Electrochemical Biosensor for Multiple Methylation-Locus Analysis Based on DNA-AuNPs and Bienzymatic Dual Signal Amplifications

DNA methylation plays a significant role in various biological events, and its precise determination is vital for the prognosis and treatment of cancer. Here, we proposed an ultrasensitive electrochemical biosensor for the quantitative analysis of multiple methylation-locus in DNA sequence via DNA anchoring the gold nanoparticles (DNA-AuNPs) and bienzyme dual signal amplifications. After the target DNA captured by the DNA-AuNPs of the biosensor, the methyl-CpG binding protein MeCP2 could specifically conjugate to the methylation-loci in the double-stranded DNA. Successively, the glucose oxidase (GOD) and horseradish (HRP) co-labeled antibody captured the His tagged MeCP2, which leads to a cascade enzymatic catalysis of the substrates to yield a detectable electrochemical signal. Both the two strategies, including the high content of DNA-AuNPs and the associated catalysis of bienzyme, dramatically enhanced the sensitivity of the biosensor. The response current elevated with the increasing numbers of methylation-locus, thus the multiple methylated DNA was identified by detecting the corresponding current signals. This method could detect the methylated target as low as 0.1 fM, and showed a wide linear range from 10 - 15 M to 10 - 7 M. Besides, the long-term stability and repeatability of the biosensor were also validated. The prepared electrochemical immunosensor exhibits ultrasensitivity through the bienzyme labeling process, which can be applied for the detection of DNA methylation with low concentration.


Introduction
DNA methylation has always been the cutting-edge of epigenetic studies, which plays a significant role in cellular development, genomic stability, gene expression and regulation. In eukaryotes, DNA methylation occurs mainly at the carbon-5 position of cytosine residues at CpG dinucleotides, resulting in the formation of the 5-methylcytosine (5-mC). Normally, a majority of CpG regions in the promoter regions of tumor suppressor genes are unmethylated, once the aberrant methylation occurs, these genes are transcriptionally repressed and inactivated, which is considered as an early warning of tumorigenesis [1]- [8]. Therefore, the determination of DNA methylation can benefit the assessment and classification of tumors. Traditional DNA methylation assays, such as bisulfite sequencing (BS-seq), restriction endonucleases (RE) analysis, high-performance liquid chromatography (HPLC), mass spectrometry (MS), DNA methylation microarrays [9]- [13], generally demand time-consuming, intricate processes or expensive large-scale instruments, which extremely limit their utilization in the analytical and clinical applications. However, electrochemical biosensors offer superior features owing to its simplicity, portability, sensitivity and fast response [14] [15] [16]. Up to now, a number of electrochemical strategies have been established for the detection of DNA methylation. For instance, Geng and Bao utilized the DNA methyltransferases (DNA MTase) to catalyze the formation of 5-mC coupled with the specific cleavage of methylation-sensitive endonucleases HpaII, which could cleave the unmethylated DNA and lead to the decrease of electrochemical signals of methylene blue (MB) to obtain the methyltransferase detection limit of 0.04 U/mL with the DNA concentration of 1 μM [17]. Using 5-mC antibody, Wang et al. built an electrochemical biosensor which can easily detect the MTase activity and DNA methylation with a detection limit of 0.5 μM [18]. Xu et al. also presented a MTase activity detection approach based on the signal amplification of enzyme-modified on the gold nanoparticles to obtain a lower detection limit of 0.17 pM [19]. However, most of reports achieved DNA methylation levels through the enzymatic reactions conducted by DNA MTase without the accurate detection of methylation-locus directly.
As an ideal methylation-specific recognition molecule, the methyl-CpG binding domain (MBD) proteins could be utilized for the quantitative analysis of methylation patterns due to its superior methylation-loci specificity. It is reported that the MBDs include at least the MeCP2, MBD1, MBD2 and MBD4.
They are capable of binding methylation-locus in double-stranded through their functional MBD domain, indicating that MBDs could be used to serve as the specific recognition unit for the detection of DNA methylation-locus [20] [21].
Immunoassay exhibits high specificity and sensitivity in which the employed immunomolecules were labeled with certain enzyme as molecular labels. Horseradish peroxidase (HRP) is known for its robust capability to catalyze various chemical oxidations involving hydrogen peroxide (H 2 O 2 ), and it has been extensively employed in electrochemical immunoassays by transforming the redox reaction process into a detectable electrochemical signal [22] [23] [24]. Glucose oxidase (GOD) is also widely used as the signal tracer which specifically catalyzes the oxidation of glucose to gluconic acid, while O 2 was reduced to H 2 O 2 [25] [26]. Theoretically, these two enzymes could be co-labeled on the antibody molecule as amplifying labels. The GOD is catalytically linked to HRP to form bienzymatic cascade scheme, which could produce a further enhanced response signal rather than the one that labeled with mono-enzyme.
The purpose of this study, which involves the methylation-loci recognition unit of MeCP2 and dual signal amplification from powerful content of DNA anchoring gold nanoparticles (DNA-AuNPs) coupled with bienzyme labeled antibody, is to present an ultrasensitive electrochemical biosensing method for the quantitative analysis of multiple methylation-locus in particular sequence without the need for bisulfite treatment or gene amplification. The electrodeposited AuNPs could enlarge the specific surface area of the electrode, which effectively promote the electron transfer on the electrode and increase the anchoring sites for the self-assembling of the thiolated DNA probes via strong Au-S bond [27] [28] [29]. Thus, the DNA-AuNPs has the potential ability for the enhancement of sensitivity. Also, the bienzyme labeling system could conduct the robust cascade catalysis to produce a further amplified signal and therefore enhanced the sensitivity of the biosensor. As illustrated in Scheme 1, the thiolated singlestranded DNA probe was first self-assembled on the surface of the AuNPsmodified electrode. After the hybridization with the complementary oligonucleotides, the conjugation of the His-tagged MeCP2 was performed. Thereafter, the bienzyme labeled antibody binds to the His-tag of MeCP2 via immunoreactions. In the buffer solution containing glucose and hydroquinone, the GOD could catalyze the glucose substrate to generate H 2 O 2 by the reduction of ambient oxygen. Then the in situ generated H 2 O 2 could be rapidly consumed by the nearby HRP. The combination of GOD and HRP formed bienzymatic cascade catalysis to initiate the oxidation of hydroquinone, generating a detectable amplified oxidation current. The current signal was directly related to the amount of methylation-loci, therefore allowing the precise determination of multiple methylation-locus. Impressively, owing to the superior methylation-loci specificity of MeCP2 and dual signal amplification procedures, this designed method could be a reliable tool for the accurate detection of DNA methylation. Scheme 1. Schematic illustration of the electrochemical biosensing method for the detection of DNA methylation.

Materials
Hydrogen tetrachloroaurate trihydrate (HAuCl 4 •3H 2 O), Tris-EDTA (TE, pH 7.4) buffer solution and tris (2-carboxyethyl) phosphine (TCEP) were obtained from BBI Life Science Corporation. Bovine serum album (BSA) was purchased from Shanghai Solarbio Bioscience & Technology Co. Ltd., Recombinant human methyl-CpG-binding protein 2 (MeCP2) was from novoprotein. MeCP2 was produced by mammalian expression system and the target gene encoding Met1-Ser486 was expressed with 6 × His tag at the C-terminus. Anti-6 × His tag mouse monoclonal antibody, GOD and HRP were obtained from Shanghai Sangon Biotechnology Co. Ltd. The oligonucleotide sequences (Table 1)   Probe DNA S -HS-(CH2)6-GCG CGC TGG GTG GGC GCC GCG GCG CT Target DNA S0 AGC GCC GCG GCG CCC ACC CAG CGC GC Target DNA S1 AGC GCC GCG GCG /i5med C/CC ACC CAG CGC GC *5' end of probe DNA S was modified with -HS, and the i5med represents the methylated cytosine base. The methylation site of the target DNA was located two bases away from the 5' end, and the multiple methylation sites were separated randomly with several other bases.
All electrochemical measurements were performed on a CHI660D electrochemical workstation with a conventional three-electrode system: platinum wire as auxiliary electrode, Ag/AgCl electrode as reference electrode, and a gold electrode as working electrode. The morphology of nanoparticles and probe immobilization were estimated using scanning electron microscope (SEM, crossbeam 340 zeiss, Germany) and atomic force microscopy (AFM, IPC-208B, Chongqing University), respectively.

Preparation of bienzyme co-labeled antibody
The co-labeling of HRP and GOD on the anti-His tag antibody was dealt with a two-stage process. To begin with, the labeling of GOD was performed using the glutaraldehyde cross-link method [30] [31]. The reaction mixture consisted of 1 mg/mL antibodies and 2 mg GOD in 1.25% glutaraldehyde solution. Subsequently, the GOD labeled antibodies were coupled to HRP using the sodium periodate procedure through their carbohydrate moieties [32].

UV-vis spectrophotometer analysis
The coupling products were identified by ultraviolet and visible (UV-vis) scanning at a range of 200 -800 nm. As can be seen in Figure 1, two absorption peaks are observed at the wavelength of 403 nm and 275 nm, representing the UV absorption spectra of HRP/antibody and GOD/antibody, respectively [32] [33] [34]. Due to the combined modification of antibody with these two enzymes, there was shift of absorption peak at 403 nm and 275 nm, which confirmed an efficient bienzyme modification to the antibody.
Morphological characterization of AuNPs/Au and ssDNA/AuNPs/Au According to the SEM image (Figure 2(a)), it can be observed that the AuNPs are spherical and in good distribution with an average particle size of 32 nm, so the superficial area of the electrode was enlarged. The AuNPs/Au and ssDNA/AuNPs/Au were compared by AFM. Figure 2  probe. The AFM parameters have been evaluated for 1000 × 1000 nm and 2000 × 2000 nm surface area. It is significant that there are morphological differences between both the films. The view of AuNPs/Au (Figure 2(b)) shows uniformly deposited homogeneously dispersed AuNPs on electrode, which showed consistent particle sizes with those of SEM. After the modification of ssDNA probe, it can be observed the high brightness white dots which were marked by a white circle and the surface morphology became rougher (Figure 2(c)), giving the increased height of ssDNA anchoring on the surface of the AuNPs/Au films.
Electrochemical characterization of the biosensor EIS was employed to characterize the electrode fabrication process. As depicted in Figure 3 After the MeCP2 protein conjugated to the DNA methylation-loci, the assembled protein could also block the transfer on the electrode, so the impedance value continued to elevate (curve e). We also employed CV to monitor each step of the fabricating process ( Figure  3(b)). The CV curve showed a pair of distinct redox peaks, indicating that the electron transfer of [Fe(CN) 6 ] 3−/4− on the electrode surface. The bare gold showed a well-defined redox peak with the current of 0.625 μA (curve a). The separation of peak-to-peak (ΔEp) was 0.077 V. After the AuNPs were electrodeposited on the bare gold surface, the redox peak current increased with the decreased ΔEp (curve b). This phenomenon was caused by the excellent conductivity of AuNPs, which increased the electrode effective surface and improved the electron transfer rate. However, the redox peak current of [Fe(CN) 6 ] 3−/4− decreased when the DNA probe was immobilized through the Au-S bond due to the electrostatic repulsion force between the negatively charged [Fe(CN) 6 ] 3−/4− and the negatively charged DNA (curve c), thereby decreasing the redox peak current and enlarging the distance between two peaks. In short, the electrode surface had been successfully modified with this probe. Then, the redox peak current further decreased after the probe DNA hybridized with target sequences (curve d) due to the number of negative charges on the gold electrode surface continued to rise, so the electron transfer ability of [Fe(CN) 6 ] 3−/4− was further attenuated, which then reduced the redox peak current. Subsequently, when MeCP2 protein conjugated with the methylated DNA, the redox peak current decreased obviously (curve e). The current decrease can be attributed to the steric hindrance caused by the large volume of MeCP2 protein, which blocked the diffusion of [Fe(CN) 6 ] 3−/4− towards electrode surface and decreased the electron transfer rate. Collectively, each modification step of the electrochemical biosensor was successful. This was in accordance with the results of other studies [18] [19]. The CV results correspond to the EIS for each step of the electrode modification process, which confirmed the successful modification steps on the working electrode.
Optimization of experimental conditions In order to prove the electrocatalytic effect of the bienzyme system, we conducted a comparison test by using three kinds of labeled antibodies with GOD/anti-His tag, HRP/anti-His tag and GOD-HRP/anti-His tag, respectively. The tests were investigated through the DPV signal to 0.1 μM target sequence with one methyaltion-loci. As shown in Figure 5, the bienzyme system shows a strong oxidation current of about 6.37 μA (curve c), increased to 3.2 fold than the monoenzyme HRP (curve b). In contrast, an extremely low current signal was obtained when the anti-His tag antibody was merely modified with GOD (curve a), indicating that the oxidation of hydroquinone by H 2 O 2 was inefficient without HRP. These results indicate that the bienzyme co-labeling procedure was successful and could considerably enhance the sensitivity of the biosensor.
Sensitivity, repeatability and stability of the biosensor We evaluated the DPV responses of different DNA concentrations with only one methylation-loci. Figure 6(a) indicates that the peak currents are increasing along with increased concentration of target DNA across the range of 10 −15 M to 10 −7 M, and the linear relationship between DPV peak current and the logarithm  with a correlation coefficient (R) of 0.989 (Figure 6(a)). Comparing to the previous assays (Table 2), our method is proven to be a sensitive tool for the detection of DNA methyaltion. The repeatability of the proposed method was conducted by detecting the target DNA with one methylation-loci for five repeat measurements. The relative standard deviation (RSD) value was 4.6%, which is lower than previously reported studies [35] [36] [37]. The results illustrate an ideal repeatability of the proposed method.
The long-term stability of the biosensor was also investigated. We stored the prepared biosensors in pH 7.4 PBS at 4˚C and detected every seven days. The current responses were stable, and finally retained 93.7% of its initial redox current after storage of 28 days.

Conclusion
In this work, we successfully fabricated an enhanced electrochemical biosensor for the quantitative analysis of multiple methylation-locus in particular sequence with MeCP2 as the specific recognition unit. A lower detection limit of 0.1 fM was achieved owing to the dual signal amplification of the AuNPs and bienzymatic   This work 1 × 10 −15 -1 × 10 −7 1 × 10 −17 / cascade catalysis. The repeatability and stability were also validated. Comparing with other biosensing approaches based on conventional bisulfate conversion or methylation restriction enzyme cleavage, our presented method is much easier, cheaper and more time-saving, indicating that our method possesses great prospect for the clinic application in the early diagnosis of cancer and other methylation-related diseases.