A signal-off electrogenerated chemiluminescence biosensing platform

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A signal-off electrogenerated chemiluminescence biosensing platform based on the quenching effect between ferrocene and Ru(bpy)32+-functionalized MOFs for the detection of methylated RNA Wanqiao Bai, Aiping Cui, Meizhou Liu, xuezhi qiao, Yan Li, and Tie Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02569 • Publication Date (Web): 15 Aug 2019 Downloaded from pubs.acs.org on August 16, 2019

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Analytical Chemistry

A signal-off electrogenerated chemiluminescence biosensing platform based

on

the

quenching

effect

between

ferrocene

and

Ru(bpy)32+-functionalized MOFs for the detection of methylated RNA Wanqiao Bai1, Aiping Cui1, Meizhou Liu1, Xuezhi Qiao2, Yan Li1* and Tie Wang2*

1. Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of the Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an 710069, PR China 2. Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China

*: Corresponding Authors. * Tel/Fax: +86-29-81535026; E-mail: [email protected]. * Tel/Fax: +86-10-82362042; E-mail: [email protected].

1

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ABSTRACT: N6-methyladenine (m6A), one of the most common chemical modifications of eukaryotic RNA, participates in many important biological processes. An effective strategy for the quantitative determination of m6A is of great significance. Herein, we used methylated microRNA-21 (miRNA21) as the model target to propose a simple and sensitive electrogenerated chemiluminescence (ECL) biosensing platform to detect a specific m6A RNA sequence. This strategy is based on the fact that the anti-m6A-antibody can specifically recognize and bind to the m6A site in

the

RNA

sequence,

resulting

in

a

quenching

effect

between

Ru(bpy)32+-functionalized metal-organic frameworks and ferrocene. Luminescent metal-organic frameworks (Ru@MOFs) not only act as ECL indicators but also serve as nanoreactors for the relative ECL reactions owing to their porous or multichannel structure, which overcomes the fact that Ru(bpy)32+ is easily released when used for aqueous-phase detection, thus enhancing the ECL efficiency. Moreover, the ECL method has fewer modification steps and uses only one antibody to recognize the target RNA sequence, which simplifies the operation process and reduces the detection time, presenting a wide linear range (0.001 to 10 nM) for m6A RNA determination with a low detection limit (0.0003 nM). Additionally, this developed strategy was validated for m6A RNA detection in human serum. Thus, the ECL biosensing method provides a new method for m6A RNA determination that is simple, highly specific, and sensitive.

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N6-methyladenosine (m6A) has been identified as one of the most abundant RNA modifications, and it is a dynamic and reversible process of RNA methylation modification that omnipresent in RNA of eukaryotic and nuclear-replicating viruses.1,2 Cumulative studies have provided evidence that variations in the m6A levels provide diverse biological functions in mammals, such as cellular heat shock response,3 embryogenesis,4 transcription splicing,5 nuclear export,6 circadian rhythm,7 protein translation control,8 and proliferation/differentiation of stem cells.9 The regulatory mechanism of m6A modification may be involved in diseases, especially tumorigenesis.10 Therefore, a simple, rapid, and sensitive method for m6A RNA determination is of biological importance. However, the content of m6A in isolated RNA has been estimated to be very low with only approximately 3-5 m6A sites per messenger RNA (mRNA), let alone in the total RNA.11,12 The low abundance of m6A increases the difficulty of its accurate detection. Moreover, the methylation of adenosine does not impede its pairing with thymidine or uracil, making it hardly detectable with reverse transcription-based methods. Although m6A was first discovered in 1974, due to technical limitations, the detection of m6A, especially the quantification of m6A, or even the identification of m6A from the single base level has been progressing slowly for a long time. Until now, different techniques for m6A RNA detection have been proposed. In early studies, some traditional techniques, including capillary electrophoresis,13 thin layer chromatography,14 paper chromatography,15 reversed-phase high-performance liquid chromatography,16 and high-performance liquid chromatography17 have been reported for m6A detection. However, these techniques require labeling RNA with radioactive P32 or require costly and large instruments; however, m6A methylation is 3



detected at the whole level with the detection result often being unsatisfactory. In 2012, two studies proposed the approach of immunoprecipitation followed by a high-throughput sequencing technique, which identified the methylation levels of m6A in human and mouse for a wide range and with high throughput at the transcriptional level.2,18 Afterwards, some other techniques based on sequencing were also developed in succession. Although these techniques provided a qualitative analysis of the hypermethylated mRNA region, complicated multistep procedures, time-consuming detection or the requirement of well-trained operators considerably limit their applications for m6A RNA detection with rapidness. Electrogenerated chemiluminescence (ECL) biosensing method, owing the merits of high sensitivity, easy operation, good controllability, and the equipment is simple and low-cost,19 has attracted considerable attention and offers new opportunities for applications in the determination of biomolecules (such as DNA,20,21 microRNA,22,23 DNA methylation,24,25 and proteins26,27). Metal-organic frameworks (MOFs) are emerging and promising porous crystalline materials with a well-defined crystal structure, adjustable micropores/channels, large surface area, high load capacity for agents, and chemical and thermal stability,28-30 and they have attracted tremendous attention from researchers. These porous organic-inorganic hybrid materials have been exploited for chemical sensing,31 gas storage/separation,32 heterogeneous catalysis,33,34 and transporting/releasing of drugs,35 etc. Currently, the synthesis and applications of functionalized MOFs for the detection of various targets has become another research hot topic in this field.36-39 For instance, Ge et al. fabricated a novel electrochemical biosensor using gold nanoparticle-functionalized Cu-MOFs to immobilize DNA strands for signal reporting, meanwhile, the functionalized Cu-MOFs with excellent catalytic activity also acted as catalyst for the 4


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oxidization of glucose, which resulted to highly sensitive detection of miRNA-155.40 Yuan’s group prepared N-(aminobutyl)-N-(ethylisoluminol) (ABEI)-functionalized MOFs which contained abundant luminescent molecule of ABEI can serve as ECL indicator for mucin1 determination on cancer cells, which provided an improved analytical

performance.41

Shao

et

al.

recently

in

situ

synthesized

Ru(bpy)3Cl2-functionalized MOFs thin film on the electrode surface, which encapsulated numerous Ru(bpy)3Cl2 within the MOFs framework thus can be employed as ECL indicator for the detection of cardiopathy-related protein, which showed excellent ECL behavior.42 Cao’s group constructed a quenching ECL immunosensor using resonance energy transfer between Au@SiO2 nanoparticles and Ru@UiO-67 for the determination of insulin.43 Positively charged Ru(bpy)32+ luminophore was packaged into the electronegative UiO-67 MOFs structure which served as ECL donor, meanwhile, Au@SiO2 nanoparticles acted as ECL acceptor. Because there were suitable overlaps between the emission spectrum of ECL donor and the ultraviolet absorption spectrum of ECL acceptor, resulting in the occurrence of resonance energy transfer between them, which decreased the ECL intensity. Furthermore, the incorporation of Ru(bpy)32+ enhanced both the luminescence lifetime and the quantum yield of the luminophore due to the space limitation effect of MOF micropores.44 Therefore, functionalized MOFs showed great potential in field of biosensing application. To our knowledge, detection of m6A RNA based on Ru(bpy)32+-functionalized MOFs has not been reported yet. In this study, methylated microRNA-21 (miRNA21) was chosen as the model target because miRNA21 is a typical identified miRNA sequence which plays an up-regulating role in various physiological activities and has been widely used in related microRNA researches,45-47 and Ru(bpy)32+-functionalized MOFs (Ru@MOFs) 5



served as the ECL indicator and nanoreactor, thus a simple and sensitive “signal-off” ECL biosensing platform was fabricated to quantitatively detect the m6A RNA sequence based on the quenching of luminescent MOFs by ferrocene (Fc) and the high specificity between the m6A antibody and m6A RNA. In this protocol, luminescent Ru@MOFs were synthesized with trimesic acid (H3BTC) as the ligand and zinc nitrate as the Zn2+ source, and luminescent Ru(bpy)32+ was incorporated into the MOF structure through a one-pot hydrothermal method. The luminophore of Ru(bpy)32+ was encapsulated into the cavities and adsorbed on the surface of the polyhedral MOF structure. The incorporation of Ru(bpy)32+ into the MOF structure can prolong the residence time of the ECL indicator on the electrode surface, which overcomes the drawback that Ru(bpy)32+ is easily released when used for aqueous phase detection; additionally, the multipore/channel structure of MOFs can supply a reaction location for relevant ECL reactions, which can substantially enhance the ECL efficiency and stability. To expand the application of functionalized MOFs in the aqueous phase, carboxylic carbon nanotubes (CNTs) were used to prepare Ru@MOFs/CNT composites for improving the electric conductivity and dispersity, at the same time, increasing the loading amount of m6A antibody due to the abundant carboxyl of the functionalized CNTs. The -COOH at the edge of the Ru@MOFs was activated and conjugated with the m6A antibody. The m6A site in the target RNA sequence can be specifically recognized and captured by the m6A antibody through an immunoreaction between the antibody and antigen. When a complementary DNA sequence modified with Fc at the end of 3’ (Fc-DNA) was introduced, it could hybridize with the target m6A RNA to form a double helix structure, which brought Fc near the ECL indicator on the surface of the electrode, leading to a decreased ECL signal intensity as a result of the high quenching efficiency of ferrocene on 6


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Ru(bpy)32+. Thus, m6A RNA was quantitative detected according to the decrease in ECL intensity. This platform was fabricated without considerable modification, which simplified the detection steps compared with conventional ECL detection. EXPERIMENTAL SECTION The reagents, materials and apparatus are displayed in the Supporting Information. Preparation of the Ru@MOFs and Ru@MOFs/CNTs composites. The synthesis of the Ru@MOFs was performed through a solvothermal process that was reported previously with minor modifications.48 Briefly, 80 mg of H3BTC, 200 mg of Zn(NO3)2·6H2O and 20 mg of Ru(bpy)3Cl2·6H2O were mixed into 6 mL of EtOH/DMF/H2O solution (V(EtOH) : V(DMF) : V(H2O)=1 : 1 : 1) followed by 15 min of ultrasonic treatment. Then, the mixture was settled into a Teflon-lined stainless-steel autoclave and underwent reaction at 105 ℃ for 14 h. The resulting yellow/ white products were collected by a centrifuge and washed with EtOH/DMF/H2O solution three times, and finally, the products were dried at 60 ℃ overnight. Then, 100 mg of the obtained Ru@MOFs were added into 10 mL of the CNT aqueous solution (cCNTs =1 mg/mL) and sonicated for another 2 h to obtain a well-dispersed Ru@MOFs/CNTs solution. After centrifuged and washed, the Ru@MOFs/CNT powder was collected. Then, the obtained Ru@MOFs/CNTs were redispersed into 2 mL of a 0.1% chitosan solution to immobilize the composites on the surface of the electrode in a more stable manner. Fabrication of the ECL Biosensing Platform (Ru@MOFs/CNTs/Ab). First, the surface of a glass carbon electrode (GCE) was cleaned by polishing with 0.3 and 0.05 μm Al2O3 successively. After it was dried, 9 μL of the Ru@MOFs/CNT dispersion was covered on the surface of the GCE. Subsequently, 10 μL of EDC/NHS (100 mM: 7



25 mM) solution was used to activate the carboxyl group in the Ru@MOFs for 1 h. After removing redundant reactants, the dried modified electrode was immersed in 10 μL of m6A antibody (10 μg·mL-1) for 30 min. Then, 10 μL of 0.25% bovine serum albumin (BSA) was added to the surface of the modified GCE at 4 ℃ to block the remaining active sites for 30 min. Followed by rinsing with 10 mM PBS buffer, 10 μL of TE buffer containing different amounts of target methylated RNA were dripped onto the electrode surface for 30 min at 37 ℃. Then, the surface of the electrode was rinsed thoroughly with PBS to remove the unreacted m6A RNA. Finally, the modified GCE was incubated with 10 μL PBS that contained 1 μM of Fc-DNA for hybridization with target RNA for 2 h. Subsequently, the modified electrode was rinsed with 10 mM PBS buffer to eliminate redundant Fc-DNA. Then, the obtained modified electrode served as the working electrode. ECL Determination (Ru@MOFs/CNTs/Ab/m6A/Fc-DNA). ECL measurements were carried out using a commercial ECL system with a scanning range of 0 ~ 1.3 V at a scanning rate of 50 mV·s-1, and the voltage was set to −800 V. The modified GCE served as the working electrode, and a platinum wire and an Ag/AgCl electrode acted as the auxiliary electrode and reference electrode, respectively. The test solution was 1 mL of PBS (0.1 M, pH 7.4) with 50 mM tripropylamine (TPA) as the coreactant. Electrochemical impedance spectroscopy (EIS) was performed using a CHI 660D electrochemical station in PBS (10 mM, pH 7.4) that contained 5 mM Fe(CN)63−/4− and 0.1 M KCl with a frequency range of 10−1 to 105 Hz. RESULTS AND DISCUSSION Principle of m6A RNA Detection. As illustrated in Scheme 1, first, Ru@MOFs/CNTs were coated on the surface of GCE, which generated a very strong initial ECL signal when a positive voltage was applied to the electrode. Because the 8


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incorporation of Ru(bpy)32+ into the MOF structure can prolong the residence time of the ECL indicator on the electrode surface, which overcomes the drawback when Ru(bpy)32+ simply adsorbed on the surface of the electrode used for aqueous phase detection, the multipore/channel structure of MOFs can supply a reaction location for ECL reactions, which can substantially enhance the ECL efficiency and stability. Second, due to the abundant -COOH groups in Ru@MOFs/CNTs, the m6A antibody can be firmly bonded onto the GCE surface through a reaction between the carboxyl and amino groups. Third, the m6A antibody can specifically recognize the m6A site and capture target RNA due to the immunoreaction between the antibody and antigen. Finally, when a complementary DNA sequence modified with ferrocene at the end of 3’ (Fc-DNA) was introduced, it could hybridize with target m6A RNA to form a double helix structure, which brought the Fc near the ECL indicator on the surface of the electrode, leading to a decreased ECL signal intensity because of the high quenching efficiency of ferrocene on Ru(bpy)32+. Thus, m6A RNA could be quantitatively detected according to the decrease in ECL intensity.

9



Scheme 1. Schematic diagram of the ECL platform for m6A RNA determination. (A) Preparation of Ru@MOFs and Ru@MOF/CNT composites and (B) Fabrication and detection principle of the ECL biosensing platform.

Characterization of Materials. The morphologies of the Ru@MOFs and Ru@MOF/CNT composites were investigated by scanning electron microscopy (SEM). Figures 1A-C display the SEM images of the Ru@MOFs and Ru@MOF/CNT composites on different scales. From the figures, we observed that Ru@MOFs showed a uniform rod-shaped structure and dispersed uniformly on CNTs, which supplied a large “hotbed” for the Ru@MOFs. Figure 1D shows the Fourier transform infrared (FT-IR) spectrum of Ru(bpy)32+ (in red) and H3BTC (in black) and luminescent MOF (in blue). As shown, there are two significant changes that occurred after the Ru(bpy)32+ molecule was incorporated into the frameworks. In the FT-IR spectra of Ru@MOFs, the characteristic peaks (3087-2554 cm−1) assigned to carboxylic acid and free COO- (1722 and 1403 cm−1) of H3BTC disappeared, which may be due to the formation of luminescent MOFs. The strong absorption bands at 1437-1382 cm-1 and 1627-1460 cm-1 may be attributed to the vibration of carboxylate anions in H3BTC and the skeletal vibration of bipyridine/benzene in Ru(bpy)32+, respectively.49 X-ray photoelectron spectroscopy (XPS) was performed to confirm the major elemental components of the Ru@MOFs. As expected, the typical XPS spectra of the Zn, O, N, C, and Ru elements were shown; see Figures 1E-H. These characterizations confirmed the successful incorporation of Ru(bpy)32+ into the synthesized MOF structure. Additionally, as presented in Figure S1, the elemental mapping distributions of the single Ru@MOF polygon also confirmed the primary components of Zn and Ru in the functionalized MOFs. The Ru element was derived from Ru(bpy)32+ molecules, which indicated that the luminophor was homogeneously distributed in the framework structure. Moreover, after Ru(bpy)32+ incorporation, the 10


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framework integrity of these MOFs was maintained, as evidenced by the X-ray diffraction (XRD) patterns (Figure S2), indicating that the incorporation of Ru(bpy)32+ did not affect the original morphology. Furthermore, the ζ potentials of the different materials were measured to understand their charge distribution in solution. As illustrated in Figure S3, after incorporating with Ru(bpy)32+ molecules, the surface potential of the MOFs changed from −4.6 to −2.0 due to the charge neutralization between the Ru(bpy)32+ cations and electronegative MOFs particles, which indicated the electrostatic interaction between Ru(bpy)32+ and the MOFs. To validate the incorporation and stability of the Ru@MOFs, the fluorescence of the Ru@MOFs was tested. A broad emission peak (~ 615 nm) assigned to Ru(bpy)32+ and the Ru@MOFs was observed at an excitation of 480 nm; however, no emission peak appeared for the pristine MOFs (Figure S4), which further confirmed the successful combination of Ru(bpy)32+ and the MOFs. Furthermore, the obtained Ru@MOFs were washed for different times with a water/ethanol (1:1) mixture, and the corresponding fluorescence emissions of the Ru@MOFs after washing several times almost did not change (Figure S5), which confirmed the luminescent stability of the Ru@MOFs and that no Ru(bpy)32+ leaked out.

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Figure 1. Typical SEM images of (A) Ru@MOFs and the (B, C) Ru@MOF/CNT composites. (D) FT-IR spectrum of Ru(bpy)32+ (in red), H3BTC (in black) and Ru@MOFs (in blue). XPS of the (E) full region of Ru@MOFs, (F) the Zn 2p (G) the Ru 3d and (H) the Ru 3p region, respectively.

Electrochemical Characterization and Feasibility of the ECL Sensing Platform. To verify the ECL platform can work as anticipated, EIS was conducted to monitor the changes in the modified electrode surfaces during different assembly procedures. The EIS of the electrodes observed upon the stepwise modification procedures are displayed in Figure 2A. The EIS of the bare GCE displayed an almost straight line with a small semicircle domain (curve a), indicating a low electron transfer resistance (Ret) value of the redox couple of [Fe(CN)6]3−/4−. After GCE was modified with the Ru@MOFs/CNTs, EIS revealed an increased Ret value (curve b), which showed that the Ru@MOFs/CNTs possess poor electroconductivity and that electron transfer was hampered. When the m6A antibody was further modified on the electrode, it showed a larger Ret (curve c) compared with the Ru@MOFs/CNTs/GCE because the modified m6A antibody increased the impedance. After BSA was located on the modified 12


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electrode, the Ret further increased (curve d). An obvious increase in the Ret value was obtained after the capture of the target m6A RNA (curve e), which was due to the electrostatic repulsion between the phosphate backbone of the oligonucleotides and the negatively charged redox couple probe. After being immersed in a solution of Fc-DNA, the m6A RNA on the electrode could capture Fc-DNA through hybridization; thus, the Ret value increased further. The EIS results confirmed that the ECL platform was fabricated successfully. As the ECL responses show in Figure 2B, the Ru@MOFs/CNTs/GCE showed an extremely strong ECL signal (curve a) at 1.18 V, which was due to the efficient ECL property of the Ru@MOFs. Then, it slightly decreased after the m6A antibody was bonded to the modified GCE (curve b). When BSA and m6A RNA were successively attached to the modified electrode, the ECL intensity further decreased (curve c). This is because the m6A antibody and BSA are proteins and RNA have poor electroconductivity, which impedes the electron transfer in ECL reactions. The ECL intensity reached a minimum after Fc-DNA attached to the modified GCE (curve d), which was attributed to the strong quenching effect of ferrocene on Ru(bpy)32+. These results confirmed that the proposed ECL sensing method is feasible for m6A RNA detection.

Figure 2. (A) Nyquist plots obtained from different modified electrodes: (a) bare GCE, (b) Ru@MOFs/CNTs/GCE,

(c)

m6A

antibody/Ru@MOFs/CNTs/GCE, 13


(d)

BSA/m6A


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antibody/Ru@MOFs/CNTs/GCE, (e) m6A RNA/BSA/m6A antibody/Ru@MOFs/CNTs/GCE and (f) Fc-DNA/m6A RNA/BSA/m6A antibody/Ru@MOFs/CNTs/GCE. (B) ECL behavior of (a) Ru@MOFs/CNTs/GCE,

(b)

m6A

antibody/Ru@MOFs/CNTs/GCE,

(c)

m6A

RNA

(5

nM)/BSA/m6A antibody/Ru@MOFs/CNTs/GCE, and (d) Fc-DNA/m6A RNA (5 nM)/BSA/m6A antibody/Ru@MOFs/CNTs/GCE for feasibility.

Optimization of experimental conditions. Three main parameters that affect detection performance were optimized, including the dosage of the Ru@MOF/CNT composites, antibody usage, and the reaction time for the antibody and m6A RNA. The dosage of the Ru@MOF/CNT composites had a considerable impact on the ECL performance. As observed in Figure 3A, as the volume of the Ru@MOF/CNT dispersion that was modified on GCE increased, the ECL intensity gradually increased. A high intensity ECL response was obtained when the dosage of the Ru@MOFs/CNTs increased to 9 μL. It was unhelpful to further increase the dosage to enhance the ECL intensity because the surface of GCE is not large enough to accommodate more composites and a thick ECL sensing film is not conductive to detection. Thus, we chose 9 μL as the optimal dosage. To obtain the optimum concentration of the m6A antibody, different concentrations of the m6A antibody were bonded to the Ru@MOFs/CNTs/GCE, followed by capturing 1 nM of the target m6A RNA. The ECL response was recorded and is depicted in Figure 3B, and as the concentration of m6A antibody increased from 0.01 to 10 μg/mL, the intensity of ECL gradually weakened. Upon a further increase in the concentration to 20 μg/mL, the ECL intensity tended to level off, indicating that the antibody exerted its maximum function. Therefore, 10 μg/mL of m6A antibody was used for the following measurements. Figure 3C shows the impact of the reaction time for the antibody and target m6A RNA (1 nM) on the modified electrode. When the reaction time was extended from 10 to 60 min, the ECL intensity showed a downward trend. Upon 14


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further prolonging the reaction time, the ECL intensity slowly decreased. Considering the detection efficiency, 60 min was employed.

Figure 3. Effects of (A) the dosage of the RuMOF/CNT composites (only RuMOF/CNTs modified on the GCE surface), (B) concentration of the m6A antibody, (C) and immunoreaction time between the m6A antibody and m6A RNA on the ECL intensity. The concentration of m6A RNA was 1 nM in (B) and (C).

Detection Performance. Under the optimal experimental conditions, different concentrations of methylated m6A RNA were detected using this proposed ECL sensing platform. Figure 4A displays the typical ECL response of the probe for Fc-DNA/m6A, RNA/BSA/m6A and antibody/Ru@MOFs/CNTs/GCE to various concentrations of target m6A RNA. As shown in Figure 4A, the ECL intensities presented a good linear relationship with the logarithm of the m6A RNA concentration (lg c) in the range of 0.001 to 10 nM. The linear regression equation of I = 3225.03−1843.26 lg (c/nM) (R = 0.997) was obtained for quantitative detection of the target methylated RNA, and the estimated detection limit was 0.3 pM at a signal-to-noise ratio of 3σ (σ refers the standard deviation for the blank, n = 8). In addition, a comparison of the m6A RNA detection performances using different sensing methods is displayed in Table S2 (Supporting Information). The results showed that this “signal-off” ECL strategy is promising for quantitative detection of m6A RNA. Reproducibility, Specificity and Stability of the Proposed ECL Method. The reproducibility of this ECL strategy was verified by analysis of the same 15



concentration of m6A RNA (0.1 nM) using ten electrodes prepared in parallel under the same conditions. As depicted in Figure 4B, the reproducibility of this ECL sensing platform was acceptable with a relative standard deviation (RSD) of less than 5.0%. To investigate the specificity of this ECL strategy, 0.1 nM of unmethylated RNA, single-base mismatched m6A RNA, three-base mismatched m6A RNA, methylated miRNA-155, methylated miRNA-101, target m6A RNA, and a mixed sample were used as the contrast objects to evaluate the specificity. From Figure 4C, we observe that this ECL biosensing platform almost had no quenching response for the unmethylated RNA and other methylated DNA; however, it showed a great ECL decrease for the same concentration of m6A RNA and 0.1 nM m6A RNA in a mixture with interferences, indicating the high specificity of the proposed ECL biosensing method. Figure 4D shows the ECL responses of the sensing platform under consecutive cyclic potential scanning for 15 cycles in 1 mL of PBS that contained 50 mM TPA for the determination of 0.1 nM of m6A RNA. We observed that the ECL intensity did not exhibit an obvious change, and the RSD of the ECL intensity was 2.7%. After the fabricated electrode was stored at 4 °C for three weeks, the ECL response of the sensing platform for the same m6A RNA increased by 9.3% (Figure S6), which may have been caused by the partial inactivation of m6A RNA. Therefore, this ECL biosensing platform possesses good stability.

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Figure 4. (A) ECL intensity and calibration curve for this ECL sensing platform for various quantities of m6A RNA (0.001, 0.01, 0.05, 0.1, 1, 5, and 10 nM). (B) Reproducibility of 10 biosensing electrodes towards 0.1 nM of m6A RNA. (C) Specificity of this ECL biosensing method towards a blank sample, 0.1 nM of unmethylated RNA, single-base mismatched m6A RNA, three-base mismatched m6A RNA, methylated miRNA-155, methylated miRNA-101, the target m6A RNA and a mixed sample. (D) Stability of the ECL biosensing platform towards 0.1 nM of m6A RNA under continuous cyclic potential scanning for 15 cycles.

Biological Sample Detection. The practical applicability of the proposed ECL sensing platform for methylated RNA detection was assessed using a standard addition method. The target m6A RNA at different concentrations was spiked into the pretreated serum samples. Then, the ECL platform was utilized for target m6A RNA determination in these samples. The corresponding results are listed in Table S3. The recoveries were between 88.4% and 98.3% for 0.01 nM to 1 nM in the serum samples, confirming that the analytical performance of this ECL biosensing method was not compromised in complex matrices. These results demonstrated that the 17



fabricated ECL method provides a potential analytical strategy for clinical samples. CONCLUSIONS In summary, a simple and sensitive “signal off” ECL biosensing platform was fabricated for the detection of m6A RNA based on the quenching ECL of Ru(bpy)32+-functionalized MOFs by ferrocene, as well as the recognition of the m6A antibody. In this study, Ru@MOFs were synthesized by a one-pot hydrothermal method, resulting in the incorporation of abundant Ru(bpy)32+ molecules and frameworks, which can hold the ECL indicator on the surface of the working electrode for a long time, thus enhancing the ECL efficiency. Then, composites of Ru@MOF/CNTs were utilized to fabricate the ECL sensing platform, which showed remarkable ECL performance. In addition, only one kind of antibody was used, and less modification made this strategy more efficient. Based on the specific antibody-antigen reaction and quenching effect, the ECL biosensing platform can achieve satisfying results with a high sensitivity, low detection limit, and wide detection range for m6A RNA. In addition, the proposed ECL strategy provided satisfactory reproducibility for m6A RNA determination in human serum samples, which verified its practical applicability. Furthermore, exploiting the advantages of MOFs for loading ECL luminophores provides a new strategy for the fabrication of ECL sensing systems.

ASSOCIATED CONTENT Supporting Information Supporting information includes reagents and materials (including Table S1), elemental maps for Ru@MOFs (Figure S1), XRD patterns of MOFs and Ru@MOFs (Figure S2), the ζ potential of the Ru(bpy)32+, H3BTC, MOFs and Ru@MOFs (Figure 18


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S3), fluorescence spectra of Ru(bpy)32+, MOFs and Ru@MOFs (Figure S4), fluorescence spectra of Ru@MOFs after washing for different times (Figure S5), comparison with other reported N6-methyladenosine RNA detection methods (Table S2), long-term stability of the ECL biosensing platform (Figure S6) and results of biological samples detection (Table S3).

AUTHOR INFORMATION Corresponding Authors * Tel/Fax: +86-29-81535026; E-mail: [email protected]. * Tel/Fax: +86-10-82362042; E-mail: [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by the National Natural Science Funds of China (No. 21675124, No. 21375102 and No. 201706214), the Natural Science Basic Research Plan in Shaanxi Province of China (No. 2016JM2021) and Key Laboratory of Yulin Desert Plant Resources.

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A signal-off electrogenerated chemiluminescence biosensing platform

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