Spherical Nucleic Acid Enzyme (SNAzyme) Boosted

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Spherical Nucleic Acid Enzyme (SNAzyme) Boosted Chemiluminescence miRNA Imaging Using a Smartphone Yudie Sun, Lin Shi, Qiwei Wang, Lan Mi, and Tao Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05696 • Publication Date (Web): 04 Feb 2019 Downloaded from http://pubs.acs.org on February 4, 2019

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Analytical Chemistry

Spherical Nucleic Acid Enzyme (SNAzyme) Boosted Chemiluminescence miRNA Imaging Using a Smartphone Yudie Sun, Lin Shi, Qiwei Wang, Lan Mi and Tao Li* Department of Chemistry, University of Science & Technology of China, Hefei, Anhui, 230026, China. Fax: (+86)55163601813; E-mail: [email protected] ABSTRACT: As the acute myocardial infarction (AMI) now becomes one of severe death threats to human and may abruptly occurs home and outdoors where the sophisticated equipment is not available, it is of great significance for developing facile methodologies for the point-of-care (POC) diagnosis of AMI. Towards this goal, here we build a sensing platform for chemiluminescence (CL) microRNA (miRNA) imaging with a smartphone as the portable detector, and for the first time achieve visualizing AMI-related miRNAs in real patients’ serum. We first construct a spherical nucleic acid enzyme (termed SNAzyme) derived from a dense layer of G-quadruplex (G4) DNAzyme formed on the gold nanoparticle core, which displays a ~100-fold higher catalytic activity and an improved resistance to nuclease degradation in real blood sample as compared to the G4 DNAzyme itself. These unique features endow the SNAzyme-boosted CL platform with a superior imaging performance for analyzing an AMI-related miRNA, miRNA-133a. This miRNA is employed to trigger the target-catalyzed hairpin assembly to produce a sticky dsDNA linker that captures the SNAzyme nanolabel onto the substrate. In this way, miRNA-133a is successfully detected, with a limit of detection of 0.3 pM(S/N = 3) and a high selectivity over other miRNA analogues in patients’ blood. Given its unique features in physiological environments, our SNAzyme-boosted imaging platform holds great promise for use in the POC diagnosis of AMI.

INTRODUCTION As a severe cardiovascular disease and one of major death causes,1-3 acute myocardial infarction (AMI) horribly threatens human health and therefore its diagnosis and therapy has attracted growing attentions in past years. Cardiac myocytes will respond to extracellular stimuli of ischemia after AMI, then diverse pathological gene program can be activated to reprogram gene expression.4,5 A few miRNAs, such as miRNA-133a, miRNA-499 and miRNA-1, play key roles in triggering downstream events and regulating overall cardiac function.6-8 It has been reported that the concentrations of miRNA-133a and miRNA-1 in plasma undergo a sharp increase after the onset of AMI symptoms and decrease to the normal level within 15h.9 As some specific miRNAs have been proven crucial for regulating vital activities of cardiac cells after the outbreak of AMI,10-13 unusually high levels of these miRNAs in circulation bloods are generally regarded as one hallmark of AMI especially in the early stage.14,15 For this reason, the detection of AMI-related microRNAs in human blood samples is of particular interest. Although diversities of strategies and techniques have been developed for sensing microRNAs related to various diseases,16-20 few of

them focuses on analyzing those AMI-related species in blood samples. Recently, we reported a SERS nanosensor for amplified microRNA detection and successfully applied it to real AMI patients’ bloods.21 Given that the sophisticated equipment is not always available in the AMI-occurring fields (e.g. in home and ambulance), here we seek to develop a facile sensing platform for chemiluminescence (CL) microRNA imaging using a smartphone as a portable detector that is popular in pointof-care testing (POCT).22,23 Benefiting from the advantages of simple sensing mechanism and no external light source, CL is the most promising technique for developing clinical diagnostic platforms,24,25 catalyzed by peroxidase-mimicking Gquadruplex (G4) DNAzymes with the cofactor hemin.26-29 Using a synthetic method introduced by Mirkin et al.,30 the G4 DNAzymes are usually loaded onto gold nanoparticles (AuNPs) to fabricate DNA-AuNPs constructs acting as catalytic labels.31,32 Mirkin and co-workers have demonstrated that many properties of DNA-AuNPs constructs stem from a dense nucleic acid layer,33,34 and, in fact, are independent of the cores (even coreless).35 Such a

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fundamental feature makes it reasonable to consider DNAAuNPs constructs as an important form of synthesized nucleic acids, which are termed spherical nucleic acids (SNAs)36 and differ from conventional linear nucleic acids. Since DNAzyme-AuNPs conjugates display the most importantly catalytic activity that stems from a dense layer of DNAzymes rather than the AuNPs core, this kind of SNAs are actually a nano form of nucleic acid enzymes, hereafter termed SNA enzymes (SNAzymes). A typical nanostructure of SNAzymes derived from G4 DNAzymes and AuNPs, as we synthesized in the present work, is illustrated in Figure 1A. Besides an unexpectedly larger size than the AuNPs core, the SNAzyme displays superior peroxidase activity, stability, and nuclease resistance as compared to the G4 DNAzyme itself. These features are advantageous for the CL analysis of AMI-related microRNAs in real patients’ blood samples in which nucleases and other interferent exists. In this work, we constructed the G4-AuNPs SNAzymes and employed them as catalytic nanolabels for the CL detection and imaging of microRNAs. The luminolartemisinin CL system reported for forensic bloodstain detection23 is adopted here, with a smartphone as a detector. In this way, visualization and CL imaging of an AMI related microRNA (miRNA-133a) in patients’ serum are achieved, with high selectivity for the target over other counterparts in bloods.

EXPERIMENTAL SECTION Materials and Reagents. All HPLC purified DNA oligonucleotides were ordered from Sangon Biological Company (Shanghai, China). All microRNAs used in our work were synthesized by Gene Pharma Company (Shanghai, China). The sequences of all used nucleic acids are listed in Table S1. 0.1% Diethlpyrocarbonate (DEPC)water, RNase inhibitor were also purchased from Sangon Biological Company (Shanghai, China). Magnesium acetate (MgAC2), sodium acetate (NaAc), potassium acetate (KAc) ,Chloroauric acid (HAuCl4), trisodium citrate dehydrate and sodium chloride (NaCl) were provided by Sinopharm Chemical Reagent Co., Ltd. (China). Luminol, Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) were bought from Sigma−Aldrich Co., Ltd. (Shanghai,China). Artemisinin were purchased from TCI Company (Shanghai, China). All reagents used in this study were analytical grade. All the serum used in our experiment was the serum of AMI patients. The patients’ serum was supplied by the First Affiliated Hospital of Nanjing Medical University. Instrumentation. CL intensities were collected by a LUMIstar Omega plate reader (BMG LABTECH, Germany). The gain vale was kept at 3000. Detection was achieved through a Nubia Z7 max mobile phone and the exposure time was set as 60s. Transmission electron microscope (Hitachi H-7650) and UV-vis spectrometer (cary 60) were used for structural characterization. The gel electrophoresis images were taken by a Tanon Gel Images System (Tanon-1600). Ultrapure water (>18.2 MΩ·cm) was produced by a Millipore Milli-Q gradient system.

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Polyacrylamide Gel Electrophoresis (PAGE). All of the used DNA and miRNAs were annealed at 90◦C for 10 min. Target miRNA (miRNA-133a) with varying concentrations were added into 10mM TE buffer, which contained 2 mM MgAc2 and 100 mM NaAc. The concentration of hp1 and hp2 were 0.5 µΜ and the total volume was 20 µL. After incubating at 37◦C for 2 hours, the samples were analyzed by PAGE electrophoresis (10%, w/w) in 1× TA buffer at 32V for 10 h. Preparation of SNAs and SNAzymes. Initially, 100 mL of 0.1 mM HAuCl4 aqueous was heated to boiling, then 3.5 mL of 1% sodium citrate was added rapidly. The solution was kept boiling for 30 min to synthesize 16 nM AuNPs. Then AuNPs (2 nM, 1mL) were incubated with the mixture of thiolated capture DNAs (100 μM, 6 μL) and thiolated G4 DNA (100 μM, 12 μL) over night at room temperature. Then, 2.5 μL of 3 M NaCl was added every 0.5 h to reach a final concentration of 0.5 M and kept for 21 h. Subsequently, the particles were washed three times with PBST and redispersed in 400 μL of TAE buffer. Finally, the prepared SNAs were stored at 4 °C for further use. Before the CL measurement, SNAs were allowed to fold in the presence of K+ and then complex with hemin to form the SNAzymes. Optimization of Experimental Conditions. The influences of different conditions on the catalytic effect between hemin and SNAzymes have been investigated by controlling variables. The concentration of hemin and SNAzymes remain unchanged. For pH Study, A volume of 50 μL Britton-Robinson (BR) buffer containing 2 mM artemisinin were pipetted into 50 μL of 1mM luminol and 1 μM hemin in TE buffer. For comparison, 5 nM SNAzymes were added into every control groups and incubated with hemin at 4°C overnight, the concentration of SNAzymes were calculated based on the concentration of gold by UV−Vis Spectra.37 To explore the best concentration of luminol, 50 μL of pH 12.0 BR buffer containing 2 mM artemisinin were pipetted into 50 μL of luminol with varying concentration in TE buffer (10 mM, pH 8.0). The optimal concentration of artemisinin was also studied as an important factor. The ion concentration in wells were kept as 2 mM MgAc2, 100 mM NaAc and 50 mM KAc constantly. CL Imaging with the smartphone. To illustrate the improved imaging ability of SNAzymes, comparison were made between hemin and SNAzymes. A volume of 50 μL of 2 mM artemisinin in pH 12.0 BR buffer was dripped into the wells containing different concentrations of hemin and 5 nM SNA, and the control groups contains different concentrations of hemin only. To investigate the imaging time, 50 μL of 5 μM hemin and 5 nM SNA solution in pH 8.0 TE buffer containing 1 mM luminol were added to six different wells in a row. A volume of 50 μL of 2 mM artemisinin solution was dripped into these wells every 5 minutes. Finally, detection was done after all these wells were injected oxidant. For detection of miRNA, miRNAs were also incubated with 1.5 µΜ hp1 and 1.5 µΜ hp2 at 37 °C in 10% serum for 2 h to trigger the CHA reaction. Followed by incubating the resulting solution with 5 nM SNAzymes in the biotinylated DNA modified wells for 3h at room temperature. After washing with PBST to remove extra


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Analytical Chemistry SNAzymes, a volume of 50 μL of 1 mM luminol, 50 mM KAc and 5 µΜ hemin was added into the wells and incubated overnight. For miRNA identification, 50 μL of 2.0 mM artemisinin solution in pH 12.0 BR buffer were dripped into the wells for different miRNAs at the same time. In our work, the CL images of microRNAs in serum samples were recorded with a Nubia Z7 max smartphone instead of a commerical imaging instrument with a CCD detector. An automatic mode was adopted with a exposure time of 60 s. The CL images can be processed by the software compatible with the smartphone or other imageprocessing softwares on PC. The brightness of CL images was used as the detection standard for visualizing target miRNAs. Procedures for miRNA Detection. MicroRNA detection was carried out in 96-well plates. The wells were modified with streptavidin (50 μL, 10 μg/ml) in carbonate buffer (0.05 M, pH 9.6) at 4 °C overnight. After washing, 50 μL of 5 μM biotinylated DNA was added and kept for 1h at room temperature before washing. Before CL detection, miRNA133a was first incubated with 1.5 µΜ hp1 and 1.5 µΜ hp2 in 10% serum at 37 °C for 2 h to trigger the CHA reaction. Then, the resulting solution and 5 nM SNA enzymes were added into the wells and incubated for 3 h at room temperature, followed by washing with PBST to remove extra SNAzymes. Finally, 50 μL TE buffer containing 0.5 mM luminol, 50 mM KAc and 0.1 µΜ hemin was added and incubated overnight for CL measurement.

RESULTS AND DISCUSSION Structures and properties of synthesized SNAzymes. Figure 1A depicts the synthesis and structural characteristics of our used SNAzymes made from G4 DNAzymes and AuNPs. Typically, citrate-protected AuNPs are employed as the inorganic cores to load a large number of thiolated DNAs including the G4 DNA and capture DNA (with an optimal molar ratio of 2:1, see Figure S1) onto the surface to form a dense DNA layer. The resulting DNAAuNPs constructs (i.e. SNAs)36 are next incubated in the presence of K+ and the cofactor hemin, allowing G4 DNAs to properly fold and complex with hemin thereby forming the desired SNAzymes. The synthesized SNAzymes were characterized by transmission electron microscope (TEM) and dynamic light scattering (DLS). The TEM images show that the modification of DNAs onto the surface of AuNPs did not cause any obvious changes on the morphology of particles, rather improved their monodispersity (Figure 1C, 1D vs Figure 1B). From the TEM and DLS results, it is observed that the AuNPs core displays a uniform diameter of ~16 nm (Figure 1B, 1E), as reported previously.38 However, the particle size surprisingly increases to ~31 nm after DNA modification and further to ~36 nm after complexed with hemin (Figure 1E), mainly attributed to the large hydration radius of modified DNA.39 In other words, this size shift in DLS provides an evidence for the formation of SNAzymes, which may be harnessed to be indicative of the interactions of SNAs with other ligands.


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Figure 1. Characterization of SNAzymes. (A) Schematic for the synthesis of SNAzymes. (B-D) TEM images of synthesized AuNPs (B), SNAs (C), SNAzymes (D). (E) DLS analysis of the Size distribution of AuNPs, SNAs, SNAzymes. (F) UV−vis spectra corresponding to AuNPs, SNAs and SNAzymes. (G) Kinetic curves of the CL reactions catalyzed by hemin, hemin/G4 DNAzyme and SNAzymes. (H) Comparison between the catalytic capacity of SNAzymes and G4 DNAzymes. (I) Nuclease resistance of SNAzymes and G4 DNAzymes tested in 0.1 % human blood. Besides, the corresponding UV−vis spectra with a positive absorption band near 260 nm assigned to DNA also verifiy that nucleic acids are successfully immobilized onto AuNPs (Figure 1F).40,41 Unexpectedly, after complexed with the cofactor, the hemin Soret band42 near 400 nm is not observed in the UV-vis spectra, owing to the much weaker absorption of hemin than that of AuNPs. However, the bound hemin can be straightforwardly reflected by a sharp increase in the CL intensity relative to either SNA or hemin alone when characterized in the luminolartemisinin system23 (Figure 1G). It also implies a superior peroxidase activity of the synthesized SNAzymes. For full understanding this important feature of SNAzymes, we further make a comparison between the as-prepared SNAzymes and different concentrations of G4 DNAzyme when bound to the same concentration of hemin (Figure 1H). It shows that the CL signal catalyzed by one SNAzyme particle approximates to that by 100 molecules of G4 DNAzyme, suggesting an about 100-fold signal amplification via utilizing SNAzymes as catalytic labels for the CL sensing system. According to the proportion of G4 DNA to the capture one, the density of the DNA layer of SNAzyme is estimated as ~150 nucleic acids per particle, similar to that reported previously according to the same synthesis process.43,44 In addition, the SNAzymes display an improved resistance to nuclease degradation as compared to the G4 DNAzyme itself, which was tested in real human blood after appropriate dilution (Figure 1I). This is rationally expected, as SNAs have been demonstrated to show a better nuclease-resistant ability than molecular DNAs. 45 The experimental results also demonstrate that the SNAzyme owns good stability in pH 12 alkaline buffer within 4 hours, which is enough for CL measurement. Taken together, these unique features enable the utilization of SNAzymes as novel powerful catalytic nanolabels for the CL analysis of AMI-related microRNAs especially in real blood samples. Design and optimization of the SNAzyme-boosted CL sensing platform for microRNA detection. The principle of the designed strategy for miRNA detection is shown in Scheme 1. Two well-designed hairpin DNAs, hp1 and hp2, both contain a sticky end that is complementary to the capture DNAs on the SNA and 96-well plate, respectively. The target miRNA-133a is incubated with hp1 and hp2 to trigger the catalyzed hairpin assembly (CHA) and realize target recycling. The CHA product is analyzed by native polyacrylamide gel electrophoresis (PAGE), showing that with the increasing concentration of miR133a, more and more duplex linkers are produced (Figure S2). Meanwhile, the mixture of hp1 and hp2 is continuously consumed. The results suggest that the CHA process are proceeding toward desired results. The duplex linker formed in CHA process owns two sticky ends, accordingly the SNA can be immobilized onto the plate. After incubated with hemin in the presence of potassium ion,

the SNAzymes are formed to catalyze the release of superoxide radical anion (O2•−) from artemisinin as hemin does,23 thereby promoting the CL reaction. Scheme 1. Schematic illustration of the CL detection platform for microRNA.

It has been demonstrated that hemin itself also owns a moderate catalytic ability toward luminol/artemisinin CL reaction.23 In consideration of background, we investigated the effect of major factors on the amplification ability of SNAzymes. As shown in Figure 2A, both the CL intensity of hemin and SNAzymes increase with the increasing pH. However, the SNAzymes achieve the best catalytic ability at pH 12.0, since the CL intensity gave the highest ratio at this pH (Figure 2B). High pH is favorable to CL reaction,


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Analytical Chemistry Figure 2. Influence of different factors to the catalytic performance of SNAzymes. Effect of (A) pH, (C) luminol concentration, (E) artemisinin concentration on the CL intensities; Effect of (B) pH, (D) luminol concentration, (F) artemisinin concentration on the amplification factor of CL intensity for 0nM SNAzymes versus CL intensity for 5 nM SNAzymes. while G4 is not stable at strong alkaline conditions, so a compromise condition occurred.46,47 This pH is adopted for the following optimal experiments. Besides, to obtain the best catalytic performance, the concentration of H2O2 and artemisinin were also optimized. From Figure 2C and 2D, high concentration of luminol inhibits the activity of SNAzymes, and 0.5 mM luminol is the optimal concentration. Both the CL intensity and catalytic ratio reach the maximum with 2 mM artemisinin (see Figure 2E and 2F). Finally, 0.5 mM luminol and 2 mM artemisinin in pH 12.0 buffer are chosen for the best performance of SNAzymes, under which SNAzymes achieve a catalytic activity 14-fold higher than that of hemin alone. Utilizing SNAzymes for CL Imaging with a smartphone. To illustrate the improved imaging ability of SNAzymes, we employed hemin as a comparison object because hemin itself can induce the CL signal. Figure 3 shows distinct differences in the imaging ability without and with 5 nM SNA in the presence of different concentrations of hemin. It is found that the lowest concentration of hemin for smartphone detection reach up to 6 µM (line A). However, 0.5 µM hemin can generate weak but noticeable signals with the help of 5 nM SNA due to the strong catalytic ability of the formed SNAzymes (line B). With the increasing concentration of hemin, the CL intensity of SNAzymes also increased, but accompanied by higher background from hemin.46,48

Figure 3. CL images of different concentrations of hemin (line a), 5 nM SNA plus different concentrations of hemin (line b), and time-dependent CL images of 5 µM hemin (line C), 5 nM SNA plus 5 µM hemin (line d).

The imaging time is also an important factor for practical application, smartphone detection was done every 5 minutes. Under the catalysis of SNAzymes, CL imaging can be extended to nearly 30 minutes. For such a long time, we think 5 minutes interval can also demonstrate this changing process. Besides, 5 minutes are enough for us to complete the injection and focus process. At the start, 5 µM hemin gives off a weaker light, and quickly fades and disappears within 15 minutes (line C). After incubated 5 µM hemin with 5 nM SNA, however, a bright light is observed and still visualized within 25 minutes (line D). These observations indicate that SNAzymes can significantly increase the peroxidation effect of artemisinin, resulting in higher luminescent intensity and longer luminescence time that ensure better imaging applications. CL detection and imaging of target miRNA in real samples. Various concentrations of miRNA solution were detected under the optimal conditions. Figure 4A arranges CL kinetic curve for the analysis of a serious concentrations of miRNA-133a. As the concentration of target increases, the intensity at 2 minutes increases progressively. By contrasting with the blank curve, it’s noticeable that 10 pM target can give rise to a remarkable changing of CL intensity, demonstrating a lowest detectable concentration (LDC) of 10 pM for miRNA-133a. For quantitative detection, the intensities of CL at 2 minutes are quantized as a function of the logarithm of miRNA-133a concentration and the detection was performed in both TE buffer and 10% serum. From Figure 4B, we find the platform display good anti-interference performance since the detection results in serum are in consistent with that in buffer. The calibration curve shows a linear relationship in the range of 10 pM–100 nM for this platform, with a limit of detection (LOD) of 0.3 pM (S/N = 3).

Figure 4. CL performance for microRNA assay. (A) Representative kinetic curve of the CL reaction corresponding to miR-133a with varying concentrations. (B) CL intensities at 2 minutes of detecting miRNA-133a in buffer and in 10% serum. (C) CL intensities of specific analysis for noncomplementary miRNAs (miRNA-499, miRNA-208, miRNA-328) and the mixtures of four microRNAs in 10% serum. (D) CL images for (a) blank, (b)


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miRNA-499 (c) miRNA-208, (d) miRNA-328, (e) miRNA208 and (f) the mixture of four miRNAs in 10% serum. The concentration of all used miRNAs for this study is 100 nM. The selectivity of this detection platform was investigated in 10% serum by parallel experiments between target miRNA and other AMI-related miRNAs (miRNA499, miRNA-208, miRNA-328) that coexist in the patient blood.49 The concentration of these miRNAs are 100 nM and the reaction conditions are the optimum conditions. Figure 4C exhibits the CL intensity for individual miRNAs and the mixture of four miRNAs, respectively. From the spectral detection results, we find that the CL signals are noticeable only in the presence of target miRNA, noncomplementary miRNAs and interferent in blood result in no significant changes compared to the background signal. Our sensing platform exhibits good selectivity to miRNA-133a in complex serum sample, revealing the potential identification ability for AMI. The LOD of our method is superior to most of the reported detection results of microRNA by CL, but can’t exceed some extreme levels (Table S2).50-57 Nevertheless, our method can overcome interference in serum sample, it still holds great prospect for reliable analyzing of miRNAs in some complex human tissues. Owing to the excellent selectivity and capacity of resisting disturbance of the developed sensing platform, it’s promising to develop visual identification and diagnostic tools. For imaging applications, stronger luminescence are need, while the optimal condition is only suitable for improving detection sensitivity and the CL intensity for detecting on CL apparatus is not strong enough to be detected by smartphone. For these reason, we improved the concentration of hemin to 5 µM for the smartphone detection, which is a suitable concentration to give off a faint smartphone sensible light. Then, the reliability of the CL sensor for visual identification was carried out in 10% serum by AMI-related miRNAs (Figure 4D). The CL images of miRNA-133a (panel e) and the mixture of all miRNAs (panel f) are noticeable brightening compared to noncomplementary miRNAs (panels b-d) and the background (panel a). By contrasting with the CL image of blank sample, it’s observed that 1 nM target can give rise to an obvious increase in the brightness of CL image (see below), indicating a LDC of 1 nM using the smartphone as the photodetector.(Figure S3). These results demonstrates good visual identification ability of our developed sensing platform, revealing potential for diagnosing AMI in complex cardiac systems.

Conclusions In this work, we have developed a detection and visual identification platform for miRNAs by synthesizing SNAzymes to improve the catalytic ability of luminol/artemisinin system. With the help of CHA signal amplification and SNAzymes catalytic amplification, a LDC of 10 pM and LOD of 0.255 pM for target miRNA have

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been achieved, with a linear detection range of 4 orders of magnitude, superior to most of the reported miRNA detection strategies by CL measurement. Benefiting from the strong anti-interference ability of SNAzymes, we first reported the visual identification of target miRNAs in serum sample by using smartphone directly. However, the sensitivity of our platform may bear a certain level of loss, since multiple washing steps for separating and high background from hemin for imaging. Nonetheless, our sensing platform holds great application prospects in detecting miRNAs and diagnosing disease. By introducing other amplification strategies, this detection system may extended to diverse applications.

ASSOCIATED CONTENT Supporting Information The optimization of the ratio between G4 DNA and biotinylated DNA, and sequences of oligonucleotide used in this study. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author Fax: (+86)551-63601813; E-mail: [email protected]

ACKNOWLEDGMENT Thank Prof. Liansheng Wang in the First Affiliated Hospital of Nanjing Medical University for providing the AMI patients’ serum. This work was supported by the National Key Research and Development Program of China [No. 2016YFA0201300], the National Natural Science Foundation of China [No. 21874124, No. 21575133], and the Recruitment Program of Global Experts.

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