Electrochemical immunosensor for N6-methyladenosine detection in ...

2 downloads 0 Views 951KB Size Report
Oct 27, 2016 - N6-methyladenosine (m6A), a kind of RNA methylation form and ... electrochemical method was developed for m6A detection using N6-.
Biosensors and Bioelectronics 90 (2017) 494–500

Contents lists available at ScienceDirect

Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios

Electrochemical immunosensor for N6-methyladenosine detection in human cell lines based on biotin-streptavidin system and silver-SiO2 signal amplification ⁎

Huanshun Yin, Haiyan Wang, Wenjing Jiang, Yunlei Zhou , Shiyun Ai

MARK



College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, PR China

A R T I C L E I N F O

A BS T RAC T

Keywords: RNA methylation N6-methyladenosine Electrochemical immunosensor Phos-tag-biotin Ag@SiO2 nanocomposite

N6-methyladenosine (m6A), a kind of RNA methylation form and important epigenetic event, plays crucial roles in many biological progresses. Thus it is essential to quantitatively detect m6A in complicated biological samples. Herein, a simple and sensitive electrochemical method was developed for m6A detection using N6methyladenosine-5′-triphosphate (m6ATP) as detection target molecule. In this detection strategy, anti-m6A antibody was selected as m6A recognition and capture reagent, silver nanoparticles and amine-PEG3-biotin functionalized SiO2 nanospheres (Ag@SiO2) was prepared and used as signal amplification label, and phos-tagbiotin played a vital role of “bridge” to link m6ATP and Ag@SiO2 through the two forms of specific interaction between phosphate group of m6ATP and phos-tag, biotin and streptavidin, respectively. Under the optimal experimental conditions, the immunosensor presented a wide linear range from 0.2 to 500 nM and a low detection limit of 0.078 nM (S/N=3). The reproducibility and specificity were acceptable. Moreover, the developed method was also validated for detect m6A content in human cell lines. Importantly, this detection strategy provides a promising immunodetection platform for ribonucleotides and deoxyribonucleotides with the advantages of simplicity, low-costing, specificity and sensitivity.

1. Introduction N6-methyladenosine (m6A) is a kind of important epigenetic modification, which mainly exists in the messenger RNA (mRNA) and other types of RNA (such as transfer RNA, ribosomal RNA, small nuclear RNA and long non-coding RNAs) of higher eukaryotes and some viruses (Lin and Gregory, 2014). Although about 160 types of various RNA modification have been distinguished in eukaryotic RNAs, m6A is also one of the most common and abundant RNA modification forms, which is not only involved in the biological processes of apoptosis, circadian clock, immune tolerance, stimulus responses and meiosis, but also is related to human health (Cantara et al., 2011; Dominissini et al., 2012; Harcourt et al., 2013; Machnicka et al., 2013; Schwartz et al., 2013). Though the discovery of m6A in mRNA decades ago, many bio-functions of m6A are still unclear. For solving these questions, simple and sensitive method for m6A detection is necessary. Up to now, some techniques have been developed for RNA methylation discrimination and m6A detection, such as thin-layer chromatography (Liu et al., 2013), paper electrophoresis (Kane and Beemon, 1985), paper chromatography (Canaani et al., 1979), microarray (Li et al., 2015d), reversed-phase high-performance liquid ⁎

chromatography (Xu et al., 2000), m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (Linder et al., 2015), photo-crosslinking-assisted m6A sequencing (Chen et al., 2014), m6A-specific methylated RNA immunoprecipitation with next generation sequencing (Meyer et al., 2012), capillary electrophoresis (Liebich et al., 2000), electrochemiluminescence (Lin et al., 2010). However, some of these techniques, such as thin-layer chromatography, paper electrophoresis and paper chromatography, require labeling RNA with P32, which is harmful to the operator's health. Other techniques need expensive and large instrument, complicated operation step, long detection time and skilled operator, which also limit their applications for rapid detection of m6A. Recent decade years, electrochemical technique has attracted extensive interests due to the advantages of simple operation, rapid detection, reagent-saving, miniaturized instrument, high sensitivity and selectivity. To date, electrochemical methods have been widely applied in many fileds, such as food safety (Dutta and Puzari, 2014; Najafi et al., 2014), environmental pollution (Lin et al., 2015b; Tortolini et al., 2015), immunoassay (Gao et al., 2015; Huang et al., 2015), protein phosphorylation (Wang et al., 2012; Xu et al., 2009; Yin et al., 2015a; Zhang et al., 2016), and nucleic acid detection (Li et al.,

Corresponding authors. E-mail addresses: [email protected] (Y. Zhou), [email protected] (S. Ai).

http://dx.doi.org/10.1016/j.bios.2016.10.066 Received 17 August 2016; Received in revised form 5 October 2016; Accepted 25 October 2016 Available online 27 October 2016 0956-5663/ © 2016 Elsevier B.V. All rights reserved.

Biosensors and Bioelectronics 90 (2017) 494–500

H. Yin et al.

2.3. Preparation of graphene-AuNPs nanocomposite

2015a; Wang et al., 2014; Zhou et al., 2016). More importantly, electrochemical technique has also been applied to analyze nucleic acid modification, such as DNA methylation (Li et al., 2015c; Yang et al., 2015; Yin et al., 2014; Zhu et al., 2015) and DNA hydroxymethylation (Chen et al., 2016; Zhou et al., 2015). Thus, electrochemical method should be an alternative technique for m6A detection, and several works have been done. Lin et al. developed an electrochemiluminescence method for m6A detection with high sensitivity (Lin et al., 2010). However, the detection was interfered by amino acids with low quantities (0.5 times to m6A), such as glycine, cysteine and histidine. In addition, our group fabricated an electrochemical immunosensor for RNA methylation detection. But in that work, short RNA sequence was used as detection target, and it cannot achieve the quantitative detection of single m6A residue in total RNA extracted from biological sample (Yin et al., 2015b). Thus, it is necessary to develop new electrochemical method to achieve the single m6A residue detection with high detection specificity.. Herein, a sensitive electrochemical immunosnesor was fabricated for quantitative detection of m6A because it contain the advantages of electrochemical technique and immunoassay, especially the merit of high detection specificity due to the specific interaction between antibody and antigen (Duangkaew et al., 2015; Li et al., 2015b; Liu et al., 2015). In order to obtain high detection sensitivity, silver nanoparticles (AgNPs) functionalized SiO2 (Ag@SiO2) was used as signal amplification unit due to the advantages of easy dissolution in HNO3, simple synthesis, and excellent electrochemical performance with a relatively sharp peak for AgNPs (Lin et al., 2015a; Peng et al., 2011; Shahdost-fard and Roushani, 2017; Xie et al., 2016), and good hydrophility, simple preparation process, easy surface functionalization, and good biocompatibility for carboxyl functionalized SiO2 nanosphere (Chen et al., 2015; Ensafi et al., 2016; Li et al., 2015e). In addition, phos-tag-biotin was employed as bridge to link m6A and Ag@ SiO2. For achieving the detection of m6A residue in biological samples, single m6A ribonucleotide was used as the detection target. The developed method showed high detection specificity and sensitivity. The content of m6A in mRNA of normal cell line and cancer cell line were also detected.

See Supplementary materials. TEM images of Graphene-AuNPs was also shown in Supplementary materials (Fig. S1). 2.4. Biosensors fabrication

2. Experimental

The glassy carbon electrode (GCE, d=3 mm) was first polished to a mirror-like surface using 30 nm Al2O3 slurry, and then the electrode was sonicated successively in ethanol and double distilled deionized water for 3 min, respectively. After dried under N2 blowing, 10 μL of 1 mg/mL graphene-AuNPs dispersion was dripped on GCE surface and dried under the irradiation of infrared lamp. The obtained electrode was noted as GR-Au/GCE. Then, the electrode was incubated with 10 μL of 10 mM PBS (pH 8.0) containing 5 mM MPBA for 60 min at ambient temperature in a humid cell. The obtained electrode was noted as MPBA/GR-Au/GCE. After rinsed with double distilled deionized water, the electrode was further incubated with 10 μL of 10 mM PBS (pH 7.4) containing 5 μg/mL m6A antibody for 60 min under ambient temperature. The electrode was rinsed with washing buffer for three times and noted as Ab/MPBA/GR-Au/GCE. Afterwards, 10 μL of 10 mM PBS (pH 7.4) containing different concentrations of m6ATP was dripped on the electrode surface and incubated for 50 min at 37 °C under humid environment. Then, the electrode was rinsed with washing buffer for three times and noted as m6ATP/Ab/MPBA/GRAu/GCE. Subsequently, the electrode was further incubated with 10 μL phos-tag-biotin reaction buffer containing 11 µm phos-tag-biotin for 45 min at ambient temperature. Then, the electrode was rinsed with washing buffer and marked as Biotin/m6ATP/Ab/MPBA/GR-Au/GCE. For immobilizing streptavidin, 10 μL of 10 mM PBS (pH 7.4) containing 10 μg/mL streptavidin was casted on the electrode surface, and the reaction between biotin and streptavidin was performed for 60 min at ambient temperature under humid environment. The obtained electrode was noted as SA/Biotin/m6ATP/Ab/MPBA/GR-Au/GCE. Finally, 11 μL of biotin functionalized Ag@SiO2 dispersion (1 mg/ mL) was dripped on the electrode surface and incubated for 50 min at ambient temperature in a humid cell. The obtained electrode was rinsed with washing buffer for three times and noted as Ag/SA/Biotin/ m6ATP/Ab/MPBA/GR-Au/GCE.

2.1. Reagents and solutions

2.5. Electrochemical detection

See Supplementary materials.

The electrochemical experiments were performed at a CHI660C electrochemistry workstation (Austin, USA) with three electrodes system, where the GCE or modified GCE was used as working electrode, the saturated calomel electrode as reference electrode and the platinum electrode as counter electrode. For m6A concentration detection, the fabricated electrode was first incubated with 8 mL of 10 mM HNO3 for 10 min with magnetic stirring. The solution containing Ag+ was used as detection solution, and then differential pulse stripping voltammetry (DPSV) was carried out with the potential range of −0.1–0.6 V using GCE as working electrode. The parameters of DPSV are as follows: deposition potential, −0.5 V, deposition time, 200 s, initiative potential, −0.1 V, final potential, 0.6 V, step potential, 0.004 V, amplitude, 0.05 V, pulse width, 0.05 s, pulse period, 0.2 s, quiet time, 2 s. Electrochemical impedance spectroscopy (EIS) was performed in 10 mM PBS containing 5 mM Fe(CN)63−, 5 mM Fe(CN)64− (it can also be marked as 5 mM Fe(CN)63−/4−) and 0.1 M KCl (pH 7.4) with the frequency range of 10−1 - 105 Hz.

2.2. Preparation of Ag@SiO2 nanocomposite 5 mg of carboxyl functionalized SiO2 nanospheres was dispersed into 50 mL deionzed water with the aid of ultrasonication for 30 min. Then, the dispersion was added into a 250 mL round-bottom flask with magnetic stirring. After that, 10 mL of 3 mM AgNO3 was added into the round-bottom flask and the mixture dispersion was further stirred for 1 h. Subsequently, 5 mL of 6 mM sodium citrate was added quickly into the flask. After boiling for 30 min, the obtained suspension was centrifuged with 8000 rpm for 20 min. Then, the sediments were collected and washing with deionzed water for three times. After dried in vacuum, 1 mg of Ag@SiO2 nanocomposite was dispersed in 0.5 mL of 10 mM PBS (pH 7.4) under ultrosonication. Then 0.5 mL of the mixture solution of 50 mM EDC and 100 mM NHS was added and the dispersion was incubated for 1 h with shaking. Subsequently, 1 mL of Amine-PEG3-Biotin (5 mg/mL) was introduced into the dispersion and reacted for 1 h. After centrifugation, the sediment was incubated with 1 mL of 0.1 mM ethanolamine for 1 h to remove the carboxyl residues on SiO2. Finally, the biotin functionalized Ag@SiO2 nanocomposite was collected by centrifuging with 8000 rpm and the obtained sediment was dried under vacuum. TEM images of SiO2 and Ag@SiO2 were shown in Supplementary materials (Fig. S1).

2.6. Cell cultural and total RNA extraction Cell lines were cultured in DMEM (Dulbecco's modified Eagle's medium) (Gibco-BRL, USA) supplemented with 10% (v/v) fetal bovine serum (Invitrogen, New Zealand) in a CO2 (5%) incubator at 37 °C. The cultured cells were washed three times with normal saline. Total RNA 495

Biosensors and Bioelectronics 90 (2017) 494–500

H. Yin et al.

Scheme 1. (A) Preparation of Ag@SiO2. (B) Schematic illustration of biosensor fabrication and electrochemical detection of m6A.

from cells was extracted using TRIzol reagent according to the manufacturer's recommended protocol. mRNA was extracted from total RNA using mRNA Isolation Kit (Roche) according to the recommended protocol.

Ag and the concentration of m6A, the quantitative detection of m6A can be achieved.

3. Results and discussion

The electrode fabrication process was characterized by EIS technique and the results were shown in Fig. 1. The bare GCE presents a welldefined semi-circle in high frequency region with the interface electron transfer resistance (Ret) of 293 Ω (curve a). After immobilization of graphene-AuNPs, the Ret value decreased significantly and only a straight line can be observed (curve b), indicating a diffusion-controlled process. The decreased Ret can be ascribed to the immobilization of graphene-AuNPs, which increases the active surface area of GCE and facilitates the diffusion of the redox probe of Fe(CN)63−/4−. Then, a slightly increased Ret is obtained after the GR-Au/GCE was incubated with MPBA (curve c). It can be attributed to negative charged boric acid of MPBA, which blocked the diffusion of negative charged redox probe of Fe(CN)63−/4−. Afterwards, the Ret value increased significantly when

3.2. EIS characterization

3.1. Detection strategy In this work, a sensitive and selective electrochemical method is proposed for m6A detection based on anti-m6A antibody, phos-tagbiotin and Ag@SiO2 nanocomposite, where anti-m6A antibody is used as m6A recognition and capture reagent, phos-tag-biotin is used as a bridge to connect m6ATP and streptavidin, and Ag@SiO2 is used as signal amplification unit. In addition, Gr-Au was selected as electrode substrate material because it has good conductivity, large specific surface area and excellent biocompatibility (Yin et al., 2015c; Zhao et al., 2015). As illustrated in Scheme 1, after immobilization of Gr-Au on GCE, MPBA can be self-assembled on electrode surface through the formation of Au-S bond. Then, based on the chemical reaction between boric acid of MPBA and glycosyl of anti-m6A antibody, the antibody can be captured on the electrode surface. Afterwards, m6ATP (N6methyl-2′-deoxyadenosine-5′-triphosphate) can be further captured through the specific immunoreaction between m6A and anti-m6A antibody, where the phosphate group in m6ATP is away from the electrode. Phos-tag is a kind of organic chelating agent with specific recognition ability for phosphate group in the presence of Zn2+ or Mn2+(Kinoshita et al., 2013). Therefore, phos-tag-biotin is employed to play a “bridge” role for connecting phosphate group and streptavidin, and the immobilized streptavidin can further capture biotin functionalized Ag@SiO2 nanocomposite as signal amplification unit. As shown in Fig. S1B, one SiO2 nanosphere can load many AgNPs, which can increase the immobilization amount of AgNPs on the electrode surface. As a result, the electrochemical signal can be improved, which can achieve the signal amplification. After the fabricated electrode is immersed into 0.1 M HNO3 solution, the Ag nanoparticles can be oxidized to form Ag+. Then, the Ag+ can be electrochemically deposited on GCE surface and Ag can be electrochemically oxidized in detection buffer. Based on the relationship between the oxidation peak current of

Fig. 1. The Nyquist plot of different electrodes in 10 mM PBS containing 5 mM Fe(CN)63−/4− and 0.1 M KCl (pH 7.4) with the frequency range of 10−1–105 Hz. (a) GCE, (b) GR-Au/GCE, (c) MPBA/GR-Au/GCE, (d) Ab/MPBA/GR-Au/GCE, (e) m6ATP/ Ab/MPBA/GR-Au/GCE, (f) Biotin/m6ATP/Ab/MPBA/GR-Au/GCE, (g) SA/Biotin/ m6ATP/Ab/MPBA/GR-Au/GCE, (h) Ag/SA/Biotin/m6ATP/Ab/MPBA/GR-Au/GCE.

496

Biosensors and Bioelectronics 90 (2017) 494–500

H. Yin et al.

m6ATP/Ab/MPBA/GR-Au/GCE was directly treated with SA and Ag@SiO2. It was clearly that the electrochemical response was weak and almost no oxidation peak could be observed (curve d, Fig. 2). This phenomenon demonstrated that Ag@SiO2 could not be captured on the electrode surface due to the absence of bridging unit of phos-tag-biotin.

the electrode was incubated with anti-m6A antibody (curve d). There is no doubt that this increase is caused by the modification of antibody on the electrode surface, and the huge volume of antibody further hinders the diffusion of the redox probe. After capture of m6ATP, the Ret value increased greatly (curve e) due to the strong electrostatic repulsion effect between phosphate group of modifier and the redox probe. Subsequently, the Ret value increased successively when phos-tagbiotin (curve f) and strepavidin (curve g) are captured on the electrode surface, respectively. It can be explained as the fact that the immobilized phos-tag-biotin and strepavidin impede the diffusion of Fe(CN)63−/4− to electrode surface and decrease the electron transfer rate. After the immobilization of Ag@SiO2, the diameter of semi-circle increased a little (curve h), indicating that the Ret value slightly increased. It can be ascribed to the –OH on SiO2, which blocked the diffusion of the redox probe due to the electrostatic repulsion. According to the change of the Ret value after different modification process, the successful fabrication of the biosensor can be confirmed.

3.4. Optimization of detection conditions In order to achieve the high detection sensitivity, several parameters were optimized, such as antibody concentration, antibody immobilization time, m6ATP immobilization time, phos-tag-biotin concentration, phos-tag-biotin reaction time and Ag@SiO2 immobilization time. Fig. 3A illustrated the effect of antibody concentration on the electrochemical response of the biosensor. The electrochemical response increased when antibody concentration was changed from 0.1 to 5.0 μg/mL. Then, the electrochemical response only increased a little when further enhancing antibody concentration, which can be ascribed to the saturated immobilization of antibody. Thus 5.0 μg/mL was chosen as the optimal concentration for antibody. The effect of antibody immobilization time was also investigated. As shown in Fig. 3B, the electrochemical response increased with extending the immobilization time from 5 to 60 min, and a plateau was achieved when further prolonging the immobilization time. With long immobilization time, more antibodies can be captured on the electrode surface, which can lead to the high immobilization efficiency of m6ATP. As a result, more Ag@SiO2 can be captured on the electrode surface and resulted in an increasing electrochemical response. However, when further prolonging the immobilization time, the immobilization of antibody tended to saturate, resulting a plateau for electrochemical response. Therefore, 60 min was selected in this work. For m6A detection, the high efficiency of m6A capture can greatly improve the detection sensitivity. So the effect of m6ATP immobilization time on the electrochemical response was investigated. As presented in Fig. 3C, the electrochemical response of the biosensor increased with extending the immobilization time from 5 to 50 min. Then the electrochemical response plateaued when further prolonging the immobilization time. With increasing the immobilization time, the immobilization amount of m6ATP also increased, which could lead to the increased immobilization amount of Ag@SiO2. Based on it, a large electrochemical signal can be achieved. However, for long immobilization time, the capture of m6ATP could be saturated and a stable electrochemical response can be obtained. Herein, 50 min was used as the optimal condition. As a kind of important “bridge-link” reagent, the phos-tag-biotin concentration and phos-tag-biotin reaction time were optimized. As seen in Fig. 3D, the electrochemical response of the biosensor increased with changing the concentration of phos-tag-biotin from 0.1 to 11 µm. Under the fixed immobilization time, more phos-tagbiotin can be immobilized on the electrode surface with high concentration, and lead to a stronger electrochemical signal. However, the electrochemical response of the biosensor tends to level off when the concentration of phos-tag-biotin was higher than 11 µm. Considering the detection efficiency, 11 µm was used. Fig. 3E showed the effect of phos-tag-biotin immobilization time. According to the results, 45 min was chosen. Ag@SiO2 was used in this work as signal amplification unit, and its immobilization amount can also influence the detection sensitivity. Thus Ag@SiO2 immobilization time was optimized. As presented in Fig. 3F, the optimal immobilization of 50 min was employed.

3.3. Detection feasibility assay In order to testify the detection feasibility, several experiments were performed and the detection results were shown in Fig. 2. No redox peak was observed for SA/Biotin/m6ATP/Ab/MPBA/GR-Au/GCE (curve a) after it was immersed into 10 mM HNO3 solution for 10 min with stirring. This result indicated that the immobilized materials were electrochemical inactivity in the selected potential range. However, for Ag/SA/Biotin/m6ATP/Ab/MPBA/GR-Au/GCE (cm6ATP=10 nM, curve b), an obviously electrochemical oxidation peak was obtained at 0.29 V, which could be attributed to the electrochemical oxidation behavior of electrodeposited Ag on GCE surface. It also indicated that the signal amplification unit was successfully captured on the electrode surface through the interaction between biotin and strepavidin. For further proving the detection feasibility, the electrochemical response of the biosensor with different concentrations of m6ATP was compared. As also seen in Fig. 2, the electrochemical oxidation signal increased apparently when m6ATP concentration changed from 10 (curve b) to 100 (curve c) nM, which confirmed that the change of m6ATP concentration could cause accordingly the change of the electrochemical response of Ag. The above results also proved that the developed method could be used for m6A detection. Phos-tag-biotin is used as an important “bridge” role in this work and it can connect the detected target molecule of m6ATP and the signal amplification unit of Ag@SiO2. For verifying this crucial function of phos-tag-biotin, a control experiment was performed, where

Fig. 2. Different pulse voltammograms of GCE in 10 mM HNO3 after different electrodes were immersed into this HNO3 solution for 10 min with stirring. (a) SA/ Biotin/m6ATP/Ab/MPBA/GR-Au/GCE. (b) and (c), Ag/SA/Biotin/m6ATP/Ab/MPBA/ GR-Au/GCE, where m6ATP concentration were 10 and 100 nM for (b) and (c), respectively. (d) m6ATP/Ab/MPBA/GR-Au/GCE was directly incubated with SA and Ag@SiO2 successively.

3.5. Detection performances Under the optimal experiment conditions, the relationship between m6ATP concentration and the oxidation peak current of Ag was investigated. As presented in Fig. 4A, the oxidation peak current 497

Biosensors and Bioelectronics 90 (2017) 494–500

H. Yin et al.

Fig. 3. Effect of antibody concentration (A), antibody immobilization time (B), m6ATP immobilization time (C), phos-tag-biotin concentration (D), phos-tag-biotin reaction time (E) and Ag@SiO2 immobilization time (F) on the electrochemical response of the biosensor. m6ATP concentration was 10 nM.

Detection specificity is an important property for a kind of analytical method. In order to investigate the specificity of the developed method for m6A detection, 11 nucleotides (ATP, CTP, GTP, UTP, dATP, dCTP, dGTP, dTTP, 5mdCTP, m6dATP, and 5hmdUTP) were selected as interferents. As shown in Fig. 4B, except m6dATP, other 10 kinds of nucleotides do not interfere with the detection of m6ATP with very weak electrochemical signal. As for m6dATP, there is m6A structure in m6dATP molecule, similar with m6ATP (Inset of Fig. 4B). Therefore, the m6A antibody can also

enhanced with increasing the concentration of m6ATP. The oxidation peak current presented a linear relationship with m6ATP concentration in the range of 0.2–500 nM (Inset of Fig. 4A). The linear regression equation can be expressed as Ipa (μA)=0.84 logc (nM)+0.98 (R=0.9956) and the limit of detection was calculated to be 0.078 nM (S/N=3). The detection limit of the fabricated biosensor was lower than that obtained at electrochemiluminescence biosensor (0.77 nM) and the linear range was comparable with that work (1.9–3900 nM) (Lin et al., 2010). 498

Biosensors and Bioelectronics 90 (2017) 494–500

H. Yin et al.

Fig. 5. The relative expression of m6A in different human cell lines.

interference of m6dATP, the total RNA was first extracted from the cell lysate, and then mRNA was further isolated. Subsequently, mRNA was incubated with 1 µm poly(dT)20, 50 unit/mL RNase H and 100 μg/ mL RNase A for 2 h at 37 °C. Afterwards, 10 μL of the digested solution was used to fabricate the biosensor to detect the content of m6A. As shown in Fig. 5, the electrochemical responses of HepG-2 and MCF-7 were higher than that at HEK239T, indicating a higher concentration of m6A in HepG-2 and MCF-7. For further proving the accuracy of the developed method in biological sample detection, the m6A content in the extracted total RNA samples was also compared using EpiQuik M6A RNA Methylation Quantification Kit (Epigentek, USA). The results also demonstrated the high concentration of m6A in cancer cell, indicating the detection reliability of our developed method. 4. Conclusions Fig. 4. (A) Differential pulse voltammograms of the electrochemical immuniosensor with different concentrations of m6ATP. a-i: 0.2, 0.5, 1, 5, 10, 50, 100, 250, 500 nM. Inset graph, the relationship between the electrochemical response and the logarithm value of m6dATP concentration. The data was the average value of three detections (n=3). (B) The electrochemical response of the biosensor with 50 nM of different kinds of nucleotides.

In summary, an electrochemical immunoassay was constructed for m6A detection using Gr-Au as signal amplification unit, anti-m6A antibody as m6A recognition reagent, phos-tag-biotin as bridging reagent and Ag@SiO2 as signal label and signal amplification unit. With the dual signal amplifications, the fabricated biosensor showed wide linear range from 0.2 to 500 nM with a low detection limit of 0.078 nM. In addition, the developed method also presented good detection selectivity based on the specific immunoreaction. Moreover, the fabricated biosensor was further successfully applied to detect m6A in biological samples with high detection accuracy, indicating that this work maybe provide a new platform for the investigation of m6A biofunctions.

recognize and capture m6dATP and interfere with the detection of m6ATP. However, m6dATP is a kind of deoxyribonucleotide, which can only exit in DNA. And m6ATP is a kind of ribonucleotide, which can only exit in RNA. So m6dATP will not interfere with the detection of m6ATP content in RNA as long as the extraction of total RNA from sample. Based on these results, the developed method presented good selectivity for m6ATP detection. Reproducibility is another key parameter for analytical technique. For testifying the reproducibility of the developed method, seven biosensors were fabricated independently with m6ATP concentration of 100 nM. And the electrochemical response was compared and the relative standard deviation (RSD) for the seven measurements was 5.21%, indicating that the developed method possessed good detection reproducibility. For stability investigation, Ab/MPBA/GR-Au/GCE was used as substrate electrode. After the substrate electrode was immersed in 10 mM PBS (pH 7.4) and stored in refrigerator for one week, it was used to fabricate biosensor for 100 nM m6dATP detection. The result indicated that it could retain 92% of its original current.

Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21375079), the Natural Science Foundation of Shandong province, China (No. ZR2014BQ029), and the Project of Development of Science and Technology of Shandong Province, China (No. 2013GZX20109). Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.10.066.

3.6. Sample assay

References

In order to verify the applicability of the fabricated biosensor in biological samples, the content of m6A in human hepatoma cell line HepG-2, human breast carcinoma cell line MCF-7, and human embryonic kidney cell HEK239T were detected. For avoiding the

Canaani, D., Kahana, C., Lavi, S., Groner, Y., 1979. Nucleic Acids Res. 6, 2879–2899. Cantara, W.A., Crain, P.F., Rozenski, J., McCloskey, J.A., Harris, K.A., Zhang, X., Vendeix, F.A., Fabris, D., Agris, P.F., 2011. Nucleic Acids Res. 39, D195–D201. Chen, K., Lu, Z., Wang, X., Fu, Y., Luo, G.-Z., Liu, N., Han, D., Dominissini, D., Dai, Q.,

499

Biosensors and Bioelectronics 90 (2017) 494–500

H. Yin et al.

65, 281–286. Liu, N., Parisien, M., Dai, Q., Zheng, G., He, C., Pan, T., 2013. RNA 19, 1848–1856. Machnicka, M.A., Milanowska, K., Oglou, O.O., Purta, E., Kurkowska, M., Olchowik, A., Januszewski, W., Kalinowski, S., Dunin-Horkawicz, S., Rother, K.M., 2013. Nucleic Acids Res. 41, D262–D267. Meyer, K.D., Saletore, Y., Zumbo, P., Elemento, O., Mason, C.E., Jaffrey, S.R., 2012. Cell 149, 1635–1646. Najafi, M., Khalilzadeh, M.A., Karimi-Maleh, H., 2014. Food Chem. 158, 125–131. Peng, J., Feng, L.-N., Ren, Z.-J., Jiang, L.-P., Zhu, J.-J., 2011. Small 7, 2921–2928. Schwartz, S., Agarwala, S.D., Mumbach, M.R., Jovanovic, M., Mertins, P., Shishkin, A., Tabach, Y., Mikkelsen, T.S., Satija, R., Ruvkun, G., 2013. Cell 155, 1409–1421. Shahdost-fard, F., Roushani, M., 2017. Biosens. Bioelectron. 87, 724–731. Tortolini, C., Bollella, P., Antonelli, M.L., Antiochia, R., Mazzei, F., Favero, G., 2015. Biosens. Bioelectron. 67, 524–531. Wang, C.-L., Wei, L.-Y., Yuan, C.-J., Hwang, K.C., 2012. Anal. Chem. 84, 971–977. Wang, M., Fu, Z., Li, B., Zhou, Y., Yin, H., Ai, S., 2014. Anal. Chem. 86, 5606–5610. Xie, H., Wang, Q., Chai, Y., Yuan, Y., Yuan, R., 2016. Biosens. Bioelectron. 86, 630–635. Xu, G., Schmid, H., Lu, X., Liebich, H., Lu, P., 2000. Biomed. Chromatogr. 14, 459–463. Xu, X., Nie, Z., Chen, J., Fu, Y., Li, W., Shen, Q., Yao, S., 2009. Chem. Commun. 6946, 6948. Yang, Z., Wang, F., Wang, M., Yin, H., Ai, S., 2015. Biosens. Bioelectron. 66, 109–114. Yin, H., Sun, B., Zhou, Y., Wang, M., Xu, Z., Fu, Z., Ai, S., 2014. Biosens. Bioelectron. 51, 103–108. Yin, H., Wang, M., Li, B., Yang, Z., Zhou, Y., Ai, S., 2015a. Biosens. Bioelectron. 63, 26–32. Yin, H., Zhou, Y., Yang, Z., Guo, Y., Wang, X., Ai, S., Zhang, X., 2015b. Sens. Actuators B: Chem. 221, 1–6. Yin, P.T., Shah, S., Chhowalla, M., Lee, K.-B., 2015c. Chem. Rev. 115, 2483–2531. Zhang, Q., Li, Z., Zhou, Y., Li, X., Li, B., Yin, H., Ai, S., 2016. Sens. Actuators B: Chem. 225, 151–157. Zhao, H., Ji, X., Wang, B., Wang, N., Li, X., Ni, R., Ren, J., 2015. Biosens. Bioelectron. 65, 23–30. Zhou, Y., Yang, Z., Li, X., Wang, Y., Yin, H., Ai, S., 2015. Electrochim. Acta 174, 647–652. Zhou, Y., Yin, H., Li, J., Li, B., Li, X., Ai, S., Zhang, X., 2016. Biosens. Bioelectron. 79, 79–85. Zhu, B., Booth, M.A., Shepherd, P., Sheppard, A., Travas-Sejdic, J., 2015. Biosens. Bioelectron. 64, 74–80.

Pan, T., 2014. Angew. Chem. Int. Ed. 54, 1587–1590. Chen, S., Dou, Y., Zhao, Z., Li, F., Su, J., Fan, C., Song, S., 2016. Anal. Chem. 88, 3476–3480. Chen, X., Zhang, Q., Qian, C., Hao, N., Xu, L., Yao, C., 2015. Biosens. Bioelectron. 64, 485–492. Dominissini, D., Moshitch-Moshkovitz, S., Schwartz, S., Salmon-Divon, M., Ungar, L., Osenberg, S., Cesarkas, K., Jacob-Hirsch, J., Amariglio, N., Kupiec, M., 2012. Nature 485, 201–206. Duangkaew, P., Tapaneeyakorn, S., Apiwat, C., Dharakul, T., Laiwejpithaya, S., Kanatharana, P., Laocharoensuk, R., 2015. Biosens. Bioelectron. 74, 673–679. Dutta, R.R., Puzari, P., 2014. Biosens. Bioelectron. 52, 166–172. Ensafi, A.A., Zandi-Atashbar, N., Rezaei, B., Ghiaci, M., Taghizadeh, M., 2016. Electrochim. Acta 214, 208–216. Gao, J., Guo, Z., Su, F., Gao, L., Pang, X., Cao, W., Du, B., Wei, Q., 2015. Biosens. Bioelectron. 63, 465–471. Harcourt, E.M., Ehrenschwender, T., Batista, P.J., Chang, H.Y., Kool, E.T., 2013. J. Am. Chem. Soc. 135, 19079–19082. Huang, J., Tian, J., Zhao, Y., Zhao, S., 2015. Sens. Actuators B: Chem. 206, 570–576. Kane, S.E., Beemon, K., 1985. Mol. Cell. Biol. 5, 2298–2306. Kinoshita, E., Kinoshita-Kikuta, E., Koike, T., 2013. Anal. Biochem. 438, 104–106. Li, B., Pan, G., Avent, N.D., Lowry, R.B., Madgett, T.E., Waines, P.L., 2015a. Biosens. Bioelectron. 72, 313–319. Li, F., Han, J., Jiang, L., Wang, Y., Li, Y., Dong, Y., Wei, Q., 2015b. Biosens. Bioelectron. 68, 626–632. Li, W., Liu, X., Hou, T., Li, H., Li, F., 2015c. Biosens. Bioelectron. 70, 304–309. Li, Y., Wang, Y., Zhang, Z., Zamudio, A.V., Zhao, J.C., 2015d. RNA 21, 1511–1518. Li, Z., Wang, Y., Ni, Y., Kokot, S., 2015e. Biosens. Bioelectron. 70, 246–253. Liebich, H., Lehmann, R., Xu, G., Wahl, H., Haring, H.-U., 2000. J. Chromatogr. B: Biomed. Sci. App. 745, 189–196. Lin, D., Mei, C., Liu, A., Jin, H., Wang, S., Wang, J., 2015a. Biosens. Bioelectron. 66, 177–183. Lin, H., Li, M., Mihailovič, D., 2015b. Electrochim. Acta 154, 184–189. Lin, S., Gregory, R.I., 2014. Nat. Cell Biol. 16, 129–131. Lin, Z., Wang, W., Jiang, Y., Qiu, B., Chen, G., 2010. Electrochim. Acta 56, 644–648. Linder, B., Grozhik, A.V., Olarerin-George, A.O., Meydan, C., Mason, C.E., Jaffrey, S.R., 2015. Nat. Methods 12, 767–772. Liu, J., Wang, J., Wang, T., Li, D., Xi, F., Wang, J., Wang, E., 2015. Biosens. Bioelectron.

500