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A Novel Ternary Electrochemiluminescence System Based on Rubrene Microrods as Luminophore and Pt Nanomaterials as Coreaction Accelerator for Ultrasensitive Detection of MicroRNA from Cancer Cells Jia-Li Liu, Zhi-Ling Tang, Ying Zhuo, Yaqin Chai, and Ruo Yuan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01812 • Publication Date (Web): 07 Aug 2017 Downloaded from http://pubs.acs.org on August 7, 2017

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Analytical Chemistry

A Novel Ternary Electrochemiluminescence System Based on Rubrene Microrods as Luminophore and Pt Nanomaterials as Coreaction Accelerator for Ultrasensitive Detection of MicroRNA from Cancer Cells Jia-Li Liu, Zhi-Ling Tang, Ying Zhuo*, Ya-Qin Chai, Ruo Yuan* Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715,China.

*

Corresponding authors at: Tel.: +86 23 68252277, fax: +86 23 68253172.

E-mail addresses: [email protected] (Y. Zhuo), [email protected] (R. Yuan).

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ABSTRACT As the only endogenous co-reactant in ECL system, the dissolved O2 was the ideal candidate due to the mild reaction and easy operation, but compared to S2O82-, the dissolved O2 with weaker redox activity suffers from the poor enhancement effect of luminophore, which restricted the further application in bioanalysis. Here, a high-intense ECL signal was gained by the employing of Pt nanomaterials as co-reaction accelerator to generate more intermediate of dissolved O2 to promote the co-reaction efficiency. Based on a new ternary ECL system of Pt nanomaterials as the co-reaction accelerator, dissolved O2 as co-reactant, and a neotype rubrene microrods as luminophore, an efficient “on-off-on” solid-state ECL switch platfrom was designed for ultrasensitive microRNA (miRNA) detection with a background reduction strategy of ferrocene-labeled single-stranded DNA (Fc-DNA) as a quencher. In the presence of miRNA 141, the Pt nanoparticles labeled hairpin (HP1/PtNPs) was opened to produce plenty of Pt nanoparticles labeled output DNA (S1/PtNPs) and release the miRNA-141 to participate in the next cycle. Then, the S1/PtNPs were captured on the surface of the electrode by the complementary strand to obtain the super “signal on” state with extremely high ECL signal. This novel solid-state ECL platform exhibited excellent sensitivity from 10 aM to 100 pM with a detection limit of 2.1 aM, which provided a new approach for ultrasensitive ECL bioanalysis. KEYWORDS: rubrene, electrochemiluminescence, ternary system, Pt nanomaterials, co-reaction accelerator.

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INTRODUCTION

Electrochemiluminescence (ECL), as a promising analytical technique in the biosensing field, has received considerable concern due to the intrinsic advantages of low background, wide dynamic range and high sensitivity.1-3 Usually, the individual ECL luminophore is unable to provide a strong enough signal due to the annihilation of themselves.4 Since the C2O42- was first reported as co-reactant to react with the ECL luminophore for achieving a significantly enhanced ECL signal than that of the annihilation ECL process, the ECL binary system of ECL luminophore and co-reactant has become fashionable because the addition of co-reactant (e.g C2O42-, TPrA, S2O82-) in the detection solution is the most simple and effective way to enhance luminous efficiency.5-7 As one of the most classical ECL co-reactant, S2O82has been extensively studied based on the various kinds of ECL luminophore, such as the Ru(II) complex,8,9 nanoclusters,10 perylene derivative11 and so on. Generally, the ECL reaction mechanisms of S2O82- as co-reactant could be described as that the radical of luminophore reacted with the oxidant intermediates of SO4•− which was produced by the electrochemical reduction of the S2O82− to induce an excited state. Therefore, the ECL intensity was closely related to the amount of SO4•−. In the previous work, we had developed the new ternary ECL system by introducing a novel co-reaction accelerator (such as semicarbazide,12,13 aminobenzene14) to react with S2O82− to generate massive SO4•−, which promoted the ECL reaction rate of the co-reactant of S2O82− and luminophore to amplify the ECL signal significantly.

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However, at present the co-reaction accelerators were only applied to the ECL system of S2O82− as co-reactant, which limited the generality and universality of the ternary ECL system. Compared with the strong oxidative co-reactant of S2O82−, dissolved O2 as an endogenous co-reactant possessed great advantages of the mild reaction and easy operation which has been studied on the ECL emission of CdTe, CdS, and perylene derivative etc.15-17 Nevertheless, due to the relatively weaker reaction activity of dissolved O2 than that of S2O82−, the ECL potential in those works was usually required more negative than -1.5 V to obtain strong enough ECL intensity, which might introduce more interference or make electrode damaged. Herein, an efficient ternary ECL system was developed by employing Pt nanomaterials as efficient co-reaction accelerator of dissolved O2, which not only enhanced the reaction rate of the ECL luminophore and dissolved O2, but also made the ECL potential positively shift to -1 V to reduce the effect caused by the extremely negative potential. Rubrene (Rub) is a classic ECL luminophore18,19 due to the high fluorescence quantum yields with near 100% and relatively low potential to gain the radical cation.20 In the earlier research, the ECL of Rub was weak and dependent on the organic solvent because of the structure of polycyclic aromatic hydrocarbons, which restricted the application in bioanalysis for it requires an aqueous environment. Until 2014, Bard’s group reported the ECL of Rub in an aqueous solution with an oil-in-water emulsion containing Rub as the luminophore and TPrA as the co-reactant.21 Although the ECL signal of Rub was measured in the emulsion system, it still suffers from the problem that the emulsions could only be stable for hours. 4


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Recently, our group has constructed a series of solid-state ECL binary platforms by immobilizing Ru(bpy)32+22 or perylene14 as luminophore on the electrode surface to cut down the consumption of expensive luminophore, and boost the ECL signal. Herein, based on the Pt nanoflowers (PtNFs) in-situ reduced on the rubrene micronods (RubMRs) as co-reaction accelerator to improve the reaction rate of RubMRs and dissolved O2, the ECL luminous efficiency of RubMRs was enhanced significantly to construct an ultrasensitive solid-state ECL ternary platform.

In this study, RubMRs as luminophore, dissolved O2 as co-reactant and Pt nanomaterials as co-reaction accelerator were first developed as a novel ECL ternary system to construct an efficient “on-off-on” ECL switch platform for ultrasensitive detection of the microRNA (miRNA) from cancer cells. Besides, the target-induced cyclic enzymatic amplification (TICEA) was performed to further improve the sensitivity. As depicted in Scheme 1, the PtNFs as co-reaction accelerator were first in-situ reduced on the RubMRs to obtain a multifunctional composite nanomaterials which not only provided the active sites for DNA probe immobilization, but also exhibited a strong ECL signal as the first “signal-on” state. Then, a background reduction strategy of ferrocene-labeled single-stranded DNA (Fc-DNA) as a quencher was introduced for the “signal-off” state. In the presence of target miRNA 141, the TICEA (the details were showed in Supporting Information 1.2) was operated to generate numerous output DNA labeled with Pt nanoparticles (S1/PtNPs). Finally, PtNPs, the co-reaction accelerator, promoted the reaction rate of RubMRs and dissolved O2 to obtain a significant enhancement of ECL intensity which achieved a 5



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super “signal on” state. Compared to the traditional “on-off-on” switch platform, the super “signal on” state possessed stronger ECL signal than that of the first “signal on” state, achieving a significantly enhanced sensitivity, because the S1/PtNPs not only replaced the Fc-DNA to recover the ECL signal, but also produced more reactive oxygen species (ROS) from the dissolved O2 to react with RubMRs for a remarkable enhancement of ECL intensity. Thus, the proposed ECL biosensor achieved ultrasensitive detection of miRNA-141 with a detection limit of 2.1 aM. This novel solid-state ECL ternary platform introduced the PtNPs as co-reaction accelerator of dissolved O2 to construct the ultrasensitive ECL biosensors, which broke a new path for the detection of biological molecules.

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Scheme 1. Schematic illustration of (A) the synthesis of the PtNFs@RubMRs, (B) the fabrication of the biosensor and (C) the possible reaction mechanism of the RubMRs.

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EXPERIMENTAL SECTION

Preparation of PtNFs@RubMRs. The rubrene micronods (RubMRs) were synthesized by a reprecipitation method as described in Scheme 1. Firstly, 5 mg rubrene powder was dissolved in 5 mL tetrahydrofuran (THF) to vigorously stirred for 5 min. Then the orange solution was rapidly injected into 10 mL of SDS solution (5×10-3 M) to stir for half an hour, and ultrasonicated for 15 min. After centrifugation, the RubMRs were collected and washed several times by the deionized water. Subsequently, Pt nanoparticles (PtNPs) were in-situ reduced at the surface of RubMRs to serve as the seed to grow the Pt nanoflowers (PtNFs). Briefly, 350 µL of 1% H2PtCl6 aqueous solution was mixed with 15 mL of RubMRs to agitate for 60 min. Under vigorous stirring, 200 µL of freshly prepared 50 mM ice-cold NaBH4 solution was added in the above solution. Lately, the color of the solution changed from croci to claybank, implying the PtNPs was capped on the RubMRs. After centrifugation, the sediment was re-dispersed in 15 mL SDS solution with 375 µL of 1% H2PtCl6 aqueous solution. While the solution was constantly stirred, 0.2 mL of 1 M ascorbic acid (AA) was added to induce the growth of the PtNFs on the RubMRs. Finally, the above solution was stirred overnight to form PtNFs@RubMRs. The synthesis of HP1/PtNPs. Firstly, the PtNPs were synthesized based on previous reports with minor modifications.24 In brief, 350 µL of 1% H2PtCl6 aqueous solution was diluted to 15 mL with deionized water, and 1 mL of 50 mM sodium citrate solution was mixed with the aqueous solution in a glass beaker. Then 0.5 mL of 30 mM NaBH4 solution was added in the above solution to obtain the PtNPs. 8


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Subsequently, 200 µL of 2 µM HP1 with amino group were mixed with the PtNPs to stir for 12 h at 4 ºC for complete reaction, and the PtNPs labeled HP1 (HP1/PtNPs) were obtained by the interaction of the Pt-N covalent bond. Then, the non-reacted HP1 were removed by centrifugation and washing. The HP1/PtNPs were dispersed in 1 mL deionized water for further use.

Fabrication of Biosensor. Firstly, the bare GCE was pretreated as our previous method.25 Then, 4 µL of the PtNFs@RubMRs was dropped on the polished GCE and dried at room temperature to form a stable film. After that, 10 µL of capture probe (CP1) with amino group was incubated on the modified electrode overnight at 4 ºC. When rinsed by deionized water, the modified electrode was immersed in 1 mM HT for 1 h to block the non-specific sites. Finally, 10 µL of 2 µM Fc-DNA was incubated on the electrode to hybridize with the CP1.

Measurement Procedure. The target-induced cyclic enzymatic amplification (TICEA) was performed in a centrifuge tube as follows. Briefly, 100 µL of 1 × NE buffer 4 containing various concentrations miRNA-141 standard solution (or the extraction of 22Rv1), 2 µM HP1/PtNPs, and 100 U/mL T7 Exo were incubated for 90 min at 25 ºC to obtain the reaction solution containing the output DNA (S1/PtNPs). After that, 10 µL of the reaction solution was added on the biosensor to incubate at 37 ºC for 2 h. Then the biosensor was rinsed by deionized water to remove the uncombined S1/PtNPs and other materials. At last, the ECL signal of the biosensor was measured with a MPI-A ECL analyzer in 2 mL 0.1 M PBS (pH 7.4). And the ECL

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potential ranged from -1 to 1.2 V with the voltage of the photomultiplier tube (PMT) at 800 V.

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RESULTS AND DISCUSSION Morphology Characterization of Rubrene Nanomaterials. The morphologies of the prepared rubrene nanomaterials were characterized by scanning electron microscopy (SEM). As illustrated in Figure 1A, the SDS capped RubMRs prepared by reprecipitation method showed an uniform size of rod-like structures with lengths of 4±1 µm and widths of 900±200 nm. Compared with the pure RubMRs, the in-situ reduction of PtNFs onto the RubMRs interface were shown in Figure 1B. The micronods became irregular, and plenty of PtNFs grew on the RubMRs surface. In addition, X-ray photoelectron spectroscopy (XPS) of PtNFs@RubMRs were investigated to analyze the elements in that nanomaterials. As exhibited in Figure 1C, the characteristic peaks of O1s, C1s, S2p and Pt4f were clearly acquired in the PtNFs@RubMRs XPS spectra. The peaks at 531.9 eV, 283.5 eV, and 168 eV in Figure 1D, could be assigned to O1s, C1s, S2p, respectively, which manifested the presence of the rubrene and SDS. Moreover, the XPS doublet of Pt4f (71.5 eV and 75.4 eV) indicated the existence of the metallic Pt0 which confirmed the PtNFs on RubMRs. According to the elemental analysis results, we concluded that the PtNFs@RubMRs were successfully synthesized. Besides, the HRTEM images of PtNPs@RubMRs and PtNFs@RubMRs were shown in Supporting Information 1.3, which were identical to the morphologies characterized by SEM.

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Figure 1. SEM images of (A) RubMRs and (B) PtNFs@RubMRs, XPS characterization for (C) the full region of PtNFs@RubMRs, (D) the different elements of O1s region, C1s region, S2p region and Pt4f region.

UV−Vis Absorption and Luminescence Characterization of Rubrene. The optical properties of rubrene were characterized by a UV-vis spectrometer and fluorescence spectrophotometer. As displayed in Figure 2A, the rubrene in tetrahydrofuran (THF) exhibited a broad band at 420-550 nm in the UV-vis absorption spectra. However, after the rubrene was capped by SDS to synthesize the RubMRs, the characteristic absorption peaks of rubrene experienced a slight bathochromic shift from 463, 492, 526 nm to 464, 498, 542 nm, respectively, which implied that the rubrene molecules aggregated to nanoparticles (J-aggregates).26 The fluorescence (FL) 12


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spectra and ECL emission spectra of RubMRs were also portrayed in Figure 2B. The fluorescence emission peak was observed at around 554 nm when the excitation wavelength was 491 nm. Compared to the FL spectra of 554 nm, the ECL spectra of RubMRs with the emission peak of 570 nm exhibited a slight red-shift, which ascribed to the self-absorption, and instrument effects.27,28 The FL spectra and FL microscopic image of RubMRs were also characterized. As illustrated in Figure 2C, the fluorescence excitation wavelengths of RubMRs were at 463 nm, 491 nm, 526 nm (curve a), and the emission were at 554 nm (curve b), respectively. Under the excitation wavelengths of 488 nm, the RubMRs showed good fluorescence performance (Figure 2D), and the fluorescence image were in good agreement with the SEM image.

Figure 2. (A) UV-vis absorption spectra of (a) rubrene in tetrahydrofuran (THF), (b) RubMRs in

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deionized water. (B) The FL spectra (a) of RubMRs with the 491 nm excitation wavelengths and ECL emission spectra (b) of RubMRs. (C) Fluorescence (a) excitation spectra (Em=554 nm) and (b) emission spectra (Ex=491 nm) of RubMRs. (D) Fluorescence microscopic image of RubMRs.

Possible ECL Mechanism of the PtNFs@RubMRs. To evaluate the ECL mechanism of the PtNFs@RubMRs, the synchronous CV and ECL of different modified electrodes were measured with the cyclic potential scanning from -1.0 to 1.2 V under the different conditions. As shown in Figure 3, the RubMRs modified electrode in N2-saturated showed no clear redox peaks (Figure 3A, curve a), and the ECL signal of RubMRs was negligibly weak (Figure 3B, curve a), which ascribed to few dissolved O2 in solution. In the air-saturated pH 7.4 PBS, the curve b in Figure 3A showed a reduction wave at about - 0.64 V, which was attributed to the reduction of O2.29 The corresponding ECL signal (Figure 3B, curve b) was about 43 times higher than that of the RubMRs in N2-saturated PBS, indicating that the dissolved O2 might generate reactive oxygen species (ROS) as the reactive intermediate to react with rubrene radical cation (Rub•+) generating the excited state (Rub*). Subsequently, the RubMRs modified electrode was swept in N2-saturated PBS containing 320 mM H2O2 which roughly equaled to the saturated concentration of dissolved O2 under the standard atmospheric pressure at room temperature.30 The ECL intensity (Figure 3B, curve c) was stronger than that of RubMRs modified electrode in the air-saturated PBS. The results suggested that both the dissolved O2 and H2O2 could act as the efficient co-reactant to generate ROS (such as O2•-, OH•) as the intermediate to react with the RubMRs. After the in-situ growth of PtNFs on the RubMRs, the 14


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PtNFs@RubMRs modified electrode was investigated in air-saturated PBS. The curve d in Figure 3A showed an enhanced peak current at about -0.64 V and the ECL intensity zoomed to the maximum of 16070 a.u (Figure 3B, curve d) ,which was about 14 times compared to that of the independent RubMRs without the PtNFs (Figure 3B, curve b). This phenomenon implied that the PtNFs could facilitate the reduction of dissolved O2 producing more ROS31 to interact with Rub•+ to enhance the ECL reaction efficiency. Consequently, the possible mechanism of ECL ternary system could be outlined in the following routes:

In order to explore the ROS in the ECL system, superoxide dismutase (SOD) as a scavenging factor to eliminate O2•- and L-cysteine as an efficient scavenger of both O2•- and OH• were employed. When the PtNFs@RubMRs swept in the air-saturated PBS containing 4 U mL-1 SOD, the corresponding CV wave showed a peak at about -0.9 V (Figure 3A, curve e), however, the ECL intensity decreased by 82.9 % (Figure 3B, curve e) due to the scavenging of superoxide radical (O2•-) by SOD. In addition, 15



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the presence of 1 mM L-cysteine in air-saturated PBS completely quenched the ECL signal (Figure 3B, curve f), which attributed to the consumption of both OH• and O2•by L-cysteine. These results demonstrated that the OH• and O2•- reacted with Rub•+ together to generate excited state species (Rub*). Consequently, the possible ECL mechanism of different electrodes could be depicted in Figure C.

Figure 3. (A) CV waves, (B) ECL-potential profiles and (C) The possible ECL mechanism of different electrodes: RubMRs in the N2-saturated (curve a), air-saturated (curve b), 320 mM H2O2 + N2-saturated (curve c), PtNFs@RubMRs/GCE in air-saturated (curve d), 4 U mL-1 SOD + air-saturated (curve e), and 1 mM L-cysteine + air-saturated (curve f).

Optimization of the Reaction Conditions. In order to obtain the optimal

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experimental conditions, two important experimental parameters were investigated. The effect of the hybridization time between the CP1 and Fc-DNA of the proposed biosensor was shown in Figure 4A. With the growth of incubation time between the CP1 and Fc-DNA, the ECL intensity declined rapidly which was attributed to more Fc-DNA were combined to the electrode. Nevertheless, the ECL signal tended to be stable when the incubation time was 3 h, which was treated as the optimal hybridization time at last. Meanwhile, as shown in Figure 4B, with the increasing reaction time of the TICEA, the ECL signal increased, and trended to a constant value. At the time of 2 h, the maximum ECL response was observed. However, the ECL signal decreased slightly with longer reaction time. Therefore, the optimal reaction time of the TICEA in the following experiments was 2 h.

Figure 4. The optimization of (A) the hybridization time between the CP1 and Fc-DNA of the proposed biosensor. (B) The reaction time of the TICEA at 100 fM miRNA-141. Error bars, SD, n = 3.

Characterization of the ECL immunosensor. To confirm the feasibility of the

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method, we investigated the ECL intensity-time responses stepwise to characterize the modification process of the proposed biosensor fabrication. As depicted in Figure 5A, the bare GCE displayed no ECL signal (curve a) in the absence of luminophore. When the PtNFs@RubMRs was decorated on the electrode, an intensive ECL emission peak of 12047 a.u. (curve b) was observed. After immobilizing with the CP1 and HT, the ECL intensity decreased sequentially (curve c, curve d) on account of the insulation of CP1 and HT. As expected, an obvious decrease in ECL intensity (curve e) was obtained after hybridizing with the Fc-DNA, which was attributed to the quench effect of ferrocene. However, when the electrode was incubated with the S1/PtNPs, an enhanced ECL signal was detected again. The reason was that the output DNA hybridized with CP1 to displacement the Fc-DNA, and the PtNPs labeled on the output DNA would accelerate the ECL reaction rate of dissolved O2 and rubrene. Cyclic voltammetry (CV) was also used to verify the successful stepwise fabrication of the biosensor in the presence of [Fe(CN)6]3−/4−. As shown in Figure 5B, the PtNFs@RubMRs modified GCE showed a much higher redox peak currents (curve b) than that of the bare electrode (curve a), ascribing to the excellent electric conduction of PtNPs on the RubMRs. When the CP1 was incubated on the modified electrode, a decreased redox peak current (curve c) was obtained due to the repulsion effect between [Fe(CN)6]3−/4− and the double strands DNA with negative charge. As expected, after blocking with 1 mM HT, the CV responses (curve d) declined on account of the hindrance of HT. When the Fc-DNA was hybridized with the CP1, an ulteriorly decrease of peak current (curve e) was found. Finally, the peak current

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(curve f) was higher than that of curve e, the reason might be that PtNPs might promote the electron transfer. Furthermore, the results of the CV characterization were consistent with the EIS measurements (the details were showed in Supporting Information 1.3).

Figure 5. (A) ECL characterization of the modified electrodes implemented in PBS (pH 7.4). (B) CV of the stepwise modified electrodes performed in PBS (pH 7.4) containing 5.0 mM [Fe(CN)6]3−/4− with the scan range of -0.2 to 0.6 V. (a) bare GCE, (b) PtNFs@RubMRs/GCE, (c) CP1/PtNFs@RubMRs/GCE,

(d)

HT/CP1/PtNFs@RubMRs/GCE,

(e)

Fc-DNA/HT/CP1/PtNFs@RubMRs/GCE, (f) S1/PtNPs/Fc-DNA/HT/CP1/PtNFs@RubMRs/GCE.

Detection of MiRNA-141 with the Biosensor. Under the optimum conditions, the property of the proposed biosensor was investigated. As displayed in Figure 6A, the ECL signal increased with the increasing of concentrations of miRNA-141 in the incubation solution. The calibration plot exhibited a favourable linear relationship between the changes in the ECL intensities (∆IECL) and the logarithm value of miRNA-141 ranged from 10 aM to 100 pM, with a correlation coefficient of 0.9977 (Figure 6B). The regression equation was ∆I = 26062.6 + 1547.54 lgc, with the

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detection limit of 2.1 aM at a signal-to-noise ratio (S/N) of 3, which was more sensitive than those previously reported detection of miRNA-141. The comparison results between the proposed method and other reported assays were listed in Table 1.

Figure 6. (A) ECL intensity-time curves of the proposed biosensor with different concentrations of miRNA-141. (B) The calibrating plot of the proposed biosensor for miRNA-141 assay. Error bars, SD, n = 3. Table 1. Comparison of Different Methods for MicroRNA Detection detection

signal amplification strategy

linear range

method fluorescence

detectio

ref

n limit DSN-assisted target recycling

100 pM~100 nM

100 fM

32

amplification fluorescence

non-enzymatic target recycling

0.1 pM~10 nM

80 fM

33

SWV

DSN-assisted target recycling

5.0 fM~50 pM

4.2 fM

34

PEC

Exo-III assisted target recycling

0.25 fM~0.25 nM

83 aM

35

ECL

DNA Walking Machine

5 fM~500 pM

1.51 fM

36

ECL

Target-Cycling Process

10 aM to 100 pM

2.1 aM

This work

Application of the MiRNA Biosensor in the Tumor Cells. The ECL assay was 20


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performed in 22Rv1 (human prostate carcinoma cells) and MCF-7 (human breast cancer cells) extracts which were disposed by a RNA extraction kit after cell counting to demonstrate the capability of the proposed ECL biosensor. Then, the sensitivities were investigated with the cell concentration from 10 cells to 106 cells. As shown in Figure 7, when the cell concentrations of the 22Rv1 increased from 10 cells to 106 cells, the ECL response increased gradually. While the extraction of MCF-7 cell exhibited low ECL signal with the increase of the cell concentration from 10 cells to 106 cells. The results indicated the higher expression of miRNA-141 in the 22Rv1 cells than the MCF-7 cell, which was well conform to other literature.32

Figure 7. Application of the proposed biosensor in human prostate carcinoma cells (22Rv1) and human breast cancer cells (MCF-7), respectively: (a) 10 cells, (b) 1×102 cells, (c) 1×103 cells, (d) 1×104 cells, (e) 1×105cells, and (f) 1×106 cells.

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Selectivity and Stability of the MicroRNA Biosensor. In order to evaluate the selectivity of the proposed biosensor, the effects of three other miRNAs including miRNA-21, miRNA-199a, miRNA-155 on the ECL intensity were measured. Different reaction solutions containing miRNA-141 (100 pM), miRNA-21 (10 nM), miRNA-155 (10 nM), miRNA-199a (10 nM), and the mixture (containing miRNA-141 (100 pM), miRNA-21 (10 nM), miRNA-199a (10 nM), and miRNA-155 (10 nM)), respectively, were incubated for 90 min at 25 ºC. After reaction, the reaction solutions were incubated with the modified electrodes. As displayed in Figure 8A, compared with the blank, the other three miRNAs showed few effect on the ECL intensity. Moreover, the ECL signal of the biosensor incubating with the mixture solution exhibited similar ECL signal to that incubating with the miRNA-141, which implied that the interfering substances had no remarkable influence on the response to miRNA-141. The above results suggested that the proposed method had high selectivity and specificity for the detection of miRNA-141.

Besides, the stability of the biosensor in ECL detection was also demonstrated by detecting 10 aM miRNA-141 under a continuous scan for 9 cycles in 0.1 M PBS (pH 7.4). As depicted in Figure 8B, the ECL biosensor exhibited barely constant signals and the relative standard deviation (RSD) was 1.415%, indicating excellent stability of the proposed biosensor.

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Figure 8. (A) The specificity of the ECL biosensor with different miRNA. Error bars, SD, n = 3. (B) The ECL-time curve of the ECL biosensor under a continuous scan for 9 cycles in PBS (pH = 7.4) in the presence of 10 aM miRNA-141.

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CONCLUSIONS A novel ternary ECL “on-off-on” switch platform was constructed based on Pt nanomaterials as co-reaction accelerator of dissolved O2 for ultrasensitive miRNA-141 detection in the present work. With the help of the PtNFs, a high ECL intensity was obtained as the first “signal-on” state by accelerating the reaction rate of the RubMRs and dissolved O2. Remarkably, the super “signal on” state was obtained through the PtNPs labeled on the output DNA as co-reaction accelerator, which possessed higher ECL signal than first “signal-on” state to achieve extremely high sensitivity. Therefore, on the basis of the novel ternary ECL system, the ECL biosensor for miRNA-141 detection displayed low detection limit, and high selectivity. Inspired by the employment of Pt nanomaterials as co-reaction accelerator to greatly boost the ECL intensity, the proposed ECL ternary system has provided an efficient way to implement ultrasensitive bioanalysis. ASSOCIATED CONTENT Supporting Information Reagents and apparatus, experimental details for the target-induced cyclic enzymatic amplification (TICEA) the HRTEM images of nanomaterials, and EIS characterization of the ECL biosensor were supplied in Supporting Information. AUTHOR INFORMATION * Corresponding authors Tel.: +86 23 68252277, fax: +86 23 68253172. E-mail addresses: [email protected] (Y. Zhuo), [email protected] (R. Yuan).

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Notes The authors declare no competing financial interest.

Acknowledgements This work was financially supported by the NNSF of China (21675129, 21675130, 21575116), and the Fundamental Research Funds for the Central Universities (XDJK2015A002), China.

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For TOC only

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Ternary Electrochemiluminescence System Based ... - ACS Publications

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