DNA Octahedron-Based Fluorescence Nanoprobe for Dual Tumor

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Article Cite This: Anal. Chem. 2018, 90, 12059−12066

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DNA Octahedron-Based Fluorescence Nanoprobe for Dual TumorRelated mRNAs Detection and Imaging Lin Zhong,† Shuxian Cai,† Yuqing Huang, Litian Yin, Yuling Yang, Chunhua Lu,* and Huanghao Yang* MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350116, People’s Republic of China Anal. Chem. 2018.90:12059-12066. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 10/31/18. For personal use only.

S Supporting Information *

ABSTRACT: With the development of biotechnology, the detection of cancer biomarkers has been a promising approach for cancer diagnosis and therapy. Herein, we reported a DNA octahedron-based fluorescence nanoprobe, which was capable of detecting and imaging of two kinds of tumor-related mRNAs in living cells simultaneously. The DNA nanoprobe was constructed of eight single-stranded DNAs, in which two oligonucleotides (recognition sequences) were modified with quenchers (BHQ2 and BHQ3) and the adjacent sequences were modified with fluorophores (Cy3 and Cy5), respectively. In the presence of targets, the recognition sequences could dissociate from the nanoprobe skeleton by strand displacement reaction and the fluorescence was recovered accordingly. With the modification of AS1411 aptamer, the nanoprobe could internalize cancer cells more efficiently and distinguish cancer cells from normal cells. In addition, the nanoprobe exhibited good stability, biocompatibility, selectivity, and responded quickly to the targets as well. The DNA nanoprobe was designed in the formation of octahedron and may provide an inspiration for multidetection of cancer biomarkers based on the DNA nanotechnology. Fortunately, a group of fluorescence probes that can be internalized into cells are developed by scientists to provide opportunities to detect and image mRNAs in living cells. For example, Mirkin et al. constructed multiplexed nanoflares from gold nanoparticles (AuNPs) and spherical nucleic acid (SNA) to detect mRNA in living cells.16 Tang et al. utilized AuNPs and molecular beacons (MBs) to establish a four-color nanoprobe for multiplexed detection and imaging of intracellular mRNAs.17 Other than these approaches, many tetrahedron-based DNA fluorescence nanoprobes were developed owing to the features of the nanostructure, such as biocompatibility,18,19 biostability,20,21 and easily multiple modification properties.22−24 For instance, Tan et al. developed a three-dimensional DNA amplifier, which can operate inside living cells in response to a specific mRNA target.25 Zhang et al. constructed a DNA tetrahedron-based multicolor nanoprobe to detect and image three tumor-related mRNAs in living cells.26 Therefore, it is of great significance to develop and establish more valuable DNA nanostructures as probes for biological application. Herein, we proposed a new kind of DNA fluorescence nanoprobe based on the octahedron structure. DNA

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ith the development of biology, a wide variety of cancer biomarkers have been discovered, such as nucleic acids, proteins, sugars, small metabolites, and cytogenetic and cytokinetic parameters, as well as entire tumor cells found in the body fluid.1 Tumor-related mRNA is a kind of important cancer biomarker, of which the type and expression levels are closely related to the occurrence and development of the tumor.2,3 Up to now, an increasing number of tumor-related mRNAs have been identified, such as TK1 mRNA, c-Met mRNA, MUC1 mRNA, GalNAc-T mRNA, survivin mRNA and so on.2,4−6 In addition, the expression levels of tumorrelated mRNAs always change, accompanied by the development and metastasis progression of cancer.7 The overlapped expression of the same mRNAs could take place in terms of different cancers, such as prostate cancer, breast cancer, and so on.8−11 Moreover, different kinds of specific mRNAs not only express in cancer cells, but also express in normal cells in different amount.2 As a result, it is of great value to develop effective methods to detect and image multiple tumor-related mRNAs at the same time in living cells for cancer diagnosis and therapy. The widely used approaches for mRNA detection include real-time polymerase chain reaction (RT-PCR)12,13 and microarray analysis,14,15 which need to extract mRNA from cell lysates, are time-consuming, and cannot reflect the realtime change of mRNA expression levels in living cells. © 2018 American Chemical Society

Received: June 23, 2018 Accepted: September 18, 2018 Published: September 18, 2018 12059

DOI: 10.1021/acs.analchem.8b02847 Anal. Chem. 2018, 90, 12059−12066

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Figure 1. Schematic illustration of tumor-related mRNA targets detected by DNA octahedron-based fluorescence nanoprobe. The DNA nanoprobe is constructed of eight oligonucleotides, in which two recognition sequences are modified with quenchers (BHQ2 and BHQ3) and the adjacent sequences are modified with fluorophores (Cy3 and Cy5), respectively. In the presence of the targets, the recognition sequences could dissociate from the skeleton due to the hybridization with the targets, and the fluorescence signals would restore afterward.

hydroxide (NaOH), and hydrochloric acid (HCl) were purchased from China National Pharmaceutical Group Corporation (Shanghai, PRC); Cell Counting Kit-8, tamoxifen, and β-estradiol were purchased from Sigma-Aldrich (Shanghai, PRC); Gel Stain (10000×) was purchased from Trans Gen Biotech Co., Ltd. (Beijing, PRC). Cell culture products were purchased from GIBCO. IP Lysis Buffer was purchased from Thermo Fisher Scientific (Rockford, IL, U.S.A.). All the chemicals were of analytical grade and used without further purification. Ultrapure water was obtained from a Milli-Q water purification system (Millipore Corp., Bedford, MA) with resistivity of 18.2 MΩ·cm. DNA oligonucleotides (the sequences of oligonucleotides are shown in Table S1 in the Supporting Information) were synthesized and purified by TAKARA Biotechnology (Dalian, PRC) and Sangon Biotechnology Co., Ltd. (Shanghai, PRC). The human breast cancer cell line MCF-7 and the normal immortalized human mammary epithelial cell line MCF-10A were purchased from Committee on Type Culture Collection of the Chinese Academy of Sciences. Instruments. The assembling of DNA nanoprobe was performed on C1000 Touch Thermal Cycler from Bio-Rad Laboratories. CCK-8 results were acquired on SH-1000 Lab microplate reader. The fluorescence intensities were acquired on a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies Inc.). Gel images were captured using a ChemiDoc System from Bio-Rad Laboratories. Confocal microscopy assay was operated on a laser-scanning confocal microscopy system (NIKON, A1, Japan). Flow cytometric assay was performed on BD FACSCanto (BD Bioscience, U.S.A.).

octahedron may provide more opportunities in cancer detection and diagnosis due to its complication in structures, flexibility of design, and multiple binding sites.27,28 We hope that the present approach could further extend the application of DNA nanotechnology in biological field. As shown in Figure 1, the nanoprobe was constructed from eight oligonucleotides, in which two oligonucleotides (recognition sequences) were modified with quenchers (BHQ2 and BHQ3) to target GalNAc-T mRNA and TK1 mRNA, and other two sequences were modified with fluorescence molecules (Cy3 and Cy5), respectively. The GalNAc-T mRNA and TK1 mRNA, corresponding to β-1,4-N-acetylgalactosaminyl-transferase (GalNAc-T) and thymidine kinase 1 (TK1), are important biomarkers in cancer cells.2,29−31 In the absence of the targets, the fluorophores were quenched by the adjacent quenchers, while the recognition sequences would hybridize with the targets completely in the presence of the targets. Afterward, the recognition sequences would dissociate from the skeleton, and in consequence, the corresponding fluorescence signals were restored. The nanoprobe possessed good biostability and biocompatibility as well. Furthermore, the DNA nanoprobe was designed with two AS1411 aptamer sequences, which could bind with nucleolin overexpressed on the surface of cancer cells and then accelerate the internalization of the nanoprobe into the cancer cells.32−34 Based on these, the asprepared DNA nanoprobe was capable of detecting and imaging two kinds of tumor-related mRNAs in living cells simultaneously.

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EXPERIMENTAL SECTION Materials. Tris(hydroxymethyl) aminomethane (Tris), sodium chloride (NaCl), potassium chloride (KCl), sodium 12060

DOI: 10.1021/acs.analchem.8b02847 Anal. Chem. 2018, 90, 12059−12066

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(8 × 103 cells/well) were dispersed within replicate 96-well microtiter plates to a total volume of 200 μL well−1 and were incubated at 37 °C in a 5% CO2 incubator for 24 h. Then the original medium was removed, and the MCF-7 cells were incubated with fresh medium containing DNA nanoprobe (0, 50, and 100 nM) for 6, 12, and 24 h. Ultimately, the cells were washed with 1× PBS for three times and 10 μL CCK-8 solutions were added into each well. After incubation for 1−2 h, the absorbance was measured at 450 nm with a SH-1000 Lab microplate reader. Confocal Microscopy Assay. For confocal microscopy assay, MCF-7 or MCF-10A cells were seeded in Petri dishes and cultured overnight at 37 °C for 24 h in a 5% CO2 incubator. The DNA fluorescence nanoprobe (100 nM) was, respectively, delivered into MCF-7 and MCF-10A cells in culture medium at 37 °C in 5% CO2. Then the cells were washed with PBS several times to remove the excess samples after incubating for 4 h. Finally, the confocal fluorescence images of the cells were obtained on a laser-scanning confocal microscopy system with different laser transmitters. To evaluate the expression levels of tumor-related mRNA, MCF-7 cells were divided into three groups, in which one group without treatment was served as control, the other groups were treated with β-estradiol (10−8 M) and tamoxifen (10−6 M), respectively, for 48 h. The confocal microscopy assay was performed as described above with the DNA fluorescence nanoprobe (100 nM). To demonstrate the targeting ability of AS1411 aptamer modified nanoprobe, MCF-7 cells were treated with DNA fluorescence nanoprobe with or without AS1411 aptamer (100 nM), respectively, for 4 h. The confocal microscopy assay was performed as described above. Flow Cytometric Assay. MCF-7 cells or MCF-10A cells were seeded with media in Petri dishes and cultured at 37 °C for 24 h in a 5% CO2 incubator. After being incubated with DNA fluorescence nanoprobe (100 nM) in culture medium at 37 °C in 5% CO2 for 4 h, cells were detached from culture dishes using trypsin solution. The solution containing treated cells was centrifuged at 1000 rpm for 4 min and resuspended in PBS for three times. Flow cytometric assay was performed by BD FACSCanto Flow Cytometer (BD Bioscience, U.S.A.) using fluorescent channel Cy3 (543 nm excitation) and fluorescent channel Cy5 (633 nm excitation). To evaluate the expression levels of tumor-related mRNA, MCF-7 cells were divided into three groups, one group of which without treatment was served as control, the other groups were treated with β-estradiol (10−8 M) and tamoxifen (10−6 M), respectively, for 48 h. Then, the cells were incubated with DNA fluorescence nanoprobe (100 nM) in culture medium at 37 °C in 5% CO2 for 4 h, and the flow cytometric assay was performed as described above.

Preparation of DNA Nanoprobe. The DNA fluorescence nanoprobe was assembled from single-stranded DNAs through base-pairing interaction. Briefly, eight ssDNA strands (A-Cy5, B, C-Cy3, A-BHQ3, C-BHQ2, D, E, and F, the sequences of oligonucleotides are shown in Table S1 in the Supporting Information) were mixed at exactly equimolar concentrations in a buffer solution (10 mM Tris, 250 mM NaCl, 100 mM KCl, pH 8.0). The mixture was heated to 95 °C for 10 min, then cooled down to 4 °C slowly at a speed of −1 °C per 3 min and stored at 4 °C for further use. Kinetics Assay. The DNA nanoprobe (100 nM) was mixed with two tumor-related mRNA targets (200 nM) respectively, then the fluorescence intensity was determined by Cary Eclipse Fluorescence Spectrophotometer (Kinetic Model). The fluorescence of Cy3 was excited at 545 nm and measured at 566 nm; the fluorescence of Cy5 was excited at 645 nm and measured at 666 nm. Hybridization Experiment. The DNA fluorescence nanoprobe (100 nM) was incubated with the two complementary targets (TK1 target, GalNAc-T target) simultaneously, with increasing concentrations of the DNA targets (0, 10, 20, 30, 40, 80, 100, and 120 nM). After incubation for 30 min, the fluorescence intensities of Cy5 and Cy3 were measured at appropriate excitation wavelengths. All experiments were repeated at least three times. Specificity Experiment. Different kinds of oligonucleotide targets (200 nM) including TK1 target, GalNAc-T target, cmyc target, survivin target and c-raf-1 target were spiked in 250 μL hybridization buffer (10 mM Tris, 250 mM NaCl, 100 mM KCl, pH 8.0) containing 100 nM DNA nanoprobe, respectively. After incubation for 30 min, fluorescence intensities of Cy5 and Cy3 were recorded by Cary Eclipse fluorescence spectrophotometer. All experiments were repeated at least three times. Cell Lysis. The lysis solution (IP Lysis Buffer) contains 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol. MCF-7 cells (1 × 106) were washed with PBS and detached from the flask with trypsin, then resuspended in ice-cold lysis buffer. The mixture was incubated for 30 min on ice and then centrifuged at 12000 g at 4 °C for 20 min. Finally, the supernatant was collected and stored at −20 °C for further use. Nuclease Stability. The DNA nanoprobe (250 nM) was mixed with FBS (10%, v/v) and incubated at 37 °C for 0, 3, 6, 9, 12, and 24 h, and then the mixtures were run on a 1% agarose gel and imaged by ChemiDoc System from Bio-Rad Laboratories. The DNA nanoprobe (250 nM) was mixed with the MCF-7 cell lysate (10%, v/v) at 37 °C and incubated for different periods of time (0, 3, 6, 9, 12, and 24 h). Finally, the mixtures were analyzed by 1% agarose gel electrophoresis, as described above. The DNA fluorescence nanoprobe (100 nM) was mixed with FBS (10%) and incubated at 37 °C for 30 min. Then TK1 target (200 nM) and GalNAc-T target (200 nM) were added into the mixture and reacted for 10 min, and the fluorescence spectra of Cy5 and Cy3 were obtained by Cary Eclipse fluorescence spectrophotometer for 0, 1, 2, 3, 4, 5, 6, 7, and 8 h. All experiments were repeated at least three times. CCK-8 Assay. MCF-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum, penicillin, and streptomycin, and incubated at 37 °C in a 5% CO2 incubator. The CCK-8 assay was adopted to study the cytotoxicity of the DNA nanoprobe. Briefly, MCF-7 cells

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RESULTS AND DISCUSSION Characterization of the DNA Nanoprobe. To verify the successful formation of the DNA nanoprobe, eight singlestranded DNAs, including two recognition sequences targeting TK1 and GalNAc-T target without modification, were employed to synthesize the nanostructure. And the formation of DNA nanoprobe was demonstrated by 1% agarose gel electrophoresis. As shown in Figure S1 in the Supporting Information, the migration movement decreased from lane 1 to lane 8 in sequence, with the addition of DNA strands. Besides, lane 8 showed that the DNA octahedron-based nanostructure 12061

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Figure 2. Fluorescent characterization of the DNA nanoprobe (100 nM) by adding various concentrations of DNA targets (0−120 nM). (a, b) Fluorescence spectra after adding different amount of TK1 target and GalNAc-T target (0−120 nM). (c) and (d) are the fluorescence intensity to concentration plots of (a) and (b), respectively. Error bar represents the standard deviation from three independent assays.

Figure 3. Stability of the DNA fluorescence nanoprobe treated with or without FBS (10%, v/v). (a, c) Fluorescence changes of the nanoprobe treated with FBS (red trace) or without FBS (black trace) for 0−8 h. (b, d) Fluorescence spectra of the nanoprobe by hybridizing with TK1 target (200 nM) and GalNAc-T target (200 nM), respectively, after treating with FBS (10%, v/v) for 8 h. TK1 target (Cy5), EX/EM: 645 nm/666 nm; GalNAc-T target (Cy3), EX/EM: 545 nm/566 nm.

migrated more slowly than the previous strands, which demonstrated that the octahedron-based nanoprobe was formed with high efficiency. In Vitro Studies of the Nanoprobe. To investigate the response ability of the nanoprobe toward the two DNA targets,

kinetic studies were carried out with the perfectly matched DNA targets of the tumor-related mRNAs, respectively. As shown in Figure S2 in the Supporting Information, the fluorescence signals of Cy5 and Cy3 were monitored in vitro before and after addition of the DNA targets in kinetic model, 12062

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Figure 4. Intracellular imaging of TK1 and GalNAc-T mRNA using the DNA nanoprobe. (a) MCF-7 and (b) MCF-10A cells were incubated with the nanoprobe (100 nM) for 4 h at 37 °C. The two mRNAs were recorded by Cy5 with 640 nm excitation, and Cy3 with 561 nm excitation, respectively. Scale bar: 50 μm.

comparison with the control groups, the restored fluorescence signals of the nanoprobe treated with FBS or cell lysate showed no significant difference after the addition of DNA targets (Figure 3 and Figure S6 in Supporting Information). These results confirmed that the DNA nanoprobe was stable enough against degradation, which was important for performing detection and imaging in living cells. Stability of the Nanoprobe. Before the application of the DNA nanoprobe in vitro detection and imaging of tumorrelated mRNAs, the stability of DNA nanoprobe was studied with 10% fetal bovine serum (FBS). As shown in the agarose gel electrophoresis (Figures S4 and S5, in Supporting Information), the band of the DNA nanostructure remained almost unchanged after incubation with FBS (10%, v:v) or MCF-7 cell lysate (10%, v/v) for 24 h, reflecting the nanoprobe possessed good biostability against degradation. Furthermore, the Cy3 and Cy5 fluorescence intensities of the DNA nanoprobe almost unchanged by incubating with the FBS (10%, v/v) or MCF-7 cell lysate (10%, v/v) for 0 to 8 h (Figure 3, and Figure S6 in Supporting Information). By comparison with the control groups, the restored fluorescence signals of the nanoprobe treated with FBS or cell lysate showed no significant difference after the addition of DNA targets. (Figure 3, and Figure S6 in Supporting Information). These results confirmed that the DNA nanoprobe was stable enough against degradation, which was important for performing detection and imaging in living cells. CCK-8 Assay. The well biocompatibility is another crucial property for the application of the DNA nanoprobe in vitro detection and imaging. Therefore, the CCK-8 assay was performed on human breast cancer cell line MCF-7 to study the cytotoxicity of the nanoprobe. The viability of MCF-7 cells was evaluated by the absorbance of CCK-8 at 450 nm after incubation with DNA nanoprobe. As can be seen in Figure S10 in the Supporting Information, the cell viabilities were around 100% with different concentrations of the nanoprobe (50 and 100 nM) for different period of time (6, 12, and 24 h). These results confirmed that the nanoprobe was biocompatible in

and the fluorescence intensities of Cy5 and Cy3 increased around 4.97-fold and 3.06-fold in the presence of two DNA targets. Apart from these, the fluorescence intensity increased rapidly with the addition of TK1 target and GalNAc-T target separately within 5 min, and the fluorescence signal corresponding to each DNA target did not exert influence on another target, indicating that the nanoprobe were capable of responding to the two targets efficiently and independently. Afterward, the changes of fluorescence of the nanoprobe with the increasement of the concentration of the DNA targets were investigated. As shown in Figure 2, the fluorescence intensities of the DNA nanoprobe changed linearly with the increasement of the DNA targets from 0 to 120 nM, proving that the hybridization between nanoprobe and DNA targets led to fluorescence recovery. Additionally, the detection limit was calculated to be 3.03 nM for TK1 target and 1.09 nM for GalNAc-T target. Besides, the other three targets (c-myc target, survivin target and c-raf-1 target) were adopted to evaluate the selectivity of the nanoprobe. Compared with the background fluorescence, the fluorescence restoration of fluorophores exhibited no obvious changes in the presence of the other targets and increased remarkably to the specific target (Figure S3, in Supporting Information). All the results proved that the nanoprobe was able to detect TK1 target and GalNAc-T target at the same time. Stability of the Nanoprobe. Before the application of the DNA nanoprobe in vitro detection and imaging of tumorrelated mRNAs, the stability of DNA nanoprobe was studied with 10% fetal calf serum (FBS). As shown in the agarose gel electrophoresis (Figures S4 and S5, in Supporting Information), the band of the DNA nanostructure remained almost unchanged after incubation with FBS (10%, v/v) or MCF-7 cell lysate (10%, v/v) for 24 h, reflecting the nanoprobe possessed good biostability against degradation. Furthermore, the Cy3 and Cy5 fluorescence intensities of the DNA nanoprobe almost unchanged by incubating with the FBS (10%, v/v) or MCF-7 cell lysate (10%, v/v) for 0 to 8 h (Figure 3, and Figure S6 in Supporting Information). By 12063

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Figure 5. Intracellular imaging of different levels of TK1 mRNA in MCF-7 cells. The left group was the control group. The middle group was treated with β-estradiol (10−8 M), and the right group was treated with tamoxifen (10−6 M) for 48 h. The three groups were incubated with the nanoprobe (100 nM) for 4 h at 37 °C. Images were obtained with excitation at 640 nm. Scale bars: 50 μm.

This result proved the fact that the AS1411 aptamer did make contribution to the targeting ability of DNA nanoprobe. All of these demonstrated that the DNA nanoprobe was capable of distinguishing the cancer cells from normal cells through fluorescence imaging and flow cytometry quantification, depending on the difference of tumor-related mRNA between the two cells. Evaluation of the mRNA Expression Levels. The expression levels of tumor-related mRNA are accompanied by the progression or metastasis of cancer cells. Hence, it is critical to determine the level of tumor-related mRNA in living cells for cancer detection and therapy. We further studied the ability of the nanoprobe to monitor the expression level of the tumor-related mRNA in living cells. As reported before, the βestradiol and tamoxifen were able to accelerate and reduce the expression of TK1 mRNA;39,40 thus, they were adopted to modulate the expression level of TK1 mRNA. MCF-7 cells were divided into three groups, one group was treated with βestradiol to up-regulate the TK1 mRNA expression level and another group was treated with tamoxifen to down-regulate the TK1 mRNA expression level, respectively. The third untreated group was used as control. Then confocal microscopy assay was performed. As shown in Figure 5, the fluorescence intensity of cells treated with β-estradiol was stronger than that of the untreated group, while the fluorescence intensity of cells treated with tamoxifen was lower than that of the untreated ones. The fluorescence intensity was accord with the expression level of TK1 mRNA expression in MCF-7 cells, which indicated that the nanoprobe was able to monitor the changes of tumor-related mRNA in cancer cells. To further confirm the proposed nanoprobe can monitor varied levels of mRNA in MCF-7 cells with drug stimuli, flow cytometry assay was carried out. As shown in Figure S9 in the Supporting Information, the tamoxifen-treated group displayed the modest fluorescence intensity of Cy5. While fluorescence intensity of Cy5 from β-estradiol treated group was stronger than that of the untreated group. Results from flow cytometry assay were in

living cells and was an approving candidate for intracellular detection and imaging. Intracellular Imaging of the Nanoprobe. As reported before, thymidine kinase 1 (TK1) is a pyrimidine metabolic pathway enzyme involved in salvage DNA synthesis, and thus is associated with cell division and is proposed to be a biomarker for tumor growth.29−31 The β-1,4-N-acetylgalactosaminyl-transferase (GalNAc-T) is a key factor in the biosynthetic pathway of gangliosides GM2/GD2, which are oncofetal glycolipids found elevated in expression on the surface of various types of cancer cells.2,35 The TK1 mRNA and GalNAc-T mRNA, corresponding mRNAs of TK1 and GalNAc-T enzyme, are overexpressed in MCF-7 cells. Hence, MCF-7 cells and MCF-10A cells were chosen as positive group and control group to investigate the imaging capacity of the DNA nanoprobe against TK1 mRNA and GalNAc-T mRNA in living cells. First, confocal microscopy assay was carried out to investigate the imaging capacity of the DNA nanoprobe. As shown in Figure 4, a strong red fluorescence signal of Cy5 (TK1 mRNA) and a green fluorescence signal of Cy3 (GalNAc-T mRNA) were observed from MCF-7 cells after incubating with the nanoprobe (100 nM) for 4 h, while MCF10A cells exhibited both faint fluorescence signals under the same condition. Then flow cytometry was performed to further confirm the results. As can be seen in Figure S7 in the Supporting Information, the percentage of dual fluorescence signals (Cy5 and Cy3) of MCF-7 cells (81.4%) was evidently higher than that of MCF-10A (1.40%) after incubated with equal amount of nanoprobe. These phenomena were consistent well with the levels of mRNA expression as described above. Moreover, the AS1411 aptamer could easily form a dimeric G-quadruplex structure in the presence of K+ and bind with nucleolin overexpressed on the surface of cancer cells with high affinity and specificity.36−38 As shown in Figure S8, the fluorescence signals of Cy3 and Cy5 of MCF-7 cells treated with DNA fluorescence nanoprobe with AS1411 aptamer were stronger than that without AS1411 aptamer. 12064

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line with that of confocal microscopy assay, validating the ability of the nanoprobe to monitor different levels of tumorrelated mRNA in living cells.

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CONCLUSIONS In summary, we have developed a new kind of DNA octahedron-based fluorescence nanoprobe, which could realize detection and imaging of two kinds of tumor-related mRNAs in living cells. With the accurate design, the DNA nanoprobe was formed with high efficiency, which was confirmed by the agarose gel electrophoresis. Attributing to the complete hybridization reaction between the recognition sequences with the targets, the nanoprobe responded with high efficiency and good selectivity. In addition, the nanoprobe exhibited good structural stability and biocompatibility. Modified with AS1411 aptamers, the nanoprobe was internalized into the cancer cells more efficiently and then distinguished the cancer cells from the normal cells better. Importantly, this nanoprobe could not only distinguish cancer cells from normal cells, but also monitor the different expression levels of TK1 mRNA. As a result, we believed that the new kind of DNA nanoprobe could provide an opportunity to create biosensors for cancer diagnosis and therapy.

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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.8b02847. Supporting table and supporting figures (PDF).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Huanghao Yang: 0000-0001-5894-0909 Author Contributions

† L.Z. and S.C. contributed equally to this work. All authors have given approval to the final version of the Manuscript.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (Nos. 21775025, U1705281, U1505221, 21475026, and 21635002) and Natural Science Foundation of Fujian Province of China (Nos. 2015H6011 and 2018J01687)

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DOI: 10.1021/acs.analchem.8b02847 Anal. Chem. 2018, 90, 12059−12066