Multiplexed Detection and Imaging of Intracellular mRNAs Using a

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Multiplexed Detection and Imaging of Intracellular mRNAs Using a Four-Color Nanoprobe Wei Pan, Tingting Zhang, Huijun Yang, Wei Diao, Na Li,* and Bo Tang* College of Chemistry, Chemical Engineering and Materials Science, Engineering Research Center of Pesticide and Medicine Intermediate Clean Production, Ministry of Education, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Normal University. Jinan 250014, People’s Republic of China S Supporting Information *

ABSTRACT: Simultaneous detection and imaging of multiple intracellular biomarkers hold great promise for early cancer detection. Here, we introduce a four-color nanoprobe that can simultaneously detect and image four types of mRNAs in living cells. The nanoprobe composed of gold nanoparticles functionalized with a dense shell of molecular beacons, which can identify multiple intracellular mRNA transcripts. It shows rapid response, high specificity, nuclease stability, and good biocompatibility. Intracellular experiments indicate that the nanoprobe could effectively distinguish cancer cells from their normal cells, even some mRNAs are overexpressed in normal cells. Moreover, it can identify the changes of the expression levels of mRNA in living cells. The current strategy could provide more-accurate information for early cancer detection and availably avoid false positive results.

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Fluorescent imaging analysis provides new opportunities to detect and image intracellular mRNAs. Based on this, many fluorescent probes have been designed and synthesized for mRNA detection and imaging in living cells.10−12 Nevertheless, much attention has been paid on the detection of one or two mRNAs,13−17 which may give “false positive” results, since mRNA transcripts are heterogeneously expressed in cancer cells and some mRNA targets are also overexpressed in normal cells. Recently, our group reported a multicolor nanoprobe based on nanoflares that can simultaneously detect and image three types of mRNAs in living cells.18 But the fluorescence signals of nanoflares were produced through a competitive hybridization of the mRNA transcripts and the recognition sequences hybridized with reporter sequences, which is relatively difficult and a slow process in living cells. Therefore, there is an urgent need to develop rapid and effective platforms for simultaneous detection of multiple intracellular targets. To overcome this limitation and further improve the accuracy of early cancer detection, we develop a four-color

ancer is a ubiquitous and devastating disease that affects the lives of millions of people worldwide.1 The chances of survival for cancer patients can be enormously improved if the disease is diagnosed early. A successful treatment for the disease heavily depends on the biomarkers for early diagnosis of the presence and progression of cancer. Identifying the alterations of the biomarkers at the genetic level in living cells has great value in understanding the biological processes and mechanisms of the disease and is becoming increasingly imperative for cancer detection, prevention, and therapy.2,3 As a specific biomarker, mRNA is currently being developed for use in the diagnosis and treatment of cancer, because the changes of expression levels are associated with the tumor burden and malignant progression.4−6 In recent years, several methods have been utilized for mRNA detection, such as real-time polymerase chain reaction (RT-PCR)7 and microarray analysis.8 However, these techniques are mainly employed for determination of the mRNA expression in bulk samples and are unable to identify the cell-to-cell mutations. Significantly, the biological processes of cancer are not only associated with bulk mRNA expression but also are highly dependent on cell-to-cell variations in mRNA.9 Therefore, it is of great importance to develop effective approaches for mRNA detection in living cells. © 2013 American Chemical Society

Received: August 25, 2013 Accepted: October 2, 2013 Published: October 2, 2013 10581

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Scheme 1. Schematic Illustration of the Four-Color Nanoprobe for Detection of Multiple Intracellular mRNAs

HL-7702 were obtained from the Committee on Type Culture Collection of the Chinese Academy of Sciences. Instruments. Transmission electron microscopy (TEM) was carried out on a JEM-100CX II electron microscope. Absorption spectra were measured on a Pharmaspec UV-1700 ultraviolet−visible (UV-Vis) spectrophotometer (Shimadzu, Japan). Fluorescence spectra were obtained with FLS-920 Edinburgh Fluorescence Spectrometer with a xenon lamp and 1.0 cm quartz cells at the slits of 3.0/3.0 nm. All pH measurements were performed with a pH-3c digital pH-meter (Shanghai LeiCi Device Works, Shanghai, China) with a combined glass-calomel electrode. Absorbance was measured in a microplate reader (RT 6000, Rayto, USA) in the MTT assay. Confocal fluorescence imaging were performed with a TCS SP5 confocal laser scanning microscopy (Leica Co., Ltd. Germany) with an objective lens (20×). RT-PCR was carried out with an ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City, CA). Synthesis of Oligonucleotides. All DNA oligonucleotides used to prepare and test mRNA targets were synthesized and HPLC purified by TAKARA Biotechnology (Dalian, China) and Sangon Biotechnology Co., Ltd. (Shanghai, China). The sequences of the involved oligonucleotides are listed in Table S1 in the Supporting Information. The thiolated oligonucleotides were reduced with tris(2-carboxyethyl) phosphine hydrochloride (TCEP·HCl) before they were assembled on the surface of AuNPs. Preparation of Gold Nanoparticles. AuNPs (13 nm in size) were prepared using the sodium citrate reduction method that has been previously reported.23 All glassware was cleaned in aqua regia (HCl/HNO3, 3:1), rinsed with H2O, and ovendried before the experiments. Then 100 mL HAuCl4 (0.01%) was heated to boiling with vigorous stirring, after that 2.0 mL trisodium citrate (1%) was added under stirring. The color of the solution turned from pale yellow to colorless and finally to burgundy. Boiling was continued for an additional 10 min. The colloid was stirred until the solution reached room temperature. Then, it was filtered through a 0.45 μm Millipore membrane filter. Transmission electron microscopy (TEM) images (Figure S1a in the Supporting Information) indicated the particle sizes are 13 ± 2 nm (100 particles sampled). The prepared AuNPs were stored at 4 °C. MB Structure. The potential secondary structure of MB was predicted using UNAfold (www.idtdna.com). It indicated

nanoprobe based on gold nanoparticles (AuNPs) and molecular beacons (MBs). To the best of our knowledge, this is the first example that a nanoprobe was successfully applied for detection and imaging of four types of biomarkers in living cells. As a promising candidate, molecular beacon (MB) was designed to form stem-loop structured oligonucleotides with reporter fluorophore and quencher.19−22 In the presence of targets, the loop sequences hybridize with the target sequences and the hairpin structure was forced to open. It can rapidly respond with the targets and effectively produce the fluorescence signals with low background. The AuNPs then were functionalized by a gold−thiol bond with a dense monolayer of four types of MBs, which specifically target four mRNAs involved in many cancer cells. In the absence of the targets, the fluorophores are in close proximity to the AuNPs, leading to the quenching of the fluorescence of dyes by fluorescence resonance energy transfer (FRET). When the nanoprobe encounters with the complementary targets, a hybrid of probe−target was formed and the hairpin was opened, separating the fluorophores and AuNPs, recovering the fluorescence and revealing the presence of the targets. (See Scheme 1.)

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EXPERIMENTAL SECTION Materials. Bovine insulin, 3-(4,5-dimethyl-thiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Company; deoxyribonuclease I (DNase I) was purchased from Solarbio Science and Technology Co., Ltd. (Beijing, PRC); hydrogen tetrachloroaurate(III) (HAuCl4· 4H 2 O, 99.99%), trisodium citrate (C 6 H 5 Na 3 O 7·2H 2 O), MgCl2, and KCl were purchased from China National Pharmaceutical Group Corporation (Shanghai, PRC). Cell culture products, unless mentioned otherwise, were purchased from GIBCO. All the chemicals were of analytical grade and used without further purification. Sartorius ultrapure water (18.2 MΩ cm) was used throughout the experiments. DNA oligonucleotides were synthesized and purified by TAKARA Biotechnology (Dalian, PRC) and Sangon Biotechnology Co., Ltd. (Shanghai, PRC). The human breast cancer cell line MCF-7 was purchased from KeyGEN Biotechnology Company (Nanjing, PRC), the normal immortalized human mammary epithelial cell line MCF-10A was purchased from Shanghai Bioleaf Biotechnology Company (Shanghai, PRC); human hepatocellular liver carcinoma cell line HepG2 and human hepatocyte cell line 10582

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that the “stem and loop” conformation had formed for all four MBs. Preparation of the Nanoprobe. Equimolar MBs (labeled Alexa Fluor 405, Alexa Fluor 488, Cy3, and Cy5) were mixed and then added to a solution of AuNPs (1 nM) with a final concentration of 30 nM each and shaken overnight. After 12 h, SDS solution (10%) was added to the mixture and the final concentration of SDS was 0.1%. Phosphate buffer (0.1 M; pH 7.4) was added to the mixture to achieve 0.01 M phosphate concentration and the NaCl concentration of the mixture was slowly increased to 0.1 M over an eight-hour period. The nanoprobe solution then was centrifuged (13 500 g, 30 min) and resuspended in phosphate buffered saline (PBS) three times. The nanoprobe then was sterilized using a 0.22 μm acetate syringe filter and resuspended in PBS with a concentration of 3 nM as stock solution stored at 4 °C. The nanoprobe was diluted to a certain concentration for use in all subsequent experiments. The concentration of AuNPs was determined by measuring their extinction at 524 nm (ε = 2.7 × 108 L mol−1 cm−1). Quantitation of Each MB Loaded on the Nanoprobe. The four MBs loaded on AuNPs were quantified according to the published protocol.24 The mercaptoethanol (ME) was added (final concentration = 20 mM) to the probe solution (1 nM). After being incubated overnight with shaking at room temperature, the MBs were released. The released MBs then were separated via centrifugation and the fluorescence was measured with a fluorescence spectrometer. The fluorescence of Alexa Fluor 405-labeled MB was excited at 405 nm and measured at 420 nm; the fluorescence of Alexa Fluor 488labeled MB was excited at 488 nm and measured at 515 nm; the fluorescence of Cy3-labeled MB was excited at 550 nm and measured at 560 nm; and the fluorescence of Cy5-labeled MB was excited at 648 nm and measured at 688 nm. The fluorescence was converted to molar concentrations of MB by interpolation from a standard linear calibration curve that was prepared with known concentrations of MB with identical buffer pH, ionic strength, and ME concentrations (see Figure S3 in the Supporting Information). By dividing molar concentrations of each MB by the original nanoprobe concentration, the amount of MBs per nanoprobe was calculated. Specificity Experiment. The complementary DNA targets for every MB and other targets were spiked in 1 mL hybridization buffer containing 1 nM nanoprobe, while the DNA target concentrations were 200 nM. All experiments were repeated at least three times. Kinetics. The nanoprobe (1 nM) was hybridized with four perfectly matched targets (200 nM), then the fluorescence intensity was determined with increasing time (0, 5, 10, 15, 20, 30, 40, 50, 60 min). The fluorescence of Alexa Fluor 405 was excited at 405 nm and measured at 420 nm; the fluorescence of Alexa Fluor 488 was excited at 488 nm and measured at 515 nm; the fluorescence of Cy3 was excited at 550 nm and measured at 560 nm and the fluorescence of Cy5 was excited at 648 nm and measured at 688 nm. Hybridization Experiment. For multiplexed analyte detection, the nanoprobe (1 nM) was incubated with the four complementary targets, respectively, with increasing concentrations of the DNA targets (0, 5, 10, 15, 20, 30, 40, 50, 90, 100, 120, 150, 200 nM). After incubation for 1 h at 37 °C, the fluorescence was monitored at appropriate excitation wavelengths. All experiments were repeated at least three times.

Nuclease Assay. Two groups of nanoprobe (1 nM in buffer) were placed in a 96-well fluorescence microplate at 37 °C. After allowing the samples to equilibrate (10 min), 1.3 μL of DNase I in assay buffer (2 U/L) was added to one group. The fluorescence of these samples was monitored for 60 min. After that 200 nM DNA targets were added into the two samples with incubation for 1 h at 37 °C in parallel, the fluorescence was measured at appropriate excitation wavelengths after the solution was cooled to room temperature. Cell Culture. All the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and were supplemented with 10% fetal bovine serum and 100 U/mL 1% antibiotics penicillin/streptomycin and maintained at 37 °C in a 100% humidified atmosphere containing 5% CO2 at 37 °C. MTT Assay. MTT assay was performed to study the cytotoxicity of the nanoprobe. MCF-7 cells (1 × 106 cells/well) were dispersed within replicate 96-well microtiter plates to a total volume of 200 μL well−1. Plates were maintained at 37 °C in a 5% CO2/95% air incubator for 24 h. After the original medium has been removed, the MCF-7 cells were incubated with naked AuNPs (1 nM), nanoprobe (1 nM) for 3, 6, 12, and 24 h. The cells incubated with the culture medium only served as controls. The cells then were washed with PBS for three times and 100 μL MTT solutions (0.5 mg mL−1 in PBS) were added to each well. After 4 h, the remaining MTT solution was removed, and 150 μL of DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured at 490 nm with a RT 6000 microplate reader. Confocal Fluorescence Imaging. In a comparative experiment of cancer cells and normal cells, all cells were plated on chamber slides for 24 h. The nanoprobe (1 nM) then was respectively delivered into MCF-10A, MCF-7, HepG2, and HL-7702 cells in a DMEM culture medium at 37 °C in 5% CO2 for 3 h. The cells were examined by confocal laser scanning microscopy (CLSM) with different laser transmitters. In the experiments for the expression levels of tumor mRNA, one group of MCF-7 cells was treated with β-estradiol (10−8 mol/ L) and the other group of MCF-7 cells was treated with tamoxifen (10−6 mol/L) for 48 h. One group of MCF-7 cells without treatment was served as control. Other steps performed as described above using the nanoprobe (1 nM). The cells then were monitored by CLSM with 405-nm excitation. RT-PCR. Total RNA from sorted cells was extracted with the RNeasy Mini Kit (Qiagen, Valencia, CA). cDNA synthesis was performed using an iScript kit (Bio-Rad). RT-PCR was carried out with SYBR Green I (Qiagen) on an ABI PRISM 7000 sequence detection system. Relative level of mRNA was calculated from the quantity of mRNA PCR products and the quantity of GAPDH PCR products. The primers used in this experiment were as follows: TK1 forward, 5′TATGCCAAAGACACTCGCTAC-3′; TK1 reverse, 5′GCAGAACTCCACGAT-GTCAG-3′; survivin forward, 5′TCCACTGCCCCACT-GAGAAC-3′; survivin reverse, 5′TGGCTCCCAGCCTTCCA-3′; c-myc forward, 5′TCGGGTAGTGGAAAACCAGCAGCCT-3′; c-myc reverse, 5′-CCTCCTCGTCGCAGTAGAAATA-3′; GalNAc-T forward, 5′-CCAAGACCTTCCTCCGTTAT-3′; GalNAc-T reverse, 5′-AACCGTTGGGTAGAAGCG-3′; GAPDH forward, 5′-GGGAAACTGTGGCGTGAT-3′; and GAPDH reverse, 5′GAGTGGGTGTCGCTGTTGA-3′. 10583

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Figure 1. Kinetics of the four-color nanoprobe. The nanoprobe (1 nM, black curve) was hybridized with four perfectly matched targets (red curve); target concentrations are 200 nM. (a) TK1 (Alexa Fluor 405, blue emission at 420 nm); (b) survivin (Alexa Fluor 488, green emission at 515 nm); (c) c-myc (Cy3, yellow emission at 560 nm); and (d) GalNAc-T (Cy5, red emission at 668 nm).

Figure 2. Fluorescence intensity of the nanoprobe (1 nM) in the presence of various concentrations of DNA targets (0−200 nM) measured with different excitation wavelength, respectively: (a) Alexa Fluor 405 labeled MB1, targeting TK1 mRNA; (b) Alexa Fluor 405 labeled MB2, targeting survivin mRNA; (c) Cy3 labeled MB3, targeting c-myc mRNA; and (d) Cy5, labeled MB4 targeting GalNAc-T mRNA.

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RESULTS AND DISCUSSION Preparation and Characterization of the Four-Color Nanoprobe. To simultaneously detect and image multiple mRNAs in living cells , four types of MBslabeled as Alexa Fluor 405, Alexa Fluor 488, Cy3, and Cy5were designed to target TK1 mRNA, survivin mRNA, c-myc mRNA, and GalNAc-T mRNA, respectively. (The details of the sequences and dyes are shown in Table S1 in the Supporting Information.) AuNPs 13 nm in diameter were prepared for the nanoprobe, because such large-sized AuNPs exhibit strong surface plasmon resonance (SPR) absorption and can be employed as efficient quenchers.25−27 Moreover, AuNPs with

this size can be densely modified with many oligonucleotides at a single particle, thus providing the opportunity to construct the multicolor nanoprobe.24,28,29 The TEM images of AuNPs and nanoprobe (AuNPs functionalized with the four types of MBs) are shown in Figure S1 in the Supporting Information. The border of the AuNPs was clear while that of the nanoprobe was ambiguous, which was caused by the functionalization of MBs on the surface of AuNPs. The UV−vis absorption spectra showed that the maximum absorption of the AuNPs was observed at 519 nm and it was red-shifted to 524 nm for the nanoprobe, which further confirmed that the AuNPs were successfully assembled with MBs (see Figure S2 in the 10584

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Figure 3. Nuclease stability of the nanoprobe in the presence or absence of DNase I. Fluorescence curve of the nanoprobe (1 nM) in buffer without DNase I (black trace) or in the presence of DNase I (red trace) as a function of time. Insets: fluorescence spectra after hybridization of the nanoprobe with DNA targets in the presence of DNase I (red curve) and absence of DNase I (black curve): (a) the trace and curve for TK1 target measured with 405-nm excitation, (b) the trace and curve for survivin target measured with 488-nm excitation, (c) the trace and curve for c-myc target measured with 550-nm excitation wavelength, and (d) the trace and curve for GalNAc-T target measured with 648-nm excitation wavelength.

intracellular mRNAs, the nuclease stability of the nanoprobe was investigated under physiological condition with fluorescence spectroscopic analysis. In the following experiments, enzyme deoxyribonuclease I (DNase I), a common endonuclease,31 was used to evaluate the nuclease stability of the nanoprobe. Figure 3 showed that the fluorescence intensities of the nanoprobe treated with DNase I was not obviously changed, compared with the case without DNase I. However, the fluorescence intensities of the two solutions for four MBs were all increased greatly after hybridization with the equivalent DNA targets (Figure 3, insets). The results demonstrated the nanoprobe possessed high resistance to nuclease and further confirmed that the fluorescence recovery was indeed due to the hybridization of the nanoprobe and targets instead of nuclease degradation. MTT Assay. The cytotoxicity of the nanoprobe was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide) assay in human breast cancer cell line (MCF-7) as an example. The absorbance of MTT at 490 nm is dependent on the degree of activation of the cells. Then cell viability was expressed by the ratio of absorbance of the cells incubated with the nanoprobe to that of the cells incubated with culture medium only. As shown in Figure S5 in the Supporting Information, the cell viabilities were all more than 90% when incubated with free AuNPs and the nanoprobe (1 nM) for different incubation time. The results demonstrated that the nanoprobe showed almost no cytotoxicity or side effects in living cells. It confirmed that the nanoprobe was an approving candidate to be applied in intracellular cancer diagnosis. Intracellular Imaging of the Nanoprobe. The nanoprobe was then applied to simultaneously detect multiple mRNAs in normal immortalized human mammary epithelial cell line (MCF-10A) and MCF-7 cells where the TK1, survivin, c-myc, and GalNAc-T mRNA transcripts were all overex-

Supporting Information). Quantification of MB surface loading by fluorescence30 shows that each AuNP carries ∼13 Alexa Fluor 405 labeled MB1, targeting to TK1 mRNA; ∼14 Alexa Fluor 488 labeled MB2, targeting to survivin mRNA; ∼15 Cy3 labeled MB3, targeting to c-myc mRNA; and ∼13 Cy5 labeled MB4, targeting to GalNAc-T mRNA. Details of the characterization are provided in Figure S3 in the Supporting Information. In Vitro Studies of the Nanoprobe. To evaluate the properties of the nanoprobe for the simultaneous detection of four DNA targets, binding studies were carried out with the perfectly matched DNA targets of the MBs. Single-base mismatched DNA targets and other DNA targets were also used under identical conditions. Figure S4 in the Supporting Information showed that every MB was specifically bound to its own DNA target and generated a fluorescence signal that was 5-fold to 7-fold higher. By comparison, the signals did not obviously change in the presence of other targets and were of comparable magnitude to background fluorescence. These results suggested that the nanoprobe was capable of revealing the presence of specific targets but not for other targets as well as single-base mismatched DNA targets. It demonstrated that the nanoprobe retained the high sequence specificity offered by the conformational constraint of stem-loop structures. Kinetic studies showed that the nanoprobe responded rapidly to the perfectly matched DNA target within 5 min (Figure 1). As can be seen in Figure 2, the fluorescence intensity of each MB increased as the DNA target concentrations increased from 0 to 200 nM, thus indicating that the hybridization of nanoprobe and DNA targets led to fluorescence recovery. Moreover, the fluorescence intensity is correlated positively with the concentrations of DNA targets in a concentration-dependent manner. Nuclease Stability of the Probe. Before performing the nanoprobe for simultaneously detecting and imaging multiple 10585

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Figure 4. Intracellular imaging of TK1 mRNA, survivin mRNA, c-myc mRNA, and GalNAc-T mRNA under CLSM. MCF-7, MCF-10A, HepG2, and HL-7702 cells were incubated with the nanoprobe (1 nM) for 3 h at 37 °C. The four mRNAs were recorded by Alexa Fluor 405 with 405-nm excitation, Alexa Fluor 488 with 488-nm excitation, Cy3 with 543-nm excitation, and Cy5 with 633-nm excitation, respectively. Scale bars = 100 μm.

pressed.32−35 When the MCF-7 cells were incubated with the nanoprobe (1nM) for 3 h, a strong blue fluorescence signal for TK1 mRNA, a green fluorescence signal for survivin mRNA, a yellow fluorescence signal for c-myc mRNA, and a red fluorescence signal for GalNAc-T mRNA were observed under confocal laser scanning microscopy (CLSM) (see Figure 4). However, all four fluorescence signals were very low after MCF-10A cells were incubated with the nanoprobe under the same conditions, suggesting that the nanoprobe can be employed to differentiate breast cancer cells from normal breast cells. The bright-field images confirmed that the cells were viable in the imaging experiments. The results of RT-PCR revealed the relative expression levels of the four types of mRNAs in MCF-7 cells were all higher than that in MCF-10A cells. (See Figure S6 in the Supporting Information.) The results were consistent with the fluorescence confocal imaging results and further indicated that the fluorescence signals of the nanoprobe were correlated very well with the levels of mRNA expression. Another couple of cellshuman hepatocellular liver carcinoma cell line (HepG2) and human hepatocyte cell line (HL-7702)was also chosen to evaluate the nanoprobe for simultaneously detecting multiple mRNAs. As shown in Figure 4, after HepG2 cells being incubated with the nanoprobe, strong blue, green, yellow, and red fluorescence signals for the four relative mRNAs were observed under CLSM, which was similar to the case in MCF-7 cells. Interestingly, when HL-7702 cells were incubated with the nanoprobe, the phenomenon was greatly different from the

case observed in normal breast cells. The blue and yellow fluorescence signals were very faint, while the green and red fluorescence signals were very strong, showing that the expression of survivin and GalNAc-T mRNAs in HL-7702 was also very high. The RT-PCR results further demonstrated that the relative levels of c-myc and TK1 mRNAs in HepG2 cells were higher than that in HL-7702 cells, while the levels of survivin and GalNAc-T mRNAs were comparable in HL-7702 and HepG2 cells, suggesting that the GalNAc-T and survivin mRNAs were also overexpressed in HL-7702 cells (see Figure S7 in the Supporting Information). The results revealed that multiplexed detection of intracellular mRNAs could avoid false positive results produced by identifying one or two mRNAs. Furthermore, these results showed that the nanoprobe was capable of distinguishing cancer cells from normal cells. Evaluation of the mRNA Expression Levels. The ability to evaluate relative mRNA levels in living cells is significant for understanding the biological processes and mechanisms of cancer. Because the expression levels of mRNA in cancer cells are various, in different stages of tumor progression. Furthermore, it is pivotal to determine the relative expression levels of mRNA for cancer detection, prevention, and therapy. The ability of the nanoprobe to identify the changes of mRNA expression level in living cells was then studied. The relative levels of TK1 mRNA expression in MCF-7 cells were modulated by down-regulation with tamoxifen36 and upregulation with β-estradiol.37 The MCF-7 cells were separated into three groups in parallel. One group was treated with 10586

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Notes

tamoxifen, to decrease the TK1 mRNA expression, and another group was treated with β-estradiol to increase the TK1 mRNA expression. The untreated one was used as a control. Next, the nanoprobe was incubated with the treated and untreated cells, respectively. As can be seen in Figure S8 in the Supporting Information, the fluorescence intensity showed a decrease in the tamoxifen-treated MCF-7 cells and an increase in the βestradiol-treated MCF-7 cells, compared to the untreated cells. The bright-field images (see Figures S8d, S8e, and S8f in the Supporting Information) suggested that the cells were viable throughout the imaging experiment. RT-PCR results further confirmed that tamoxifen decreased the level of TK1 mRNA and β-estradiol increased the level of TK1 mRNA in MCF-7 cells (see Figure S9 in the Supporting Information). It indicated that the nanoprobe was able to detect the changes of the expression levels in cancer cells.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the 973 Program (No. 2013CB933800), the National Natural Science Foundation of China (Nos. 21227005, 21035003, 21375081, and 21105059), Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20113704130001), and Program for Changjiang Scholars and Innovative Research Team in University.

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CONCLUSION In conclusion, we have demonstrated a four-color nanoprobe, based on gold nanoparticles (AuNPs) and molecular beacons (MBs), that can be used for the simultaneous detection and imaging of four types of mRNAs in living cells. To the best of our knowledge, this is the first time that imaging four intracellular biomarkers could be achieved using a nanoprobe. The four-color nanoprobe makes use of the advantages of AuNPs, such as highly efficient quenching for multiple fluorophores, good nuclease stability in living cells and the ease of intracellular uptake. The nanoprobe could signal the presence of target mRNAs with the open of stem-loop structure of MBs, which lead to an increase in fluorescence. It demonstrated that the nanoprobe retained the high sequence specificity offered by the conformational constraint of stemloop structures. Kinetic studies suggested that the target binding and response of the nanoprobe was rapid. With the increase of the DNA target concentrations, the fluorescence intensity of the nanoprobe increased gradually for each of the DNA targets. It indicated that the nanoprobe could produce the fluorescence signals and reflect the relative abundance of mRNA in a target concentration-dependent manner. The nuclease stability results revealed the nanoprobe possessed high resistance to nuclease degradation and further verified that the fluorescence recovery was attributed to the hybridization of the nanoprobe and targets rather than the nuclease degradation. Intracellular experiments showed that the nanoprobe could successfully distinguish cancer cells from normal cells, even if two mRNAs were overexpressed in normal cells. Moreover, the nanoprobe can detect the changes of the expression levels of mRNA in living cells, which would be advantageous for estimating the stage of tumor progression and making treatment decisions. This novel approach could offer integrated and reliable information for early detection of cancer and prevent false positive results caused by detection of one or two mRNAs. We hope that this strategy can be extended to detect and image multiple biomarkers in living cells.

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REFERENCES

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S Supporting Information *

Supporting table and supporting figures. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*E-mail: [email protected]. 10587

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