Gold Nanocluster (Au NC)-Based

Feb 28, 2018 - A novel fluorescence resonance energy transfer (FRET)-based platform using polydopamine nanospheres (PDANSs) as energy acceptors and du...
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Article Cite This: Anal. Chem. 2018, 90, 4039−4045

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Polydopamine Nanosphere/Gold Nanocluster (Au NC)-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, and 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 S Supporting Information *

ABSTRACT: A novel fluorescence resonance energy transfer (FRET)-based platform using polydopamine nanospheres (PDANSs) as energy acceptors and dual colored Au NCs as energy donors for simultaneous detection of multiple tumorrelated 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 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 and 3.6 pM (3σ) for miRNA-21 and let-7a, which was almost 20 times lower than that without DNase I. Additionally, semiquantitative determination of miRNA-21 and let-7a can also be realized through photovisualization. Most importantly, serums from normal and breast cancer patients can be visually and directly discriminated without any sample pretreatment by confocal microscope experiments, demonstrating promising potential for auxiliary clinical diagnosis.

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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 several decades. A series of nanomaterials including graphene oxide (GO), carbon nanotubes (CNTs), gold nanoparticles (AuNPs), MoS2 nanosheets, and recently developed metal− organic 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, a complicated preparation process, or cytotoxicity, restricting their potential in real applications. In addition, traditional energy donors such as organic dyes or semiconductor quantum dots (QDs) also suffered from non-negligible shortcomings, including poor photostability, easy photobleaching, or high toxicity.23 Thus, the development of new effective energy donor and acceptor pairs is of high significance to overcome these disadvantages. As a kind of biopolymerized material with tunable diameters,

he 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 a 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 miRNA 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 and expensive instrumentation still restrict their further application.11 Recently, fluorescent methods for multiple tumor-related miRNA 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 is inevitable. Therefore, simultaneous fluorescent © 2018 American Chemical Society

Received: December 16, 2017 Accepted: February 28, 2018 Published: February 28, 2018 4039

DOI: 10.1021/acs.analchem.7b05253 Anal. Chem. 2018, 90, 4039−4045

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Analytical Chemistry

Synthesis of PDANSs. PDANSs were prepared according to a previous report with some modfications.24 In brief, 0.1 g of dopamine hydrochloride was added to a mixture of 100 mL of Tris-buffer (10 mM) and 40 mL of isopropyl alcohol with continuous stirring for 48 h in the 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 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 boiling 4.0 mL solution of 3.0 mM HAuCl4 under vigorous agitation for 10 min and then cooled down to room temperature, and 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 of HAuCl4 were added to 43.5 mL of ultrapure water. The mixture was stirred at 70 °C for 24 h, and the orange emitting Au NCs (575 nm) were obtained. 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 °C for 1 h. Then, 180 μL of blue emitting Au NCs (445 nm) were added into the proceeding solution with continuous stirring at 37 °C 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 °C for 1 h. Then, 180 μL of orange emitting Au NCs (575 nm) were added into the proceeding solution with continuous stirring at 37 °C for 4 h. Simultaneous Detection of miRNA-21 and let-7a. First, 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 °C 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.

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 et al. have utilized polydopamine coated gold nanoparticles 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 proven to be good candidates as FRET energy donors for biosensing.11 Moreover, with the help of enzyme-assisted target recycling, the detection signal can be effectively amplified, which is beneficial for improving the detection sensitivity.29−31 To the best of our knowledge, the integration of polydopamine nanospheres with multicolored Au NCs for developing FRET-based platforms for simultaneous detection of multiple miRNAs has not been explored yet, which includes the combination of FRET-based platforms with enzyme-assisted target recycling amplification. Herein, we develop a novel PDANSs/Au NCs-based FRET nanoplatform with DNaseI-assisted target recycling amplification for dual color simultaneous detection of multiple tumorrelated microRNAs. Compared with the traditional FRETbased 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 that of carbon nanotubes (CNTs) and molybdenum disulfide (MoS2) and comparable to that of 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 when 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.

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EXPERIMENTAL SECTION Chemicals. Dopamine hydrochloride, HAuCl4·3H2O (hydrogen tetrachloroaurate trihydrate), DNase I, glutathione (GSH), proline (Pro), isopropanol, N-hydroxylsuccinimide sodium salt (NHS), and 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC) were purchased from Sigma-Aldrich (U.S.A.). DNA and miRNA sequences were synthesized by Sangon, and the sequences are 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 the Institutional Review Board of Eighth Peoples’ Hospital of Qingdao. Instruments. All fluorescence spectra were recorded by an F-7000 fluorescence spectrometer (Hitach, Japan). The morphologies of all samples were characterized by a JEM2100 high-resolution transmission electron microscope (HRTEM) with an accelerating voltage of 200 kV(Hitach, Japan). Infrared (IR) spectra were acquired by using a FT/IR410 Fourier transform infrared spectrophotometer (JASCO, Japan). Zeta potentials were measured by a Zetasizer Nano-ZS (Malvern Instruments, Malvern, U.K.). Confocal images were obtained by an Olympus FV1000.

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 donors and PDANSs were chosen as the energy acceptors for constructing the FRET-based platform. For a typical simultaneous detection of multiple targets in a FRET-based platform, it is of high significance to use a single excitation to reduce the background noise as well as improve sensitivities as opposed to multiple excitations.13 As 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 an average diameter of 250 nm showed no aggregation even after being stored for 2 weeks, demonstrating their high stability (Figure S1). IR spectra revealed that the characteristic peaks of monomer dopamine between 500 and 4040

DOI: 10.1021/acs.analchem.7b05253 Anal. Chem. 2018, 90, 4039−4045

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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,D, the absorption intensity of the characteristic peaks at 1555 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 Clet‑7aDNA 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 average particle diameter. As the average diameter of PDANSs can be controlled by controlling polymerization time, we synthesized six kinds of PDANSs with average diameters of 65, 90, 135, 184, 250, and 325 nm, respectively, by varying the polymerization time from 8, 12, 24, 36, 48, to 72 h, respectively (Figure 3A−F). DLS results also demonstrate the good monodispersity

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.

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 the successful synthesis of PDANSs (Figure 1D), which was consistent with previous reports.24,33 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

Figure 3. (A) TEM image of PDANSs with average diameters of (A) 65, (B) 90, (C) 135, (D) 184, (E) 250, and (F) 325 nm, respectively. (G) Fluorescence intensity of the Au NCs after adding the same concentrations of PDANSs with different average diameters (using Au NCs 445 as a model). Figure 2. (A) 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 (445 nm), (b) AuNCs(445 nm)/C miRNA‑21 DNA, and (c) AuNCs(445 nm)/ CmiRNA‑21DNA/EDC-NHS. (D) IR spectra of (a) the AuNCs(575 nm), (b) AuNCs(575 nm)/Clet‑7aDNA, and (c) AuNCs(575 nm)/ Clet‑7aDNA/EDC-NHS.

of the differently sized PDANSs (Figure S3), which is consistent with the TEM results. As shown in Figure 3G, PDANSs with an average diameter of 250 nm exhibited the best fluorescence quenching ability than the other ones. Also, compared with the common quenching materials such as GO, CNTs, and MoS2, the fluorescence quenching ability of PDANSs with an average diameter of 250 nm was better 4041

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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, miRNA remained intact in the samples, in spite of the presence of the DNase I (lanes 5 and 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 was 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,C), 1 min of incubation time (Figure S5B,D) 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. 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 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 and

than that of CNTs and MoS2 and comparable to GO (Figure 3H). Principle of the FRET-Based Platform for the Assay of Multiple miRNAs. The mechanism of this FRET-based platform for the dual color assay of multiple miRNAs with DNaseI-assisted target recycling signal amplification was illustrated in Figure 4A. First, the probes (P1+P2) displayed

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

low fluorescence intensity after incubation with PDANSs, indicating the efficient adsorption and quenching ability of PDANSs (Figure 4B-a). After adding target miRNAs (miRNA21 and let-7a), blue and orange fluorescence simultaneously recovered at 445 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 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 pair of probes and repeat the cyclic cleavage reaction, leading to a prominent fluorescence enhancement, which confirmed DNaseI-assisted target recycling signal amplification. It is noteworthy that the 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 that a single-stranded 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

Figure 5. (A) Fluorescence spectra of the mixture of P1, P2 and PDANSs with increasing concentrations of miRNA-21 and let-7a (in the presence of 20 U DNase I). (B) Linear curve for miRNA-21 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 semiquantitative 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). 4042

DOI: 10.1021/acs.analchem.7b05253 Anal. Chem. 2018, 90, 4039−4045

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Furthermore, the specificity for miRNA-21 and let-7a detection was investigated by introducing three kinds of miRNA sequences, including complementary miRNA (miR21 and let-7a), one-base mismatched miRNA (S1 for miR-21 and let-7f for let-7a), and noncomplementary 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 noncomplementary miRNA specifically by the DNaseI-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) Visual Discrimination between Serums from Breast Cancer Patients and Healthy People. Considering the significance of multiple miRNA 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 the late stage of breast cancer patients, who have already been diagnosed by the standard method of a chemiluminescent microparticle immunoassay in the 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 min, confocal microscope images 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 fluorescence can be observed after adding serums from breast cancer patients to the proposed platform (Figure 7B). However, no obvious fluorescence can be

575 nm simultaneously increased with excellent linear correlations, respectively. The fluorescence intensity was found to be linear with the 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,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 semiquantitative 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 an increase in 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 an increase in 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). 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 differentiate these two microRNAs by different colored fluorescence recovery emission. As shown in Figure 6A, when miRNA-21

Figure 7. Laser confocal microscope images of the fluorescence quenched mixtures of P1, P2and PDANSs in the presence of serums from (A) healthy people and (B) breast cancer patients. Figure 6. (A) Fluorescence spectra of the recovered fluorescence with the increasing concentrations of (A) pure miRNA-21 and (B) pure let7a. (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).

observed after adding serums from 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 and 575 nm were both obviously increased after adding the serums from the breast cancer patient to the proposed platform. However, no obvious recovered fluorescence 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,

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. 4043

DOI: 10.1021/acs.analchem.7b05253 Anal. Chem. 2018, 90, 4039−4045

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(5) Chen, T.; Wu, C. S.; Jimenez, E.; Zhu, Z.; Dajac, J. G.; You, M.; Han, D.; Zhang, X.; Tan, W. Angew. Chem., Int. Ed. 2013, 52, 2012− 2016. (6) Peng, Y. F.; Yi, G. S.; Gao, Z. Q. Chem. Commun. 2010, 46, 9131−9133. (7) Zhou, W.; Tian, Y. F.; Yin, B. C.; Ye, B. C. Anal. Chem. 2017, 89, 6120−6128. (8) de Bang, T. C.; Shah, P.; Cho, S. K.; Yang, S. W.; Husted, S. Anal. Chem. 2014, 86, 6823−6826. (9) Ge, Z. L.; Lin, M. H.; Wang, P.; Pei, H.; Yan, J.; Shi, J. Y.; Huang, Q.; He, D. N.; Fan, C. H.; Zuo, X. L. Anal. Chem. 2014, 86, 2124− 2130. (10) Xu, Z. Q.; Liao, L. L.; Chai, Y. Q.; Wang, H. J.; Yuan, R. Anal. Chem. 2017, 89, 8282−8287. (11) Xu, S. H.; Feng, X. Y.; Gao, T.; Liu, G. F.; Mao, Y. N.; Lin, J. H.; Yu, X. J.; Luo, X. L. Anal. Chim. Acta 2017, 983, 173−180. (12) Pan, W.; Zhang, T.; Yang, H.; Diao, W.; Li, N.; Tang, B. Anal. Chem. 2013, 85, 10581−10588. (13) Pan, W.; Li, Y. L.; Wang, M. M.; Yang, H. J.; Li, N.; Tang, B. Chem. Commun. 2016, 52, 4569−4572. (14) Li, L.; Feng, J.; Liu, H. Y.; Li, Q. L.; Tong, L. L.; Tang, B. Chem. Sci. 2016, 7, 1940−1945. (15) Jin, Z. W.; Geißler, D.; Qiu, X.; Wegner, D.; Hildebrandt, N. Angew. Chem., Int. Ed. 2015, 54, 10024−10029. (16) Cui, L.; Lin, X. Y.; Lin, N. H.; Song, Y. L.; Zhu, Z.; Chen, X.; Yang, C. Y. J. Chem. Commun. 2012, 48, 194−196. (17) Lee, J. H.; Kim, J. A.; Jeong, S.; Rhee, W. J. Biosens. Bioelectron. 2016, 86, 202−210. (18) Sun, X. X.; Fan, J.; Zhang, Y. P.; Chen, H. L.; Zhao, Y. Q.; Xiao, J. X. Biosens. Bioelectron. 2016, 79, 15−21. (19) Qian, Z. S.; Shan, X. Y.; Chai, L. J.; Chen, J. R.; Feng, H. Chem. Eur. J. 2014, 20, 16065−16069. (20) Yang, Y. J.; Huang, J.; Yang, X. H.; Quan, K.; Xie, N. L.; Ou, M.; Tang, J. L.; Wang, K. M. Chem. Commun. 2016, 52, 11386−11389. (21) Zhu, C.; Zeng, Z.; Li, H.; Li, F.; Fan, C.; Zhang, H. J. Am. Chem. Soc. 2013, 135, 5998−6001. (22) Zhu, X.; Zheng, H.; Wei, X.; Lin, Z.; Guo, L.; Qiu, B.; Chen, G. Chem. Commun. 2013, 49, 1276−1278. (23) Wang, Y. H.; Gao, D. Y.; Zhang, P. F.; Gong, P.; Chen, C.; Gao, G. H.; Cai, L. T. Chem. Commun. 2014, 50, 811−813. (24) Qiang, W. B.; Li, W.; Li, X. Q.; Chen, X.; Xu, D. K. Chem. Sci. 2014, 5, 3018−3024. (25) Qiang, W. B.; Hu, H. T.; Sun, L.; Li, H.; Xu, D. K. Anal. Chem. 2015, 87, 12190−12196. (26) Choi, C. K. K.; Li, J. M.; Wei, K. C.; Xu, Y. J.; Ho, L. W. C.; Zhu, M. L.; To, K. K. W.; Choi, C. H. J.; Bian, L. M. J. Am. Chem. Soc. 2015, 137, 7337−7346. (27) Xu, S. H.; Lu, X.; Yao, C. X.; Huang, F.; Jiang, H.; Hua, W. H.; Na, N.; Liu, H. Y.; Ouyang, J. Anal. Chem. 2014, 86, 11634−11639. (28) Xu, S. H.; Gao, T.; Feng, X. Y.; Fan, X. J.; Liu, G. F.; Mao, Y. N.; Yu, X. J.; Lin, J. H.; Luo, X. L. Biosens. Bioelectron. 2017, 97, 203−207. (29) Ma, F.; Liu, W. J.; Zhang, Q. Y.; Zhang, C. Y. Chem. Commun. 2017, 53, 10596−10599. (30) Mao, Y.; Liu, M.; Tram, K.; Gu, J.; Salena, B. J.; Jiang, Y. Y.; Li, Y. F. Chem. - Eur. J. 2015, 21, 8069−8074. (31) Ren, W.; Zhang, Y.; Chen, H. G.; Gao, Z. F.; Li, N. B.; Luo, H. Q. Anal. Chem. 2016, 88, 1385−1390. (32) Xu, S. H.; Wu, Y. F.; Sun, X. M.; Wang, Z. Q.; Luo, X. L. J. Mater. Chem. B 2017, 5, 4207−4213. (33) Liu, Y. L.; Ai, K. L.; Liu, J. H.; Deng, M.; He, Y. Y.; Lu, L. H. Adv. Mater. 2013, 25, 1353−1359. (34) Liang, M. J.; Chen, Y. L.; Zhang, H. J.; Niu, X. Y.; Xu, L. F.; Ren, C. L.; Chen, X. G. Analyst 2015, 140, 6711−6719. (35) Zhuang, M.; Ding, C.; Zhu, A.; Tian, Y. Anal. Chem. 2014, 86, 1829−1836. (36) Lin, L. S.; Cong, Z. X.; Cao, J. B.; Ke, K. M.; Peng, Q. L.; Gao, J.; Yang, H. H.; Liu, G.; Chen, X. ACS Nano 2014, 8, 3876−3883. (37) Xie, Y.; Lin, X. Y.; Huang, Y. S.; Pan, R. J.; Zhu, Z.; Zhou, L. J.; Yang, C. Y. J. Chem. Commun. 2015, 51, 2156−2158.

determination of the concentrations of miR-21and let-7a in serums from healthy people and breast cancer patients 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 prospects for auxiliary clinical diagnosis of breast cancer.

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CONCLUSIONS In summary, a novel PDANSs/Au NCs-based FRET nanoplatform with DNaseI-assisted target recycling amplification for dual color simultaneous detection of multiple tumor-related microRNAs was developed. Here, PDANSs are used as an 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. In addition, semiquantitative determination of miRNA-21 and let-7a by photovisualization can also be realized. Furthermore, without any sample pretreatment, the proposed strategy can provide an auxiliary method for rough diagnosis of breast cancer. It is expected that the present study may greatly contribute to multiple tumor marker detection and cancer diagnostics.

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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.7b05253.

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Additional information as noted in text (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; Fax: +86-532-84022681. ORCID

Shenghao Xu: 0000-0003-3908-1525 Xiliang Luo: 0000-0001-6075-7089 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS 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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REFERENCES

(1) Giljohann, D. A.; Mirkin, C. A. Nature 2009, 462, 461−464. (2) Li, J. B.; Tan, S. B.; Kooger, R.; Zhang, C. Y.; Zhang, Y. Chem. Soc. Rev. 2014, 43, 506−517. (3) Li, L.; Feng, J.; Liu, H. Y.; Li, Q. L.; Tong, L. L.; Tang, B. Chem. Sci. 2016, 7, 1940−1945. (4) Giljohann, D. A.; Seferos, D. S.; Daniel, W. L.; Massich, M. D.; Patel, P. C.; Mirkin, C. A. Angew. Chem., Int. Ed. 2010, 49, 3280−3294. 4044

DOI: 10.1021/acs.analchem.7b05253 Anal. Chem. 2018, 90, 4039−4045

Article

Analytical Chemistry (38) Jin, Z. W.; Geißler, D.; Qiu, X.; Wegner, K. D.; Hildebrandt, N. Angew. Chem., Int. Ed. 2015, 54, 10024−10029. (39) Zhen, S. J.; Xiao, X.; Li, C. H.; Huang, C. Z. Anal. Chem. 2017, 89, 8766−8771. (40) Zhu, Y.; Qiu, D.; Yang, G.; Wang, M. Q.; Zhang, Q. J.; Wang, P.; Ming, H.; Zhang, D. G.; Yu, Y.; Zou, G.; Badugu, R.; Lakowicz, J. R. Biosens. Bioelectron. 2016, 85, 198−204. (41) Asaga, S.; Kuo, C.; Nguyen, T.; Terpenning, M.; Giuliano, A. E.; Hoon, D. S. B. Clin. Chem. 2011, 57, 84−91.

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DOI: 10.1021/acs.analchem.7b05253 Anal. Chem. 2018, 90, 4039−4045