFERENCES

(1) Hanahan, D.; Weinberg, R. A. Cell 2000, 100, 57−70. (2) Thompson, M. A.; Casolari, J. M.; Badieirostami, M. P.; Brown, O.; Moerner, W. E. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 17864− 17871. 5129

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Article

Analytical Chemistry (3) Almeida, M. I.; Reis, R. M.; Calin, G. A. Mutat. Res., Fundam. Mol. Mech. Mutagen. 2011, 717, 1−8. (4) Dong, H. F.; Zhang, J.; Ju, H. X.; Lu, H. T.; Wang, S. Y.; Jin, S.; Hao, K. H.; Du, H. W.; Zhang, X. J. Anal. Chem. 2012, 84, 4587−4593. (5) Schwarzenbach, H.; Hoon, D. S. B.; Pantel, K. Nat. Rev. Cancer 2011, 11, 426−437. (6) Helmchen, F.; Denk, W. Nat. Methods 2005, 2, 932−940. (7) Wu, C. C.; Cansiz, S.; Zhang, L. Q.; Teng, I. T.; Qiu, L. Q.; Li, J.; Liu, Y.; Zhou, C. S.; Hu, R.; Zhang, T.; Cui, C.; Cui, L.; Tan, W. H. J. Am. Chem. Soc. 2015, 137, 4900−4903. (8) Li, N.; Chang, C. Y.; Pan, W.; Tang, B. Angew. Chem., Int. Ed. 2012, 51, 7426−7430. (9) Pan, W.; Zhang, T.; Yang, H.; Diao, W.; Li, N.; Tang, B. Anal. Chem. 2013, 85, 10581−10588. (10) Li, L.; Feng, J.; Liu, H. Y.; Li, Q. L.; Tong, L. L.; Tang, B. Chem. Sci. 2016, 7, 1940−1945. (11) Namekawa, S. H.; Lee, J. T. Nat. Protoc. 2011, 6, 270−284. (12) Deng, R.; Tang, L.; Tian, Q.; Wang, Y.; Lin, L.; Li, J. Angew. Chem., Int. Ed. 2014, 53, 2389−2393. (13) Qiu, L.; Wu, C.; You, M.; Han, D.; Chen, T.; Zhu, G.; Jiang, J.; Yu, R.; Tan, W. J. Am. Chem. Soc. 2013, 135, 12952−12955. (14) Liao, X. J.; Ju, H. X. Chem. Commun. 2015, 51, 2141−2144. (15) Dong, H. F.; Ju, H. X.; Lei, J. P.; Zhi, F.; Wang, H.; Guo, W. J.; Zhu, Z.; Yan, F. Angew. Chem., Int. Ed. 2012, 51, 4607−4612. (16) Li, S.; Xu, L. G.; Ma, W.; Wu, X. L.; Sun, M. Z.; Kuang, H.; Wang, L. B.; Kotov, N. A.; Xu, C. L. J. Am. Chem. Soc. 2016, 138, 306− 312. (17) Liao, X. J.; Wang, Q. B.; Ju, H. X. Chem. Commun. 2014, 50, 13604−13607. (18) Aldaye, F. A.; Palmer, A. L.; Sleiman, H. F. Science 2008, 321, 1795−1799. (19) Lin, C.; Liu, Y.; Yan, H. Biochemistry 2009, 48, 1663−1674. (20) Zheng, J.; Yang, R. H.; Shi, M. L.; Wu, C. C.; Fang, X.; Li, Y.; Li, J.; Tan, W. H. Chem. Soc. Rev. 2015, 44, 3036−3055. (21) Wu, C. C.; Cansiz, S. N.; Zhang, L. Q.; Teng, I. T.; Qiu, L. P.; Li, J.; Liu, Y.; Zhou, C. S.; Hu, R.; Zhang, T.; Cui, C.; Cui, L.; Tan, W. H. J. Am. Chem. Soc. 2015, 137, 4900−4903. (22) Wu, H. X.; Cisneros, B. T.; Cole, C. M.; Devaraj, N. K. J. Am. Chem. Soc. 2014, 136, 17942−17945. (23) Cheglakov, Z.; Cronin, T. M.; He, C.; Weizmann, Y. J. Am. Chem. Soc. 2015, 137, 6116−6119. (24) Chen, T. T.; Tian, X.; Liu, C. L.; Ge, J.; Chu, X.; Li, Y. F. J. Am. Chem. Soc. 2015, 137, 982−989. (25) Seferos, D. S.; Giljohann, D. A.; Hill, H. D.; Prigodich, A. E.; Mirkin, C. A. J. Am. Chem. Soc. 2007, 129, 15477−15479. (26) Qian, R. C.; Cao, Y.; Long, Y. T. Anal. Chem. 2016, 88, 8640− 8647. (27) Giljohann, D. A.; Seferos, D. S.; Patel, P. C.; Millstone, J. E.; Rosi, N. L.; Mirkin, C. A. Nano Lett. 2007, 7, 3818−3821. (28) Qian, R. C.; Ding, L.; Yan, L. W.; Lin, M. F.; Ju, H. X. J. Am. Chem. Soc. 2014, 136, 8205−8208. (29) Sun, M. Z.; Xu, L. G.; Fu, P.; Wu, X. L.; Kuang, H.; Liu, L. Q.; Xu, C. L. Adv. Funct. Mater. 2016, 26, 7352−7358. (30) Xu, L. G.; Zhao, S.; Ma, W.; Li, S.; Wu, X. L.; Kuang, H.; Wang, L. B.; Xu, C. L. Adv. Funct. Mater. 2016, 26, 1602−1608. (31) Yang, Y. J.; Huang, J.; Yang, X. H.; Quan, K.; Wang, H.; Ying, L.; Xie, N. L.; Ou, M.; Wang, K. M. J. Am. Chem. Soc. 2015, 137, 8340− 8343. (32) Zhao, X. L.; Xu, L. G.; Sun, M. Z.; Ma, W.; Wu, X. L.; Kuang, H.; Wang, L. B.; Xu, C. L. Small 2016, 12, 4662−4668. (33) Mohimen, A.; Dobo, A.; Hoerner, J. K.; Kaltashov, I. A. Anal. Chem. 2003, 75, 4139−4147. (34) Falick, A. M.; Maltby, D. A. Anal. Biochem. 1989, 182, 165−169. (35) Papadopoulou, E.; Bell, S. E. J. Angew. Chem., Int. Ed. 2011, 50, 9058−9061. (36) Zhai, W. L.; Li, D. W.; Qu, L. L.; Fossey, J. S.; Long, Y. T. Nanoscale 2012, 4, 137−142.

(37) Zheng, J.; Jiao, A. L.; Yang, R. H.; Li, H. M.; Li, J. S.; Shi, M. L.; Ma, C.; Jiang, Y.; Deng, L.; Tan, W. H. J. Am. Chem. Soc. 2012, 134, 19957−19960. (38) Koshiol, J.; Wang, E.; Zhao, Y. D.; Marincola, F.; Landi, M. T. Cancer Epidemiol., Biomarkers Prev. 2010, 19, 907−911. (39) Campuzano, S.; Torrente-Rodríguez, R. M.; López-Hernández, E.; Conzuelo, F.; Granados, R.; Sánchez-Puelles, J. M.; Pingarrón, J. M. Angew. Chem., Int. Ed. 2014, 53, 6168−6171. (40) Dubertret, B.; Calame, M.; Libchaber, A. J. Nat. Biotechnol. 2001, 19, 365−370. (41) Shimron, S.; Cecconello, A.; Lu, C. H.; Willner, I. Nano Lett. 2013, 13, 3791−3795. (42) Albrecht, M. G.; Creighton, J. A. J. Am. Chem. Soc. 1977, 99, 5215−5217. (43) Brown, R. J. C.; Milton, M. J. T. J. Raman Spectrosc. 2008, 39, 1313−1326. (44) Yin, P.; Choi, H. M. T.; Calvert, C. R.; Pierce, N. A. Nature 2008, 451, 318−322. (45) Zhang, D. Y.; Turberfield, A. J.; Yurke, B.; Winfree, E. Science 2007, 318, 1121−1125. (46) Chithrani, B. D.; Ghazani, A. A.; Chan, W. C. W. Nano Lett. 2006, 6, 662−668. (47) Fang, P. P.; Li, J. F.; Yang, Z. L.; Li, L. M.; Ren, B.; Tian, Z. Q. J. Raman Spectrosc. 2008, 39, 1679−1687. (48) Barhoumi, A.; Zhang, D. M.; Tam, F.; Halas, N. J. J. Am. Chem. Soc. 2008, 130, 5523−5529. (49) Konorov, S. O.; Schulze, H. G.; Piret, J. M.; Blades, M. W.; Turner, R. F. B. Anal. Chem. 2013, 85, 8996−9002. (50) Leung, E.; Kannan, N.; Krissansen, G. W.; Findlay, M. P.; Baguley, B. C. Cancer Biol. Ther. 2010, 9, 717−724. (51) Geltmeier, A.; Rinner, B.; Bade, D.; Meditz, K.; Witt, R.; Bicker, U.; Bludszuweit-Philipp, C.; Maier, P. PLoS One 2015, 10, e0134999. (52) (a) Degliangeli, F.; Kshirsagar, P.; Brunetti, V.; Pompa, P. P.; Fiammengo, R. J. Am. Chem. Soc. 2014, 136, 2264−2267. (53) Chan, H. M.; Chan, L. S.; Wong, R. N. S.; Li, H. W. Anal. Chem. 2010, 82, 6911−6918.

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DOI: 10.1021/acs.analchem.7b00697 Anal. Chem. 2017, 89, 5124−5130