Simple and Sensitive Quantification of MicroRNAs via PS@Au

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Simple and Sensitive Quantification of MicroRNAs via PS@Au Microspheres -Based DNA Probe and DSN -Assist Signal Amplification Platform Qian Zhao, Jiafang Piao, Weipan Peng, Yang Wang, Bo Zhang, Xiaoqun Gong, and Jin Chang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16733 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018

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ACS Applied Materials & Interfaces

Simple and Sensitive Quantification of MicroRNAs via PS@Au Microspheres -Based DNA Probe and DSN -Assist Signal Amplification Platform Qian Zhaoa‡, Jiafang Piaoa‡, Weipan Penga, Yang Wanga, Bo Zhanga, Xiaoqun Gonga*, Jin Changa*

a

School of Life Sciences, Tianjin University and Tianjin Engineering Center of Micro-Nano

Biomaterials and Detection-Treatment Technology (Tianjin), Tianjin 300072, China

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Identifying the microRNA (miRNA) expression level can provide critical information for early diagnosis of cancers or monitoring the cancer therapeutic efficacy. This paper focused on a kind of gold nanoparticles coated polystyrene microbeads (PS@Au microspheres)-based DNA probe as miRNA capture and duplex specific nuclease (DSN) signal amplification platform based on RGB value read-out for miRNAs detection. In virtue of the outstanding selectivity and simple experimental operation, 5′- fluorochrome -labeled molecular beacons (MBs) were immobilized on PS@Au microspheres via their 3′-thiol, in the wake of the fluorescence quenching by nanoparticle surface energy transfer (NSET). Target miRNAs were captured by the PS@Au microspheres -based DNA probe through DNA/RNA hybridization. DSN enzyme subsequently selectively cleaved the DNA to recycle the target miRNA and release of fluorophores, thereby triggering the signal amplification with more free fluorophores. The RGB value measurement enabled a detection limit of 50 fM, almost 4 orders of magnitude lower than PS@Au microspheres-based DNA probe detection without DSN. Meanwhile, by different dyes encoding, miRNA-21 and miRNA-10b were simultaneously detected in the same sample. Considering the ability for quantitation, high sensitivity and convenient merits, the PS@Au microspheres -based DNA probe and DSN signal amplification platform supplied valuable information for early diagnosis of cancers.

KEYWORDS: miRNA, PS@Au microspheres, MBs, DSN, signal amplification


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INTRODUCTION As a typical kind of small-sized (approximately 22 nucleotides) noncoding RNA molecules, microRNAs (miRNAs), have important implications as regulators in post-transcriptional for gene expression.1, 2 The aberrant (up- or down-regulated) expression of miRNAs often associated with the occurrence of tumor progression and metastasis.3-5 Proverbially, the pervasive and fatal diseases of cancer robbed millions of lives on a world scale.6 Early diagnosis of cancer is of great value in improving the survival rate of cancer patients, and biomarkers detection plays an important role.7, 8 Nowadays, identifying the amounts of biomarkers in genetic level performs important functions in understanding the biological processes and shows great promise for early diagnosis of cancers or monitoring the cancer therapeutic efficacy.9, 10 Among them, miRNA-21 and miRNA-10b are often found overexpressed in breast, liver, and pancreatic cancers, so the quantification of miRNAs expression is adopted for the diagnosis and monitoring the therapy of cancer.11 Northern blotting,12, 13 quantitative reverse transcription polymerase chain reaction (qRT-PCR)14, 15

and oligonucleotide microarray16,

17

as conventional strategies, are broadly applied in the

detection of miRNAs. Nevertheless, the major drawbacks for these assays, such as needing trained personnel with an expensive apparatus (qRT-PCR), complicated procedures (qRT-PCR, Microarray, Northern blotting), long assay time (Microarray), low sensitivity (Microarray, Northern blotting) and poor reproducibility (Microarray), triggered the development of new approaches for convenient (e.g. no complex separation steps), sensitive, selective and easy operation (without complicated preparation procedures) for multiplexed (simultaneous measurement of several miRNAs) detection. In recent years, a number of alternative sensing assays with improved sensitivity, selectivity and convenience based on the incorporation of nanomaterials have attracted prominent interest. For instance, quantum dot,18 carbon nanotubes,19 magnetic microparticle,20 Fe3O4@Ag core-shell microspheres,21 gold-nanoparticles (AuNPs),22 silver nanoclusters,23 and so on, which have improved detection sensitivity with shortened assay time. Among them, as a promising candidate, AuNPs could effectively trigger the strong fluorescence quenching of fluorescent molecules due to the nanoparticle surface energy transfer (NSET) phenomenon.24, 25 Molecular beacons (MBs) is a kind of oligonucleotide sequences, which consist of stem-loop structure and fluorophore-quencher



pair.26 The shouting distance between fluorophore and quencher initiated the quenching of fluorescence through fluorescence resonance energy transfer (FRET). The addition of target molecule, which could hybridize with the complementary loop sequence of the MBs, formed stronger intermolecular hybridization and forced the open of hairpin structure, accompanied by the remarkable restoration of fluorescence. Depending on the outstanding selectivity and simple experimental operation, the MBs is widely used in the gene sequencing.27 In view of above-mentioned of AuNPs triggered-NSET phenomenon, the AuNPs are often employed to supersede the quencher to proceed the quench of fluorophore. Recently, in order to improve the detection sensitivity of miRNAs, duplex-specific nuclease (DSN), as a promising candidate, has been intensely employed to ameliorate signal amplification effects in miRNA analysis, profiting from the preference of selectively cleaving DNA in DNA-RNA heteroduplexes or DNA-DNA duplex but keeping the RNA strand in perfect condition.28 The released miRNAs again formed a new heteroduplex structure and triggered a next round of target cyclic reaction. Due to the signal amplification through DSN-assisted target recycling, it has been widely introduced to incorporate with different types of nano-biosensors, such as magnetic relaxation switch sensors,20 colorimetric sensors,29 electrochemical sensors,30 fluorescence sensors31 and so on. Moreover, the cleavage function for DNA just occurred when there were perfectly matched duplexes with at least 12 nucleotides, resulting in an excellent specificity for the assays.32 In general, the DSN-based miRNAs assays have superiority regarding sensitivity and specificity. Federica et al firstly combined AuNP and DSN for microRNA detection strategy for the absolute quantification of miRNAs based on enzymatic processing of DNA-functionalized gold nanoparticles (AuNPs), with concomitant development of a fluorescence signal, resulting in the LOD of 0.2 fM.22 In this paper, we constructed a kind of PS@Au probe combining the advantage of MBs (excellent selectivity and simplified experimental procedure) and PS@Au microbeads (three dimensional microbeads provide a high capacity for loading MBs and strong NSET between surface AuNPs and fluorescent molecules), as well as the DSN-based signal amplification for one-step detection of multiple miRNAs (miRNA-21 and miRNA-10b) detection. The generated AuNPs on the surface of the PS microbeads through the reduction of chloroauric acid by polydopamine (PDA), performed not merely as the linker to immobilize the MBs on the surface of


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with 3′-thiol through Au-S bond, but also as the fluorescence quencher to realize turn-off fluorescence of 5′- fluorochrome -labeled MBs. The PS microbeads provided a high capacity to carry numerous AuNPs to load more MBs and be helpful for the improvement of detecting sensitivity. When the target miRNAs were present, DNA-RNA heteroduplexes formed and DSN started to circularly cleave the DNA, leading to the separation of numerous fluorophores from surface AuNPs with turn-on fluorescence. The released fluorophores were characterized by RGB read-out for the quantitative detection of miRNA-21, as well as the different dye coded miRNA-10b in one step. So, our system may allow for the high sensitive and convenient detection of miRNAs without complex separation and multiple assay steps.

EXPERIMENTAL SECTION Materials and Instrumentation. Polystyrene microspheres (1.7 µm in diameter, PS microspheres) were obtained from BaseLine (Tianjin, China). Tris-(2-carboxyethyl) phosphine hydrochloride (TCEP), sodium dodecyl sulfate (SDS), Hydrogen tetrachloroaurate (III) (HAuCl4·4H2O) and sodium citrate were purchased from Sigma-Aldrich. Dopamine, phosphate buffer (PB, pH 7.4), phosphate buffered saline (PBS, pH 7.4), potassium chloride (KCl), mercaptoethanol

(2-ME),

magnesium

chloride

(MgCl2),

sodium

chloride

(NaCl),

Tris(hydroxymethyl)aminomethane (Tris), Tris-HCl buffer and hydroxylamine hydrochloride (NH2OH-HCl) were obtained from Aladdin Industrial Corporation. Duplex-specific nuclease (DSN), master buffer and stop solution were provided from Evrogen Joint Stock Company. The deionized water with a resistivity of 18.2 MΩ-cm was employed in the experiment. HPLC-purified, synthetic oligonucleotides used in this work were purchased from Sangon Biotech (Shanghai, China). Diethy pyrocarbonate (DEPC) treated water was used in the preparation of aqueous solutions. Base sequences of the oligonucleotides are listed in Table S1. The signal was acquired by Azure C600 (the Azure Biosystems, Inc.) Synthesis of the Composite Microspheres: Gold Nanoparticles Coated Polystyrene Microbeads (PS@Au Microspheres). To prepare PS@Au microspheres, firstly, 5 mL of as-prepared dopamine solution (1 mg/mL) was mixed with 5 mL of PS microspheres (2 mg/mL) under vigorous stirring for 1 h at room temperature. Secondly, 1 mL of Tris solution (110 mM) was added into the mixture and keeping stirred for other 3 h. The products were washed three



times at 8 000 rpm for 8 min and re-dispersed in 10 mL water, followed by adding 48 µL HAuCl4·4H2O (20 mM) and under vigorous stirring overnight. The above washing steps were repeated. Finally, 20 mM 120 µL HAuCl4·4H2O and 20 mM 120 µL NH2OH-HCl solutions were added and incubation for 20 min. Purified by centrifugation at 8000 rpm for 8 min for three times to remove impurities so as to obtain the PS@Au microspheres solution. PS@Au Microspheres -Based DNA Probe (PS@Au Probe). Prior to labeling on the surface of PS@Au microspheres of the oligonucleotide, the thiol groups were activated by the freshly prepared TCEP for 1 min.33 20 µL of 10 µΜ MBs (Cy5 modified on 5′ and thiol modified on 3′) were activated according to the freshly prepared TCEP (20 µL 1 mM) for 1 h, and then was added into the solution of 500 µL PS@Au microspheres with the final concentration of 5 mg/mL PS@Au. After 4 h, SDS solution and PB buffer (pH 7.4) were also introduced into the mixture. Aqueous NaCl (2.0 M with 0.01M PBS) was then added batch-wisely to bring its concentration gradually increased to 0.3 M. The mixed solution was again incubated overnight to achieve an efficient coupling between the MBs and PS@Au microspheres. The final products were purified and suspended in the hybridization buffer. Feasibility Experiment. In order to verify whether the MBs-21 successfully attached to PS@Au microspheres, 2 µL mercaptoethanol was added and the detection signals were monitored using an Azure C600 device after 20 min. 10 µL miRNA-21 (10-8 M) and 0.2 µL DSN in 1 µL of 10 × DSN buffer was added into 20 µL PS@Au microspheres -based DNA probe respectively. The solution was incubated at 45 °C for 60 min and 5 µL stop solution (EDTA) was added as terminators for the reaction. The signals were monitored using an Azure C600 device. Target miRNA-21 Detection with DSN-Amplified PS@Au Probe -Based Sensor. 10 µL miRNA-21 was mixed with 20 µL PS@Au probe, with various final concentrations of the targets of 1×10-6 M to 1×10-11 M. Then incubation for 1 h at 45 °C and signals acquisition was measured. 10 µL of various concentration of miRNA-21 (final concentration, 5×10-9 M-5×10-14 M) was added into the mixture solution (total volume of 21.2 µL) consists of 20 µL of PS@Au probe, 1 µL of 10 × DSN buffer (50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, and 1 mM DTT), 0.2 µL 1 U/µL DSN in 50% glycerol. The solution was incubated at 45 °C for 60 min, allowing for the target miRNA recycling assisted by DSN. Finally, 5 µL stop solution (EDTA) was added as terminators for the reaction. The detection signals were monitored.


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Optimization of Temperature and Time of Reaction. The reaction temperature and time of the DSN-amplified PS@Au probe sensor was evaluated under various operating temperatures (25 °C, 35 °C, 45 °C, 55 °C and 70 °C) and time (0 min, 24 min, 40 min, 90 min and 120 min) by through the detection of miRNA-21(1×10-10 M) as an example. The RGB value after reaction was recorded to obtain an optimized temperature and time for the sensor. Specificity Investigation of the Sensor. The specificity experiments were performed by incubating 5 µL miRNA-21 (1×10-10 M) and different kinds of miRNAs (NC miRNA-21, miRNA-200b, Let-7d, miRNA-141) at the same concentration of 1×10-9 M with 20 µL PS@Au probe, allowing for the miRNAs recycling assisted by DSN and the detection signals were monitored. Base sequences of those miRNAs are listed in Table S1. Detection of MiRNA-21 and MiRNA-10b Under Their Coexistence in One Sample. A series of miRNA-21standard solutions (5 µL with concentration from 5×10-13 M to 5×10-9 M) with a certain amount of miRNA-10b (5 µL, 1×10-10 M) were added in the mixture solution (total volume of 21.2 µL) consisting 20 µL of PS@Au probe, 1 µL of 10 × DSN buffer and 0.2 µL of 1 U/µL DSN enzyme. Meanwhile, a series of miRNA-21 standard solutions (10 µL with concentration from 5×10-13 M to 5×10-9 M) without miRNA-10b were added in the mixture solution. The final solution was incubated at 45 °C for 60 min, the detection signals were monitored by an Azure C600 device after the addition of stop solution. MBs-10b (Cy3 modified on 5′ and thiol modified on 3′) were used for the preparation of PS@Au microspheres -based DNA probe for the detection of miRNA-10b, according to the preparation of probe for the detection of miRNA-21 as mentioned above. A series of miRNA-10b standard solutions (5 µL with concentration from 10-10 M to 10-14 M) with a certain amount of miRNA-21 (5 µL, 1×10-10 M) and without miRNA-21 were detected with the same procedure as above. Three kinds of probes, miRNA-21 probe labeled by Cy5, miRNA-10b probe labeled by Cy3 and the mixture probe (contain these two probes) were designed, which were numbered as probe 1, probe 2 and probe 3. Then, 5 µL of miRNA-21 (1×10-10 M) and 5 µL of miRNA-10b (1×10-10 M) were added into these three probes (20 µL) respectively. Afterwards, 1 µL of 10 × DSN buffer and 0.2 µL of 1 U/µL DSN enzyme were added. Under different excitation light (Cy5 excitation, Cy3 excitation and combined excitation), the detection signals were monitored using an Azure C600



device. MiRNAs Detection in Real Samples. To assess the applicability of the developed sensor for miRNAs analysis in real samples, target miRNAs were monitored using standard addition in spiked serums, which were received from the General Hospital of Tianjin Medical University. The miRNAs were added into the serum (10 dilution) with a certain concentration of 10×10-10 M. The detection of the samples was identical to the preceding procedure.

RESULTS AND DISCUSSION Mechanism of the DSN-Amplified PS@Au Probe -Based Sensor. The working principle of the PS@Au microspheres -based DNA probe and DSN -assist signal amplification platform was illustrated in Scheme 1. 5′- Cy5 -labeled and 3′- SH -labeled MBs were immobilized on PS@Au microspheres through Au-S bond by self-assemble, and the fluorescence on the hairpin structure was significantly quenched by the vicinity of the fluorophores to the AuNP surface via the NSET effect. In the presence of target miRNAs, the PS@Au microspheres -based DNA probe could effectively capture the miRNAs through DNA/RNA hybridization, accompanied by the open of stem-loop structure and fluorophores away from the AuNPs with the partially restored fluorescence. The DSN enzyme possesses strong preference for cleaving DNA in DNA-RNA hybrid duplexes with at least 12 bp and is scarcely active toward single-stranded (ss) DNA, RNA, or dsRNA, so the DSN was further introduced to assist target recycling and amplify the signal output. The formed miRNA/DNA heteroduplexes between MBs and miRNA immediately became the substrate for the DSN, leading to the further fluorescence recovery and release of the captured miRNA, which could bind to another MBs and initiate a next round of target-recycling amplification to set free more Cy5 into solution with a stronger fluorescent signal. Moreover, the DSN-amplified PS@Au probes -based sensor enabled multiple targets detection using MBs labeled with different fluorophores. The RGB value was introduced as the signal read-out to evaluate the detection performance of miRNA in this paper.


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Scheme 1. Schematic diagram of the PS@Au microspheres -based DNA probe and DSN -assist signal amplification platform for the detection of miRNA-21.

Fabrication and Characterization of PS@Au Microspheres. The fabrication of PS@Au microspheres was shown in Figure 1. Firstly, the monodisperse PS microspheres with a diameter of approximately 1.7 µm were dispersed homogeneously in the aqueous solutions. Secondly, in order to harvest PS@Au microspheres, the introduced dopamine was self-polymerized to form the coating layer spontaneously, which adhered firmly to the surface of PS microspheres as the linkage between the PS microspheres and AuNPs.34 As shown in Figure 1a2, the markedly coarser surface indicated the triumphantly PDA coating on the surface of PS. Afterwards, the Au3+ was reduced to AuNPs under the reduction of PDA, profiting from the large numbers of catechol groups with excellent reduction ability. Finally, hydroxylamine hydrochloride was introduced for further reduction35 to obtain dense AuNP coating PS microspheres as displayed in Figure 1a4, c4, which proved the dense deposition of AuNPs on the PS microspheres, as well as the X-ray diffraction patterns (XRD) characterization in Figure S1.



Figure 1. The synthesis procedure of PS@Au microspheres. (a) Corresponding SEM imagines, (b) Schematic illustration of synthesis procedure, (c) Corresponding TEM imagines.

Preparation and Feasible Verification of the PS@Au Probe. To fabricate the nucleic acid probe (PS@Au probe), we first immobilized the MBs (Cy5 modified on 5′ and thiol modified on 3′) onto the surface of PS@Au microspheres via Au-S bond according to the mirkin’s method.36 The PS@Au microspheres and MBs were incubated overnight to achieve an efficient coupling, afterwards, the dissociative MBs with high fluorescence was washed away. As shown in Figure 2a, the Cy5 labeled on MBs was quenched by the surface AuNP of PS@Au microspheres with negligible RGB value. By addition of 2-ME, which could selectively cleave the Au-S bond, there was strong recovery fluorescence generated from the released MBs with high RGB value, proving the triumphant immobilization of MBs on PS@Au microspheres. To evaluate the feasibility of the DSN-amplified PS@Au probe -based sensor, the miRNA-21 was introduced as the targeting molecule, as it has been proved that miRNA-21 is a potential tumor marker over-expressed in some tumors, such as brain tumor, colorectal cancer, breast cancer, and pancreatic cancer.36 The miRNA-21 sequence perfectly matched with the MBs, and their hybridization led to the open of stem-loop structure and high RGB value as demonstrated in Figure 2b. As the DSN was employed, the signal amplification was verified for miRNA-21 detection, according to Figure 2b, a significant increase of RGB value due to the DSN-assisted


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cyclic release of the miRNA-21 as discussed previously was realized. These results clearly suggested that the signal enhancement with the involvement of the DSN in the assay and the DSN-amplified PS@Au probe -based sensor was capable to improve detection sensitivity, which is the major parameter for the designed nucleic acids testing method. These results clearly suggested that the signal enhancement with the involvement of the DSN in the assay and the DSN-amplified PS@Au probe -based sensor was capable to improve detection sensitivity, which is the major parameter for the designed nucleic acids testing method.

Figure 2. Principle feasibility verification based on the PS@Au probe. (a) Measurement of RGB value of 20 µL PS@Au probe after the addition of 2 µL 2-ME. (b) RGB value of PS@Au probe after the incubation with miRNA-21(10-8 M) in the presence and absence of 0.2 µL DSN enzyme.

Optimization of DSN-Amplified PS@Au Probe -Based Sensor. The feature of our sensor for the miRNA-21 detection was DSN-oriented signal amplification to improve the detection sensitivity. Incubation time and temperature, as two foremost parameters for the DSN performance, were optimized. Firstly, the influence of incubation temperature, including 25 °C, 35 °C, 45 °C, 55 °C, 70 °C, was studied. As shown in Figure 3a, the maximum ∆RGB value was obtained at 45 °C, while a higher or lower temperature yielded the decreased ∆RGB value due to the

inefficient hybridization between DNA and miRNA or reduced activity of DSN enzyme. In general, approximate to 5-10 °C lower temperature than Tm (59 °C) was testified to be the optimal hybridization temperature.38 Therefore, 45 °C is selected as the optimal temperature.

Then, we next evaluated the optimized incubation time. In Figure 3b, the ∆RGB value



gradually increased with the extension of the incubation time from 0 to 60 min. Nevertheless, after 60 minutes, a significant decrease of the ∆RGB value from 60 to 120 min was observed. The reason may be that the further released Cy5 was gradually quenched by the PS@Au microspheres.

On account of the working of DSN, the MBs on the surface of PS@Au microspheres were cleaved, resulting in the more exposed AuNPs. As shown in Figure S2, the bare PS@Au microspheres led to stronger quenching performance than PS@Au probe. Therefore, in the following studies, the detection of miRNA was carried out at 45 °C for 60 min.

Figure 3. Optimization of miRNA Assay based on PS@Au probe. (a) The incubation temperature (with 60 min incubation time) and (b) incubation time (at 45 °C) in the presence of miRNA-21 (5×10-10 M).

Properties of the DSN-Amplified PS@Au Probe -Based Sensor. To investigate the signal amplification effect of the DSN, the experiments with/without DSN were both carried out under the optimized conditions. In order to quantitatively evaluate the contents of miRNA-21 in the samples, herein, we measured the change of RGB values (∆RGB, ∆RGB = RGB1-RGB0, RGB0 represents the RGB value of the blank experiment, RGB1 represents the RGB value of the experimental sample). The tonality of the solution was quantitatively determined by an Azure C600 device. As shown in Figure 4a①, 4b, the MBs was opened by the miRNA-21 with one-step detection in the absence of DSN, resulting in the effectively attenuated NSET effect and the recovery of fluorescent signal. The minimum detectable concentration reached to 1×10-10 M. While, in the presence of DSN (Figure 4a② and 4c), ∆RGB value gradually augmented with the increasing concentration of target miRNA-21. Parallel tests further disclosed that DSN-amplified sensor possessed higher detecting sensitivity with the limit of detection (LOD) of miRNA-21 about 5×10-14 M as intended, exceeding the LOD of the PS@Au probes -based sensor without


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DSN by more than at least 4 orders of magnitude. A calibration curve was obtained by plotting ∆RGB value of solutions against various concentrations of miRNA-21 (Figure 5a). A good linear relationship between ∆RGB value was observed in the range between concentrations of 1×10-12 M to 1×10-9 M, as descripted in equation of ∆RGB = 32.2739+ 0.03634c (R2 = 0.9939).

Figure 4. Development and validation of DSN-amplified PS@Au probe -based sensor compared with that without DSN in the detection of miRNA-21. (a) Schematic illustration of our DSN-amplified sensor and detection without DSN; (b) Plotting ∆RGB versus varying concentrations (1×10-11 M to1×10-6 M) of miRNA-21 based on PS@Au probe without DSN; (c) Plotting ∆RGB versus varying concentrations (5×10-14 M-5×10-9 M) of miRNA-21 based on DSN-amplified PS@Au probe.

The specificity was also studied to evaluate the interference of other miRNAs to the sensor, by employing the miRNA-21 (1×10-10 M) as the target miRNAs, NC miRNA-21 (1×10-9 M), miRNA-200b (1×10-9 M), Let-7d (1×10-9 M), miRNA-10b (1×10-9 M), miRNA-141 (1×10-9 M) as control groups, which were mismatched with the ssDNA probes. As illustrated in Figure. 5b, there



were negligible ∆RGB in NC RNA-21, miRNA -200b, Let-7d, miRNA-10b and miRNA -141 groups against with the blank test (in the absence of the target miRNA-21). While, the presence of the target miRNA-21 at even low concentrations also led to a significant enhanced ∆RGB signal. These results demonstrated that our DSN-amplified PS@Au probe -based sensor possessed a high selectivity toward the target miRNAs against other mismatched miRNAs.

Figure 5. Calibration curve establishment and detection specificity evaluation. (a) The linear response between the ∆RGB values and the concentration of miRNA-21 using DSN-amplified PS@Au probe; (b) Specificity tests: ∆RGB value of various interfering miRNAs (1×10-9 M NC miRNA-21, 1×10-9 M miRNA-200b, 1×10-9 M Let-7d, 1×10-9 M miRNA-10b, 1×10-9 M miRNA-141) and 1×10-10 M miRNA-21. Error bars show the standard deviations of three independent measurements.

Simultaneous Detection of the Coexisting miRNA-21 and miRNA-10b. To test the multiplex capability of the developed DSN-amplified PS@Au probe sensor for tumor- associated nucleic acids analysis, miRNA-21 and miRNA-10b, which were present abnormal expression in liver cancer, gastric cancer, breast cancer, glioma and pituitary adenoma tissues, and closely related with tumor invasion and distant metastasis39, were monitored by our designed assay. The simultaneous detection of the two markers had a higher specificity and clinical practical value for early diagnosis and evaluation of the prognosis of cancer,40 as well as greatly improved the detecting rate and saved the cost and time. In order to investigate the influence of miRNA-10b to miRNA-21detection, a series of different concentration (from 5×10-13 M to 5×10-9 M) of miRNA-21mixed with 1×10-10 M of miRNA-10b were assayed. With the help of DSN, the ∆RGB value had positive correlation with the concentration of miRNA-21, coincided with the detection result of miRNA-21 in the absence of


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miRNA-10b as shown in Figure 6a. So, the existence of miRNA-10b hardly influenced the detection of miRNA-21. Meanwhile, the almost same trend happened between ∆RGB values with different concentration of miRNA-10b with or without miRNA-21. The optimization of incubation temperature (25 °C, 35 °C, 45 °C, 55 °C and 65 °C) for miRNA-21 detection under the coexistence of miRNA-10b in one sample was shown in Figure S4. To further investigate the simultaneous detection of the coexisting miRNA-21 and miRNA-10b, we designed three kinds of probes, miRNA-21 probe labeled by Cy5, miRNA-10b probe labeled by Cy3 and the mixture probe, which were numbered as probe 1, probe 2 and probe 3. It clearly displayed that when hybridization and DSN-amplified tests were carried out with a mixture of miRNA-21 and miRNA-10b, probe 1 and probe 2 responded only to the specific target with negligible crosstalk as indicated in Figure 6c. These results indicated that the detection platform could realize the analysis of multiple tumor associated miRNAs simultaneously.

Figure 6. Detection of miRNA-10b and miRNA-21 in one sample. (a) Comparison for the detection of miRNA-21 in the presence or absence of miRNA-10b; (b) Comparison for the detection of miRNA-10b in the presence or



absence of miRNA-21. (c)The mixture of miRNA-10b and miRNA-21 was added into the probe 1, probe 2 and probe 3 respectively. Histogram was the combined result of Cy5 excitation and Cy3 excitation.

Investigation of the Practicability for the Real Samples. To test the applicability of the developed sensor for miRNA analysis in real samples, target miRNA-21 was monitored using standard addition in spiked serum as previous reports.41-43 Firstly, miRNA-21 with a concentration of (10×10-10 M) was added into the serum samples obtained from five healthy volunteers, and then detected by our DSN-amplified PS@Au probe -based sensor. A determined content was obtained according to the calibration curve of miRNA-21 in Figure S5. It was worth celebrating that the analytical results and theoretical value were roughly concordant as displayed in in Figure 7a. Meanwhile, the analytical recoveries for the five miRNA-21 spiked human serum samples were determined to be in the range of 92.27−121.9% and 92.4−127% for miRNA-10b spiked human serum samples as shown in Table S2. The detection of miRNA-10b in serums was identical to the preceding procedure with miRNA-21 and the results were shown in Figure 7b and Table S2. The RSD were almost blow 15% and P values were greater than 0.05 (Table S3-5). Although the number of serum samples is limited in the present study, the preliminary results data implies that our method achieved good anti-interference ability and could provide a potential analytical tool for the monitoring of miRNAs in real biological samples for early diagnosis of cancers.

Figure 7. Serum analysis of different samples from different healthy volunteers. standard addition method. (a) Results for the determination of miRNA-21. (b) Results for the determination of miRNA-10b.

CONCLUSIONS Herein, by taking advantage the specific cleaving activity of DSN and superior properties of


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AuNPs coated PS microbeads, we constructed a kind of PS@Au microspheres -based DNA probe with DSN-based signal amplification and demonstrated its application for rapid and ultrasensitive detection of miRNAs in one step. Three-dimensional PS microbeads provided a high capacity to load numerous AuNPs, which could effectively quench the fluorescence of the conjugated MBs via NEST. After the cleavage of DNA in DNA/ target RNA hybridization by DSN, the target recycling signal amplification was realized. The cleaved fluorophore in solution was direct read out in the means of ∆RGB values, which was proportional to the amount of target miRNA. Moreover,

multiplexed

miRNAs

detection

was

achieved

by

designing

different

fluorophore-labeled MBs. In view of the successfully monitoring of miRNA in serum, the assay strategy may open new opportunities in early detection of cancers.

ASSOCIATED CONTENT Supporting Information Available Supplementary material (including the oligonucleotides sequences employed in this work; X-ray diffraction patterns (XRD) characterization of PS microspheres and PS@Au microspheres， quenching performance between the PS@Au microspheres and PS@Au probe; optimization of the amount of DSN; optimization of incubation temperature under the coexistence of miRNA-10b and miRNA-21 in one sample; calibration curve of ∆RGB value vs. concentration of miRNA-21 and miRNA-10b; detection of miRNA-21 using PS@Au microspheres with two different Au nanoparticles' size; enzymatic activity of DSN; the detection recoveries of miRNA-21 and miRNA-10b from human serums, statistical analysis of miRNA-21 and miRNA-10b from human serums) is available.

AUTHOR INFORMATION Corresponding Authors *(X.G.) E-mail: [email protected]. *(J.C.) E-mail: [email protected].

Author Contributions ‡

Q.Z. and J.P. have equal contribution to this paper.

Notes The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank Professor Dingbin Liu (School of Chemistry, Nankai University) for providing access to some equipment used in this study. The authors gratefully acknowledge that this work was financially supported by the National Natural Science Foundation of China (31600800), Tianjin Natural Science Foundation (15JCQNJC03100, 17JCQNJC09100), the National Key Research and Development Program of China (2017YFA0205100).

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Diagram of the PS@Au microspheres -based DNA probe and DSN -assist signal amplification platform for the detection of miRNA-21.


Simple and Sensitive Quantification of MicroRNAs via PS@Au

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