Dual-signal amplified photoelectrochemical assay for DNA methyltransferase activity based on RGO-CdS:Mn nanoparticles and a [email protected] network

Journal Pre-proof Dual-signal amplified photoelectrochemical assay for DNA methyltransferase activity based on RGO-CdS:Mn nanoparticles and a CdTe@DNA network Jing Luo, Lichao Fang, Huamin Liu, Quanjing Zhu, Hui Huang, Jun Deng, Feixue Liu, Yan Li, Junsong Zheng

PII:

S0925-4005(19)31465-0

DOI:

https://doi.org/10.1016/j.snb.2019.127266

Reference:

SNB 127266

To appear in:

Sensors and Actuators: B. Chemical

Received Date:

14 March 2019

Revised Date:

6 October 2019

Accepted Date:

9 October 2019

Please cite this article as: Luo J, Fang L, Liu H, Zhu Q, Huang H, Deng J, Liu F, Li Y, Zheng J, Dual-signal amplified photoelectrochemical assay for DNA methyltransferase activity based on RGO-CdS:Mn nanoparticles and a CdTe@DNA network, Sensors and Actuators: B. Chemical (2019), doi: https://doi.org/10.1016/j.snb.2019.127266

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier.

Dual-signal amplified photoelectrochemical assay for DNA methyltransferase activity based on RGO-CdS:Mn nanoparticles and a CdTe@DNA network

Jing Luo1,2,‡, Lichao Fang1,‡, Huamin Liu1,2, Quanjing Zhu1, Hui Huang1, Jun Deng1,

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Feixue Liu1, Yan Li1,*, Junsong Zheng1,*

Department of Clinical and Military Laboratory Medicine, College of Medical

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Laboratory Science, Army Medical University, 30 Gaotanyan Street, Shapingba

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District, Chongqing 400038, PR China.

Department of Materials and Energy, Southwest University, 2 Tiansheng Street,

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* Corresponding authors

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Beibei District, Chongqing 400715, PR China.

Equal contribution by the first two authors.

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‡

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E-mail addresses: [email protected] (J. Zheng), [email protected] (Y. Li)

Highlights 

A dual-signal amplified photoelectrochemical method was developed for the assay of DNA methyltransferase activity.



GR-CdS:Mn nanoparticles was used as substrate materials to firstly enhance 1

the photocurrent. 

CdTe@DNA network used as a signal material was to further enhance the photocurrent and sensitivity.

Abstract DNA methyltransferase (DNA MTase) has been shown to catalyze DNA

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methylation, which is closely associated with cancers. Therefore, developing

detection technology for DNA MTase activity is significant for early diagnosis of

cancer. Herein, a new and ultrasensitive photoelectrochemical (PEC) biosensor was

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fabricated to analyze DNA MTase activity using a dual-signal amplified system,

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where RGO-CdS:Mn nanoparticles and a CdTe@DNA network were used as photoelectric materials. RGO-CdS:Mn nanoparticles were first modified on a gold

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electrode as substrate materials. Then, probe DNA and target DNA with a recognition

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sequence for DNA methylation were assembled on the electrode. After the process of methylation and digestion, the methylated dsDNA could be reserved and further

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hybridized with a CdTe@DNA network, leading to a stronger photocurrent. As expected, the PEC responses were in proportion to the logarithm of M.SssI MTase

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concentration from 0.01 to 80 U/mL with a detection limit of 0.0071 U/mL. This proposed method showed ideal detection ability for DNA MTase activity and could provide a new idea as well as technology in cancer diagnosis. Abbreviations 5-mC, 5-methylcytosine; AA, ascorbic acid; BSA, bovine serum albumin; DNA

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MTase, DNA methyltransferase; EIS, electrochemical impedance spectroscopy; GO, graphene oxide; PEC, photoelectrochemical; Ret, electron-transfer resistance; RGO, reduced graphene oxide; RSD, relative standard deviation; SD, standard deviation; TAA, thioacetamide. Keywords: photoelectrochemical biosensor, DNA methyltransferase activity,

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RGO-CdS:Mn nanoparticles, CdTe@DNA network

1. Introduction

DNA methylation is an important epigenetic modification, that plays a crucial

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role in mammalian development, including gene transcription, gene imprinting and

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gene expression [1-3]. Aberrant DNA methylation can inactivate the tumor suppressor gene and silence gene transcription, resulting in various cancers, such as

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hepatocellular carcinoma, squamous cell lung cancer and leukemia [4-5]. It is well

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known that DNA methylation, which usually refers to 5-mC, is catalyzed by DNA methyltransferase (DNA MTase) [6]. Thus, the DNA MTase activity assay is

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significant for the prediction and therapy for an early diagnosis of cancer. Recently, numerous methods for DNA MTase activity detection have been

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proposed, including high-performance liquid chromatography [7], rolling circle amplification [8], a light scattering technique [9], electrogenerated chemiluminescence [10,11], a colorimetric approach [12,13] and an electrochemical method [14-16]. Although these methods successfully detect the DNA MTase activity, most of them suffer from shortcomings, such as tedious pretreatment processes,

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time-consuming, complex labeling processes and low sensitivity. Therefore, it is urgently needed to develop a sensitive, novel and rapid method for accurately analyzing DNA MTase activity. The photoelectrochemical (PEC) method is a newly developed method on the basis of electrochemical technology and has attracted increasing attention [17,18]. Compared with previous methods, the PEC assay has its own remarkable superiority,

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such as small background interference, high sensitivity, simplicity and rapid response

[19-21]. Thus, the PEC biosensor is the ideal means of detecting DNA MTase activity. In fabricating PEC biosensors, the selection of photoactive material is the most

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vital consideration. The most commonly used photoactive materials are

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semiconductor nanoparticles, such as SnO2 [22], TiO2 [23], CdSe [24] and CdS [25]. With the further study of photoactive materials, it has been found that composite

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materials has stronger photocurrent than single semiconductor nanoparticles, which is

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due to a formation of heterojunctions that can improve the photovoltaic properties [26,27]. Based on this principle, we synthesized a new nanocomposite material

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RGO-CdS:Mn NPs, by doping Mn2+ in CdS nanoparticles and then immobilizing them on a reduced graphene oxide (RGO) platform. The advantages of this

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nanocomposite material are as follows: (1) the doped Mn2+ can not only induce charge separation but also hinder electron-hole recombination by creating of a new energy level. (2) the RGO can enhance charge separation and accelerate electron transport. Additionally, RGO can also load more CdS:Mn NPs because of its large surface area and thus can perform an enhanced photocurrent.

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Simultaneously, to further enhance the photocurrent response and improve sensitivity, we successfully synthesized a CdTe@DNA network via a condensation reaction between the -NH2 of DNA and the -COOH of CdTe QDs to join together with more CdTe QDs, which had never been reported in other studies. As CdTe QDs had a narrow gap band compared with the RGO-CdS:Mn NPs, an electron transfer was available from the CdTe QDs to the RGO-CdS:Mn NPs with the assistance of AA,

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which was used as an electron donor and produced an enhanced photocurrent (Scheme. 1). Thus, the sensitivity could be further improved.

Herein, we proposed a new ultrasensitive PEC method for a DNA MTase activity

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assay using a dual-signal amplified system of RGO-CdS:Mn NPs and a CdTe@DNA

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network (Scheme. 1). RGO-CdS:Mn NPs were first modified on a gold electrode as substrate materials. Next, probe DNA S1 and BSA were sequentially captured by the

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modified electrode. Then target DNA S2 was hybridized with probe DNA S1 to form

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dsDNA with a recognition sequence for DNA methylation. After a process of methylation and digestion, the methylated dsDNA could be reserved and further

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hybridized with the CdTe@DNA network, which resulted in a stronger photocurrent. Therefore, with the assistance of signal amplification, this approach is capable of

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detecting DNA MTase activity sensitively. Scheme. 1

2. Experimental procedure 2.1 Materials and reagents All DNA sequences (Table S1 in Supplementary Information), phosphate

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buffered saline (PBS, pH 7.4), tris (hydroxymethyl) aminomethane (Tris), L-ascorbic acid (AA) and ethylenediaminetetraacetic acid (EDTA) were purchased from Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). Sodium borohydride (NaBH4), tellurium dioxide (TeO2), N-hydroxysuccinimide (NHS), polyvinyl pyrrolidone (PVP, Mw=40000), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), manganese (II) chloride tetrahydrate (MnCl2·4H2O), bovine serum albumin

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(BSA) and thioacetamide (TAA) were received from Sigma-Aldrich (USA). Graphene oxide (GO, thickness: 0.55-1.2 nm, layers: < 3) was purchased from Nanjing

XFNANO Materials TECH Co., Ltd. M.SssI MTase and HpaII endonuclease were

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this work with a resistivity of 18.2 MΩ·cm.

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purchased from New England BioLabs (USA). Distilled water was used throughout

2.2 Apparatus

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The characterization of the samples was carried out by transmission electron

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microscope (TEM, JEM-1400 Plus). The ratio of Mn/Cd in RGO-CdS:Mn was characterized by EDX mounted on a scanning electron microscope (SEM,

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JSM-6510LV, Japan). Electrochemical impedance spectroscopy (EIS) was characterized by a CHI660D electrochemical workstation (Shanghai Chen Hua

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Instruments, China). The photocurrent response was measured on a PEC apparatus (PEAC 200A, Tianjin Aida Heng Sheng technology co. LTD, Tianjin, China). 2.3 Synthesis of RGO-CdS:Mn Nanoparticles The RGO-CdS:Mn nanoparticles were synthesized by the following steps according to some previous methods with modifications [28,29]. First, GO (5 mg)

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was diluted with 5 mL of double distilled water to obtain dispersion by ultrasonication in a water bath. Next, 0.2000 g of PVP and the GO dispersion (200 μL, 1 mg/mL) were dissolved in 50 mL of distilled water with continuous stirring. Then, 0.0917 g CdCl2 with 0.0050 g MnCl2·4H2O and 0.1000 g TAA were added. TAA was able to reduce GO to RGO. The mixed solution was reacted at 80 ºC for 2 h with gentle stirring. Finally, the RGO-CdS:Mn nanoparticles were obtained by centrifuging at

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11000 g three times and vacuum drying.

2.4 Preparation of TGA-capped CdTe QDs and CdTe@DNA S3 network

TGA-capped CdTe QDs were synthesized using a reported method with slight

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modifications [30]. Briefly, 0.0500 g NaBH4 and 1.5960 g TeO2 were dissolved in

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10 mL distilled water to acquire a fresh NaHTe solution. Another mixed solution containing CdCl2 (1 mM) and TGA (1.8 mM) was prepared and adjusted to a pH of 11.

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Then, N2 was bubbled to the mixed solution to remove O2 for 30 min. After that, fresh

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NaHTe solution was added and continued to react at 80 ºC for 5 h under gentle stirring. After purification, the CdTe QDs were re-dispersed in ultrapure water and

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kept at 4 ºC. Then, 5’-amino group-modified DNA S3 (1 μM, 40 μL) was first activated by a 40 μL solution of 10 mM EDC and 20 mM NHS for 30 min. Next, an

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equal volume of CdTe solution was added and reacted at 2 h at 37 ºC with gentle stirring. The acquired solution was called the CdTe@DNA network. 2.5 Fabrication of PEC biosensor A mirror-like gold electrode (GE) with a diameter of 2 mm was obtained from a previous report [31]. Then, RGO-CdS:Mn NPs (10 μL, 3 mg/mL) were coasted on the

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GE. After drying in the air, the GE was dipped in a 10 μL Tris-HCl buffer (0.1 M NaCl, 0.3 M TGA) for 5 h at 4 ºC to introduce -COOH on the surface of the electrode [32]. Thus, the TGA-capped RGO-CdS:Mn NPs/GE was obtained. The GE/RGO-CdS:Mn NPs modified electrode was dipped in a solution of 10 mM EDC and 20 mM NHS for 30 min in order to activate -COOH. Then, the RGO-CdS:Mn NPs modified GE was immediately incubated with a 10 μL probe DNA

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S1 (1 μM) for 12 h at 4 ºC by an EDC/NHS coupling reaction. After blocking with

1% BSA solution for 30 min , 10 μL of target DNA S2 (1 μM) was hybridized with probe DNA S1 at 37 ºC for 2 h and the electrode was prepared for the process of

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methylation and digestion.

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The methylation process was conducted in a 10 μL stock buffer (160 μM SAM, 0-150 U/mL of M.SssI MTase) for 2 h at 37 ºC. Next, 10 μL 80 U/mL HpaII which

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was used to cleave the unmethylated dsDNA was performed for 2 h at 37 ºC. Finally,

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10 μL CdTe@DNA network was used as a signal amplifier and reacted for 40 min at 37 ºC.

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2.6 PEC detection

The photocurrent response was performed on a PEC apparatus using a

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three-electrode system, which consisted of an Ag/AgCl electrode, a Pt wire counter electrode and a 2 mm diameter GE. The PEC response was detected in 0.1 M PBS buffer (0.1 M AA, pH 7.4) under conditions of a potential of 0.0 V and the white light which was turned on and off every 10 s. 3. Results and discussion

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3.1 Characterization of RGO-CdS:Mn, CdTe and CdTe@DNA The morphology of RGO-CdS:Mn NPs, CdTe QDs and CdTe@DNA network were characterized by transmission electron microscope (TEM). As shown in Fig. 1A, CdS:Mn NPs were modified on the RGO layer evenly and tightly. As a control, we synthesized the RGO layer (Fig. 1B) and CdS NPs (Fig. 1C) by the same method. It can be seen clearly that the RGO had a sheet structure and the CdS NPs were

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uniformly dispersed with a diameter of approximately 70 nm. These results further illustrated the successful synthesis of RGO-CdS:Mn NPs. Fig. 1D shows the EDX

spectrum. In the spectrum, both Cd and S showed strong peaks, while Mn had much

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weaker peaks compared with Cd and S. The molar ratio of Mn/Cd was approximately

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5%. As can be seen in Fig. 1E, CdTe QDs were each dispersed and had a diameter of approximately 40 nm. Fig. 1F shows the TEM imagine of CdTe@DNA network,

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indicating the success of synthesis of CdTe@DNA network by condensation reaction

DNA strands.

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Fig. 1.

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between the -NH2 of DNA and the -COOH of CdTe QDs and the hybridization of

3.2 EIS characterization of biosensor

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Each step in fabricating the PEC biosensor was characterized by EIS. Fig. 2A

showed that the GE had a very small impedance value (curve a), which was caused by good electron transfer ability of the bare gold electrode. When the RGO-CdS:Mn NPs were assembled onto the GE (curve b), the Ret value increased moderately, indicating that the nanoparticles were modified on the GE surface successfully and the electron

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transfer rate was reduced. Subsequently, when probe DNA S1 was immobilized with the RGO-CdS:Mn NP-modified GE (curve c), the Ret value increased obviously, which could be ascribed to an electrostatic repulsion that obstructed of electron transfer. After blocked by BSA (curve d), the Ret value increased significantly because electron transfer was hindered by the BSA proteins. When target DNA S2 hybridized with probe DNA S1 (curve e), the Ret value further increased, suggesting

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that dsDNA impeded the electron transfer process. After performing the process of

methylation and digestion (curve f), the Ret value increased slightly. However, the Ret value decreased when the electrode was only treated with HpaII (curve h). This could

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be explained by the successful digestion of unmethylated DNA by the HpaII

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endonuclease successfully. Finally, when the CdTe@DNA network was captured on the electrode surface (curve g), the Ret value increased dramatically, indicating that

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the modification of the network structure further increased the steric hindrance.

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3.3 PEC characterization of biosensor

The photocurrent response for the fabricated biosensor was presented in Fig. 2B.

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The GE showed no photocurrent (curve a), indicating no PEC activity with the GE. After the RGO-CdS:Mn NPs were coated on the GE surface (curve b), the

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photocurrent response increased obviously because of its good photoelectric activity. Subsequently, when probe DNA S1 was immobilized with the RGO-CdS:Mn NP-modified GE (curve c), the photocurrent response diminished gradually due to its poor charge transfer ability. Then, BSA and target DNA S2 were assembled sequentially (curve d and e), and the photocurrent response became successively

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weaker because the electron transfer from the AA to the RGO-CdS:Mn was obstructed by these biomolecules. After performing the process of methylation and digestion (curve f), the photocurrent response slightly changed. Finally, when the CdTe@DNA network was immobilized (curve g), a dramatic increase in the photocurrent response was gained because of the strong electron transfer between the RGO-CdS:Mn NPs and the CdTe QDs. In addition, the photocurrent response was slightly weaker than

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that of RGO-CdS:Mn/GE when the electrode was only treated with HpaII (curve h). Fig. 2. 3.4 Optimization of experimental conditions

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To confirm the most suitable molar ratio of Mn/Cd, which has great influence to

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the photoelectric properties of RGO-CdS:Mn NPs due to the formation of an intermediate valence band, the RGO-CdS:Mn NPs-modified electrodes with different

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amounts of Mn2+ (molar ratio of Mn/Cd was from 0% to 10%) were prepared for

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measurement. Fig. 3A demonstrates that the photocurrent increased obviously accompanied by the enhancement of molar ratio of Mn/Cd. When it exceeded 5%, the

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photocurrent began to gradually decrease. This result could indicate that excessive intermediate levels can lead to the passivation of nanoparticles. Therefore, the best

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molar ratio of Mn/Cd was 5%. The photocurrent intensity was also closely related to the concentration of the

RGO-CdS:Mn NPs. Fig. 3B shows that the photocurrent increased gradually with changing concentrations from 1 mg/mL to 3 mg/mL. When further increasing the concentration, the photocurrent diminished. This might be because the RGO-CdS:Mn

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sheet was too thick to transport the charge. Thus, 3 mg/mL RGO-CdS:Mn NPs were used in this experiment. To confirm the best concentration of HpaII endonuclease, different concentrations of HpaII endonuclease were prepared to digest the unmethylated dsDNA. Fig. 3C shows that with a higher concentration of HpaII endonuclease, more unmethylated dsDNA would be digested, which resulting in a lower photocurrent.

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However, it seemed to be steady after 80 U/mL. Therefore, 80 U/mL of HpaII was chosen in this experiment.

The optimal hybridization time of the CdTe@DNA S3 network was also

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important for the photocurrent signal. Fig. 3D shows the relationship between

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photocurrent and different hybridization times. With longer hybridization time, a larger photocurrent was obtained. However, when the hybridization time was beyond

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40 min, the photocurrent gradually diminished. This could be explained by the fact

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that the CdTe@DNA S3 network fell off from the electrode because of the excessive hybridization time. Thus, 40 min of hybridization time was selected as the optimal

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condition for the CdTe@DNA S3 network. Fig. 3.

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3.5 PEC performance with M.SssI MTase Under optimum conditions, electrodes treated with different concentrations of

DNA methyltransferase were prepared for PEC detection. Fig. 4A showed the photocurrent response of M.SssI MTase over a range of 0-150 U/mL. With the increase of its concentration, more dsDNA was methylated and could be reserved for

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further capture with the CdTe@DNA network. The Fig. 4B showed a linear regression equation of I = 0.9868+0.2693lg[c] (R2=0.9973) in the concentration range from 0.01 to 80 U/mL with a detection limit of 0.0071 U/mL, which was obtained from Mean + 3SD (Mean was the average PEC responses of the blank and SD was the standard deviation of detection responses of the blank). Additionally, the analytical performances were compared with those in some previous reports (Table 1),

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indicating acceptable application potentials for effective and sensitive estimation of M.SssI MTase activity.

Table 1

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3.6 Specificity, repeatability and stability studies

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Fig. 4.

The specificity was confirmed by measuring the photocurrent of the prepared

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electrodes using three different types of target DNA sequences. As shown in Fig. 5A,

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one-mismatched target DNA had a much weaker photocurrent response than complementary target DNA. In addition, the photocurrent response of the

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non-complementary target DNA and the blank DNA was almost the same. The results showed excellent specificity of the PEC biosensor, which could effectively distinguish

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the complementary DNA sequence and mismatched DNA sequence. The repeatability was assessed by measuring the photocurrent of five

independently prepared electrodes which treated with 1 U/mL M.SssI MTase. As shown in Fig. 5B, the relative standard deviation (RSD) was 5.7%, meaning that the PEC biosensor had acceptable repeatability.

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The stability was also evaluated by measuring the photocurrent of the prepared electrodes which treated with 10 U/mL M.SssI MTase. As shown in Fig. 5C, the prepared electrodes were stored in the refrigerator at 4 ℃ and were removed to detect their own photocurrent response after one week and one month, respectively. The prepared electrodes after one week and one month lost approximately 1.3% and 8.9% of their initial response respectively, which indicated good stability.

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Fig. 5.

3.7 The recovery test of the PEC biosensor for real sample analysis

To test the selectivity of this designed PEC biosensor for DNA methylation

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detection in complex biological matrixes, which is significant for clinical application,

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a recovery experiment was conducted using different concentrations of DNA methyltransferase (0.1, 10, 40 and 80 U/mL) in diluted human serum samples. The

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diluted human serum samples were obtained from healthy volunteers. As shown in

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Table 2, the recovery rate of the added DNA methyltransferase varies from 96.78% to 104.68%, with the RSD ranging from 2.14% to 6.45%. These results indicating that

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the various biomolecules such as proteins and DNA in human serum had little interference in the experiments, which further illustrated that the proposed PEC

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biosensor has good selectivity and could be further applied to clinical diagnosis Table 2

4. Conclusion In this work, a dual-signal amplified photoelectrochemical DNA biosensor was used to analyze M.SssI MTase activity based on RGO-CdS:Mn nanoparticles and a

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CdTe@DNA network. With the assistance of signal amplification, this designed PEC biosensor presented excellent detection sensitivity with detection limit as low as 0.0071 U/mL (S/N=3). Compared with other works, this developed method is simple to operate and does not require a sophisticated instrument or tedious label process. Thus, this proposed method can provide a new idea as well as technical means for

Declaration of Interest Statement

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The authors declare no competing financial interest.

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cancer diagnosis.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China

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(No. 81572078, No. 81873982 and No. 81401722) and the Third Military Medical

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University Medical Creative Research Foundation (SWH2016JCYB-62 and

Notes

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SWH2016JCYB-33).

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The authors declare no competing financial interest.

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Biographies Jing Luo is a combined training postgraduate of the Army Medical University and Southwest University. Her current interests are fabricating photoelectrochemical biosensers in DNA methylation analysis.

Lichao Fang is an associated professor of Clinical and Military Laboratory Medicine, College of Medical Laboratory Science, Army Medical University. Her current

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interests are fabricating biosensers in DNA methylation analysis.

Huamin Liu is a combined training postgraduate of the Army Medical University and Southwest University. Her current interests are fabricating photoelectrochemical

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biosensers in DNA methylation analysis.

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Quanjing Zhu is a postgraduate of Clinical and Military Laboratory Medicine, College of Medical Laboratory Science, Army Medical University. Her current

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interests are fabricating photoelectrochemical biosensers in DNA methylation analysis.

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Hui Huang is an associated professor of Clinical and Military Laboratory Medicine, College of Medical Laboratory Science, Army Medical University. His current

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interests are DNA methylation analysis.

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Jun Deng is an associated professor of Clinical and Military Laboratory Medicine, College of Medical Laboratory Science, Army Medical University. His current interests are DNA methylation analysis.

Feixue Liu is a lecturer of of Clinical and Military Laboratory Medicine, College of Medical Laboratory Science, Army Medical University. Her current interests are DNA methylation analysis. 23

Yan Li is a senior experimentalist of Clinical and Military Laboratory Medicine, College of Medical Laboratory Science, Army Medical University. Her current interests are fabricating photoelectrochemical and electrochemical biosensors in DNA methylation analysis.

Junsong Zheng is a professor of Clinical and Military Laboratory Medicine, College of Medical Laboratory Science, Army Medical University. His current interests are

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fabricating photoelectrochemical and electrochemical biosensors in DNA methylation

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analysis.

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Figure caption

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Scheme. 1. Schematic illustration of the PEC assay for DNA MTase activity based on

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dual-signal amplified system of RGO-CdS:Mn nanoparticles and CdTe@DNA.

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Fig. 1. TEM images of (A) RGO-CdS:Mn nanoparticles, (B) RGO layer, (C) CdS NPs, (E) CdTe QDs, (F) CdTe@DNA network and (D) the EDX spectrum of

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RGO-CdS:Mn nanoparticles.

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Fig. 2. EIS (A) and PEC (B) of each step in fabricating the biosensor: (a) bare gold electrode; (b) RGO-CdS:Mn/GE; (c) S1/RGO-CdS:Mn/GE; (d)

BSA/S1/RGO-CdS:Mn/GE; (e) S2/BSA/S1/RGO-CdS:Mn/GE; (f) HpaII(80

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U/mL)/M.SssI MTase(150 U/mL)/S2/BSA/S1/RGO-CdS:Mn/GE; (g)

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CdTe@S3/HpaII(80 U/mL)/M.SssI MTase(150 U/mL)/

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S2/BSA/S1/RGO-CdS:Mn/GE; (h) HpaII(80 U/mL)/S2/BSA/S1/RGO-CdS:Mn/GE

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Fig. 3. Optimization of the bioassay conditions based on the PEC responses. (A) the molar ratio of Mn2+; the concentration of RGO-CdS:Mn nanoparticles (B) and the

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HpaII (C); (D) the hybridization time of the CdTe@DNA network.

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Fig. 4. (A) The PEC response of the biosensor with different concentrations of M.SssI MTase, from a to j: 0, 0.01, 0.1, 1, 10, 20, 40, 80, 100 and 150 U/mL. (B) The linear calibration curve of the different concentrations of M.SssI MTase from 0.01 to 80

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U/mL.

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Fig. 5. (A) The PEC response of the biosensor with different types of target DNA: S2

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noncomplementary target DNA; (B) the PEC response of five independent biosensors with 1U/mL M.SssI MTase; and (C) the PEC response of the biosensor with different

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storage time.

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Table Caption

Linear range

Detection limit

(U/mL)

(U/mL)

Colorimetric method

0.8-40

0.4

33

Electrochemiluminescence

1-120

0.05

34

Surface plasmon resonance

0.5-50

0.09

Electrochemistry

0.05-120

0.03

Electrochemistry

0.05-120

0.025

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Table 1. Comparison of different methods for the detection of M.SssI MTase activity.

Photoelectrochemistry

0.1-50

Photoelectrochemistry

1-60

Photoelectrochemistry

0.01-80

Reference

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Methods

36 37

0.035

38

0.316

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0.0071

This work

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Table 2. Experimental results of the recovery test in a diluted human serum (n=3) calculated

Selectivity

RSD

(U/mL)

(U/mL)

(%)

(%)

0.1

0.102

102.00

3.12

10

9.845

98.45

2.14

40

38.71

96.78

4.34

80

83.74

104.68

6.45

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Added

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