Colorimetric detection of methyltransferase activity based on the enhancement of CoOOH nanozyme activity by ssDNA

Accepted Manuscript Title: Colorimetric Detection of Methyltransferase Activity Based on the Enhancement of CoOOH Nanozyme Activity by ssDNA Authors: Zhi-Mei Li, Xiao-Li Zhong, Shao-Hua Wen, Li Zhang, Ru-Ping Liang, Jian-Ding Qiu PII: DOI: Reference:

S0925-4005(18)32041-0 https://doi.org/10.1016/j.snb.2018.11.085 SNB 25683

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

4 September 2018 16 November 2018 16 November 2018

Please cite this article as: Li Z-Mei, Zhong X-Li, Wen S-Hua, Zhang L, Liang RPing, Qiu J-Ding, Colorimetric Detection of Methyltransferase Activity Based on the Enhancement of CoOOH Nanozyme Activity by ssDNA, Sensors and amp; Actuators: B. Chemical (2018), https://doi.org/10.1016/j.snb.2018.11.085 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Colorimetric Detection of Methyltransferase Activity Based on

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the Enhancement of CoOOH Nanozyme Activity by ssDNA

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Zhi-Mei Li,a,1 Xiao-Li Zhong,a,1 Shao-Hua Wen,a Li Zhang,a Ru-Ping Liang,a,* JianDing Qiua,b*

College of Chemistry, Nanchang University, Nanchang 330031, China

b

Environmental Protection Materials and Equipment Engineering Technology Center

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a

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of Jiangxi, Department of Materials and Chemical Engineering, Pingxiang University,

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211 Pingan North Road, Pingxiang 337055, Jiangxi, China

*Corresponding authors. Tel/Fax: +86-791-83969518.

authors contributed equally to this work.

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1These

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E-mail: [email protected] (R.-P. Liang), [email protected] (J.-D. Qiu).

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This is the first report on the peroxidase-like activity of CoOOH nanoflakes for the detection of MTase activity.

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The peroxidase-like activity of CoOOH nanoflakes can be improved by adsorbing ssDNA.

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The colorimetric sensor with a low detection limit of 0.069 U/mL for M.SssI MTase activity detection.

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The colorimetric sensor allows detection of M.SssI MTase in cell lysates

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of lung cancer cells (A549).

ABSTRACT

A label-free colorimetric assay for MTase activity based on the nanozyme activity of

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cobalt oxyhydroxide (CoOOH) nanoflakes and the difference in affinity of the

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nanoflakes with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA)

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has been developed. In this strategy, ssDNA can enhance the nanozyme activity of

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CoOOH nanoflakes, which could be attributed to the electrostatic attraction and π-π

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stacking interaction between 3,3',5,5'-tetramethylbenzidine (TMB) and ssDNA. The proposed method presents a wide linear range from 0.08-50 U/mL and a low detection

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limit (LOD) of 0.069 U/mL. Additionally, this colorimetric sensor also has been proved to detect MTase in the cell lysates of lung cancer cells (A549) with good

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recovery and reproducibility. Furthermore, the novel strategy can be applied for highthroughput evaluation and screening the inhibitors for M.SssI MTase by using

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representative anticancer drugs as model inhibitors, therefore, the colorimetric sensor can not only achieve the purpose of sensitively detecting the activity of MTase, but also has the potential in clinical diagnostics and drug discovery. Keywords: DNA methylation; methyltransferase; colorimetric method; cobalt oxyhydroxide; nanozyme.

1.

Introduction

DNA methylation, refers to the process of methyl groups transferring from Sadenosine methionine (SAM) to the target adenine or cytosine residues, which is

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closely related to the activity of DNA methyltransferase (MTase) [1-3]. Aberrant methylation induces changes in the structure of microbes, affecting protein

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interactions, delaying gene expression, and inducing tumorigenesis [4,5]. Hence, the construction of a simple, low-cost and sensitive DNA MTase activity assay sensor is

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of great significance for the diagnosis and treatment of methylation-related cancers,

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which have aroused people’s research interests [6]. So far, many types of methods

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have been exploited to detect the activity of DNA MTase, including polymerase chain

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reaction (PCR) [7], high performance liquid chromatography (HPLC) [8], radioactive labeling [9,10], fluorescence methods [11,12], electrochemical biosensors [13-15],

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colorimetric assay [16,17] and so on.

Among these methods, colorimetric strategies have been received extensive

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attention due to its simplicity, rapidity, and visualization in detecting DNA MTase

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activity. For example, many colorimetric methods for detecting MTase based on the aggregation (blue) and dispersion (red) morphology of gold nanoparticles (Au NPs) have been reported [16,18-21], these colorimetric sensors prefer to use Au NPs as

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detection signals due to the simple preparation process and visible color changes of Au NPs. However, Au NPs are prone to aggregation and produced false positive signals in complex detection systems, which limit its practical application [22-25]. To avoid the disadvantages of the Au NPs-based MTase assay described above, the colorimetric sensors have been designed which were based on hemin/G-quadruplex

DNAzyme to mimick horseradish peroxidase (HRP) for visual detection of MTase activity [17,26], However, these methods often requires complicated DNA strand design due to the need to form a G-quadruplex. Since the peroxidase-like catalytic activity of ferroferric oxide (Fe3O4) nanoparticle was first reported [27], the studies of nanomaterials as mimic enzymes have attracted much attention [28-32]. Nanozymes

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are a class of mimetic enzymes that not only have the unique properties of

nanomaterials, but also have catalytic functions [33-36]. They have the characteristics

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of high catalytic efficiency, stability, and large-scale preparation, which are widely used in medicine, chemistry, biology and other fields [37-40]. The meso-SiO2@Fe3O4

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and DNA template Ag/Pt nanoclusters (NCs) with nanozyme activity have been

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reported for colorimetric detection of DNA MTases activity [41,42]. However, the

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preparation of meso-SiO2@Fe3O4 and DNA template Ag/Pt NCs were a tedious and

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time-consuming process, it is still a challenge to develop a nanozyme material with a simple preparation process for MTase detection.

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Nowadays, with the rapid development of nanotechnology synthesis and application, two-dimensional (2D) nanomaterials have been increasingly used to

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prepare sensors due to their unique structure and excellent physicochemical properties [43,44]. Recently, CoOOH nanoflakes have been emerged as the 2D hexagonal

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transition metal oxyhydroxides nanomaterials, and show great potential in molecular detection. The main advantage of CoOOH nanoflakes over other 2D nanomaterials is

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that it is available to prepare the nanoflakes in a short time under mild conditions and without expensive instruments. The synthesized nanoflakes are composed of edge– sharing octahedral CoO6 layers with good stability [45]. It has been reported that CoOOH nanoflakes not only can adsorb ssDNA selectively to distinguish ssDNA and dsDNA [46, 47], but also have intrinsic peroxidase-like activity and have been applied

to detect glucose and ascorbic acid [48,49]. At present, the research and application of CoOOH nanoflakes are still one of the hotspots of scientific research. However, the application of CoOOH nanoflakes to detect MTase activity has not been reported. Herein, a simple and label-free colorimetric strategy has been developed based on CoOOH nanoflakes for rapid detection of M.SssI MTase activity. In this strategy, the

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nanoflakes can adsorb ssDNA selectively but not dsDNA, the substrate TMB and

ssDNA can be attracted with each other by aromatic stacking and electrostatic

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attraction. Therefore, in absence of M.SssI MTase, the ssDNA produced by

endonuclease can make TMB close to the nanoflakes and oxidize TMB to oxTMB

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rapidly in the presence of H2O2, thereby increasing the catalytic activity of CoOOH

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nanoflakes, the solution shows dark blue. While in the solution of M.SssI MTase, the

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original dsDNA can be methylated and protected to be dsDNA structure, the solution

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keeps the gray blue. The activity of M.SssI MTase could be detected by observing changes in the color of the oxidation reaction as well as UV–vis absorption spectra.

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Scheme 1 illustrates the detection mechanism of the colorimetric strategy.

Experimental section

2.1.

Chemicals and Instrument

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2.

Scheme 1

The oligonucleotides contain the sequence of 5'-CCGG-3' (Table S1 in ESI†) were

designed by ourselves and gotten from Sangon Biotechnology Co., Ltd.

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(Shanghai,China). Cobalt chloride (CoCl2), sodium hypochlorite (NaClO), sodium hydroxide (NaOH), hydrogen peroxide (H2O2), sodium acetate (CH3COONa) and acetic acid (CH3COOH) were commercially available from Shanghai Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). M.SssI MTase (contains 10×NEBuffer 2), S-Adenosylmethionine (SAM), restriction endonuclease HpaII

(contains 10×Cutsmart buffer) were gotten from New England Biolabs. Ltd (Beijing, China) and used as received. 3,3',5,5'-tetramethylbenzidine (TMB), 5-azacytidine (5Aza), and 5-aza-2’-deoxycytidine (5-Aza-dC) were available from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were commercially available from Thermo Scientific

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HyClone (MA, USA). All experiments were used ultrapure water (18.2 MΩ) from a Millipore Milli-Q system.

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UV-2450 spectrophotometer (Shimadzu, Japan) was used to record the UV– visible absorption spectra. Morphological measurements were measured by field scanning

electron microscopy (FE-SEM, SU-8010, Hitachi)

and

the

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emission

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transmission electron microscopy (TEM) were conducted on a JEM-2010 (JEOL, Japan). X-ray powder diffraction (XRD) was recorded on a Bruker D8 powder

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diffractometer in the 2θ range 10-80°. The X-ray photoelectron spectroscope (XPS, VG Multilab 2000X instrument, Thermal Electron, USA) was used to analyst the XPS

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spectrum. The Fourier transform infrared (FT-IR) spectrum was acquired on a Thermo Nicolet 5700 spectrometer.

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2.2.Synthesis of two-dimensional CoOOH nanoflakes The hexagonal CoOOH nanoflakes were prepared as reported methods [46, 50].

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Briefly, 250 μL of 1.0 M NaOH sample were added to 1 mL of 10 mM CoCl2 solution with vigorous whisking, and the resulting mixture was sonicated for 60 s.

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Subsequently, 50 μL 0.9 M NaClO solution were added and then sonicationed for 15 min. The excess reactants were removed by centrifugation and the precipitate was washed three times with ultrapure water. The entire synthesis processes were carried out at room temperature. The synthesized CoOOH nanoflakes could be dried in a vacuum oven for a series of characterization.

2.3.Activity assay of M.SssI MTase To obtain dsDNA, 10 μM DNA1 and 10 μM DNA2 were mixed together in TrisHCl buffer. The methylation experiments were conducted in 20 μL of 1×NEBuffer 2 (10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9) containing 80 μM SAM, 200 nM dsDNA and with 0, 0.08, 0.1, 0.2, 1, 2, 4, 10, 20, 40,

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50 U/mL of M.SssI MTase, respectively. The mixture was reacted in a water bath at

37 ° C for 3.5 hours. After completing the methylation reaction, the above solutions

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were heated at 65°C for 20 minutes to inactivate the MTase. Then, 20 U/mL HpaII

was added into the above solution and then reacted at 37°C for another 2 hours in

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order to initiate the cleavage reaction. Next, 1 μg/mL of CoOOH nanoflakes was

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added and incubated for 30 min at 37 °C in a water bath to trigger adsorption of

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ssDNA on the CoOOH nanoflakes by electrostatic interaction. Subsequently, 100 mM

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acetate buffer (pH 4.0) was injected into the above solution, so that the oxidation reaction was performed under weak acid conditions. 0.8 mM TMB in

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dimethylsulfoxide (DMSO) solution, 40 mM H2O2 were added and incubated for another 60 min. The absorption spectrum was measured by an UV–vis spectrometer.

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The selectivity of this strategy was further studied by using Dam MTase as a potential interference enzyme. In the selectivity experiment, 30 U/mL Dam MTase was used to

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replace M.SssI MTase. The other procedures were similar to M.SssI MTase activity detection. 1×NEBuffer 2 was used instead of MTase as a control experiment. The

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concentrations mentioned here are the final concentrations of the corresponding solution. 2.4. Evaluation of colorimetric assay for M.SssI MTase inhibition screening The experimental steps for investigating the effect of inhibitors on M.SssI MTase activity was similar to that of the MTase activity assay, except that different amounts

of inhibitors were added during methylation. The methylation of dsDNA was performed in 1×NEBuffer 2 containing 80 μM SAM, 200 nM dsDNA, 30 U/mL M.SssI, and various amounts of the inhibitors and incubated at 37 °C. The other experimental steps were consistent with the described above. The following equation: (A − A0) / (A1 − A0) was used to calculate the relative activity of M.SssI MTase.

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Among them, A0 represents the absorption intensity of 0 U/mL M.SssI MTase, while A1 and A represent the absorption intensity of 30 U/mL M.SssI MTase before and

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after the incubation of inhibitor, respectively. 2.5.Preparation of cell lysates

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A549 cells (human lung malignant adenocarcinoma cells, 1×104 cells) were

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incubated in DMEM medium supplemented with 15% FBS under a humid

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atmosphere with 5% CO2 at 37 °C. When the cell number reached approximately 80%

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of the cell culture flask (normally cultured for approximately 3 days), they were washed 3 times with PBS buffer solution (pH 7.4) to remove non-adherent cells. After

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the adherent cells were digested with trypsin, 3 mL of culture medium (containing 15% FBS) was injected and the resulting mixture was centrifuged at 1000 rpm for 3 min.

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The precipitation was washed thrice with 2 mL of PBS buffer solution. Subsequently, 1 mL of lysis buffer was used to treat the cells for 20 min at 0 °C. Then the cell lysate

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was clarified by centrifuging, and the resulting supernatant was stored at -20 °C.

Results and discussions

3.1.

Characterization of the CoOOH nanoflakes

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TEM image (Fig. 1A) shows that the prepared nanoflakes are mainly hexagonal in

shape which is consistent with the report [48]. The UV−vis absorption spectra of Fig. 1B reveal that after formation the CoOOH nanoflakes, the absorption peak of CoCl2 solution disappears at 512 nm, and a new absorption band appears at 410 nm, which is

the characteristic absorption peak of CoOOH nanoflakes [47]. The structure and chemical composition of synthetic nanoflakes are analyzed by FT-IR spectrum (inset in Fig. 1B), XRD pattern (Fig. 1C) and XPS spectrum (Fig. 1D). In the FT-IR spectrum of the CoOOH, the peak at 3421 cm-1 is contributed to the bond stretching of the O-H. The band at 1635 cm-1 is ascribed to the Co-O double bond, and the peak

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at 584 cm-1 is due to the Co-O2- complex. The peaks at 20.18°, 38.98°and 50.66° in XRD pattern correspond to the planes of (003), (012), and (018) of CoOOH

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nanoflakes, which agree well with the standard JCPDS card (No. 07–0169). The XPS

spectrum shows that the cobalt in the synthesized nanoflakes exhibits the oxidation

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state of Co(III) (779.5 eV), and there is no Co(II) oxidation state impurity, which

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further confirms the formation of CoOOH nanoflakes. According to previous reports

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[46,49,51], all these results show that hexagonal CoOOH nanoflakes have been

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successfully prepared.

Fig. 1

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3.2.The feasibility analysis of the strategy for M.SssI MTase activity detection To investigate the feasibility of this assay strategy, the experiments have been

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conducted under different conditions and the corresponding absorption spectra are shown in Fig. 2. When only TMB and H2O2 are present, the absorption intensity is

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weak (curve a), the TMB oxidation rate is slow. Compared with curve a, the absorption intensity of the curve b is obviously enhanced due to the presence of

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CoOOH nanoflakes, which indicates that the nanoflakes possess intrinsic peroxidaselike activity to catalyze the oxidation of TMB. When dsDNA (curve c) or both HpaII and M.SssI MTase (curve d) are added, their absorption intensities can’t be changed compared to curve b. In the absence of dsDNA, there are no specific recognition sites for HpaII and M.SssI MTase, the addition of the two enzymes does not affect the

activity of CoOOH nanoflakes. Curve e shows the strongest absorption intensity compared with other curves, this is because in the absence of M.SssI MTase, ssDNA produced by HpaII cleavage of dsDNA can be adsorbed onto CoOOH nanoflakes by electrostatic interaction and combined with more TMB, enhanced peroxidase activity of CoOOH nanoflakes in the presence of H2O2. Contrarily, when M.SssI MTase is

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introduced, the dsDNA is methylated, to retain its double-stranded structure even if in

the solution of HpaII, and can’t be adsorbed on the surface of CoOOH nanoflakes, so

the color change corresponding to the absorption spectra.

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Fig. 2

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the catalytic activity of the nanoflakes does not be changed (curve f). The inset shows

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3.3.Optimization of assay conditions

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In order to achieve optimal sensing performance for the designed colorimetric

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sensor, a series of experimental conditions have been optimized. The effect of CoOOH nanoflakes concentrations has been investigated on the system (Fig. S1A in

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ESI†). The values of A0 (the absorption intensity of 0 U/mL M.SssI MTase) and A (the absorption intensity of 30 U/mL M.SssI MTase in solution) gradually increase,

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with increasing CoOOH nanoflakes concentration, A0/A ratio reaches the maximum at the nanoflakes concentration of 1 μg/mL, so this concentration was used in the

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subsequent experiments. The concentrations of dsDNA, HpaII and SAM, are crucial in the sensing system and play key roles in DNA methylation and digestion. In order

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to obtain the optimal concentration of dsDNA, the amount of dsDNA is gradually increased in the optimization experiment (Fig. S1B in ESI†), when the concentration of dsDNA reaches 200 nM, the maximum difference in absorbance before and after the addition of the MTase can be obtained, which indicates that the optimal concentration of dsDNA is 200 nM. Similarly, the optimal concentrations of SAM

and HpaII for this system are obtained, which are 80 μM and 20 U/mL, respectively (Fig. S1C and Fig. S1D in ESI†). Besides, the concentrations of TMB and H2O2 are also optimized, which are significant factors that influence the oxidation reaction (Fig. S2A and Fig. S2B in ESI†). In order to obtain a low background signal and a sensitive detection limit, the final optimized TMB and H2O2 concentrations are 0.8 mM and 40

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mM, respectively. All reactions are performed at 37°C. When one parameter is changed, the others are at their optimal value.

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Additionally, the effect of methylation time and oxidation reaction time has been

studied on the system. As shown in Fig. S2C (in ESI†), the results of optimized

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experiments for methylation time show the plateau at 210 minutes, demonstrating that

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the methylation reaction was completed at this time. The curves of absorbance intensity with oxidation reaction time show that the signal to noise ratio reaches the

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maximum at 60 minutes (Fig. S2D in ESI†). So 210 and 60 minutes are selected as the optimum methylation and oxidation reaction time, respectively. Acetate buffer

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(pH 4) is applied to the oxidation reaction (Fig. S3 in ESI†). 3.4. Assay of M.SssI MTase activity

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The detection performances of the designed sensor have been carried out under optimal experimental conditions (Fig.3A). Without M.SssI MTase, dsDNA are totally

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cleaved into ssDNA catalyzed by HpaII, which enhances obviously CoOOH nanoflakes catalytic activity with the strongest absorption intensity. The absorption

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intensity (adsorbance at 370 nm) decreases with increasing the amount of M.SssI MTase, which mainly because the more dsDNA are methylated and can’t be cleaved by HpaII, the less ssDNA are produced, and results in less TMB to bind to CoOOH nanoflakes by interacting with ssDNA. The inset in Fig.3A shows that as the amount of M.SssI MTase increases, the color of the samples gradually fades (from left to

right). Therefore, the activity of M.SssI MTase can be detected by visible color changes of the oxidation reaction as well as UV–vis absorption spectra. Additionally, a good linear relationship can be obtained between the absorption intensities and the logarithm of M.SssI MTase concentrations from 0.08 to 50 U/mL (Fig.3B). The correlation coefficient is 0.992, and the corresponding regression equation is A = -

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0.0681logC + 0.22136. According to the rule of 3/k, the detection limit of 0.069 U/mL is better than the previously reported assays (Table 1). The CoOOH nanoflakes

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have the advantages of simple and rapid synthesis steps, do not require expensive instruments. Moreover, as a material with peroxidase-like activity, they can

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distinguish ssDNA and dsDNA, which is not available in many other materials. Based

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on these excellent properties of the nanoflakes, the sensor for colorimetric detection

Table 1

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Fig. 3

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of MTase with excellent selectivity and sensitivity were successfully designed.

Dam MTase has been employed as an interfering enzyme to evaluate the

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selectivity of the sensor system, which can methylate the palindrome sequence of 5’GATC-3’ on the N6-adenine in the presence of methyl donor [52]. As can be seen

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from Fig.4, the solution at the presence of Dam MTase without M.SssI MTase shows

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high absorption intensity, which is similar to the control experiment. The phenomenon is caused by that the Dam MTase can’t catalyze the methylation of the sequence of 5’-CCGG-3’. The results show that the designed colorimetric sensor has

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good selectivity to detect the activity of M.SssI MTase. The CoOOH nanoflakes did not change its peroxidase activity after several months of storage, and maintained good response to M.SssI MTase. The results suggest that the CoOOH nanoflakes are stable and can be used for M.SssI MTase assay after long-term storage. Fig. 4

3.5.M.SssI MTase sensing in cell lysates A significant challenge in the determination of MTase activity is to achieve the detection in complex biological sample. A large number researches show that MTase is overexpressed in cancer cells such as human colon cancer, breast cancer, lung cancer, and gastric cancer. To confirm the availability of proposed method in complex

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biological samples, the sensor has been used to detect MTase in cell lysates of A549 cells (lung cancer). The cell lysates are diluted by 1×NEBuffer 2 in a ratio of 1:10 and

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spiked with M.SssI MTase. As seen from Fig. S4, the absorption intensity in the cell lysates is consistent with that in NEBuffer 2. The recoveries are in the range of 97.0%

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~ 101.7% and with the relative standard deviations (RSDs) ≤ 5.2%, which indicates

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that this method can also be used in complex biological samples.

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3.6.Evaluation of M.SssI MTase activity inhibition

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To validate the feasibility of our strategy in screening of M.SssI MTase inhibitors, two typical anticancer drugs: 5-Aza and 5-Aza-dC are chosen as model inhibitors [53].

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As can be seen from Fig.5, the activity of M.SssI MTase can be gradually inhibited with increasing the concentration of 5-Aza (Fig. 5A) and 5-Aza-dC (Fig. 5B). The

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IC50 value means that the concentration of the inhibitor requires to reducing enzyme activity by 50%, and the values are calculated to be 0.39 and 4.2 μM for 5-Aza-dC

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and 5-Aza, respectively, which are similar to the previous reports [54, 55]. These results demonstrate that this proposed strategy can be applied for screening of M.SssI

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MTase inhibitors.

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Fig. 5

Conclusions

In conclusion, a novel strategy based on ssDNA enhances the catalytic activity of CoOOH nanoflakes for label-free colorimetric assay of M.SssI MTase activity have

been developed. The hexagonal CoOOH nanoflakes with peroxidase activity were prepared by a simple ultrasonic method in a short time. Compared with the traditional DNA methylation detection methods, this proposed method exhibited the advantages of label-free, simple operation, low cost, excellent selectivity and low detection limit. Additional, the developed label-free colorimetric strategy has great potential for

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determining M.SssI MTase activity in practical samples such as cell lysates.

Moreover, the colorimetric sensor has been demonstrated to be applied to rapid screen

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of M.SssI MTase inhibitors, which may help to discovery methylation-related anti-

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cancer drugs.

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Biographies

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Zhi-Mei Li received her doctor degree from Nanchang University in 2008. Presently, she is an associate professor in College of Chemistry, Nanchang University, China.

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Her research interest is enzyme analysis.

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Xiao-Li Zhong is a MS candidate in College of Chemistry, Nanchang University. Her current researches are nanomaterial and bioanalysis.

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Shao-Hua Wen received his PhD degree from Nanchang University in 2018. His main research interests are biosensing and nanotechnology.

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Li Zhang received his PhD degree from Southwest University in 2010. Presently, he is an associate professor in College of Chemistry, Nanchang University. His main research interests are biosensing, nanotechnology, and fluorescence analysis.

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Ru-Ping Liang received her PhD degree from Sun Yat-Sen University in 2004. Presently, she is a professor in College of Chemistry, Nanchang University. Her main research interests are chemical modified electrode, electrochemiluminescence, and molecular recognition. Jian-Ding Qiu is a professor in College of Chemistry, Nanchang University, China. He received his PhD in analytical chemistry from Sun Yat-Sen University of China in

2004. He served as a postdoctoral research associate at the Nanjing University and Hokkaido University, respectively. His current research interests include bioanalysis, nanotechnology, environmental analysis, and bioinformatics.

Acknowledgments

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We greatly appreciate the supports of the National Natural Science Foundation of China (21475056, 21675078, 21775065 and 21365015) and the Natural Science

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Foundation of Jiangxi Province (20171BAB203016).

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Figure Captions Fig. 1 (A) The TEM image of CoOOH nanoflakes, (B) UV-vis absorption of CoCl2 aqueous solution (red line) and CoOOH solution (black line), inset in Fig.B is the FT-

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IR spectrum of CoOOH, (C) the XRD pattern of CoOOH and (D) XPS spectrumof Co (2p) regions for CoOOH nanoflakes.

CoOOH+TMB+H2O2;

(c)

dsDNA+CoOOH+TMB+H2O2; (e)

(d)

HpaII+

dsDNA+HpaII+CoOOH+TMB+H2O2;

(f)

U

M.SssI+CoOOH+TMB+H2O2;

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Fig. 2 The absorption spectra for a series of experiments: (a) TMB+H2O2; (b)

N

dsDNA+HpaII+ M.SssI+CoOOH+TMB+H2O2. 1 μg/mL CoOOH, 0.8 mM TMB, 40

A

mM H2O2, 200 nM dsDNA, 20 U/mL HpaII, 80 μM SAM and 30 U/mL M.SssI were

M

used in the experiment. The insets of a-f are corresponding to the curves a-f. Fig. 3 (A) Absorption spectra with different concentrations of M.SssI MTase (from

ED

top to bottom the concentrations of M.SssI MTase are 0, 0.08, 0.1, 0.2, 1, 2, 4, 10, 20, 40, 50 U/mL, respectively), the inset shows the color change corresponding to the

PT

absorption spectra. (B) The absorption peak intensities corresponding to different

CC E

concentrations of M.SssI MTase. The inset represents the linear relationship of absorption intensities with the logarithm of the M.SssI MTase concentrations. Fig. 4 Selectivity of the CoOOH nanoflakes-based colorimetric sensor. Both the

A

concentrations of M.SssI and Dam are 30 U/mL. The control sample contains 0 U/mL M.SssI. Fig. 5 The inhibition effects of different amounts of 5-Aza (A) and 5-Aza-dC (B) on M.SssI MTase activity. The concentration of M.SssI is 30 U/mL.

Scheme 1 Schematic illustration of the CoOOH nanoflakes-based colorimetric sensor strategy

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Scheme 1

A

CC E

PT

ED

M

A

N

U

Fig. 1

Fig. 2

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A

N

U

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Fig. 3

A

CC E

PT

ED

Fig. 4

Fig. 5

A ED

PT

CC E

IP T

SC R

U

N

A

M

Table 1 Detection performance comparison of our strategy to detect MTase activity with other methods Intracellular detection

Ref.

No

[56]

0.1-40

0.06

Yes

[57]

0.1-450

0.05

No

0.02-50

0.0067

No

[59]

1.0-120

0.05

Yes

[60]

0.01

No

[61]

0.001-100

7.23×10-4

No

[62]

0.1-100

0.03

No

[54]

0-50

0.01

Yes

[63]

0.8-24

0.4

No

[17]

0-40

0.73

No

[41]

0.2-50

0.08

No

[55]

0.1-30

0.14

No

[52]

0.08-50

0.069

Yes

Our work

ED

PT

CC E A

SC R

U

0.01-5

IP T

Detection limit (U/mL) 0.02

M

Electrochemistry DNA- Au NPs network as signal amplification unit Electrochemistry uracil-specific excision reagent digestion induced Gquadruplex formation Electrochemistry signal amplification of GO Electrochemistry HCR signal amplification ECL glucose oxidase mimicking AuNPs enhanced ECL of CdS quantum dots Fluorometry dsDNA-templated CuNPs with endonucleaseassisted signal transduction system Fluorometry HCR and metal iondependent DNAzyme recycling Fluorometry fluorescence quenching of GO Fluorometry exonucleasemediated target recycling Colorimetry methylationblocked cascade amplification strategy Colorimetry the keypad lock of duplex DNA modified mesoSiO2@Fe3O4 Colorimetry strand displacement amplification Colorimetry unmodified Au nanorods with enzyme-linkage reactions Colorimetry ssDNA enhances the nanozyme activity of the CoOOH nanoflakes

Linear range (U/mL) 0.075-30

N

Strategy

[58]

A

Method

A

CC E

PT

ED

M

A

N

U

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AgNPs: silver nanoparticles; AuNPs: gold nanoparticles; HCR: hybridization chain reaction; ECL: electrochemiluminescence; CuNPs: copper nanoparticles; ScGFP/GO: supercharged green fluorescent protein/graphene oxide; GO: graphene oxide; ssDNA: single stranded DNA; CoOOH: cobalt oxyhydroxide.