Dual-miRNA-Propelled Three-Dimensional DNA Walker for Highly Specific and Rapid Discrimination of Breast Cancer Cell Subtypes in Clinical Tissue Samples

生物分析 化学 纳米技术 图书馆学 计算机科学 材料科学
作者
Yanan Wu,Yating He,Miaomiao Han,Di Zhao,Bojun Liu,Kun Yuan,Hongzhi Sun,Hong‐Min Meng,Zhaohui Li
出处
期刊:CCS Chemistry [Chinese Chemical Society]
卷期号:5 (7): 1561-1573 被引量:26
标识
DOI:10.31635/ccschem.022.202202051
摘要

Open AccessCCS ChemistryRESEARCH ARTICLES2 Aug 2022Dual-miRNA-Propelled Three-Dimensional DNA Walker for Highly Specific and Rapid Discrimination of Breast Cancer Cell Subtypes in Clinical Tissue Samples Yanan Wu, Yating He, Miaomiao Han, Di Zhao, Bojun Liu, Kun Yuan, Hongzhi Sun, Hong-Min Meng and Zhaohui Li Yanan Wu College of Chemistry, Institute of Analytical Chemistry for Life Sciences, Henan Joint International Research Laboratory of Green Construction of Functional Molecules and Their Bioanalytical Applications, Zhengzhou Key Laboratory of Functional Nanomaterial and Medical Theranostics, Zhengzhou University, Zhengzhou 450001 , Yating He College of Chemistry, Institute of Analytical Chemistry for Life Sciences, Henan Joint International Research Laboratory of Green Construction of Functional Molecules and Their Bioanalytical Applications, Zhengzhou Key Laboratory of Functional Nanomaterial and Medical Theranostics, Zhengzhou University, Zhengzhou 450001 , Miaomiao Han College of Chemistry, Institute of Analytical Chemistry for Life Sciences, Henan Joint International Research Laboratory of Green Construction of Functional Molecules and Their Bioanalytical Applications, Zhengzhou Key Laboratory of Functional Nanomaterial and Medical Theranostics, Zhengzhou University, Zhengzhou 450001 , Di Zhao College of Chemistry, Institute of Analytical Chemistry for Life Sciences, Henan Joint International Research Laboratory of Green Construction of Functional Molecules and Their Bioanalytical Applications, Zhengzhou Key Laboratory of Functional Nanomaterial and Medical Theranostics, Zhengzhou University, Zhengzhou 450001 , Bojun Liu College of Chemistry, Institute of Analytical Chemistry for Life Sciences, Henan Joint International Research Laboratory of Green Construction of Functional Molecules and Their Bioanalytical Applications, Zhengzhou Key Laboratory of Functional Nanomaterial and Medical Theranostics, Zhengzhou University, Zhengzhou 450001 , Kun Yuan College of Chemistry, Institute of Analytical Chemistry for Life Sciences, Henan Joint International Research Laboratory of Green Construction of Functional Molecules and Their Bioanalytical Applications, Zhengzhou Key Laboratory of Functional Nanomaterial and Medical Theranostics, Zhengzhou University, Zhengzhou 450001 , Hongzhi Sun College of Chemistry, Institute of Analytical Chemistry for Life Sciences, Henan Joint International Research Laboratory of Green Construction of Functional Molecules and Their Bioanalytical Applications, Zhengzhou Key Laboratory of Functional Nanomaterial and Medical Theranostics, Zhengzhou University, Zhengzhou 450001 , Hong-Min Meng *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] College of Chemistry, Institute of Analytical Chemistry for Life Sciences, Henan Joint International Research Laboratory of Green Construction of Functional Molecules and Their Bioanalytical Applications, Zhengzhou Key Laboratory of Functional Nanomaterial and Medical Theranostics, Zhengzhou University, Zhengzhou 450001 and Zhaohui Li *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] College of Chemistry, Institute of Analytical Chemistry for Life Sciences, Henan Joint International Research Laboratory of Green Construction of Functional Molecules and Their Bioanalytical Applications, Zhengzhou Key Laboratory of Functional Nanomaterial and Medical Theranostics, Zhengzhou University, Zhengzhou 450001 https://doi.org/10.31635/ccschem.022.202202051 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Accurate discrimination of cell subtypes at the molecular level is especially important for cancer diagnosis, but no current method allows rapid and precise detection of breast cancer subtypes. Herein, we developed an elegant DNA walker for direct and rapid differentiation of breast cancer cell subtypes via detection of dual-miRNAs in clinical tissue samples. This DNA nanomachine can be specifically initiated by endogenous miR-21 and miR-31, and the sensitivity was dramatically improved due to the DNAzyme-mediated signal amplification. This DNA walker enabled rapid detection of double miRNA characteristics in different breast cell lines and also distinguished the fluctuations in a single cell. Applications of this DNAzyme-based nanomachine in vivo and in clinical samples were demonstrated for efficient detection of breast cancer subtypes, making the method generally applicable for precise management of cancers. Download figure Download PowerPoint Introduction To date, although total breast cancer mortality has decreased because of the advances in early screening and effective therapy techniques, the morbidity maintains increasing and appears a younger trend.1–3 According to the Global Cancer Statistics 2020, breast cancer is the most diagnosed cancer (11.7% of total cases) and the leading cause of cancer death among women worldwide.4 Breast cancer is highly heterogeneous, including luminal A, luminal B, HER2-positive, and triple-negative breast cancer.5,6 For instance, triple-negative breast cancer is associated with high metastatic risk, high invasiveness, poor prognosis, and high mortality.7–9 Therefore, precise and rapid discrimination of breast cancer subtypes can enable more effective therapeutic options, which is of great significance for substantial improvement of patient survival, especially for triple-negative breast cancer. As is well known, immunohistochemistry (IHC) is the gold standard method for classifying breast cancer subtypes through the detection of different receptors, such as the estrogen receptor, progesterone receptor, and human epithelial growth factor receptor-2.10,11 However, the accuracy and sensitivity of IHC is often affected by the heterogeneity of antibodies, and its operation is rather cumbersome and complicated. Therefore, developing a reliable, rapid, and user-friendly approach for breast cancer identification is an ever-increasing need. In recent years, diagnostic techniques have focused on an individual patient's biological information, including DNA, RNA, proteins, or cells.3,12–15 In addition to the genomic and proteomic biomarkers, noncoding miRNA expression levels also have been discovered to be extremely informative for classifying cell subtypes.16–18 Recent work has revealed that miR-21, an oncogene with antiapoptotic potential, is highly overexpressed in breast cancer cell types while miR-31 is reported to be a metastasis suppressor in breast cancer via targeting SATB2 and downregulating in the most aggressive triple-negative breast cancers.19–21 Therefore, developing efficient methods for simultaneous detection of miR-21 and miR-31 can provide new perspectives for precise discrimination of breast cancer subtypes. DNA circuits, which can effectively integrate various recognition units, transduce the signal and report the results in an intelligent manner, provide a natural connection between molecular recognition and information processing.22–26 For example, Han's group developed some DNA molecular computation platforms for the assay of miRNAs and protein profiles for cancer diagnosis.27–29 Nevertheless, only a few examples were successfully used in classifying diseases in clinical samples due to the following limitations: (1) The one-signal input and output strategy may lead to false-positive signals caused by nuclease digestion or unspecific binding events. (2) Some representative tandem nanosystems involve multiple freely diffusible molecular modules in solution, leading to a quite slow response. Accordingly, we sought to implement a programmed DNA platform that logically operates on miR-21 and miR-31 in specific cells and achieves highly reliable and rapid diagnostic results for breast cancer. To the best of our knowledge, this is the first report of a DNA nanomachine for the smart and simultaneous detection of miR-21 and miR-31 for rapid discrimination of luminal A breast cancer and triple-negative breast cancer. Motivated by this, we here designed a dual-miRNA activated 3D DNA walker for differential diagnosis of breast cancer subtypes with high accuracy and quick detection speed. As shown in Scheme 1a–c, both substrate strands with cleavage RNA sites (S) and Mg2+-specific DNAzyme (Dz) silenced by a locking strand (L) are attached on gold nanoparticles (AuNPs). The L is designed to respond to specific intracellular miR-21 and miR-31. Carboxyfluorescein (FAM) and cyanine5 (Cy5) are conjugated on the end of S and Dz, respectively, to enable real-time monitoring of the operation of the DNA walker. In the presence of miR-21 and miR-31, a series of strand-displacement reactions make the L release from LDz, and the free Dz then hybridizes to S and cleaves to S. The energy derived from the cleavage of the RNA chimeric DNA substrate affords the demand for the Dz to move from one S to another, realizing the autonomic and processive walking along the AuNP. These cleaved fluorophores labeled DNA fragments are separated from AuNP, resulting in fluorescence enhancement, and offering amplified signal generation for detection of two target miRNAs. In addition, it must be noted that all elements assembled on one AnNP offer intensive local concentrations, rapid reaction rates, and high reaction efficiency due to their proximity on the AuNP. The performance of the DNA walker is demonstrated in normal, malignant, and metastatic breast tissue in living mice. Additionally, the unexpected identification ability of the DNA walker was observed in clinical samples, including Luminal A, triple-negative, para-carcinoma, and normal breast tissues. Scheme 1 | (a) Schematic illustration of the fabrication of dual-miRNA-responsive dual-color DNA walker. (b) Intracellular operation process of DNA walker. (c) Dual-color using two miRNAs for cell subtype identification. Download figure Download PowerPoint Experimental Methods Reagents Synergy Brands (SYBR) Gold, Hoechst 33342, lipofectamine 3000, and LysoTracker Red were bought from Invitrogen (Thermo Fisher Scientific, Waltham, USA). All DNA oligonucleotides, acrylamide/Bis solution (30% (w/v)), phosphate-buffered saline (PBS), and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). These DNA oligonucleotides were high performance liquid chromatography-purified, and detailed information about them is listed in Supporting Information Table S1. Chloroauric acid (HAuCl4·4H2O) was provided by Shanghai Energy Chemical (Shanghai, China). Trisodium citrate was acquired from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Preparation of DNA walker The gold nanoparticles (13 nm) were prepared based on previously reported strategies.30,31 The assembly of LDz duplexes and S strands into AuNPs were also prepared according to the previous literature.32,33 Dz-Cy5 and L strands were mixed in the ratio of 1∶1 in PBS buffer (2.7 mM KCl, 137 mM NaCl, 10 mM phosphate, pH 7.4), and then the mixture was annealed by incubating it at 95 °C for 5 min, followed by slowly cooling it down to room temperature for 3 h to ensure sufficient hybridization. Then the LDz duplexes were mixed with the S-FAM in a ratio of 1∶4, and the mixture was added to 200 μL AuNPs solution. After being frozen at −20 °C for 3 h and thawed at room temperature, unmodified DNAs was removed by centrifugation at 13,000 rpm for 30 min and washed twice with PBS. The DNA walker was stored in darkness at 4 °C until it was used. The concentration of AuNPs and DNA walker was determined by UV–vis absorption spectra. Gel electrophoresis analysis The DNA reaction was characterized by a gel electrophoresis experiment under 37 °C for 2 h with a concentration of 1 μM. Different samples (10 μL) were mixed with 2 μL 6 × loading buffer and 3 μL SYBR Gold. Then these mixtures were loaded into 12% denatured polyacrylamide gel electrophoresis (PAGE). The PAGE was implemented at 80 V for 40 min and imaged a Bio-Rad Gel Doc™ EZ imager. In vitro fluorescence experiments Two miRNA targets with the same concentration were first mixed with 2 nM DNA walker. The mixture was incubated at 37 °C for 1 h, and then the corresponding emission spectrum of FAM and Cy5 was recorded. For the kinetic study, the fluorescence change of related samples was monitored at 37 °C (excitation, 488 nm; emission, 520 nm), and the time interval was set as 10 min. Cell culture and confocal fluorescence imaging MCF-7 cells (a malignant human breast cell line) were grown in an Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% Fetal Bovine Serum (FBS). MDA-MB-231 cells (a metastatic human breast cancer cell line) were cultured in Leibovitz's L-15 supplemented with 10% FBS. MCF-10A cells (normal human mammary epithelial cells) were cultured in a mammary epithelial cell medium (MEpiCM). All confocal fluorescence imaging of cells was acquired on a laser scanning fluorescence microscope with a 63 × oil-immersion objective. The Cy5 and FAM fluorescence imaging was carried out in the red channel with 633 nm excitation and in the green channel with 488 nm excitation, respectively. Excitation of Hoechst 33342 was carried out at 405 nm. The cells were seeded in 20 mm glass-bottomed dishes and grown for 24 h. The cells were treated with a 2 nM DNA walker at 37 °C for 4 h, subsequently the cells were washed three times with PBS before being imaged. In order to modulate the expression of miRNA in MCF-7 cells, synthetic anti-miR-21 or anti-miR-31 with a given concentration was transfected by lipofectamine 3000. Then, the cells were incubated with 2 nM DNA walker for 4 h, after which they were washed three times with PBS. The cells were imaged under a confocal laser scanning microscope. In vivo fluorescence imaging Seven-week-old female athymic BALB/c (BALB/c-nu) mice were used to build up the tumor xenograft model. MCF-7/MDA-MB-231 cells in PBS were subcutaneously implanted to their right back flank with a density of 5 × 106 cells/mouse. After a week, the mice were used for in vivo imaging. The mice were intravenously injected with the Cy5 and Cy7 labeled DNA walker. Fluorescence imaging was taken at an exposure time of 0.1 s with excitation at 640 and 740 nm. After injection with DNA walker, the real-time fluorescence imaging was monitored. After imaging, the tumors of the mice in each group were extracted. After being fixed with 4% paraformaldehyde and dehydrated with 30% sucrose, tumor tissues were embedded in frozen optimal cutting temperature medium and cut into sections at a thickness of 10 μm using a cryostat (Leica, Wetzlar, Germany). Then the sections were stained with Hoechst for 15 min at 37 °C in the dark. The sections were washed with PBS and then imaged under a confocal laser scanning microscope. All mouse experimental procedures were approved by the Laboratory Animal Center of Henan Province. Analytical performance of DNA walker in clinical samples Tissue samples were cut into 10 μm thick sections. After incubation with DNA walker at 37 °C for 1 h, the sections were washed with PBS and then imaged by a confocal laser scanning microscope. Detailed data of clinical breast cancer tissue sections are listed in Supporting Information Table S2. Results and Discussion Design and characterization of the DNA walker The sensing principle of the designed DNA walker is illustrated in Figure 1a and Supporting Information Figure S1. Both S and LDz were assembled on the 13 nm AuNPs and labeled FAM and Cy5 were quenched by the AuNPs. Meanwhile, the catalytic activity of DNAzyme was inactive due to locking by L. In our design, L is composed of three domains: gray, yellow, and red. The gray domain consists of a 10-thymine region that is conjugated to the AuNPs. Yellow and red domains are the locking regions, whose sequence design relies on the target-to-initiate nanomachine operation. In the presence of miR-21 and miR-31, miR-21 hybridizes to the yellow domain and forms a complex of LDz-miR-21, thus partially releasing Dz (step 1). Then, the subsequent combining between miR-31 and the red domain enables the complete liberation of Dz from the LDz-miR-21 complex, along with the recovery Cy5 signal (step 2). The released Dz initiates the operation of the Dz with the cofactor Mg2+, resulting in FAM fluorescence enhancement (step 3). The free Dz subsequently hybridizes to another S, causing the cleaving number of S (step 4). Finally, the Dz releases from the AuNPs and recovers the Cy5 fluorescence (step 5). During this cyclic process, a very small number of miR-21 and miR-31 can initiate the cleavage of many FAM-labeled S, providing an amplified detection of targets. When incubated with miR-21 or miR-31, the reaction will stop at the first step or no reaction will occur. Therefore, the dual-miRNAs specifically induced amplification of the model with dual-signal outputs. Figure 1 | (a) Schematic illustration of the working principle of dual-miRNA-driven DNA walker. (b) Schematic diagram of the DNA reaction in nanomachine. I: Dual-miRNAs hybridizes L in LDz duplex and displaces Dz through strand displacement reaction; II: active Dz binds and catalyzes S. (c) Gel electrophoresis imaging of the scheme І. Lane 1: S; lane 2: miR-21; lane 3: miR-31; lane 4: L; lane 5: Dz; lane 6: LDz; lane 7: LDz + miR-21; lane 8: LDz + miR-31; lane 9: LDz + miR-21 + miR-31; lane 10: LDz + S. (d) Gel electrophoresis analysis of scheme ІІ. lane 1: Dz; lane 2: S; lane 3: Dz + S. The "+" and "−" represent the presence and absence of the relevant component, respectively. Download figure Download PowerPoint To prepare DNA walker, 13 nm AuNPs were chosen as the nanocarriers because they can efficiently quench fluorophores and possess high DNA-loading capacity and excellent membrane penetrability. AuNPs were functionalized with LDz duplex and S via frozen treatment ( Supporting Information Figure S2). The transmission electron microscopy images and UV–vis absorption spectra suggested that AuNPs displayed spherical morphology and red-shifted from 518 to 524 nm after attaching LDz and S. Meanwhile, the fluorescence of fluorophore was also quenched by AuNPs. Furthermore, by recording the fluorescence of 2-mercapto-ethanol treated DNA walker, the loading ability for single AuNP was approximately 62 S. These results confirmed that the DNA walker was successfully prepared. To investigate whether the DNA reaction can perform as expected, every step was studied by the gel electrophoresis experiment. The whole system is divided into two parts, and the schemes are described in Figure 1b: (I) dual-miRNAs recognizes L in LDz duplex and releases free Dz; (II) active Dz binds and cleaves S. As shown in Figure 1c, a distinct Dz band was appeared when miR-21 and miR-31 were both added; otherwise, it remained inactive. However, only adding S to the LDz duplex did not produce any new bands, meaning that there was no signal leakage. The scheme (II) is the downstream reaction of scheme (I). We observed that the addition of Dz to the S could produce an evident cleavage product (Figure 1d). Meanwhile, the operation of the whole reaction was demonstrated by gel electrophoresis analysis ( Supporting Information Figure S3). The results suggest that dual-miRNA can trigger stand replacement reaction and activate Dz to cleave S. Together, these data demonstrate that the DNA walker works as anticipated. The fluorescence performances of DNA walker in vitro The sensitivity of DNA walker to target miRNAs was first evaluated in vitro, as described in Figure 2a. The DNA walker displayed weak fluorescence in the absence of two targets. However, FAM fluorescence was elevated with the coexistence of miR-21 and miR-31 (Figure 2b). When DNA walker was treated with miR-21 alone or both miR-21 and miR-31, Dz departed from the AuNP surface and restored the Cy5 signal (Figure 2b). These results were consistent with the results of fluorescence images of DNA walker responsive to miRNAs (Figure 2c). Then, Dz-to-S ratio and the concentration of metal cofactor was optimized, and the largest signal-to-background noise ratio was achieved with 1:4 of Dz-to-S ( Supporting Information Figure S4) and 20 mM Mg2+ ( Supporting Information Figure S5). Under the optimized conditions, the limit of detection was calculated to be 20.7 pM for miR-21 (S/N = 3), which is almost 103 lower than the average abundance of biologically relevant miRNAs ( Supporting Information Figures S6 and S7). To investigate the reaction rate of DNA walker, a real-time fluorescence assay was performed on both a DNA nanomachine and a free probe (Figure 2d). As shown in Figure 2e, a fast and high fluorescence change of DNA walker was noticed while a much slower fluorescence response was observed for the free probe. The higher reaction rate of DNA walker may be attributed to the confinement of multiple modules in a compact space of the AuNPs and the subsequent evident increased collision frequency. Figure 2 | (a) Schematic diagram of the DNA walker with different inputs. (b) Fluorescence emission spectra of DNA walker treated with different miRNA under excitation wavelength of 488 and 633 nm. (c) The fluorescence images acquired with an IVIS spectrum imaging system of nanomachine response to miR-21 and miR-31. The "+" and "−" represent the presence and absence of the relevant component, respectively. (d) Schematic and (e) real-time kinetic analysis of the DNA walker and free probe (with the BHQ-1 instead of AuNP) responding to dual-miRNAs. Download figure Download PowerPoint Then the selectivity of the DNA walker was evaluated by exploring six single-base variants in two miRNAs of varying base positions. The results showed that the fluorescence increase resulting from the miR-21 and miR-31 is significantly larger than those increases from the six variants at the same concentration ( Supporting Information Figure S8), which indicates that far fewer DNA walkers are opened by the mismatched variants. Thus, DNA walker has reliable specificity to target miRNAs. In addition, a control nanoprobe was further designed, in which two nucleotides were mutated (AG to GC) in the catalytic core. As displayed in Supporting Information Figure S9, no fluorescence increase was detected under the same conditions. These results further demonstrated that the fluorescence increase of the DNA walker arises from its specific ability to recognize the target sequences. In addition to independent sensing ability, stability is also critical to the achievement of intracellular miRNA assay. The stability of DNA walker was evaluated in PBS, 10% FBS as well as the cell culture medium. The results showed that no obvious fluorescence increases were detected after addition of DNA walker into these matrices for 8 h ( Supporting Information Figure S10), which indicated that DNA walker possessed high resistance to nuclease and nonspecific targets. Furthermore, the hydrodynamic sizes of these nanomachines remained unchanged after incubation with PBS and culture medium for 8 h ( Supporting Information Figure S11), which was also consistent with results from the fluorescence assay. The cytotoxicity of the DNA walker was also carefully investigated. From the MTT assay results, DNA walker has little effect on the proliferation of MCF-7 cells ( Supporting Information Figure S12). Encouraged by the superior stability in complexed living systems and excellent biocompatibility, DNA walker holds promise for accurate detection of targets in living cells, in vivo, and even in clinical samples. Specific discrimination breast cancer cell subtypes Prior to the intracellular experiments, the cellular uptake ability was evaluated. As described in Supporting Information Figure S13, the real-time fluorescence imaging results showed increased FAM fluorescence and reached the maximum level after 4 h. Because the efficient escapement of DNA walker from the lysosome is crucial for cell imaging, intracellular distribution of DNA walker was also investigated. Thus, the colocalization analysis using DNA walker without Cy5 labeled and organelle-specific fluorescence dyes (LysoTracker Red and Hoechst 33342) was performed. As shown in Supporting Information Figure S14, after MCF-7 cells were treated with DNA walker for 4 h, the fluorescence colocalization imaging showed that the green and red with a Pearson's coefficient was 0.218, confirming that the efficient escape of the intact DNA walker from the lysosome. In addition, nuclear colocalization experiments showed that green and blue with a Pearson's coefficient was 0.014, demonstrating that the DNA walker could not access the nucleus and enter the cytoplasm. Based on the above studies, we wondered if DNA walker could accurately distinguish different cell types. The intracellular imaging performance of DNA walker was investigated in malignant (noninvasive) MCF-7, metastatic (highly invasive) MDA-MB-231, and nontumorigenic MCF-10A. As shown in Figure 3a, the malignant MCF-7 cells exhibited strong green and red fluorescence due to high expression levels of miR-21 and miR-31. At the same time, MDA-MB-231 cells exhibited detectable red signal and neglible green signal (Figure 3b). The reason is that during the tumor metastasis, the concentration of miR-31 expression is drastically downregulated in MDA-MB-231 cells. In addition, the MCF-10A cells, which have a high expression level of miR-31 and a low expression level of miR-21. As expected, there was almost no signal detected. Moreover, the flow cytometric quantification assay was also performed and showed that the FAM signal in MCF-7 cells was 8.61- and 8.76-fold higher that in the MDA-MB-231 and MCF-10A cells, respectively (Figure 3c,e). Meanwhile, the Cy5 fluorescence intensity in MDA-MB-231 cells was 1.21- and 6.34-fold higher that in the MCF-7 and MCF-10A cells, respectively (Figure 3d,f). Furthermore, the results detected by DNA walker in different breast cells were consistent with the qRT-PCR results ( Supporting Information Figure S15), suggesting the automata functions for accurate tumor progression discrimination. Figure 3 | Images of different breast cell lines treated by DNA walker. (a) Schematic and (b) confocal fluorescence images of different breast cell lines treated with DNA nanomachine. І: MCF-7 cells; ІІ: MDA-MB-231 cells; ІІІ: MCF-10A cells. The corresponding statistical histogram of mean fluorescence intensity is shown. Scale bar: 20 μm. Flow cytometry analysis and corresponding normalized fluorescence intensity of FAM (c, e) and Cy5 (d, f) channels of the cells from (a). Download figure Download PowerPoint To check if DNA walker could accurately identify different cancer cell subclasses in complex samples, MCF-7 cells were mixed with an equal number of MDA-MB-231 cells for recognition tests (Figure 4a). The flow cytometry data validated that FAM signal in MCF-7 cells was much higher than that in MDA-MB-231 cells, and the two Cy5 signals were almost the same (Figure 4b). The results show that two classes of breast cancer cells can clearly be discriminated with obvious difference signal gains. Further, the high specificity for distinguishing among different breast cancer subtypes was also demonstrated by fluorescent microscopy (Figure 4c). Taken together, these results confirm that DNA walker can differentiate among different cancer subtypes in complex systems. Figure 4 | Distinguishing different breast cancer cells in mixed cell populations. (a) Scheme of the experimental procedure. (b) Flow cytometry analysis of MCF-7 and MDA-MB-231 cell mixtures (1∶1). 1: Cell mixtures. 2: Cell mixtures incubated with DNA walker. (c) The fluorescent microscopy images of cell mixtures. Download figure Download PowerPoint As the expression levels of miR-21 and miR-31 at different tumor stages are varied, the capacity of the DNA walker to dynamically monitor the changes of two miRNA expression levels in MCF-7 cells was evaluated by additional anti-miR-21 and miR-
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