Sensitive Electrochemical Detection of Human Methyltransferase

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Sensitive Electrochemical Detection of Human Methyltransferase Based on a Dual Signal Amplification Strategy Coupling Gold Nanoparticle-DNA Complexes with Ru (III) Redox Recycling Hui Zhang, Huilei Dong, Guoqing Yang, Hongfei Chen, and Chenxin Cai Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03163 • Publication Date (Web): 12 Oct 2016 Downloaded from http://pubs.acs.org on October 12, 2016

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Analytical Chemistry

Sensitive Electrochemical Detection of Human Methyltransferase Based on a Dual

Signal

Amplification

Strategy

Coupling

Gold

Nanoparticle-DNA

Complexes with Ru (III) Redox Recycling

Hui Zhang*, Huilei Dong, Guoqing Yang, Hongfei Chen, Chenxin Cai Jiangsu Key Laboratory of New Power Batteries, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, Jiangsu Key Laboratory of Biomedical Materials, National and Local Joint Engineering Research Center of Biomedical Functional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210097, P. R. China.

*Correspondence author:

Hui Zhang. E-mail: [email protected]. Telephone: (025)8589-1780. Fax: (025)8589-1767.

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ABSTRACT: Effective detection of DNA methyltransferase (DNMT) activity is significant for cancer research. Herein, we developed a sensitive electroanalytical method to detect human DNA (cytosine-5)-methyltransferase1 (DNMT1) from crude lysates of cancer cells. In this assay, capture DNA having a preferred DNMT1 methylation site was immobilized on a gold electrode, then hybridized with gold nanoparticle (Au NP)-DNA complexes. The modified electrodes were equilibrated with the lysate, and then incubated with methylation-sensitive restriction enzyme. If the lysate was negative for DNMT1 activity, the Au NP-DNA complexes would be cut by the restriction enzyme, and released from the electrode. Conversely, restriction enzyme cleavage would be blocked by the fully methylated duplexes, and the Au NP-DNA complexes would remain on the electrode. Electroactive Ru(NH3)63+ was used as the signal reporter, because of its electrostatic attraction to DNA, resulting in an electrochemical signal. Since the electrochemical signal reflects the amount of Ru(III) redox and the amount of Ru(III) redox is correlated with the activity of DNMT1, the activity of DNMT1 is proportional to the electrochemical signal. The signal could be amplified by the numerous DNAs on the Au NPs, and further amplified by Ru(III) redox recycling. With this method, a detection limit down to 0.3 U/mL for pure DNMT1 and 8 MCF-7 cells was achieved. DNMT1 activities of different cell lines were also successfully evaluated.


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INTRODUCTION DNA methylation is an epigenetic modification,1 which powerfully influences gene expression in cells.2,3 In humans, the most widely studied DNA methylation is with cytosine bases. This process is catalyzed by members of the DNA methyltransferase (DNMT) family, which transfer a methyl group from S-adenosyl-L-methionine (SAM) to the target cytosine.4,5 Among the DNMT family, the maintenance DNMT, called DNMT1, the most abundant DNMT in somatic cells,6,7 has strong preference to methylate hemimethylated sites and is thought to be the enzyme primarily responsible for maintenance of the global methylation status of genomic DNA.8,9 Overexpression of DNMT1 can cause aberrant DNA methylation and aberrant DNA methylation is linked to cancer.10 Many studies have shown that DNMT1 is overexpressed in tumor cells, such as colorectal, breast carcinomas, lung, stomach, pancreas and liver cancer.11-14 Therefore, efficient detection of methyltransferase activity provides an early cancer diagnostic and helps in the screening of anticancer drugs.15 The most widely used method to determine DNMT1 activity is radiolabeling DNA with a tritium-labeled methyl group.16 But this method requires the use of radioactivity and specialized instrumentation for measurement. Other methods include separation of methylated nucleotides by HPLC.17 Fluorescence measurements and reaction of colorimetric reagents with antibodies18,19 also can be used to determine DNMT1 activity. Although these approaches have high sensitivity, they are laborious, time-consuming and require large and expensive instrumentation. Thus, an effective method for the easy detection of DNMT1 is required. Electrochemical detection schemes, compared with other methods, have the advantages of low cost, portability and high sensitivity.20-29 Recently, for the first time, Furst et al tried to use an electrochemical platform for the assessment of DNMT1 activity from human colorectal cancer cells (HCT 116) and colorectal tissur samples.3 they use two working electrodes which enabled electrochemical readout from disperse DNA monolayers with signal amplification and no necessary background correction.3,30 Their results illustrated the effectiveness of the electrochemical platform for real sample applications. However, they only detected one kind of cell’s DNMT1 activity with a semi-quantitative analysis. And their special designed two-electrode platform is complicated, limiting the practical application. 3 ACS Paragon Plus Environment


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Inspired by the pioneering works of Furst et al, in this study, we developed a new electrochemical platform to detect different cancer cells’ DNMT1 activity by using a traditional gold electrode as a sensing platform. In order to improve the sensitivity, gold nanoparticle (Au NP)-DNA complexes and Ru(III) redox recycling are adapted for dual signal amplification. Au NP-DNA complexes, first proposed by the Merkin’s group in 1996,31 have good biocompatibility,32 and compared with unmodified DNA, they are much more stable to nuclease degradation33 and have higher affinity constants for complementary nucleic acids. These distinctive properties have led to satisfactory use of Au NP-DNA complexes in amplified detection of protein,34 DNA,35 RNA,35 mRNA,36 circulating tumor cells37 and metal ions,38 as well as intracellular gene regulation.39 In this paper, Au NPs are functionalized with two different DNA sequences. One can hybridize with the DNA immobilized on the surface of the gold electrode, and the resulting Au NP-DNA complexes can capture Ru(NH3)63+, a DNA-binding and redox-active probe, which can bind to the anionic phosphate backbone of DNA strands via electrostatic forces.40 Furthermore, the resulting Ru(NH3)62+ can be cycled by a chemical reducing reagent (Fe(CN)63-) after the electro-reduction of Ru(NH3)63+ on the electrode, thus leading to the recycling of Ru(III) redox and further increase in the cathodic current signal. With the dual signal amplification strategy of the Au NP-DNA complexes and Ru(III) redox recycling, the DNMT1 activity from crude lysates of cancer cells can be detected even at low cell number, and the electrochemical signal for DNMT1 positive cells could be easily distinguished from negative cells.

EXPERMENTAL SECTION Materials and Chemicals. The chemicals, including tris(2-carboxyethyl) phosphine hydrochloride (TCEP, 98%), ethylene diamine tetraacetic acid (EDTA), glycerol (99%) and tris(hydroxymethyl) aminomethane were purchased from Aladdin (Shanghai, China). 6-mercapto-1-hexanol (MCH) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Hexaammineruthenium (III) chloride ([Ru(NH3)6]3+, RuHex) was purchased from Alfa Aesar (Massachusetts, U.S.A.). A 40% acrylamide mix solution, ammonium persulfate (APS), 1,2-bis(dimethylamino)-ethane (TEMED), and low range DNA ladder were obtained from Sangon Biotechnology Co. Ltd. (Shanghai, China). Human DNA(cytosine-5)-methyltransferase1 (DNMT1) and methylation-sensitive restriction enzyme BssHII were supplied by New England BioLabs (Ipswich, MA). The ELISA-like ‘‘EpiQuikTM 4 ACS Paragon Plus Environment

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DNMT1 Assay Kit’’ was purchased from Epigentek (New York, USA). All other chemicals were of analytical reagent grade. All the HPLC-purified oligonucleotides used here were purchased from Sangon Biotechnology Co. Ltd. (Shanghai, China), and all the oligonucleotide sequences are listed in Table S1. (see supporting information). A Tris-HCl buffer (10 mM, pH 7.4) was used as the immobilization buffer. A phosphate buffer solution (PBS) (10 mM, pH 7.4) containing 0.1 M NaCl was employed as the washing buffer. A PBS (10 mM, pH 7.4) containing 0.5 M NaCl was used as the hybridization buffer. A 50 mM Tris-HCl (pH 7.8) buffer containing 1 mM EDTA and 5% glycerol was employed as DNMT1 activity buffer. A 10 mM PBS (pH 7.4) buffer containing 10 mM Fe(CN)63-/Fe(CN)64- and 1 M KCl was used as the electrochemical impedance spectroscopy (EIS) buffer. A 1 mM PBS (pH 7.4) buffer containing 4 mM Fe(CN)63-, 25 µM Ru(NH3)63+ and 10 mM NaCl was used as the electrochemical buffer. Deionized water (18 MΩ/cm resistivity) obtained from a Millipore water system was used throughout the experiment. Instrumentation. Transmission electron microscopy (TEM) was observed by a JEOL-2010 transmission electron microscope operating at an accelerating voltage of 120 kV. The UV–vis absorption spectra were obtained on a Cary 5000 UV–vis–NIR spectrophotometer (Varian, USA). The EIS measurements were performed on an Autolab PGSTAT-30 potentiostat/galvanostat (Eco Chemie BV, Utrecht, The Netherlands). Differential pulse voltammetry (DPV) was carried out using a CHI760C electrochemical workstation (Chenhua, Shanghai, China). Polyacrylamide gel electrophoresis (PAGE) was performed on a JY600C electrophoresis apparatus (Beijing Junyi-dongfang electrophoresis equipment Co. Ltd., Beijing, China) and imaged on a Tanon-3500 gel image system (Shanghai, China). Cell Culture and DNMT1 Extraction. MCF-7 cells, A549 cells, HeLa cells, CCRF-CEM cells and HEK-293 cells were grown in DMEM medium (Sangon Biotechnology Co. Ltd. Shanghai, China) supplemented with 10% fetal calf serum (Sangon Biotechnology Co. Ltd. Shanghai, China), penicillin (100 µg/mL), and streptomycin (100 µg/mL) in a 37 oC incubator with 5% CO2 atmosphere. All cells were collected in the exponential phase of growth, and approximately 1 million were harvested from adherent cell culture by trypsinization, followed by washing with cold PBS and then a nuclear protein extraction kit (Sangon Biotechnology Co. Ltd. Shanghai, China. The 5 ACS Paragon Plus Environment


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cells were incubated for 15 min on ice and then centrifuged at 12000 rpm for 5 min at 4 oC. Finally, the supernatant was exchanged by a size exclusion spin column (10-kDa cutoff; Amicon) into DNMT1 activity buffer. Cell lysate was flash frozen and stored at -80oC before use. Preparation of Au NP-DNA Complexes. Gold nanoparticles were prepared using the procedures reported previously.41,42 Transmission electron microscopic (TEM) images indicated a particle size of about 14 nm. Au NP-DNA complexes were synthesized following the published protocol.18 Briefly, a volume of 150 µL, 1× 10-5 M two different thiol-modiﬁed oligonucleotides (molar ratio of S2:S3 = 1:2, 1:5, 1:7.5, 1:10 and 1:12, respectively) was activated with 3 µL of 50 mM TCEP for 2 h. The active DNA was then mixed with 1 mL of Au NPs. After standing for 16 h with gentle shaking at 37 oC. The system was buffered to a phosphate concentration of 10 mM (pH 7.4) including 0.1% sodium dodecyl sulfate (SDS). Over the course of three days, the sodium chloride concentration was brought to 0.5 M in a stepwise manner. Particles were then centrifuged for 30 min at 10000 rpm and rinsed four times (10 mM sodium phosphate, 0.1 M NaCl, pH 7.4) to remove any unbound DNA. The Au NP-DNA was finally redispersed in 1 mL PBS (10 mM, pH 7.4) containing 0.5 M NaCl and stored at 4 oC for further use. Immobilization of DNA Probe and Hybridization. Prior to immobilization, the 2 mm diameter gold electrode was pretreated as reported previously.43 For the immobilization of S1 on the cleaned gold electrode surface, a mixture of 10 µL S1 solution (10 mM) with 10 µL TCEP solution (10 mM) was first incubated for 1 h to reduce the disulfide bond at the 5’-terminus of S1 and generate a free thiol group for surface immobilization, followed by diluting the mixture to 100 µL with 10 mM Tris-HCl buffer (pH 7.4).44 The S1 was immobilized onto the surface of the gold electrode by incubating the cleaned electrode in the diluted S1 solution for 20 h. The electrode was then rinsed with Tris-HCl buffer and subsequently passivated with MCH (1 mM) for 2 h to block the unoccupied surface binding sites and displace nonspecifically bound S1 on the gold electrode surface.45 The passivated electrode was then washed and soaked in the Tris-HCl buffer twice, ready for DNA hybridization. For hybridization of Au NP-DNA with the probe DNA(S1) on the gold electrode, 10 µL of Au NP-DNA (in PBS) solution was dropped on a gold electrode and incubated for 2 h at 37 oC. After hybridization, the electrode was thoroughly rinsed with Tris-HCl buffer for electrochemical 6 ACS Paragon Plus Environment

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measurement. Electrochemical Characterization and Measurements. All the measurements were performed using a conventional three-electrode system, consisting of a modified gold working electrode, a platinum wire counter electrode and a saturated calomel (SCE) reference electrode. EIS measurements were carried out with the frequency range of 10-2 to 105 Hz, 5 mV amplitude in EIS buffer. For methyltransferase activity detection, the modified gold working electrode was incubated with different concentration of DNMT1 or cell lysates with 160 µM SAM and 100 µg/mL BSA in DNMT1 activity buffer and incubated at 37 oC for 2 h in a humidified container. Then the electrode was rinsed with Tris-HCl buffer thoroughly and scanned. Subsequently, the electrode was treated with the restriction enzyme BssHII at a concentration of 1000 U/mL at 37 oC for 2 h. Then the electrode was thoroughly rinsed with Tris-HCl buffer and scanned. Electocatalytic currents were measured with DPV (-0.5 V to +0.1 V; amplitude, 0.05 V; pulse width, 0.01 s; pulse period, 0.02 s) in electrochemical buffer. Signal changes corresponding to the activity of DNMT1 were calculated as ∆I = Iinitial- Ifinal. Error bars shown on individual figures correspond to variabilities among five independent trials of each experiment.

RESULTS AND DISCUSSION Design of the Electrochemical assay for DNMT1 activity detection. The design of the electrochemical assay for DNMT1 activity detection is illustrated in Scheme 1. The single stranded thiol-modified

probe

DNA (S1)

containing

the

preferred

DNMT1

methylation

sites

(hemimethylated 5ˊ-CG-3´ sites) were immobilized on the gold electrode through the Au-S bond, followed by the MCH blocking (step A, Scheme 1). The Au NPs modified with S2 and S3 were captured through the hybridization between S1 and S2 (step B, Scheme 1) and electrode modified with Au NP-DNA complexes/S1 could be obtained. The modified electrode was first treated with the crude lysate containing SAM (Step C, Scheme 1), and then treated with BssHII restriction endonuclease, which is a widely used restriction enzyme in the gene-specific methylation assay46 (step D, Scheme 1). The electrochemical signals were recorded before and after step D. When the S1/S2 duplexes were fully methylated by active DNMT1 in the lysate sample, the restriction endonuclease BssHII was blocked by the fully methylated duplexes, and the electrochemical signal 7 ACS Paragon Plus Environment


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was not affected. However, when the S1/S2 duplexes were not methylated in the lysate sample, the DNA could be cleaved by BssHII, significantly decreasing the amount of Au NP-DNA complexes on the gold surface, and thus diminishing the electrochemical signal. Therefore, the electrochemical signal is related to the DNMT1 activity, which establishes the basis of MTase activity assay. Since numerous DNAs were immobilized on the surface of the Au NPs, the signals could be amplified. Furthermore, the electrochemical signal could be amplified by the recycling of Ru(III) redox. Ru(NH3)63+ could bind to the negatively charged phosphate backbone of DNAs by electrostatic interactions, and electro-reduced to Ru(NH3)62+ on the electrode. The Ru(NH3)62+ could be chemically oxidized back to Ru(NH3)63+ by Fe(CN)63-, thus leading to the recycling of Ru (III), and generating an amplified electrocatalytic current.47,48 Based on the dual signal amplification strategy of Au NP-DNA complexes and Ru(III) redox recycling, the electrochemical signal for the detection of DNMT1 could be amplified remarkably.

Scheme 1. Schematic illustration of Au NP-DNA complexes and Ru (III) redox recycling dual signal amplification electrochemical assay for DNMT1 activity detection: (A) S1 modified gold electrode blocked with MCH; (B) capture of Au NP-DNA complexes; (C) the modified electrode 8 ACS Paragon Plus Environment

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treated with the crude lysate containing SAM; (D) then treated with BssHII restriction endonuclease. Characterization of Au NPs and Au NP-DNA Complexes. The TEM images of the prepared Au NPs (Figure 1A) show that Au NPs are monodispersed spherical particles with a narrow particle size distribution. The TEM images of Au NP-DNA complexes are illustrated in Figure 1B. No appreciable differences of dispersity and morphology were observed between Au NP-DNA and Au NPs. The average hydrodynamic diameters of the Au NPs and Au NP-DNA complexes obtained by DLS analysis (the insets of Figure 1A, 1B) were about 12.2 and 20.8 nm, respectively. The modification of Au NPs was also studied with UV-vis absorption spectroscopy. The UV-vis spectrum of Au NPs without modification showed an absorption peak at 520 nm (curve a in Figure 1C), a characteristic surface Plasmon resonance band of 13 nm diameter gold particles.49 This result was consistent with the value observed from the TEM images. After modification, a slight shift in the SPR peak from 520 to 524 nm was observed due to the DNA-ligands.50,51 The absorption peak at 260 nm observed in the UV-vis absorption spectroscopy of the DNA-modified Au NPs, corresponds to the absorption peak of S2 and S3 on the Au NPs, indicating the successful conjugation of S2 and S3 with Au NPs.

Figure 1. Typical TEM images of (A) the prepared Au NPs and (B) Au NP-DNA complexes. The insets show the DLS size distribution of the prepared Au NPs and Au NP-DNA complexes. (C) UV-vis absorption of (a) Au NPs and (b) Au NP-DNA complexes. Feasibility of this Strategy. To test the feasibility of this strategy, native polyacrylamide gel electrophoresis (PAGE) and DPV measurements were recorded. As show in Figure 2, PAGE was used to characterize DNA hybridization and BssHII function. S01 and S02, with DNA sequences similar to those of S1 and S2, respectively, only without a thiol group, were used. The PAGE result 9 ACS Paragon Plus Environment


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confirmed that S01 can hybridize completely with S02 (lane 4 in Figure 2) and that the restriction enzyme can cut the hemimethylated DNA efficiently (lane 5 in Figure 2), indicating the feasibility of our design.

Figure 2. Native PAGE gel of different samples. Lane 1: the standard DNA markers of 10, 15, 20, 25, 35, 50, 75, 100, 150, 200, 300 bp of DNA sequences; Lane 2: 5 µM S01; Lane 3: 5 µM S02; Lane 4: 5 µM S01 mixed with 5 µM S02, then denatured at 95 oC for 5 min followed by cooling at room temperature; Lane 5: lane 4 after treatment with 10 U / mL BssHII. DPV measurements were recorded with the electrode at different stages of the modification procedure. As shown in Figure 3A, when S1 was immobilized on the gold electrode through the Au-S bond and followed by the MCH blocking (step A, scheme 1), the current is small (Figure 3A, a). After incubating the extended working electrode with Au NP-DNA complexes (step B, scheme 1), the signal increased significantly (Figure 3A, b) because more Ru(NH3)63+ was electrostatically bound to the negatively charged phosphate backbone of DNAs. After treatment of the Au NP-DNA complex/S1 modified gold electrode with DNMT1(step C, scheme 1), the DPV value was the same as for curve b (Figure 3A, c), indicating that DNMT1 has no effect on signal detection. Further incubated with BssHII (step D, scheme 1), the signal decreased because part of the ds-DNA was fully methylated by DNMT1, and only the remaining hemimethylated ds-DNA was cleaved by BstUI, resulting in a decrease in the DPV signal (Figure 3A, d). These results show that the viability of the biosensor for evaluation of DNMT1 activity. We compared the DPV curves obtained with the Au NP-DNA complex/S1 modified gold 10 ACS Paragon Plus Environment

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electrode from different detection solutions. As shown in Figure 3B, the DPV signal of Ru(NH3)63+ and Fe(CN)63- are amplified approximately 10 fold over that of only Ru(NH3)63+ redox, indicating the great signal amplification of this assay. We also use the electrochemical impedance spectroscopy (EIS) to characterize the biosensor. (see supporting information, Figure S1)

Figure 3. (A) DPV responses with the gold electrode at different stages. (a) MCH-blocked S1-modified gold electrode; (b) capture of Au NP-DNA complexes; (c) incubation with DNMT1 (2 U/mL) and SAM (160 µM); (d) incubation with DNMT1 (2 U/mL) and SAM (160 µM), followed by incubation with BssHII (10 U/mL). The inset shows the DPV curves; (B) DPV signal obtained from the detection solution with (a) Ru(NH3)63+ only; (b) Ru(NH3)63+ and Fe(CN)63-. Optimization of the Ratio of S2:S3 and the Concentration of S1. In this paper, S2 was used to hybridize with S1 and S3 was help to increase the signal of Ru(NH3)63+. In order to gain a high hybridization efficiency, the ratio of S2:S3 binding onto the Au NPs and the concentration of S1 should be optimized. First, the probe DNA S1 with moderate concentration of 0.2 µM was immobilized on the gold electrode. Manipulation of S2 densities binding to the Au NPs was achieved with solutions containing variable ratios of S2:S3. Following the hybridization between the S1 modified gold electrode and Au NP-DNA complexes, the influence of ratio of S2:S3 was investigated by monitoring the electrochemical signal. Since the sequence of S2 is longer than that of S3, with decrease of the S2:S3 ratio, the steric hindrance can be avoid, and more S2 are exposed for hybridization with S1. As shown in Figure 4A, the current increased with decrease of the S2:S3 ratio of S2:S3 up to 1:10, further decrease in the S2:S3 ratio would result in less Ru(NH3)63+ cation binds to the S2, and then the current decreased. Therefore, the optimal ratio of S2 and S3 was 1:10. Then the ratio of S2:S3 was fixed at 1:10, and the concentration of S1 was optimized. As shown in Figure 4B, the current increased with the increasing concentration of S1 from 0.1 µM to 0.5 µM and 11 ACS Paragon Plus Environment


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reached the highest value at 0.5 µM. Further increase in the concentration would hinder the hybridization between S1 and S2, suggesting that the optimal S1 concentration was 0.5 µM.

Figure 4. (A) Optimization of the ratio of S2:S3 conjugated to the Au NPs. The ratio of 1:0 represents only S2 binding onto the Au NPs. (concentration of S1 used for electrode modification was 0.2 µM). (B) Optimization of S1 probe concentration used for self-assembly on the gold electrode. The ratio of S2:S3 conjugated to the Au NPs was 1:10. The error bars represent the standard deviation of five repetitive measurements. DNMT1 Activity Detection. Under the optimal experimental conditions, DNMT1 with different concentrations and SAM were added to the reaction system to evaluate the analytical performance of the electrochemical platform. As shown in Figure 5A, the DPV signal increased with increasing DNMT1 concentration. This finding indicates that with the increase of DNMT1 concentration, more hemimethylated substrates were methylated by active DNMT1, thus blocking the cleavage site of the BssHII endonuclease, resulting in an increase in DPV current. Figure 5B showed a linear relationship between the variation of the DPV current and the logarithm of the concentration of DNMT1 in the range of 1 U/mL to 40 U/mL with a correlation coefficient (R) of 0.996. The linear fitting equation is ∆I = 5.928–3.339lg[c], where ∆I is the current difference, defined as ∆I =IinitialIfinal, where Iinitial and Ifinal are the DPV currents of the Au NP-DNA complex/S1 modified electrode before and after incubation with BssHII, and c is the concentration of DNMT1. The detection limit was experimentally estimated to be 0.3 U/mL, which is lower than that of the colorimetric assay and some electrochemical methods. 52-54 In addition, the reproducibility of the fabricated biosensor was tested by detecting 10 U/mL DNMT1 with 6 electrodes prepared independently with the relative standard deviation (RSD) of 12 ACS Paragon Plus Environment

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3.43%, indicating an acceptable reproducibility of the proposed strategy. Therefore, the proposed method can be used for convenient and efficient quantitative analysis of DNMT1 activity.

Figure 5. (A) Effect of DNMT1 concentration on the DPV response. The concentration of DNMT1 is (a) 0, (b) 1, (c) 2, (d) 5, (e) 10, (f) 15, (g) 20, (h) 30 and (i) 40 U/mL. (B) The linear relationship between the DPV peak current change and the logarithm of DNMT1 concentration ranging from 1 to 40 U/mL. The error bars represent the standard deviation of five repetitive measurements. Detection of DNMT1 Activity from Multiple Crude Cultured Cell Lysates. Since Au NP-DNA complexes are nontoxic to the cell and less susceptible to degradation by nuclease activity,31-38 the DNMT1 activity in cell lysates was subsequently evaluated by the proposed biosensor to testify the feasibility of this electrochemical method used in real samples. As illustrated in Figure 6, the DPV peak current changes (∆I) decreased monotonically with the logarithm of the number of MCF-7 cells. This result is in accordance with the fact that with increasing cell number, more active DNMT1 can act on the DNA substrates and more DNAs are methylated. Thus, after incubation with BssHII, cleavage is blocked, and a strong signal remains. The inset in Figure 6 shows that the signal intensity from only 20 cells can be facilely distinguished from the background. The detection limit of this approach is estimated to be equivalent to 8 MCF-7 cells (S / N = 3).



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Figure 6. The linear relationship between the DPV peak current changes and MCF-7 cell numbers (10-10 000 cells). Inset shows the peak current from the blank, 20 and 100 MCF-7 cells. The error bars represent the standard deviation of five repetitive measurements. Evaluation DNMT1 Activity and Total DNMT1 Protein Content of Different Cell Lines. To evaluate the potential of the proposed method in clinical diagnostics, we evaluated four different cancer cell lines and one normal cell line using the proposed assay. As displayed in Figure 7A, all of these cell extracts were positive for DNMT1 activity, except for the normal cells (HEK-293), consistent with the conclusion that DNMT1 is over-expressed in most known human tumors. Figure 7B shows the fold excess of DNMT1 activity in the cancer cell compared with the normal cell. The fold excess was calculated as follows: each cancer cell and normal cell were normalized for the DNMT1 concentration (Figure 5B) according to the signal change (Figure 7A). Then the cancer cell value was normalized to the normal cell, producing a ratio. If the fold excess is >1, the DNMT1 activity in the cancer cell is higher than in the normal cell. We also measured the total amount of DNMT1 with a DNMT1 assay kit, according to the manufacturer’s protocol. The resulting fold excess was calculated based on the ratio of total DNMT1 protein content in cancer cells to the total DNMT1 protein content in normal cells (Figure 7C). As show in Figure 7B and C, the electrochemical method is in good agreement with the DNMT1 kit assay. Therefore, it is feasible to apply the present method to detect the DNMT1 activity in clinical samples.


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Figure 7. (A) DPV signal changes in response to DNMT1 extracts from different kinds of cancer cell lines. (B) The fold excess activity measured by the electrochemical method shows hyperactivity of cancer cells (fold excess >1) compared to normal cells (fold excess~1). (C) The fold excess DNMT1 expression was determined with DNMT1 assay kit, which also shows higher total DNMT1 protein content in cancer cells (fold excess >1) compared to normal cells (fold excess~1). Error bars represent the standard error for three trials. All the cell numbers were 2000.

CONCLUSION In summary, based on the site-specific cleavage by methylation-sensitive restriction endonuclease, a dual signal amplified electrochemical method was developed to detect DNMT1 activity from cancer cell lysates. The dual signal amplification was realized through Au NP-DNA complexes and Ru(III) redox recycling-based electrocatalytic signal enhancement. This assay can determine as low as 8 MCF-7 cells with a linear range of 10-10 000 cells. Using this assay to analyze 4 kinds of tumor cells and 1 kind of normal cell, the overexpressions of DNMT1 were obtained from the tumor cells and the results were comparable to those of the commercial DNMT1 kit assay. This proposed strategy shows great potential for DNMT1 activity assay in DNA methylation-related clinical diagnostics and anticancer drug screening.

ACKNOWLEDGMENTS This work is supported by NSFC (21273117, 21375063 and 21335004), Program for Outstanding Innovation Research Team of Universities in Jiangsu Province, Priority Academic Program Development of Jiangsu Higher Education Institutions, and Jiangsu Government Scholarship for Overseas Studies.



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ASSOCIATED CONTENT Supporting Information Probes and sequences of the oligonucleotides used in this study (Table 1) and EIS characteristics of the biosensor (Figure S1) are available free of charge via the Internet at http://pubs.acs.org.

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Sensitive Electrochemical Detection of Human Methyltransferase

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