Accurate Electrochemistry Analysis of Circulating Methylated DNA

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Accurate Electrochemistry Analysis of Circulating Methylated DNA from Clinical Plasma Based on Paired-End Tagging and Amplifications Feng Chen, Xuyao Wang, Xiaowen Cao, and Yongxi Zhao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02572 • Publication Date (Web): 16 Aug 2017 Downloaded from http://pubs.acs.org on August 16, 2017

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Analytical Chemistry

Accurate Electrochemistry Analysis of Circulating Methylated DNA from Clinical Plasma Based on Paired-End Tagging and Amplifications Feng Chen§, Xuyao Wang§, Xiaowen Cao, Yongxi Zhao* Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, P. R. China *E-mail: [email protected]. Tel.: 86-29-82668908. ABSTRACT: Circulating methylated DNA has been a new kind of cancer biomarker, yet its small fraction of trace total DNA from clinical samples impairs the accurate analysis. Though fluorescence methods based on quantitative methylation specific PCR (qMSP) have been adopted routinely, yet alternative electrochemistry assay of such DNA from clinical samples remains a great challenge. Herein, we report accurate electrochemistry analysis of circulating methylated DNA from clinical plasma samples based on a paired-end tagging and amplifications strategy. Two DNA primers each labeled with digoxigenin (dig) and biotin are designed for the recognition and amplification of methylated DNA. Paired-end tagging amplicons and avidin-HRP molecules are successively captured on the electrode modified with Anti-Dig. Then HRP executes catalytic reaction to generate amplified signal. The design of paired-end tagging can readily integrate downstream electrochemical amplified reaction, and two heterogeneous amplifications enable high assay sensitivity. As little as 40 pg of methylated genomic DNA (~10 genomic equivalents) is well identified, and our strategy can even distinguish as low as 1% methylation level. Tumor -specific methylated DNA is clearly detected in the plasma of 10 of 11 NSCLC patients. The high clinical sensitivity of 91% (10/11) indicates the good consistency with clinical diagnosis. Excellent spatial control of electrochemistry allows simpler detection of more methylation patterns compared to fluorescence methods. The developed electrochemical assay is a promising liquid biopsy tool for the analysis of tumor-specific circulating DNA.

INTRODUCTION Methylated DNA in bodily fluids, namely circulating methylated DNA, has been proven as a new kind of cancer biomarker for early diagnosis and treatment evaluation. Despite more abundant total circulating DNA in cancer samples than normal controls, however, these DNA concentrations are as low as the level of ng/mL blood. 1,2 And tumor-related methylated DNA represents only a small fraction of the total circulating DNA, posing rigorous challenge for the accurate analysis of specific DNA methylation pattern. Currently, fluorescence-based polymerase chain reaction (PCR), including endonuclease-assisted PCR 3,4 and quantitative methylation specific PCR (qMSP), 5 is the routine method for clinical detection of methylated DNA. Compared to endonuclease digestion limited by sequence recognition sites, 6 MSP generally converts cytosine to uracil and leaves methylated cytosine (mC) unaltered with greater than 90% efficiency. 7 This treating transforms epigenetic difference into sequence change that is readily distinguished. Recently, by combining MSP with supercharged fluorescent proteins, Nie and coworkers realized reliable analysis of methylated DNA extracted from human colon carcinoma tissue samples. 8 Besides fluorescence assay methods, capillary electrophoresis, 9 colorimetry, 10 surface plasma resonance 11 and surface enhanced

Raman spectroscopy 12 are also employed to detect specific methylation pattern of DNA from tumor cell lines or tissue samples. Notably, electrochemical detection has attracted great attention due to its field portability, good spatial multiplex, intrinsic simplicity, and short analysis time. 13 Currently, several electrochemistry-based methylated DNA assays have been reported. 14-18 Yet they are limited to analyze only synthesized DNA targets due to poor assay performance, and their practice in clinical sample analysis are not explored. Electrochemistry analysis of circulating methylated DNA is still a great challenge. Excellent amplification strategy is required to further improve the sensitivity. On the other hand, electrochemical detection of pathogenic bacteria DNA, 19,20 virus DNA 21,22 and single nucleotide polymorphism 23 in clinical samples have been well demonstrated, respectively. Encouraged by these works, we believe that analyzing circulating methylated DNA by electrochemistry methods may also be practicable. In this work, we report accurate analysis of circulating methylated DNA from clinical plasma samples based on a paired-end tagging and amplifications electrochemical strategy. Two DNA primers each labeled with digoxigenin (dig) and biotin at 5' end are designed for the recognition and amplification of methylated DNA rather than un-

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methylated DNA. Paired-end tagging amplicons are immobilized on the electrode modified with Anti-Dig, raising the biotin far away from the interface. Then avidinHRP molecules are captured via the binding of biotin, and execute catalytic reaction to amplify electrochemical signal. This method is simply designed and easily operated. The design of paired-end tagging allows downstream enzymatic recycling reaction, and two heterogeneous amplifications enable high assay sensitivity for circulating methylated DNA.

EXPERIMENTAL SECTION Apparatus. PCR amplification was run on LightCycler 480 (Roche Applied Science, Mannheim, Germany). An electrochemical workstation (CHI650E, CH Instruments) was used for electrochemical impedance spectroscopy (EIS), cyclic voltammetry and amperometry detection in a conventional three-electrode cell, including a platinum counter electrode, a reference electrode (Ag/AgCl, 3 M KCl), and a gold working electrode. EIS was carried out in PBS buffer containing 5 mM K4[Fe(CN)6]/K3[Fe(CN)6] and 0.1 M KCl with a biasing potential of 0.18 V, an amplitude of 5 mV and over a frequency range from 0.1 Hz to 100 kHz. Cyclic voltammetry was performed from 0 V to 0.7 V at a scan rate of 100 mV/s. Chronoamperometric detection was carried out at 100 mV, and the steady state of HRP redox reaction could usually be obtained within 100 seconds (secs). Materials and Reagents. All chemicals were obtained from commercial sources and used without further purification. Hot Start Taq DNA Polymerase and CpG methyltransferase (MTase) M.sssI were purchased from New England BioLabs (Beijing, China). 11-mercaptoundecanoic acid (MUA), N-(3-dimethylaminopropyl)-N’ethylcarbodiimide hydrochloride (EDC) and Nhydroxysuccinimide (NHS) were from Thermo Fisher Scientific. Anti-digoxigenin (Anti-Dig) and Horseradish peroxidase-conjugated avidin (avidin-HRP) were purchased from Roche Diagnostics (Mannheim, Germany), and diluted with 1% BSA (bovine serum albumin, Solarbio, Beijing, China) in 1 × PBS. The 3,3’,5,5’tetramethylbenzidine (TMB) substrate was purchased from Neogen in the format of a ready-to-use reagent (Kblue low activity substrate, H2O2 included). All solutions were prepared with water (resistivity of 18 MΩ/cm) from a Millipore purification system (Bedford, MA, USA). The primers for p16INK4a tumor suppressor gene are designed as follows: Dig-tagged forward primer: 5'-Dig-TTATTAGAGGGTGGG GCGGATCGC-3' Biotin-tagged reverse primer: 5'-Biotin-GACCCCGAACCG CGACCGTAA-3' They are synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China). Sample Preparation. Genomic DNA form blood of normal volunteers is used as unmethylated genomic DNA. It was isolated by using the TIANamp Genomic DNA kit (Tiangen Biotech, Beijing, China). Methylated genomic

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DNA was obtained by treating normal genomic DNA with M.SssI. Briefly, aliquots of 2 μl of 10 × NEBuffer 2, 2 μl of S-adenosylmethionine (SAM, 3.2 mM), 4 U of M.SssI and 2 μg of genomic DNA extracted from healthy volunteers were added to a final volume of 20 μl, followed by incubation at 37 oC for an hour. To inactivate M.SssI, the mixture was then incubated at 65 oC for 20 min, and the solution was directly used for bisulfite treatment with an EZ DNA Methylation-Gold Kit (ZYMO Research) in accordance with the manufacturer’s instructions. Final elution was performed with 20 μl of M-elution buffer. These bisulfitetreated DNA was aliquoted and stored at -20 oC until ready for use. Blood samples from NSCLC patients were firstly centrifuged at 2000× g, and then supernatant plasma was carefully collected. 1 ml of plasma was used for DNA extraction with a Serum/Plasma Circulating DNA Kit (Tiangen Biotech, Beijing, China). A final elution volume of 20 μl was used, followed by sodium bisulfite conversion as mentioned above. In order to obtain more concentrated DNA, 10 μl of elution volume was recommended for circulating DNA from real samples. Preparation of Antibody-Coated Electrode. The gold electrode (2 mm in diameter) was cleaned according to the reported protocol. Firstly, the electrode was immersed in the ethanol solution of 1 mM MUA overnight for the coating of a MUA layer. Afterword, the electrode was rinsed with ethanol and water successively to remove the unbound MUA. The activation solution of 400 mM EDC and 100 mM NHS in 0.1 M MES (2-[N-morpholino] ethane sulfonic acid) was cast on the surface for 30 min at room temperature to activate the carboxyl group. The activated electrode surface was incubated with Anti-Dig (diluted in 1× PBS) for 30 min. The electrode was capped with plastic electrode caps in the each step in order to prevent solutions from dying up. Paired-end Tagging and Amplifications Assay. In a typical experiment, 7 μl of water, 2 μl of 10 × Standard Taq Reaction Buffer, 2 μl of dNTP (2mM), 2 μl of forward primer, 2 μl of reverse primer, DNA sample and 2 μl of Hot Start Taq Polymerase were mixed into a 20 μl reaction volume. The amplification procedure (92 oC for 15 secs, 64 oC for 20 secs and 72 oC for 15 secs) were performed for 30 cycles. After amplification, the mixture was cast on the electrodes for 30 min at the room temperature. The pairedend tagging amplicons were captured by the Anti-Dig immobilized on the surface via the antibody-antigen affinity. Then the electrodes were rinsed by W-buffer and dried with N2 gas. The captured amplicon raises a biotin molecule far away from the electrode surface, improving the molecular binding ability. The electrodes were incubated with 5 μl of avidin-HRP (1:1000 dilution in 1× PBS containing 1% BSA) for 15 min, followed by washing with W-buffer. After that, each electrode was detected by cyclic voltammetry and subsequent chronoamperometric measurement in the substrate of TMB.

RESULTS AND DISCUSSION


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The design principle of the proposed strategy is illustrated in Scheme 1. Circulating DNA is extracted from plasma, and then treated with sodium bisulfite. Unmethylated DNA is changed in sequence, whereas mC in methylated DNA remains unaltered. This bisulfite treatment results in partly complementary DNA duplexes due to the conversion of many cytosines to uracils. Dig-tagged forward primer and biotin-tagged reverse primer are designed for the recognition and amplification of methylated DNA by MSP. Notably, the reverse primer firstly anneals to one strand of methylated DNA duplex, and is extended to form completely complementary template duplex for PCR amplification. The other strand is not involved in MSP. Amplicons with paired-end tagging are exponentially accumulated. Then, they are immobilized on the Au electrode which is modified with Anti-Dig. The biotin molecules at the other end of amplicons are raised far away from the interface, enabling efficient capture of avidin-HRP molecules for catalytic amplification. In brief, HRP reduces hydrogen peroxide in the presence of TMB to generate a quantitative electrochemical signal. The design of paired-end tagging allows integrating downstream catalytic recycling reaction with MSP. Such duel amplification system enables high assay sensitivity for circulating methylated DNA with simple design and easy operation. A high clinical sensitivity of 91% (10/11 patient samples) is obtained by our method as discussed in clinical performance assessment.

As can be seen in Figure 1B and S1, the decreased Anti-Dig concentration induces a low signal to background (S/B) ratio due to fewer binding site for amplicons. And excess Anti-Dig doesn’t significantly increase the signal mainly because of limited electrode space for Anti-Dig immobilization. Thus, 200 ng Anti-Dig was used for the following experiments according to the highest S/B ratio. Additionally, the primer concentration determines yield of pairedend tagging amplicon, and may affect non-specific amplification especially primer dimers. We tested the assay performance of under different primer concentrations.

We firstly evaluate the stepwise surface modification of Au electrode. As shown in Figure 1A, the change in EIS measurements is very distinct, and the increase of Nyquist diameter indicates sequential binding of molecules on electrode surface. Then, some important conditions are investigated. The concentration of Anti-Dig on electrode surface affects the capture efficiency of pairedend tagging amplicons and the non-specific adsorption. Scheme 1. Schematic Illustration of Paired-end Tagging and Amplifications for Accurate Electrochemistry Analysis of Circulating Methylated DNA from Clinical Plasma

Figure 1. (A) Nyquist plots for the electrode successively modified with MUA, EDC/NHS, Anti-Dig and paired-end tagging amplicon. (B) The effect of Anti-Dig concentration on our electrochemical method. S and B represent the electrochemical responses in the presence and absence of methylated genomic DNA, respectively. The corresponding current curves are shown in Figure S1. (C) The effect of primer concentration on the electrochemical analysis. The corresponding current curves are shown in Figure S2.



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Increased primer induces a remarkable enhancement of current signal, whereas the background also increases (Figure 1C and S2). Owing to the highest S/B value, 200 nM of primer concentration was chosen for the following experiments. Notably, the background results from the heterodimer produced by both Dig-tagged primer and Biotin-tagged primer (Figure S3). As depicted in Figure 1C, our electrochemical method can clearly identify the slight difference in the yield of this primer dimer. Two kinds of homodimers each produced by only Dig-tagged primers or Biotin-tagged primers fail to capture avidin-HRP and Anti-Dig, respectively. Thus, they both can not lead to background current. In contrast, all these three kinds of primer dimers can be detected as interference in fluorescent dyes-based qMSP. 24-27 Actually, primer dimerinduced non-specific amplification is observed in qMSP using this set of primers (Figure S4). Yet non-specific amplification under different primer concentrations causes very similar quantification cycle (Cq) values. It indicates real-time fluorescence analysis fails to distinguish the difference of background signal. These results imply that the proposed electrochemical method may offer higher sensitivity than fluorescence qMSP. The quantitative analysis of methylated genomic DNA was subsequently evaluated. The current curves of different DNA amount in the range of 0-15 ng are shown in Figure 2A. The intensity value increases as the increasing of methylated genomic DNA. Figure 2B plots the relationship of the current intensity with the changes of methylated genomic DNA amount. It can be seen that a linear correlation between current intensity and the logarithm of DNA amount (50 pg to 5 ng) is obtained (inset of Figure 2B). The

correlation equation is I = 543.3 lgC (ng) + 1361.2 (I is the current value and C is the DNA amount) with the correlation coefficient R2 of 0.993. Based on the rule of three times the standard deviation of blank control signal, a low detection limit down to 40 pg methylated genomic DNA (about 10 genomic equivalents) is achieved. It indicates that as little as 10 copies of methylated DNA can be detected. This sensitivity is superior to those of all reported electrochemical methods and many fluorescent ones. It is well know that the amount of circulating DNA from 1 mL blood (about 0.55 mL plasma) of cancer patients is about 15 ng. Thus, our method could be applicable to the analysis of circulating methylated DNA from clinical samples. In comparison, we also investigated the sensitivity of fluorescent dyes-based qMSP using the primers in our method. As shown in Figure 2C, it can not clearly detect 50 pg methylated genomic DNA according to the similar Cq value as those of the blank control and lower DNA amount samples. These results indicate our method presents comparable and even higher sensitivity than typical fluorescence qMSP. It is well known that tumor-related methylated DNA represents very small fraction of total circulating DNA, and homologous unmethylated DNA exist abundantly as serious interference. Furthermore, DNA methylation level varies with tumor behavior, stages and therapy. 28, 29 Therefore, it is necessary to evaluate the quantitative

Figure 2. Quantitate analysis of methylated genomic DNA. (A) Electrochemical current curves of different DNA amount and (B) the changes of current intensity with DNA amount. NTC is the abbreviation of no template control (blank). The inset displays the linear correlation between current intensity and the logarithm of DNA amount ranging from 50 pg to 5 ng. (C) The qMSP analysis of different DNA amount as in (A) and (B). accuracy of our method. Methylated genomic DNA and unmethylated genomic DNA were mixed in different ratios to prepare samples of broad methylation levels (0, 1, 5, 20, 50, and 100%). The total DNA amount is fixed at 15 ng. As shown in Figure 3A, with an increasing of input methylated DNA in the mixture, corresponding increase in the current intensity is observed. According to the curve of quantification of only methylated DNA mentioned above, calculated methylation levels corresponding to input methylation levels are obtained. Figure 3B shows


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Figure 4. Clinical assay performance of circulating DNA extracted from plasma samples of NSCLC patients and normal sample. The threshold value is defined as the mean value of NTC (blank) signal plus three times its standard deviation. The patient data in Scheme 1 is from Figure 4. The standard sample indicates the one containing 15 ng methylated genomic DNA.

Figure 3. (A) Electrochemical current curves of different methylation level of genomic DNA. (B) Calculated methylation levels are plotted for the varied levels of input methylation. consistent correlation between them with correlation coefficient of 0.998. These results demonstrate the presence of abundant unmethylated DNA doesn’t impact the accurate analysis of methylated DNA by our approach. Notably, down to 1% methylation level is well identified. This performance is comparable with those of fluorescence methods. 3, 4, 8, 30 By including serial dilution methylated samples in every assay with a given total input DNA, a standard curve can be created to enable the quantitation of methylation level of unknown samples. To validate the excellent performance of our strategy in clinical patient samples with low concentrations of methylated DNA, circulating DNA extracted from plasma of NSCLC patients and normal sample were tested. Tumorspecific methylated DNA was detected with very high signal in 10 of 11 patient samples, whereas signal intensity of the other patient sample slightly exceeds those of normal sample and blank control (Figure 4). According to three times the standard deviation of blank control signal (threshold value), clinical sensitivity up to 91% (10/11) is obtained. It is higher than those of many reported works based on qMSP. 31-33

Overall, our electrochemical assay presents comparable performance to fluorescence qMSP for the analysis of clinical samples, and is superior to previously reported electrochemical methods. Simultaneous detection of different gene methylation patterns is also crucial and difficult. Multiplex qMSP can achieve this goal with many sets of primers by one-tube analysis. Corresponding fluorescence dual-labeled probes (e.g., TaqMan probes) instead of DNA binding dyes are also designed and used. Such complicated system raises the risk of non-specific amplification and false positive result. Additionally, the number of multiplex targets is typically limited to 3 or 4 in commercial qPCR systems due to spectral overlap of the fluorophores. In contrast, electrochemistry can perform multiplex detection via simple spatial control. For example, small and inexpensive screen-printed electrodes allow much higher target number than qPCR systems. Moreover, only different tagged primers are required without designing dual-labeled probes. Notably, we very recently developed a sequential discrimination-amplification electrochemical assay, and achieved reliable detection of circulating methylated DNA from clinical samples. 34 It utilizes DNA nanostructured probes to capture amplicons by DNA hybridization. The optimization of ion condition is necessary for efficient hybridization reaction nearby negatively charged DNA nanostructures. Compared to pairedend tagging and amplifications strategy, complicated probe design in this method is required for multiplex detection of methylated DNA. Our ongoing work is focused on the development of intelligent microfluidic



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electrochemical device for multiplexed detection of circulating DNA. Multiplex gene amplification and spatial electrochemistry analysis will be integrated in this device.

CONCLUSION In this work, circulating methylated DNA from clinical samples was analyzed based on a paired-end tagging and amplifications electrochemical strategy. The design of paired-end tagging allows the integration of downstream enzymatic reaction, and two heterogeneous amplifications enable high assay sensitivity. It can detect as little as 40 pg of methylated genomic DNA, and distinguish as low as 1% methylation level. Tumor-specific methylated DNA is identified in the plasma of NSCLC patients with high sensitivity of 91% (10/11 samples), indicating the good consistency with clinical diagnosis. The spatial multiplex ability of electrochemistry could realize simultaneous analysis of different methylation patterns. Therefore, our electrochemical assay is a promising liquid biopsy tool to analyze circulating tumor DNA for early diagnosis, therapy response and tumor recurrence.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional data and figures of electrochemical assay and fluorescence qMSP.

Corresponding Author *E-mail: [email protected]. Tel.: 86-29-82668908.

ORCID Yongxi Zhao: 0000-0002-1796-7651

Author Contributions §

F.C. and X.W. contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was financially supported by the National Science Foundation of China (No. 21475102 and No. 31671013), the China Postdoctoral Science Foundation (No. 2017M613102), the Fundamental Research Funds for the Central Universities and “Young Talent Support Plan” of Xi’an Jiaotong University.

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Accurate Electrochemistry Analysis of Circulating Methylated DNA

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