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Polydopamine nanospheres/ gold nanoclusters (Au NCs) based nanoplatform for dual color simultaneous detection of multiple tumorrelated microRNAs with DNase I assisted target recycling amplification Shenghao Xu, Yongyin Nie, Liping Jiang, Jun Wang, Guiyun Xu, Wei Wang, and Xiliang Luo Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05253 • Publication Date (Web): 28 Feb 2018 Downloaded from http://pubs.acs.org on February 28, 2018

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Analytical Chemistry

Polydopamine nanospheres/gold nanoclusters (Au NCs) based nanoplatform for dual color simultaneous detection of multiple tumor-related microRNAs with DNase I assisted target recycling amplification Shenghao Xu†, Yongyin Nie†, Liping Jiang†, Jun Wang†, Guiyun Xu†, Wei Wang†, Xiliang Luo†,* †

Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, P. R. China

ABSTRACT: A novel fluorescence resonance energy transfer (FRET)-based platform using polydopamine nanospheres (PDANSs) as energy acceptor and dual colored Au NCs as energy donor for simultaneous detection of multiple tumor related microRNAs with DNase I assisted target recycling amplification was developed for the first time. On the basis of monitoring the change of the recovered fluorescence intensity at 445 nm and 575 nm upon the addition of targets miRNA-21 and let-7a, these two microRNAs (miRNAs) can be simultaneously quantitatively detected, with detection limits of 4.2 pM and 3.6 pM (3σ) for miRNA-21 and let-7a, which was almost 20 times lower than that without DNase I. Additionally, semi-quantitative determination of miRNA-21 and let-7a can also realized through photo visualization. Most importantly, serums from normal and brest cancer patients can be visually and directly discriminated without any sample pre-treatment by confocal microscope experiments, demonstrating promising potential for auxiliary clinical diagnosis.

several decades. A series of nanomaterials including graphene oxide (GO), carbon nanotubes (CNTs), gold nanoparticles (AuNPs), MoS2 nanosheet and recently developed metalorganic frameworks (MOFs) have been used as energy acceptors to construct FRET based sensing platforms.18-22 Although these energy acceptors were widely applied for fluorescent biosensing, some of them exhibited poor biocompatibility and biodegradability, complicated preparation process or cytotoxicity, restricting their potential in real applications. In addition, traditional energy donor such as organic dyes or semiconductor quantum dots (QDs) also suffered from non-negligible shortcomings, including poor photo stability, easy photo bleaching or high toxicity.23 Thus, to develop new effective energy donor and acceptor pairs is of high significance to overcome these disadvantages. As a kind of biopolymerized material with tunable diameters, outstanding biocompatibility and biodegradability, polydopamine nanospheres (PDANSs) have been recently used as an effective FRET energy acceptor for the assay of biomolecules.24,25 For example, Choi CKK et al have utilized polydopamine coated gold nanoparticle as a fluorescence quencher together with DNA strands as molecular probes for multiplexed detection of two types of miRNAs.26 However, multiple excitation wavelengths are needed in their experiment, which limits the simultaneous detection of multiple targets. In addition, owing to the size-dependent emission spectra, excellent biocompatibility and stable fluorescent emitting,27,28 gold nanoclusters (Au NCs) have also been recently proved to be good candidates as FRET energy donor for biosensing.11 Moreover, with the help of enzyme assisted target recycling technique, the detection signal can be effectively amplified, which is benifical for improving the

INTRODUCTION The expression of serum microRNAs (miRNAs) has been recognized as a promising cancer diagnostic tumor marker because of their versatile potential applications for early diagnosis and treatment.1,2 It is noteworthy that many cancers are associated with multiple miRNAs and the detection of a single miRNA may cause false diagnosis which will restrict diagnostic value in clinical analysis.3 Therefore, approaches that can simultaneous detect multiple relevant miRNAs will significantly improve the accuracy and reliability for cancer diagnosis.4,5 Up to now, various methods have been developed for multiple miRNAs detection, including isothermal stranddisplacement polymerase reaction, reverse-transcription quantitative polymerase chain reaction and surface-enhanced raman spectroscopy.6-10 However, the intrinsic limitations such as time-consuming procedures, expensive instrument still restrict their further application.11 Recently, fluorescent methods for multiple tumor-related miRNAs analysis have been established owing to some advantages of unique superiority of high sensitivity, convenience and low cost.12-17 However, in most cases, multiple lasers are needed to excite each fluorophore to collect the emissive fluorescence when multiple miRNAs are simultaneously detected. Thus, the background noise originating from multiple excitation wavelengths are inevitable. Therefore, simultaneous fluorescent detection of multiple miRNAs with a single excitation wavelength is of high significance. Depending on the design of energy acceptor and donor pairs, fluorescence resonance energy transfer (FRET) based sensing platforms have gained extensive attention in the past

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detection sensitivity.29-31 To the best of our knowledge, the integration polydopamine nanospheres with multicolored Au NCs for developing FRET-based platforms for simultaneous detection of multiple miRNAs has not been explored yet, not even the combination of this FRET-based platforms with enzyme assisted target recycling amplification.

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prepared according to previous reports with some modifications.32 For blue emitting Au NCs (445 nm), 1.0 mL of 1.0 M L-proline solution was rapidly added into a boiled 4.0 mL of 3.0 mM HAuCl4 under vigorous agitation for 10 min and then cooled down to room temperature, blue emitting Au NCs (445 nm) were obtained. For orange emitting Au NCs (575 nm), 1.5 mL of 100 mM GSH and 5.0 mL HAuCl4 were added to 43.5 mL ultrapure water. The mixture was stirred at 70 °C for 24 h and the orange emitting Au NCs (575 nm) were obtained.

Herein, we develop a novel PDANSs/Au NCs based FRET nanoplatform with DNase I assisted target recycling amplification for dual color simultaneous detection of multiple tumor related microRNAs. Compared with the traditional FRET-based platforms, a series of obvious advantages of this platform make it particularly attractive: (1) As the FRET energy acceptor, the fluorescence quenching ability of PDANSs is superior to carbon nanotubes (CNTs) and molybdenum disulfide (MoS2), and comparable to graphene oxide (GO). (2) Two kinds of miRNAs can be visually and simultaneously detected by a single excitation wavelength. (3) Serums from normal and breast cancer patients can be visually discriminated by this FRET-based platform using single excitation wavelength. With these advantages, this work provides opportunities to develop economical, simple and effective FRET-based platforms for multiple tumor related miRNAs diagnostics.

Preparation of CmiR-21DNA/Au NCs (445 nm) probe (P1) and Clet-7aDNA/Au NCs (575 nm) probe (P2). For the preparation of P1, 10 µL of 10 µM CmiR-21DNA was added to 10 µL of 0.1 M EDC/NHS solution with continuous stirring at 37 oC for 1 h. Then, 180 µL of blue emitting Au NCs (445 nm) were added into the proceeding solution with continuous stirring at 37 oC for 4 h. For the preparation of P2, 10 µL of 10 µM Clet-7aDNA was added to 10 µL of 0.1 M EDC/NHS solution with continuous stirring at 37 oC for 1 h. Then, 180 µL of orange emitting Au NCs (575 nm) were added into the proceeding solution with continuous stirring at 37 oC for 4 h. Simultaneous detection of miRNA-21 and let-7a. 100 µL of P1 and 100 µL of P2 were mixed, and then 10 µL of 0.2 mg mL-1 PDANSs were added into the mixture at 37 oC to quench the fluorescence with 1 min of incubation. Then, the recovered fluorescence was recorded when 10 µL of DNase I (20 U) and different concentrations of miRNA-21 (0, 0.01, 0.05, 0.2, 0.5, 1.0, 2.0, 10, 50, 100 nM) and let-7a (0, 0.01, 0.05, 0.25, 0.5, 1.0, 2.5, 10, 50, 100 nM) were added and incubated for 60 min at 37 °C.

EXPERIMENTAL SECTION Chemicals. Dopamine hydrochloride, HAuCl4•3H2O (Hydrogen tetrachloroaurate trihydrate), DNase I, glutathione (GSH), proline (Pro) and isopropanol, Nhydroxylsuccinimide sodium salt (NHS) and 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Sigma-Aldrich (USA). DNA and miRNA sequences were synthesized by Sangon, and the sequences were shown in Table S1. Serum samples were provided by the Eighth Peoples’ Hospital of Qingdao with informed consent from the human subject. Additionally, the sample collection was approved by Institutional Review Board of Eighth Peoples’ Hospital of Qingdao.

RESULTS AND DISCUSSION Construction and characterization of the FRET-based platform. Au NCs (445 nm) and Au NCs (575 nm) were chosen as the energy donor and PDANSs were chosen as the energy acceptor for constructing the FRET-based platform. For a typical simultaneous detection of multiple targets FRETbased platform, it is of high significance to use a single excitation to reduce the background noise as well as improve sensitivities than by multiple excitations.13As shown in Figure 1A, the best emission peak intensity for both Au NCs (445 nm) and Au NCs (575 nm) was achieved when the excitation wavelength was 370 nm. Therefore, 370 nm was selected to be the optimum single excitation wavelength in the following experiments. TEM image revealed that the PDANSs (the time of polymerization is 48 h) were spherical and monodispered, with an average diameter of about 250 nm (Figure 1B), which was consistent with the dynamic light scattering (DLS) results (Figure S3E). In addition, the PDANSs with the average diameter 250 nm showed no aggregation even stored for two weeks, demonstrating their high stability (Figure S1). IR spectra revealed the characteristic peaks of monomer dopamine between 500 and 1700 cm−1 almost disappeared after polymerization,33 demonstrating the successful synthesis of PDANSs (Figure 1C). In addition, raman spectra also provided additional evidence of successful synthesis of PDANSs (Figure 1D), which was consistent with previous reports. 24,33

Instruments. All fluorescence spectra were recorded by an F-7000 fluorescence spectrometer (Hitach, Japan). The morphologies of all samples were characterized by a JEM-2100 high-resolution transmission electron microscope (HRTEM) with an accelerating voltage of 200 kV(Hitach, Japan). Infrared (IR) spectra were performed by using a FT/IR-410 Fourier transform infrared spectrophotometer (JASCO, Japan). Zeta potentials were measured by a Zetasizer Nano-ZS (Malvern Instruments, Malvern, UK). Confocal images were obtained by Olympus FV1000. Synthesis of PDANSs. PDANSs were prepared according to a previous report with some modfications.1724 In brief, 0.1 g dopamine hydrochloride was added to a mixture of 100 mL Tris-buffer (10 mM) and 40 mL isopropyl alcohol with continuous stirring for 48 h in dark. Then, the final solution was centrifuged and washed/resuspended with water for five times. At last, the precipitate was dried for further experiments. Synthesis of blue emitting Au NCs (445 nm) and orange emitting Au NCs (575 nm). Blue emitting Au NCs (445 nm) and orange emitting Au NCs (575 nm) were

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Analytical Chemistry

Figure 1. (A) Fluorescence emission spectra of the mixtures of Au NCs (445 nm) and Au NCs (575 nm) with different excitation wavelengths. Inset shows the photographs of the Au NCs (445 nm) and Au NCs (575 nm) under UV light illumination. (B) TEM image of PDANSs. (C) IR spectra of (a) PDANSs and (b) dopamine. (D) Raman spectra of (a) PDANSs and (b) dopamine.

Figure 2. (A) The absorption spectrum of PDANSs and the emission fluorescence spectra of Au NCs (445 nm) and Au NCs (575 nm). (B) TEM image of Au NCs (445 nm) and Au NCs (575 nm) adsorbed onto the PDANSs. (C) IR spectra of (a) the AuNCs (445nm), (b) AuNCs(445 nm)/CmiRNA-21DNA and (c) AuNCs(445 nm)/CmiRNA-21DNA/EDC-NHS. (D) IR spectra of (a) the AuNCs(575 nm), (b) AuNCs(575 nm)/Clet7aDNA and (c) AuNCs(575 nm)/Clet-7aDNA/EDC-NHS.

In order to verify the feasibility of the FRET-based platform, the absorption and the fluorescence experiments were performed. As shown in Figure 2A, the absorption spectrum of PDANSs and the emission fluorescence spectrum of Au NCs (445 nm) and Au NCs (575 nm) overlapped very well, demonstrating the feasibility of energy transfer from Au NCs to PDANSs. Then, CmiR-21DNA and Clet-7aDNA were modified on the Au NCs (445 nm) and Au NCs (575 nm) by the condensation reaction with EDC/NHS, respectively. Evidence for successful chemical modification was confirmed by IR spectroscopy. As shown in Figure 1C and D, the absorption intensity of the characteristic peaks at 1555 cm−1 and 1626 cm−1, which are ascribed to the N-H deformation and C=O stretching vibration, were remarkably increased, suggesting the successful attachment of CmiR-21DNA and Clet7aDNA on the surface of Au NCs (445 nm) and Au NCs (575 nm), respectively.34,35 Furthermore, the decreased zeta potentials of CmiR-21DNA and Clet-7aDNA functionalized Au NCs (445 nm) and Au NCs (575 nm) after the modification further confirmed the successful surface functionalization (Figure S2). Therefore, Au NCs can adsorb onto the surface of PDANSs due to the π−π stacking interactions between ssDNAs and PDANSs, resulting in the fluorescence quenching.36 TEM images in Figure 2B clearly verify that the modified Au NCs adsorb onto the surface of PDANSs.

In addition, we found that the fluorescence quenching efficiency of PDANSs was related to their particle average diameter. As the average diameter of PDANSs can be controlled by controlling polymerization time, we synthesized six kinds of PDANSs with the average diameter of 65 nm, 90 nm, 135 nm, 184 nm, 250 nm and 325 nm, respectively by controlling polymerization time as 8 h, 12 h, 24 h, 36 h, 48 h and 72 h, respectively (Figure 3A to F). DLS results also demonstrate the good monodispersity of the differently sized PDANSs (Figure S3), which is consistent with the TEM results. As shown in Figure 3G, PDANSs with the average diameter 250 nm exhibited the best fluorescence quenching ability than other ones. Besides, compared with the common quenching materials such as GO, CNTs and MoS2, the fluorescence quenching ability of PDANSs with the average diameter 250 nm was better than CNTs and MoS2, comparable to GO (Figure 3H).

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presence of the DNase I (lane 5 and lane 6). However, no obvious hydrolysis of the probe DNA was observed (lane 7) when mixed with PDANSs. On the contrary, when the probe DNA was mixed with its complementary miRNA, the probe DNA was completely digested even in the presence of PDANSs (lane 8), demonstrating that the probe DNA-miRNA complex had desorbed from the surface of PDANSs, and digested by DNase I. The above experiments revealed that the probe DNA could be protected from DNase I digestion when adsorbed on the surface of PDANSs. Additionally, the amounts of added PDANSs, the incubation time for fluorescence quenching, the amounts of DNase I and the incubation time for fluorescence recovery were optimized. At last, 0.2 mg/mL of PDANSs (Figure S5A and 5C), 1 min of incubation time (Figure S5B and 5D) for fluorescence quenching, 20 U of DNase I (Figure S6) and 60 min of incubation time (Figure S7) for fluorescence recovery were selected as the optimal conditions in the following experiments.

Figure 3. (A) TEM image of PDANSs with the average diameter (A) 65 nm, (B) 90 nm, (C) 135 nm, (D) 184 nm, (E) 250 nm and (F) 325 nm, respectively. (G) Fluorescence intensity of the Au NCs after adding the same concentrations of PDANSs with different average diameter (using Au NCs 445 as a model)

Principle of the FRET-based platform for the assay of multiple miRNAs. The mechanism of this FRET-based platform for dual color assay of multiple miRNAs with DNase I-assisted target recycling signal amplification was illustrated in Figure 4A. Firstly, the probes (P1+P2) displayed low fluorescence intensity after incubation with PDANSs, indicating the efficient adsorption and quenching ability of PDANSs (Figure 4B-a). After adding target miRNAs (miRNA-21 and let-7a), blue and orange fluorescence simultaneously recovered at 445 nm and 575 nm, respectively (Figure 4B-c). Therefore, simultaneous detection of multiple miRNAs can be achieved by monitoring the change of the recovered fluorescence intensity at 445 nm and 575 nm, respectively. Without target miRNAs, the presence of DNase I led to no fluorescence recovery (Figure 4B-b). By contrast, in the presence of both DNase I and target miRNAs, the probes (P1+P2) hybridized with target miRNAs and desorbed from the surface of PDANSs. The probes (P1+P2) were then digested by DNase I, followed by the release of target miRNAs to combine another probes and repeat the cyclic cleavage reaction, leading to a prominent fluorescence enhancement, which confirmed DNase I-assisted target recycling signal amplification. It is noteworthy that addition of DNase I to PDANSs+Probe does not result in an increase in fluorescence signals [by comparing Curves (a) and (b) in Figure 4B]. Previous reports have demonstrated a singlestranded DNA probe adsorbed on PDANSs can be effectively protected from enzymatic cleavage.37 Therefore, we investigated the protective properties of PDANSs for the probe DNA by using polyacrylamide gel electrophoresis. As shown in Figure S4, the probe DNA without (lane 4) or with (lane 6) target miRNA was completely digested by DNase I. Because DNase I can only catalyze the hydrolysis of DNA, and miRNA remained intact in the samples, in spite of the

Figure 4. (A) Schematic illustration of the FRET-based platform for dual color assay of multiple miRNAs with D Nase I-assisted target recycling signal amplification. (B) Fluorescence emission spectra of the probe (P1+P2) under different conditions.

Simultaneous detection of miRNA-21 and let-7a. The concentration of the target miRNAs are related to the degree of fluorescence recovery. Therefore, the miRNA-21 and let-7a can be monitored by tracking the recovered fluorescence signal at wavelengths of 445 nm and 575 nm. As shown in Figure 5A, in the presence of 20 U DNase I, with the increasing concentration of miRNA-21 and let-7a, the fluorescence intensities at 445 nm and 575 nm simultaneously increased with excellent linear correlations, respectively. The fluorescence intensity was found to be linear with the

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Analytical Chemistry concentration of miRNA-21 in the range of 0.01-2.0 nM with a detection limit of 4.2 pM, and linear with the concentration of let-7a in the range of 0.01-2.5 nM with a detection limit of 3.6 pM. The detection limit of miRNA-21 and let-7a was almost 20 times lower than that in the absence of DNase I (20 U) (Figure 5B and C). As a new miRNA-21 and let-7a sensing approach, the limit detection of this proposed assay is comparable to, or even lower than those of previously reported approaches.38-40 It is worth noting that visualization detection can also be evidently realized by this FRET-based platform. Figure 5D shows the photos of semi-quantitative determination of miRNA-21 and let-7a, respectively and simultaneously (under UV illumination). When miRNA-21 was added to the fluorescent quenched solution, the recovered blue color increased rapidly with increasing the concentration of miRNA-21 (Figure 5D-a). Similarly, when let-7a was added to the fluorescent quenched solution, the recovered orange color increased rapidly with increasing the concentration of let-7a (Figure 5D-b). If both miRNA-21 and let-7a were added to the fluorescent quenched solution, the fluorescence emission of the two distinguishing colored Au NCs (445 nm) and Au NCs (575 nm) increased simultaneously, and the solution exhibited their mixed color (Figure 5D-c).

differentiate these two microRNAs by different colored fluorescence recovery emission. As shown in Figure 6A, when miRNA-21 alone was added to the quenched solution, increasing blue fluorescence emission at 445 nm was observed. Similarly, when let-7a alone was added to the quenched solution, only orange fluorescence emission at 575 nm was increased (Figure 6B). Therefore, this FRET-based platform can easily and effectively be used to differentiate miRNA-21 and let-7a. Furthermore, the specificity for miRNA-21 and let-7a detection was inveatigated by introducing three kinds of miRNA sequences, including complementary miRNA (miR-21 and let-7a), one-base mismatched miRNA (S1 for miR-21 and let-7f for let-7a) and non-complementary miRNA (miR-16 and miR-155 for both miR-21 and let-7a). The results shown in Figure 6C revealed that the proposed FRET-based platform can distinguish the complementary miRNA from one-base mismatched and non-complementary miRNA specifically by the DNase I-assisted target recycling signal amplification method, demonstrating excellent specificity of the assay for miRNA-21 and let-7a. (sequences of S1, let-7f, miR-16, and miR-155 were shown in Table S1)

Figure 6. (A) Fluorescence spectra of the recovered fluorescence with the increasing concentrations of (A) pure miRNA-21 and (B) pure let-7a. (C) Influence of 500 nM S1, let-7f, miRNA-16 and miRNA-155 on the recovered fluorescence compared with that of miRNA-21 (100 nM) and let-7a (100 nM)

Figure 5. (A) Fluorescence spectra of the mixture of P1, P2 and PDANSs with increasing concentration of miRNA-21 and let-7a (in the presence of 20 U DNase I). (B) Linear curve for miRNA21 detection in the presence (solid line) and absence (dotted line)of 20 U DNase I. (C) Linear curve for let-7a detection in the presence (solid line) and absence (dotted line)of 20 U DNase I. (D) Photograph of semi-quantitative determination of miRNA-21 and let-7a, respectively, and simultaneously upon under UV illumination. (a) The fluorescence quenched mixtures added with various amounts of miRNA-21 (from left to right): 0, 20, 50, 75 and 100 nM. (b The fluorescence quenched mixtures added with various amounts of let-7a (from left to right): 0, 20, 50, 75 and 100 nM. (c) The fluorescence quenched mixtures added with various amounts of miRNA-21 and let-7a. The concentrations of miRNA-21 were 100, 75, 50, 20 and 0 nM, and the concentrations of let-7a were 0, 20, 50, 75 and 100 nM (from left to right).

Visual discrimination between serums from breast cancer patients and healthy people. Considering the significance of multiple miRNAs analysis in complex biological samples, we further explore this FRET-based platform to distinguish between serums from breast cancer patients and healthy people. The purpose of the serum test is to qualitatively differentiate between breast cancer and healthy human serums by confocal microscope images. Here, serum samples were obtained from late stage of breast cancer patients which have already been diagnosed by the standard method of chemiluminescent microparticle immunoassay in hospital. Serums from healthy people and breast cancer patients (20 µL) were added to 200 µL of fluorescence quenched mixtures of P1, P2 and PDANSs at 37 °C (in the presence of 20 U DNase I), respectively. After 60 minutes, confocal microscope images

Cross-reaction analysis and selectivity. Before simultaneous detection of miRNA-21 and let-7a, it is necessary to investigate whether this FRET-based platform can

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demonstrate the obvious differences. Compared with serums from healthy people, the level of let-7a and miR-21 in serums of breast cancer patients simultaneously increased.41 Therefore, blue and orange fluorescene can be observed after adding serums of breast cancer patients to the proposed platform (Figure 7B). However, no obvious fluorescene can be observed after adding serums of healthy people (Figure 7A). Thus, breast cancer and healthy human serums can be qualitatively differentiated. In addition, we also use a fluorimeter to provide quantitative measurements of the fluorescence intensity of the samples from the healthy subjects and patients. As shown in Figure S8, the recovered fluorescence intensities at 445 nm and 575 nm were both obviously increased after adding serums of breast cancer patient to the proposed platform. However, no obvious recovered fluorescene intensities were observed after adding serums of healthy people (Here, the data shown in Figure S8 were the average values of the five sets of data for five healthy people and five late-stage breast cancer patients, respectively). Moreover, determination of the concentrations of miR-21and let-7a in serums from healthy people and breast cancer patient were investigated by standard adding method (Figure S9), detailed discussions were shown in supporting information. Hence, this visual discrimination attributed to the increasing concentration of miRNA-21 and let-7a in breast cancer patients’ serums. Consequently, this FRET-based platform provides an auxiliary strategy for discriminating between breast cancer patients and healthy people, showing beneficial prospect for auxiliary clinical diagnosis of breast cancer.

contribute to multiple tumor markers detection and cancer diagnostics.

ASSOCIATED CONTENT Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

Fax: +86-532-84022681 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors gratefully acknowledge the support from the National Nature Science Foundation of China (21675093, 21505081), the Doctoral Found of QUST (010022832) and the Taishan Scholar Program of Shandong Province, China (ts20110829).

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Figure 7. Laser confocal microscope images of the fluorescence quenched mixtures of P1, P2 and PDANSs in the presence of serums from (A) healthy people and (B) breast cancer patients.

CONCLUSIONS In summary, a novel PDANSs/Au NCs based FRET nanoplatform with DNase I assisted target recycling amplification for dual color simultaneous detection of multiple tumor related microRNAs was developed. Here, PDANSs are used as energy receptor, while two kinds of dual colored Au NCs are used as energy donors. The two contrasting colored fluorescence signals can be simultaneously monitored without spectra overlap by a single wavelength excitation, and the simultaneous detection of miRNA-21 and let-7a can be easily realized. Besides, semi-quantitative determination of miRNA21 and let-7a by photo visualization can also be realized. Furthermore, without any sample pre-treatment, the proposed strategy can provide auxiliary method for rough diagnosis of breast cancer. It is expected that the present study may greatly

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