Dual Quantification of MicroRNAs and Telomerase in Living Cells

Aug 1, 2017 - The development of a unique and universal strategy for the simultaneous quantification of different types of biomolecules (i.e., nucleic...
1 downloads 10 Views 7MB Size
Download PDF
Article pubs.acs.org/JACS

Dual Quantification of MicroRNAs and Telomerase in Living Cells Wei Ma,†,‡,#,§ Pan Fu,†,‡,#,§ Maozhong Sun,†,‡,#,§ Liguang Xu,†,‡,# Hua Kuang,*,†,‡,# and Chuanlai Xu†,‡,# †

State Key Lab of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, People's Republic of China International Joint Research Laboratory for Biointerface and Biodetection and School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, People's Republic of China # Collaborative Innovationcenter of Food Safety and Quality Control in Jiangsu Province, Jiangnan University, Wuxi, Jiangsu 214122, People's Republic of China ‡

S Supporting Information *

ABSTRACT: The development of a unique and universal strategy for the simultaneous quantification of different types of biomolecules (i.e., nucleic acids and proteins) in living cells is extremely challenging. Herein, a two-signal platform, based upon surfaceenhanced Raman scattering and upconversion, for the ultrasensitive and quantitative in situ detection of microRNA (miR)-21 and telomerase in living cells is reported. In the presence of miR-21 and telomerase, the hybridization of miR-21 with a molecular beacon leads to the separation of 3,3′-diethylthiocarbamyl cyanine iodide-modified Au NR dimers, resulting in a decrease in Raman signal. Also, the target telomerase triggers elongation of the telomerase primer strands, followed by substitutional hybridization and release of upconversion nanoparticles, leading to an increase in luminescence. A linear relationship between the Raman intensities and logarithmic concentration of intracellular miR-21 between 0.021 and 22.36 amol/ngRNA is observed, and the limit of detection (LOD) was determined to be 0.011 amol/ngRNA. The luminescence data show a linear response between 0.6 × 10−12 and 31 × 10−12 IU for logarithmic concentration of intracellular telomerase with a LOD of 3.2 × 10−13 IU. These results are in good agreement with Raman and confocal imaging. Importantly, the ultrasensitive detection of miR-21 was possible due to strong plasmonic “hot spots”. This innovative two-signal approach can be utilized for the quantitative and precise detection of many types of signaling molecules in living cells and to understand the chemistry within cellular systems and its application in the diagnosis of disease.

■

INTRODUCTION MicroRNAs (miRNAs/miRs) are a class of short, single-stranded RNAs. miRs are indicators of cellular function such as cellular differentiation, proliferation, apoptosis, and hematopoiesis, etc.1 Thus, alterative expression of miRNAs was associated with a number of diseases that have been utilized as biomarkers in medical diagnostics and drug delivery.2 Currently, quantitative polymerase chain reaction (PCR),3 fluorescent probes,4−6 nanoflares (commercially sold as SmartFlares),7−11 and DNAdriven fluorescent assemblies12,13 together with fluorescencebased imaging14−17 have been designed for the detection of RNAs, including miRs. However, complicated procedures, photobleaching, intracellular stability, spontaneous autoxidation, and background autofluorescence have limited the application of some of these systems. The express level of miRs for typical cancer cells (HeLa, MCF-7, etc.) is in the range of 0.35−1.20 amol/ngRNA which is hardly quantitatively detected by these technologies. We have demonstrated that nanoparticle (NP) probes assembled into pyramids can address many of these limitations.18 Due to the plasmonic coupling of the nanostructures that comprise them, these probes exhibit intense optical activity, allowing for the ultrasensitive detection (attomolar level) of analytes in living cells,19,20 and they exhibit auxiliary upconversion (UC) luminescence (though not independent), © 2017 American Chemical Society

allowing for additional confirmatory detection of miR-21. However, multitarget (dual targets) detection is not accessible by this technology. Telomerase is a ribonucleoprotein reverse transcriptase that extends hexamer telomeric repeats (TTAGGG) using the 3′ end as a primer and deoxyribonucleoside triphosphate (dNTP) as a raw material.21,22 Telomerase is regarded as a biomarker, in which telomerase binds and elongates the telomere ends, finally responsible for the continuous and uncontrolled growth of cancer cells.23 PCR-based telomeric repeat amplification protocols are conventionally used, but relatively complicated.24 Molecular probes25,26 and electrochemiluminescence techniques23 as well as the optical sensing of telomerase activity27 have been reported. However, nonquantitative sensing processes are easily affected by environmental interference resulting in errors. The typical telomerase concentrations in cancer cells (HeLa, MCF-7, etc.) were at 10−12 IU to 10−11 IU level. Recently, we developed a chemical means for assembling particles made of Au NPs into nanostructures with well-defined multigaps, leading to a technique for measuring telomerase concentration.28,29 This approach relies on intensive surface-enhanced Raman scattering Received: April 18, 2017 Published: August 1, 2017 11752

DOI: 10.1021/jacs.7b03617 J. Am. Chem. Soc. 2017, 139, 11752−11759

Article

Journal of the American Chemical Society (SERS) generating, discrete architectures that function in living systems, but limited to one target detection. The intracellular interactions between assembled nanosystems and biomolecules are potentially important for tracing biological pathways,30−36 where initially the detection of two classes of targets is in great demand. Great progress has been made in the fabrication and assembly of NPs using components,37−39 where multiple signals are independently generated in a single discrete structure.40,41 Thus, the single-signal quantification of multiple targets or the simultaneous detection of multiple targets (limited to one type of molecule) has been established by many research groups.18,42−45 Fluorescence,46 circular dichroism (CD),47 and SERS48−50 have been studied and are useful for the detection of many types of disease biomarkers with multiple signals.51 Plasmonically active NPs and fluorescent NPs can be assembled to guide bioimaging and single-target sensing.52,53 Based on the multiple signal sensing principle, we designed assemblies of plasmonic gold nanorods (Au NRs, aspect ratio: 4.1) and upconversion nanoparticles (UCNPs, NaGdF4: Yb3+, Er3+, 19 ± 3 nm) for the generation of independent signals (via SERS and luminescence). The hybridized molecular beacons for miR-21 were localized in the “nanogaps” of Au NR dimers, while the TP strands were localized on the outer surfaces of Au NRs and hybridized with mismatched DNA-modified UCNPs, resulting in luminescence quenching via energy transfer. The hybridization of the molecular beacon to miR-21 causes the Au NRs to separate; the telomerase uses dNTP (A, G, T, C) to trigger the elongation of the TP strands and the substitutional hybridization to release the UCNPs, resulting in luminescence. Thanks to two independent DNA-correlated structures disassembling in the presence of miR-21 and telomerase, the accurate detection of the fluctuation in these two types of targets at low concentrations was achieved.

Scheme 1. Schematic Illustration of Au NR Dimer-UCNP Core−Satellite Nanostructures Used for the Simultaneous Analysis of Intracellular miR-21 and Telomerase: (A) Fabrication Routes for Au NR Dimer-UCNP Core−Satellite Nanostructures and (B) Au NR Dimer-UCNP Core−Satellite Nanostructures Used for Dual Target Detectiona

■

RESULTS AND DISCUSSION Principle of Dual-Signal Generating for Intracellular miRNA and Telomerase. Au NR dimer-UCNP core−satellite (CS) nanostructures were constructed using a DNA frame, and these structures displayed dual SERS and luminescence signals (Scheme 1). Two types of DNA backbones were designed, a 5′thiolated DNA (DNA1, DNA2)-hybridized molecular beacon (Table S1) was used for the Au NR dimer assemblies, and hybridization between 5′-thiolated TP strands, linker DNA, and 3′-thiolated mismatch DNA (Table S1) formed the satellite UCNP assemblies. For the Au NR dimer-UCNP CS nanostructures,54 the Au NR dimer core exhibited intense SERS activity with a characteristic Raman peak (493.64 cm−1), while the UCNPs exhibited luminescence (541 nm) that was extensively quenched by the Au core via the luminescence resonance energy-transfer process. The dual detection of miR-21 and telomerase was achieved based on the independent SERS and luminescence signal generating mechanism. We delivered these probes to target cells and achieved ultrasensitive and quantitative detection of two classes of disease biomarkers (i.e., miRNA and enzymes) in living cells. As depicted in Scheme 1, 3,3′-diethylthiatricarbocyanine iodide (DTTC) was employed as the SERS tag and immobilized on Au NRs via the formation of Au−SH bonds. Then, the Au NR dimers were constructed using 5′-thiolated DNA1 (containing a miR-21 complementary sequence) and 5′-thiolated DNA2 (which had sequences complementary to DNA1 at both ends) in hexadecyltrimethylammonium bromide (CTAB)−Tris buffer

a

SERS used for miR-21 detection, and luminescence used for telomerase detection.

(Table S1). The selective modification of single-strand thiol DNA on side site of Au NRs was crucial for side by side assembly, which has been investigated by our previous study.55 The hybridization of 5′-thiolated DNA1 and 5′-thiolted DNA2 resulted in Au NRs dimer assemblies with molecular beacons (miR-21 is complementary to DNA1) formed in the “nanogaps” between the Au NRs in high yield. Telomerase primer sequences were modified on the outer sides of the Au NR dimers, and UCNPs were modified with the telomerase mismatch sequence (Table S1). Then, a linker DNA sequence was used to assemble the Au dimer-UCNP CS nanostructures. Steric hindrance effect resulted in the telomerase primer hardly modified in the gap of the Au NR dimer, which can 11753

DOI: 10.1021/jacs.7b03617 J. Am. Chem. Soc. 2017, 139, 11752−11759

Article

Journal of the American Chemical Society

Figure 1. Characterization of Au NR dimer-UCNP core−satellite assemblies. (A) Representative TEM image of Au NR dimer-UCNP core−satellite assemblies in CTAB-Tris buffer. (B) EDX elemental mapping of a single Au NR dimer-UCNP core−satellite assembly .(C) UV−vis spectra, (D) SERS spectra, and (E) luminescence spectra of DTTC, UCNP (represents its mixture with DTTC), Au NRs, Au NR dimer, Au NR dimer-UCNP core− satellite assemblies, and mixture of UCNP and Au NR dimer.

Figure 2. Au NR dimer-UCNP core−satellite assemblies for miR-21 and telomerase detection in vitro. (A) SERS spectra of Au NR dimer-UCNP core− satellite responded to different concentrations of miR-21 (0, 0.5, 1, 5, 10, 50, and 100 pM) in vitro. (B) Luminescence spectra of Au NR dimer-UCNP core−satellite responded to different concentrations of telomerase (0, 0.05, 0.1, 0.5, 1, 5, and 10 IU/L) and 0.1 mM dNTPs in vitro. (C) Raman intensity (493.64 cm−1) discrimination of the nanoprobe to mismatched miR-21 (200 PM), DNase 1 (10 IU/L), SSB (1 mM), GSH (1 mM), HSA (1 mM), and 10% fetal bovine serum. (D) Luminescence intensity (541 nm) of Au NR dimer-UCNP core−satellite structures responded to TE only, dNTP only, DNase 1 (10 IU/L), SSB (1 mM), GSH (1 mM), and HSA (1 mM).

be identified in the Au NR dimer-UCNP CS structures (Figure 1A). Following treatment with thiolated poly(ethylene) glycol (PEG, MW: 5000), Au NRs and the maleimide group of the

UCNPs reacted with the thiol group of PEG to form a stable protective layer (Scheme 1), making them more resistant to aggregation in biological environments. Similarly, in order to 11754

DOI: 10.1021/jacs.7b03617 J. Am. Chem. Soc. 2017, 139, 11752−11759

Article

Journal of the American Chemical Society

Figure 3. (A) Schematics for probes 1 and 2: Probe 1 specifically reacted with the target miR-21 and could extend the telomerase primers, while probe 2 was fabricated with complementary DNA sequence that can neither react with miR-21 nor extend the telomerase primers. Intracellular SERS spectra incubated with probes (B) 1 and (C) 2 for different times. (D) Statistics of SERS signals (493.64 cm−1). Confocal images of probes (E) 1 and (F) 2 incubated with HeLa cells for different times. Scale bar = 20 μm.

facilitate high-efficiency cell membrane penetration, a cellpenetrating peptide (TAT) was coated on Au NR and UCNP that assembled in Au NR dimer-UCNP CS nanostructures.56,57 The Au NR dimer-UCNP CS nanostructures were incubated with HeLa cells, which are quintessential carcinoma cells with a

known expressed level of miR-21. The Au NR dimer linkage DNA1 reacted with miR-21 to disassemble the Au NR dimers, while the telomerase primer sequence simultaneously released UCNPs in the presence of intracellular telomerase and dNTP through a telomerase-triggered TP strand elongation process. 11755

DOI: 10.1021/jacs.7b03617 J. Am. Chem. Soc. 2017, 139, 11752−11759

Article

Journal of the American Chemical Society

relationships between the Raman intensities and logarithmic concentration of miR-21 as well as the luminescence intensities and logarithmic concentration of telomerase activities (Figure S5A,B). Stability, Selectivity, and Cytotoxicity of Au NR DimerUCNP CS Nanostructures. To demonstrate the stability and selectivity of these nanostructures, the Raman response of the Au dimer-UCNP CS nanostructures to mismatched miR-21 (200 pM) was studied, and the changes in the Raman signal were found to be negligible. In contrast, the use of miR-21 (5 pM) led to a significant reduction in Raman intensity (Figure S6A and Figure 2C). Furthermore, common coexisting biomolecules, including DNase 1 (10 IU/L), single-stranded DNA-binding protein (SSB, 1 mM), glutathione (GSH, 1 mM), and human serum albumin (HSA, 1 mM), and 10% fetal bovine serum were tested at pH 7.4 and were found to have little effect on the Raman intensities of the assemblies (Figure S6A). Moreover, DNase 1 (10 IU/L), TP (10 μM), dNTP (10 mM each), SSB (1 mM), GSH (1 mM), and HSA (1 mM) were tested for luminescence, and they did not interfere with telomerase-induced luminescence recovery (Figure S6B and Figure 2D). It was noted that the luminescence at 650 nm displayed small signal shifting as the nonspecific interaction induced effects on the luminescence resonance energy-transfer process. We used a mixture of DNase I (10 IU/L) and SSB (1 mM) as a model to determine whether the assemblies were resistant to enzymatic cleavage in an acidic environment (pH 6.0). Due to the phosphorothioate modification (Table S1), the Au dimer-UCNP CS nanostructures showed negligible luminescence recovery and strong Raman intensity after incubation for 2 h (Figure 2C,D), indicating that they were stable under biological conditions. The biocompatibility of Au dimer-UCNPs CS nanostructures was also investigated. We found that cell viability decreased with increasing concentrations of assemblies; when 5 nM assemblies were used, a viability of 93.9% was obtained (Figure S7). The time-dependent cell viability test indicated negligible cytotoxicity for up to 12 h (Figure S8). The above results suggest that these structures could be utilized for the sensing of miR-21 and telomerase in living cells. Furthermore, the disassembly of Au NR dimer-UCNP CS nanostructures was independently triggered by miR-21 and telomerase. The miR-21 triggered the disassembly of Au NR dimer, while telomerase induced disassembly of UCNP from Au NR dimer (Figure S9). The independent disassembly strategy provided avenues for dual targets detections. Cellular Uptake Study of Au NR Dimer-UCNP CS Nanostructures. The Au dimer-UCNP CS nanostructures were used as probes to examine cellular uptake. Using these Au dimer-UCNP CS nanostructures (Figure 3A), two probes were prepared: Probe 1 specifically reacted with the target miR-21 (Au NR dimer: DNA 1 and DNA2; Au NR dimer assembled with UCNP: TP, mismatch DNA, and linker DNA, see Table S1), and probe 2 was fabricated with a completely complementary sequence (Au NR dimer: DNA 2 and DNA 3, not complementary to miR-21; Au NR dimer assembled with UCNP: DNA4 and DNA5, see Table S1) and unable to react with miR-21 or extend the telomerase primers. To test the uptake efficiency of the above assemblies in living cells, the cells were incubated with 5 nM of probes 1 or 2 for different time periods (0−48 h) (Figure 3B−F). Then the cultured cells were centrifuged (1000 rpm, 3 min), followed by measuring the intracellular Raman, luminescence intensity, and confocal microscopy signals. The time-course profiles of the intracellular Raman signal from probe 1 (Raman 1) was lower

These two independent processes generated detectable SERS and luminescence signals for the quantitative detection of intracellular miR-21 and telomerase (Scheme 1). Structural and Optical Properties of Au NR DimerUCNP CS Nanostructures. As observed by transmission electron microscopy (TEM), the Au NR dimer-UCNP CS assembly was uniform and well-dispersed with a hydrodynamic diameter (Dh) of 101 ± 12 nm (Figure 1A and Figures S1−S3). The Dh of the Au dimer-UCNP CS nanostructures was much larger than that of the individual components (Au NRs or UCNPs alone or Au NRs dimers alone) (Figure S3). The Au dimer-UCNP CS nanostructures were composed of elemental Au (Au NR) in the core region and elemental Gd, F, and Er (UCNPs) in the satellite region as revealed by energy dispersive X-ray imaging (EDX) (Figure 1B). The average number of UCNP assembled on Au NR dimer was calculated to be 12 ± 3. The Au NRs possessed a sharp plasmonic band centered at 833 nm, while the plasmonic band of the Au NR dimers was blueshifted by 7 nm (centered at 826 nm) (Figure 1C). Upon nearinfrared laser irradiation (785 nm), intensive SERS was generated by the DTTC molecules immobilized in the gap region of the Au NR dimers with a locally enhanced electromagnetic field.58 We chose the strong Raman peak at 493.64 cm−1 as the characteristic peak for the quantification of miR-21; this peak originates from the strong CN stretching vibrations of DTTC. The Au NR dimers, the Au NR dimer and UCNP mixture, and the Au dimer-UCNP CS nanostructures displayed similar high SERS intensities (Figure 1D). The UCNP with DTTC mixture, DTTC-labeled Au NRs, and DTTC alone generated weak SERS signals indicating that the plasmonic coupled “hot spot” mainly contributed to the Raman enhancement. Upon irradiation with a 980 nm continuous wavelength laser at a power density of 1 W/cm2, the luminescence of the assemblies was largely quenched (the intensity decreased from 7840 to 1831, arbitrary unit) with an effective energy transfer from the UCNPs to the Au NRs (Figure 1E); the luminescence was slightly quenched for the UCNPs and the Au NR mixture. In fact, the majority of the UCNP located at the side region of Au NR, and the luminescence quenching was dominated. We speculated that the luminescence quenching effect was mainly due to coupled gap between Au NR and UCNP (14 nm, 42 bp DNA) and macromolecule (DNA) as linkage involved in energytransfer process. These results also demonstrated that the SERS was independent and did not affect the luminescence of UCNPs. The above findings suggested that the successfully assembled Au dimer-UCNP CS nanostructures displayed unique optical properties. Performance Investigation of miRNA and Telomerase Detection in Vitro. To study the reactivity of Au dimer-UCNP CS nanostructures, the miR-21 sequences, telomerase, and dNTP (A, G, T, C) were simultaneously introduced into the system. The Raman signal gradually decreased as the specific DNA hairpin captured the miR-21 sequence and triggered the dissociation of the Au NR dimer cores (Figure 2A). The luminescence gradually increased due to the specific telomeric primer extended repeats of DNA (TTAGGG) under dNTP, which triggered the dissociation of the UCNP satellites from the Au NR dimer cores (Figure 2B). To acquire an accurate limit of detection, the luminescence intensity values as a function of telomerase activity at 528, 541, and 655 nm were statistically analyzed. We found that the values obtained at 541 nm exhibited the best linear relationship against logarithmic concentration of telomerase (R2 = 0.994) (Figure S4). There were linear 11756

DOI: 10.1021/jacs.7b03617 J. Am. Chem. Soc. 2017, 139, 11752−11759

Article

Journal of the American Chemical Society

Figure 4. Au NR dimer-UCNP core−satellite assemblies used for intracellular miR-21 and telomerase simultaneous detection. (A) SERS spectra of HeLa cell transfected with Lipofectamine-RNAiMAX mixed with antisense miR-21 or different amounts of miR-21 at 37 °C with probe 1 for 24 h, then detected with Au NR dimer-UCNP core−satellite structures (excitation: 785 nm). (B) Plot of mean value of Raman intensity (493.64 cm−1) versus intracellular quantity of miR-21. (C) Confocal images of HeLa cells treated with 0, 60, 120, 180, and 260 μg mL−1 EGCG and then incubated with probe 1 for 8 h. (D) Plot of luminescence intensity versus different concentrations of intracellular telomerase. Scale bar = 20 μm.

inhibition regents (epigallocatechin gallate, EGCG) (Tables S2 and S3). After treatment, miR-21 was determined by PCR (Figures S12 and S13, Table S1), and telomerase was quantified using the enzyme-linked immunosorbent assay (ELISA) (Figure S14). It was also demonstrated that the miR-21 transfection and telomerase inhibition reagents used showed almost no cytotoxicity (Figure S15, Table S3). The Au dimer-UCNP CS nanostructures (probe 1) were then used for the quantification of miR-21 concentration and telomerase activity (Figure 4). Following incubation of the probe with HeLa cells for 8 h, the SERS intensity decreased as the amount of intracellular miR-21 increased, and the luminescence intensity increased as the activity of telomerase increased (Figure 4A−D). The mean value of both signals was linearly dependent on the logarithmic concentration of the target and had a coefficient of at least 0.99 (Figure 4B,D). The linear range of miR-21 was calculated to be 0.021−22.36 amol/ngRNA with a limit of detection (LOD) of 0.011 amol/ngRNA (67 copies/cell) (Figure 4B), which is the most sensitive intracellular analysis of miR-21 reported so far (Table S2). The closely packed “hot spot” of the plasmonic Au NR dimers resulted in ultrasensitive detection. The luminescence intensity showed a linear range between 0.6 × 10−12 and 31 × 10−12 IU for logarithmic concentration of telomerase and a LOD of 3.2 × 10−13 IU (Figure 4D). Alternatively, we found that the intracellular luminescence spectra in cell suspensions were well correlated with telomerase concentrations (Figure S16). The visible luminescence signals provided reliable detection of intracellular telomerase activity compared with those obtained in previous reports.1,59−61

than that from probe 2 (Raman 2) due to the reactivity of probe 1 with the target miR-21 (Figure 3B−D). The time-dependent increasing SERS signal of probe 2 was in agreement with the time-dependent cellular uptake effect and displayed a higher Raman intensity than probe 1 (Figure 3D). The control probe 2 did not react with miR-21 and thus generated a higher Raman intensity than probe 1, which reacted with miR-21. Similarly, probe 1 displayed a stepwise luminescence response against time periods of 0−36 h (Figures S9), and probe 2 had negligible luminescent signals since it did not react with telomerase (Figure 3F). The value, ΔRaman (ΔRaman = |Raman 2| − |Raman 1|) indicating the amount of probe 1 interacted with miR-21, continued to increase for 8 h (Figure 3B−D), and the luminescence intensity (probe 1) was strongest at 8 h and decreased after 16 h (Figure 3E and Figure S10). The probe 2 had negligible luminescence at time period of 0−36 h, indicating no reactivity to telomerase (Figure 3F). The results of the Raman and luminescence signals, together with the confocal images, demonstrated that probe 1 specifically recognized the target miR21 and extended the telomerase primers, but probe 2 can neither react with miR-21 nor extend the telomerase primers. Bio-TEM images were obtained for the two probes at 2, 8, and 24 h (Figure S11A,B), and it was shown that probe 1 was mostly dissociated ((91.5 ± 6.3)%) at 8 h, while probe 2 was not (Figure S11). These results indicate that 8 h is an optimal time for the detection of intracellular miR-21 and telomerase activity. Dual Signals Enabled Live Cell Sensor for Simultaneous Quantification of miR-21 and Telomerase. The levels of miR-21 and telomerase were regulated by miR-21 transfection regents (Lipofectamine-RNAiMAX) and telomerase 11757

DOI: 10.1021/jacs.7b03617 J. Am. Chem. Soc. 2017, 139, 11752−11759

Article

Journal of the American Chemical Society

Figure 5. (A) Raman imaging (scale bar = 10 μm) and (B) confocal images (scale bar = 20 μm) of HeLa, MCF-7, primary uterine fibroblast cells with Au NR dimer-UCNP core−satellite assemblies.

optimization of the response and stability of the DNA-driven probes will improve their application in point-of-care settings.

Two cancer cell lines (HeLa and MCF-7) and a control cell line (primary uterine fibroblasts, PCS cells: PCS-460−010) were used to test the practical applicability of our strategy. When the assembled Au NR dimer-UCNP CS hierarchical structure for cells were applied, MCF-7 cells displayed a weaker Raman signal than HeLa cells, and the PCS cells showed a very strong signal (Figure S17A). Based on a standard detection curve, the amounts of miR-21 in HeLa and MCF-7 cells were 0.39 and 1.17 amol/ ngRNA (2400 and 7020 copies/cell), respectively (Figure 4B and Figure S17B). The detection results agreed well with known miR-21 levels in previous reports.52 Raman imaging was also studied, mainly determined by the efficient SERS zone and red field of the probe.62−67 The Raman imaging results showed that the red field of the HeLa cells was more intense than that of the MCF-7 cells, but the red field of the PCS cells was greatest, consistent with the Raman spectra (Figure 5A and Figure S17A). The control groups (no probe or using probes without a SERS tag) did not display any signals upon Raman imaging (Figure S18). Based on confocal imaging, the luminescence signal for the HeLa cells was higher than that of the MCF-7 cells (Figure 5B), and the calculated telomerase activity in the HeLa and MCF-7 cells was 31 × 10−12 IU and 16.5 × 10−12 IU, respectively, which is consistent with previously reported values.26 For normal PCS cells, the calculated telomerase activity was 1.4 × 10−12 IU, too low to sense using confocal imaging (Figure 5B and Figure S19). The strong upconversion luminescence enabled highly sensitive detection in a biological matrix. These findings demonstrate that dual detection is well-suited for accurate sensing of miRNA and telomerase at low concentrations in biological samples.

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b03617. Materials and detailed experimental procedures, supplementary figures (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Chuanlai Xu: 0000-0002-5639-7102 Author Contributions §

These authors contributed equally to this paper.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work is financially supported by the National Natural Science Foundation of China (21631005, 21673104, 21522102, 21503095).

■

REFERENCES

(1) Dong, H.; Lei, J.; Ding, L.; Wen, Y.; Ju, H.; Zhang, X. Chem. Rev. 2013, 113 (8), 6207. (2) Conde, J.; Oliva, N.; Atilano, M.; Song, H. S.; Artzi, N. Nat. Mater. 2016, 15 (3), 353. (3) Ludlow, A. T.; Robin, J. D.; Mohammed, S.; Litterst, C. M.; Shelton, D. N.; Shay, J. W.; Wright, W. E. Nucleic Acids Res. 2014, 42 (13), e104. (4) Degliangeli, F.; Kshirsagar, P.; Brunetti, V.; Pompa, P. P.; Fiammengo, R. J. Am. Chem. Soc. 2014, 136 (6), 2264. (5) Zhang, J.; Fu, Y.; Mei, Y.; Jiang, F.; Lakowicz, J. R. Anal. Chem. 2010, 82 (11), 4464. (6) Li, H.; Mu, Y.; Qian, S.; Lu, J.; Wan, Y.; Fu, G.; Liu, S. Analyst 2015, 140 (2), 567. (7) Prigodich, A. E.; Randeria, P. S.; Briley, W. E.; Kim, N. J.; Daniel, W. L.; Giljohann, D. A.; Mirkin, C. A. Anal. Chem. 2012, 84 (4), 2062. (8) Seferos, D. S.; Giljohann, D. A.; Hill, H. D.; Prigodich, A. E.; Mirkin, C. A. J. Am. Chem. Soc. 2007, 129 (50), 15477. (9) Zheng, D.; Seferos, D. S.; Giljohann, D. A.; Patel, P. C.; Mirkin, C. A. Nano Lett. 2009, 9 (9), 3258.

■

CONCLUSION A universal platform using DNA-programmed Au NR dimerUCNP core−satellite assemblies as SERS- and luminescencebased probes for the simultaneous in situ quantification of microRNA and telomerase was developed. By mobilizing the assembled structures, the developed strategy provided the remarkable ability not only to visualize intracellular targets and but also to quantify miR-21 and telomerase in living cells at a LOD of 0.011 amol/ngRNA and 3.2 × 10−13 IU, respectively. These nanostructures are biocompatible, efficient, and specific for the recognition of dual targets (i.e., miRNA and enzyme) without extra cell extractions. These findings will be beneficial for accurate disease diagnostics by eliminating false positive or negative signals in diverse biological samples. Further 11758

DOI: 10.1021/jacs.7b03617 J. Am. Chem. Soc. 2017, 139, 11752−11759

Article

Journal of the American Chemical Society (10) Halo, T. L. M.; McMahon, K. M.; Angeloni, N. L.; Xu, Y.; Wang, W.; Chinen, A. B.; Malin, D.; Strekalova, E.; Cryns, V. L.; Cheng, C.; Mirkin, C. A.; Thaxton, C. S. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 17104. (11) Chinen, A. B.; Guan, C. M.; Ferrer, J. R.; Barnaby, S. N.; Merkel, T. J.; Mirkin, C. A. Chem. Rev. 2015, 115 (19), 10530. (12) Causa, F.; Aliberti, A.; Cusano, A. M.; Battista, E.; Netti, P. A. J. Am. Chem. Soc. 2015, 137 (5), 1758. (13) Esteban-Fernández de Á vila, B.; Martín, A.; Soto, F.; LopezRamirez, M. A.; Campuzano, S.; Vásquez-Machado, G. M.; Gao, W.; Zhang, L.; Wang, J. ACS Nano 2015, 9 (7), 6756. (14) Chan, H.-M.; Chan, L.-S.; Wong, R. N.-S.; Li, H.-W. Anal. Chem. 2010, 82 (16), 6911. (15) He, X.; Zeng, T.; Li, Z.; Wang, G.; Ma, N. Angew. Chem., Int. Ed. 2016, 55 (9), 3073. (16) Kim, J. K.; Choi, K.-J.; Lee, M.; Jo, M.-h.; Kim, S. Biomaterials 2012, 33 (1), 207. (17) Li, J.; Cheng, F.; Huang, H.; Li, L.; Zhu, J.-J. Chem. Soc. Rev. 2015, 44 (21), 7855. (18) Li, S.; Xu, L.; Ma, W.; Wu, X.; Sun, M.; Kuang, H.; Wang, L.; Kotov, N. A.; Xu, C. J. Am. Chem. Soc. 2016, 138 (1), 306. (19) Song, J.; Duan, B.; Wang, C.; Zhou, J.; Pu, L.; Fang, Z.; Wang, P.; Lim, T. T.; Duan, H. J. Am. Chem. Soc. 2014, 136 (19), 6838. (20) Xie, W.; Walkenfort, B.; Schlucker, S. J. Am. Chem. Soc. 2013, 135 (5), 1657. (21) Schmidt, J. C.; Zaug, A. J.; Cech, T. R. Cell 2016, 166 (5), 1188. (22) Qian, R.; Ding, L.; Ju, H. J. Am. Chem. Soc. 2013, 135 (36), 13282. (23) Zhang, H.-R.; Wu, M.-S.; Xu, J.-J.; Chen, H.-Y. Anal. Chem. 2014, 86, 3834. (24) Kim, N.; Piatyszek, M.; Prowse, K.; Harley, C.; West, M.; Ho, P.; Coviello, G.; Wright, W.; Weinrich, S.; Shay, J. Science 1994, 266 (5193), 2011. (25) Wang, J.; Wu, L.; Ren, J.; Qu, X. Small 2012, 8 (2), 259. (26) Qian, R.; Ding, L.; Yan, L.; Lin, M.; Ju, H. J. Am. Chem. Soc. 2014, 136 (23), 8205. (27) Sharon, E.; Golub, E.; Niazov-Elkan, A.; Balogh, D.; Willner, I. Anal. Chem. 2014, 86 (6), 3153. (28) Sun, M.; Xu, L.; Fu, P.; Wu, X.; Kuang, H.; Liu, L.; Xu, C. Adv. Funct. Mater. 2016, 26 (40), 7352. (29) Xu, L.; Zhao, S.; Ma, W.; Wu, X.; Li, S.; Kuang, H.; Wang, L.; Xu, C. Adv. Funct. Mater. 2016, 26 (10), 1602. (30) You, M.; Zhu, G.; Chen, T.; Donovan, M. J.; Tan, W. J. Am. Chem. Soc. 2015, 137 (2), 667. (31) Ohta, S.; Glancy, D.; Chan, W. C. W. Science 2016, 351 (6275), 841. (32) Zhou, X.; Cao, P.; Zhu, Y.; Lu, W.; Gu, N.; Mao, C. Nat. Mater. 2015, 14, 1058. (33) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382 (6592), 607. (34) Rosi, N. L.; Giljohann, D. A.; Thaxton, C. S.; Lytton-Jean, A. K. R.; Han, M. S.; Mirkin, C. A. Science 2006, 312 (5776), 1027. (35) Calabrese, C. M.; Merkel, T. J.; Briley, W. E.; Randeria, P. S.; Narayan, S. P.; Rouge, J. L.; Walker, D. A.; Scott, A. W.; Mirkin, C. A. Angew. Chem., Int. Ed. 2015, 54 (2), 476. (36) Young, K. L.; Scott, A. W.; Hao, L.; Mirkin, S. E.; Liu, G.; Mirkin, C. A. Nano Lett. 2012, 12 (7), 3867. (37) Parak, W. J.; Nel, A. E.; Weiss, P. S. ACS Nano 2015, 9 (7), 6637. (38) Song, B.; Zhong, Y.; Wu, S.; Chu, B.; Su, Y.; He, Y. J. Am. Chem. Soc. 2016, 138 (14), 4824. (39) Tian, J.; Zeng, X.; Xie, X.; Han, S.; Liew, O.-W.; Chen, Y.-T.; Wang, L.; Liu, X. J. Am. Chem. Soc. 2015, 137 (20), 6550. (40) Mark, A. G.; Gibbs, J. G.; Lee, T.-C.; Fischer, P. Nat. Mater. 2013, 12, 802. (41) Ma, W.; Kuang, H.; Xu, L.; Ding, L.; Xu, C.; Wang, L.; Kotov, N. A. Nat. Commun. 2013, 4, 2689. (42) Wu, N.; Bao, L.; Ding, L.; Ju, H. Angew. Chem., Int. Ed. 2016, 55 (17), 5220.

(43) Falconnet, D.; She, J.; Tornay, R.; Leimgruber, E.; Bernasconi, D.; Lagopoulos, L.; Renaud, P.; Demierre, N.; van den Bogaard, P. Anal. Chem. 2015, 87 (3), 1582. (44) Scott, A. W.; Garimella, V.; Calabrese, C. M.; Mirkin, C. A. Bioconjugate Chem. 2017, 28, 203. (45) Wu, Y.; Han, J.; Xue, P.; Xu, R.; Kang, Y. Nanoscale 2015, 7 (5), 1753. (46) Qiao, R.; Liu, C.; Liu, M.; Hu, H.; Liu, C.; Hou, Y.; Wu, K.; Lin, Y.; Liang, J.; Gao, M. ACS Nano 2015, 9 (2), 2120. (47) Han, B.; Zhu, Z.; Li, Z.; Zhang, W.; Tang, Z. J. Am. Chem. Soc. 2014, 136 (46), 16104. (48) Jones, M. R.; Seeman, N. C.; Mirkin, C. A. Science 2015, 347 (6224), 1260901. (49) Bodelon, G.; Montes-Garcia, V.; Lopez-Puente, V.; Hill, E. H.; Hamon, C.; Sanz-Ortiz, M. N.; Rodal-Cedeira, S.; Costas, C.; Celiksoy, S.; Perez-Juste, I.; Scarabelli, L.; La Porta, A.; Perez-Juste, J.; PastorizaSantos, I.; Liz-Marzan, L. M. Nat. Mater. 2016, 15 (11), 1203. (50) Zhang, N.; Tong, L.; Zhang, J. Chem. Mater. 2016, 28 (18), 6426. (51) Howes, P. D.; Chandrawati, R.; Stevens, M. M. Science 2014, 346 (6205), 1247390. (52) Zhao, X.; Xu, L.; Sun, M.; Ma, W.; Wu, X.; Kuang, H.; Wang, L.; Xu, C. Small 2016, 12 (34), 4662. (53) Sun, M.; Xu, L.; Ma, W.; Wu, X.; Kuang, H.; Wang, L.; Xu, C. Adv. Mater. 2016, 28 (5), 898. (54) Li, J.; Hong, C. Y.; Wu, S. X.; Liang, H.; Wang, L. P.; Huang, G.; Chen, X.; Yang, H. H.; Shangguan, D.; Tan, W. J. Am. Chem. Soc. 2015, 137 (35), 11210. (55) Ma, W.; Kuang, H.; Wang, L.; Xu, L.; Chang, W.-S.; Zhang, H.; Sun, M.; Zhu, Y.; Zhao, Y.; Liu, L.; Xu, C.; Link, S.; Kotov, N. A. Sci. Rep. 2013, 3, 1934. (56) Todorova, N.; Chiappini, C.; Mager, M.; Simona, B.; Patel, I. I.; Stevens, M. M.; Yarovsky, I. Nano Lett. 2014, 14 (9), 5229. (57) Krpetic, Z.; Saleemi, S.; Prior, I. A.; See, V.; Qureshi, R.; Brust, M. ACS Nano 2011, 5, 5195. (58) Kang, H.; Jeong, S.; Park, Y.; Yim, J.; Jun, B. H.; Kyeong, S.; Yang, J. K.; Kim, G.; Hong, S.; Lee, L. P.; Kim, J. H.; Lee, H. Y.; Jeong, D. H.; Lee, Y. S. Adv. Funct. Mater. 2013, 23 (30), 3719. (59) Duan, R.; Zuo, X.; Wang, S.; Quan, X.; Chen, D.; Chen, Z.; Jiang, L.; Fan, C.; Xia, F. J. Am. Chem. Soc. 2013, 135 (12), 4604. (60) Labib, M.; Khan, N.; Ghobadloo, S. M.; Cheng, J.; Pezacki, J. P.; Berezovski, M. V. J. Am. Chem. Soc. 2013, 135 (8), 3027. (61) Wu, H.; Cisneros, B. T.; Cole, C. M.; Devaraj, N. K. J. Am. Chem. Soc. 2014, 136 (52), 17942. (62) Aioub, M.; El-Sayed, M. A. J. Am. Chem. Soc. 2016, 138 (4), 1258. (63) Hong, S.; Chen, T.; Zhu, Y.; Li, A.; Huang, Y.; Chen, X. Angew. Chem., Int. Ed. 2014, 53 (23), 5827. (64) Huefner, A.; Kuan, W. L.; Muller, K. H.; Skepper, J. N.; Barker, R. A.; Mahajan, S. ACS Nano 2016, 10 (1), 307. (65) Lane, L. A.; Qian, X.; Nie, S. Chem. Rev. 2015, 115 (19), 10489. (66) Wang, H.; Zhou, Y.; Jiang, X.; Sun, B.; Zhu, Y.; Wang, H.; Su, Y.; He, Y. Angew. Chem., Int. Ed. 2015, 54 (17), 5132. (67) Zhang, Z.; Liu, Q.; Gao, D.; Luo, D.; Niu, Y.; Yang, J.; Li, Y. Small 2015, 11 (25), 3000.

11759

DOI: 10.1021/jacs.7b03617 J. Am. Chem. Soc. 2017, 139, 11752−11759