Biosensors and Bioelectronics 63 (2015) 458–464
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One-pot synthesis of GO/AgNPs/luminol composites with electrochemiluminescence activity for sensitive detection of DNA methyltransferase activity Hui-Fang Zhao, Ru-Ping Liang, Jing-Wu Wang, Jian-Ding Qiu n Department of Chemistry, Nanchang University, Nanchang 330031, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 23 May 2014 Received in revised form 27 July 2014 Accepted 30 July 2014 Available online 12 August 2014
DNA methyltransferases catalyze the transfer of a methyl group from S-adenosylmethionine to the target adenine or cytosine, eventually inducing the DNA methylation in both prokaryotes and eukaryotes. Herein, we developed a novel electrochemiluminescence biosensor to quantify DNA adenine methylation (Dam) methyltransferase (MTase) employing signal amplification of GO/AgNPs/luminol composites to enhance the assay sensitivity. The method was developed by designing a capture probe DNA, which was immobilized on gold electrode surface, to hybridize with azide complementary DNA to form the azideterminated dsDNA. Then, alkynyl functionalized GO/AgNPs/luminol composites as the signal probe were immobilized to azide-terminated dsDNA modified electrode via click chemistry, resulting in a high electrochemiluminescence (ECL) signal. Once the DNA hybrid was methylated (under catalysis of Dam MTase) and further cleaved by Dpn I endonuclease (a site-specific endonuclease recognizing the duplex symmetrical sequence of 5′-G-Am-T-C-3′), GO/AgNPs/luminol composites release from the electrode surface to the solution, leading to significant reduction of the ECL signal. The change of the ECL intensity is related to the methylation status and MTase activity, which forms the basis of MTase activity assay and site-specific methylation determination. This novel strategy can be further used as a universal method for other transferase determination by designing various transferase-specific DNA sequences. In addition, this method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis. & 2014 Elsevier B.V. All rights reserved.
Keywords: ECL Dam MTase GO/AgNPs/luminol Click chemistry Biosensor
1. Introduction DNA methylation, which refers to methyltransferases (MTase)catalyzed covalent addition of a methyl group to adenine or cytosine residues in the specific DNA sequence (Lee et al., 2010), is a critical process existing in both prokaryotes and eukaryotes. DNA methylation occurs at the C-5/N-4 positions of cytosine and at the N-6 position of adenine and is catalyzed by DNA MTases (Okano et al., 1999; Palmer and Marinus, 1994). A number of human diseases have been found to be associated with aberrant gene methylation (Baylin et al., 2001; Costello et al., 2000). During this aberrant methylation process, the DNA MTase acts as a crucial participator to transfer a methyl group from S-adenosylmethionine (SAM) to the N-6 position of adenine in 5′-G-A-T-C-3′ (Schmitt et al., 1997). Recent studies have testified that aberrant DNA methylation influences the interaction between DNA and n
Corresponding author. Tel./fax: þ 86 791 83969518. E-mail address: [email protected] (J.-D. Qiu).
http://dx.doi.org/10.1016/j.bios.2014.07.079 0956-5663/& 2014 Elsevier B.V. All rights reserved.
protein, and alters gene expression, which may result in tumor occurrence and tumor growth (Shames et al., 2007). Notably, the DNA MTases have been treated as a potential target for anticancer drugs (Heithoff et al., 1999; Low et al., 2001). Therefore, the development of a sensitive method for the MTase assay is of significance for both fundamental biochemical research and drug discovery. In these years, compared to high-performance liquid chromatography (Reenilä et al., 1995), radioactive labeling (Som and Friedman, 1991), fluorescence (Feng et al., 2007; Li et al., 2007a), and colorimetric analysis systems (Li et al., 2010; Zheng et al., 2013), DNA methyltransferases activity assays based on electrochemical methods have been developed quickly because of the advantages of cheap instruments, simple operation, easy separation, and high sensitivity and selectivity. Electrochemical biosensors have been designed lately for the detection of DNA methyltransferases activity by measuring the current and charge responses of redox probes conjugated during the DNA methylation processes (He et al., 2011a; Li et al., 2012a; Liu et al., 2011). Liu
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et al. reported the quantitative measurements of methyltransferase activity based on oxidation current of ferrocene (Liu et al., 2011). To amplify the electrochemical response and improve the sensitivity, thionine/GO composites labeled dsDNA for the methyltransferases assay was also designed by measuring the redox currents of thionine, yet a low sensitivity was achieved (Li et al., 2012a). A sensitive and simple signal-on electrochemical assay for detection of Dam methyltransferase (Dam MTase) activity based on DNA-functionalized gold nanoparticles (AuNPs) amplification coupled with enzyme-linkage reactions was presented (He et al., 2011a). Despite the improvement of these methods, sophisticated procedures of electroactive labeling are needed, and sometimes, the performances such as linear range and sensitivity were also limited. Therefore, it is still a challenge in developing sensitive, rapid, accurate, and simple methods for the profiling of Dam MTase activity and inhibition. Electrogenerated chemiluminescence (ECL) is a light emission process in a redox reaction of electrogenerated reactants, which combines the electrochemical and luminescent techniques (Richter, 2004). Compared to the conventional electrochemical methods and luminescence techniques, the ECL technique not only shows high sensitivity and wide dynamic concentration response range but also is potential and spatial controlled. ECL biosensor is a powerful device for ultrasensitive biomolecule detection and quantification by combining the selectivity of the biological recognition elements and the sensitivity of ECL technique and is widely used in immunoassay (Jie et al., 2008; Liu and Ju, 2008), DNA analysis (Duan et al., 2010; Hu et al., 2009), environmental detection and clinic diagnostics (Miao and Bard, 2004; Wang et al., 2009). In spite of its excellent properties, few studies have been conducted concerning the detection of DNA methyltransferase activity and inhibition by ECL methods (Li et al., 2012b, 2013). Functional nanomaterials have attracted considerable interest motivated by their promising applications ranging from chemistry to life sciences, materials, nanosciences, engineering sciences, and environmental sciences (Daniel and Astruc, 2003; Klajn et al., 2010). Gold nanomaterials possess excellent stability, good biocompatibility, ease of self-assembly and unique optical, catalytic, redox-reactive, electrochemical, and surface properties (Daniel and Astruc, 2003). To date, a series of functionalized gold nanomaterials with novel magnetic, surface-enhance Raman scattering and catalytic properties have been successfully synthesized (Debouttière et al., 2006; Kisailus et al., 2005; Qian et al., 2009). However, few studies have been reported regarding nanomaterials with novel chemiluminescent (CL) property. Recently, Chai et al. synthetized luminol functionalized AuNPs as ECL tags for ultrasensitive DNA assay (Chai et al., 2010). He et al. employed luminol– AgNPs labeling to detect M. tuberculosis DNA, and the as-prepared luminol–AgNPs exhibited much better CL/ECL activity than previously reported luminol–AuNPs (He et al., 2011b). In addition, graphene oxide (GO), a two-dimensional derivative of graphene, which consists of an atomically thin sheet of graphite, has recently attracted much attention due to its unique optical, thermal, mechanical, and electrochemical properties (Chen et al., 2012b; Dreyer et al., 2010). To further expand their application fields, the preparation of nanocomposites based GO with unique properties is very significant. For example, Cui and coworkers synthesized lucigenin functionalized Pt nanoparticles/reduced graphene oxide nanocomposites with chemiluminescence activity (He and Cui, 2012a). Then, He et al. synthesized luminol functionalized sliver nanoparticles/graphene oxide composites for “signal on” chemiluminescent detection of glutathione activity, with good sensitivity and low detection limit (He and Cui, 2012b). In spite of their excellent properties, few studies have been conducted concerning the detection of MTase activity and inhibition base on luminol
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functionalized sliver nanoparticles/graphene oxide composites as ECL labeling. In the present work, the proof-of-concept of a novel ECL biosensor for the Dam MTase activity and inhibition analysis using highly chemiluminescent GO/AgNPs/luminol composites to assemble large amount of luminol for amplifying ECL signal was demonstrated. Firstly, azide-terminated complementary DNA was hybridized on the capture probe DNA modified gold electrode surface to form azide-terminated duplex DNA modified electrode. The alkynyl functionalized GO/AgNPs/luminol composites were then assembled on the modified electrode surface via click chemistry for ECL signal generation and amplification. Once the DNA adenine methylation methyltransferase and methylationresponsive restriction endonuclease Dpn I are presented, the ECL signal would be significantly reduced by releasing GO/AgNPs/ luminol from the electrode surface into solution due to the digestion of dsDNA at the site of 5′-G-Am-T-C-3′. The ECL signal change is related to the methylation status and MTase activity, which forms the basis of MTase activity assay and site-specific methylation determination.
2. Materials and methods 2.1. Materials, apparatus and synthesis of graphene oxide sheets Materials, apparatus and synthesis of graphene oxide sheets were presented in Supplementary material. 2.2. Synthesis of alkynyl functionalized GO/AgNPs/luminol composites In a typical synthesis (He and Cui, 2012b), ethanol/H2O solution containing 9 mL absolute ethanol and 5 mL ultrapure water, 2 mL of 5 mmol/L AgNO3 and 0.33 mL of 1 mg/mL GO aqueous solution were placed in a 50 mL beaker flask fitted with a magnetic stirrer, and then 0.5 mL of 0.01 mol/L luminol was quickly added to the mixed solution under stirring and was reacted for 4 h at room temperature. During this time, the color of the reaction solution was gradually changed from light yellow to deep yellow, indicated that the GO/AgNPs/luminol composites were successfully synthesized. In this strategy, luminol was not only used as reducing agent for the effective reduction of AgNO3 to AgNPs, the excess luminol was also be used as stabilizers directly coated onto the AgNPs surface through the Ag–N covalent interaction (He et al., 2011b) and in addition used as ECL indicator immobilized on the surface of GO by π-π stacking and hydrogen bonding (He and Cui, 2012b). The obtained GO/AgNPs/luminol composites were centrifuged (12 500 rpm) twice and redispersed with 0.1 mol/L NaOH solution. 1 mM propargylamine was added to 0.2 mL GO/AgNPs/luminol composites and incubated at room temperature for 3 h to form the alkynyl functionalized GO/AgNPs/luminol composites. Then, the obtained composites were centrifuged at 12500 rpm for 10 min and the precipitates were redispersed with 200 mL click chemistry solution containing CuSO4 5H2O (1.0 μM), TBTA (1.1 μM) and sodium L-ascorbate (4.0 μM) in H2O/MeOH/THF (2:2:1 v/v/v) (Krishna and Caruthers, 2012). 2.3. Modification of gold electrode with ECL reagent via click chemistry A gold electrode (diameter of 2 mm) was polished carefully with 1.0, 0.3, and 0.05 mm α-Al2O3 powder on fine abrasive paper and then thoroughly rinsed with ethanol and water. Prior to immobilizing DNA, the gold electrode was scanned in 0.5 M H2SO4 between 0.2 and 1.55 V (vs Ag/AgCl) at 100 mV/s until a
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reproducible cyclic voltammogram peak was obtained, followed by sonication again and drying with N2. 10 μL of probe immobilization buffer containing 2 μM thiol-terminated capture DNA was dripped onto the cleaned electrode surface and incubated overnight at room temperature in humidity. The capture DNA modified electrode was rinsed with ultrapure water, followed by immersion in 1 mM MCH solution for 1 h to block the nonspecific binding sites on the electrode. After washing with PBS, 10 μL of DNA hybridization buffer containing 2 μM of azide-terminated complementary DNA was dripped onto the electrode surface at 37 °C and incubated for 2 h to form azide-terminated duplex DNA modified electrode (Chen et al., 2012a; He et al., 2011a). After rinsing with WB and drying with N2, the prepared azide-terminated duplex DNA modified electrodes were finally immersed into a click chemistry solution containing alkynyl functionalized GO/AgNPs/ luminol composites for 7 h at room temperature to obtain the GO/ AgNPs/luminol modified electrode. 2.4. ECL characterization of dam MTase-catalyzed methylation reaction and dam MTase activity detection Dam MTase-catalyzed methylation was performed by incubating the GO/AgNPs/luminol modified electrode into an assay buffer solution (50 mM Tris–HCl and 50 mM KCl, pH 7.5) containing a desired amount of Dam MTase and SAM at 37 °C for 2 h. Subsequently, the modified electrode was immersed in extension solution (20 mM Tris–HCl, 10 mM Mg(Ac)2, 50 mM KAc, pH 7.9, and 50 U/mL Dpn I) and incubated at 37 °C for 2 h. After washing with PBS thoroughly, the resulting electrode was characterized by ECL technique in a 1.0 mM H2O2 working solution. In the inhibition experiment, a desired concentration of inhibitors was also contained in the assay buffer solutions, and the procedures were similar as above.
3. Results and discussion 3.1. ECL strategy for dam MTase activity detection The developed ECL strategy in profiling the methyltransferase activity is described in Scheme 1. The thiolated-terminated capture DNA was firstly immobilized onto the gold electrode surface through the Au-S bond and then hybridized with azide-terminated complementary DNA to form a rigid azide-terminated dsDNA. Subsequently, alkynyl functionalized GO/AgNPs/luminol as the ECL signal probe was immobilized to the azide-terminated dsDNA modified electrode via click chemistry, generating a strong ECL signal. In the presence of Dam MTase and SAM, the DNA hybrid was methylated. Dpn I restriction endonuclease, which is a widely used restriction enzyme in the gene-specific methylation assay, recognizes and cleaves on the 3′ side of the modified adenine within the methylated sequence to yield DNA fragments possessing fully base-paired termini. After being digested by Dpn I, the DNA hybrid was cleaved at a specific site of 5′-G-Am-T-C-3′ and GO/AgNPs/luminol composites were released from the electrode surface into solution, resulting in significant reduction of the ECL signal. At higher activity of Dam MTase, more DNA hybrid was methylated and cleaved, and more GO/AgNPs/luminol composites were released into solution, and thus, a weaker ECL response were obtained, offering a quantitative readout of Dam MTase activity. 3.2. Characterization of GO/AgNPs/luminol composites The morphology, structure, size and dispersion of GO/AgNPs/ luminol composites were examined by TEM, XRD and UV–vis absorption. The TEM image of the GO displayed a uniform
Scheme 1. Schematic illustration of the preparation of GO/AgNPs/luminol composites and the procedures of the MTase activity assay.
structure, which appeared transparent and was folded over on one edge with isolated small fragments of graphene on its surface (Fig. 1A). After the reduction of AgNO3 by luminol onto the surface of GO, dark particles with a uniform size of 18 nm are fairly well monodispersed on the surface of GO (Fig. 1B). Compared with GO (Fig. 1C, trace a), the XRD pattern of the GO/AgNPs/luminol composites (Fig. 1C, trace b) showed new reflection peaks at 38.2°, 44.3°, 64.5°, and 77.6°, corresponding to (111), (200), (220), and (311) crystallographic orientations of fcc Ag, respectively (He and Cui, 2012b), demonstrating the successful deposition of AgNPs on GO surface. Furthermore, UV–vis spectra were also performed to illustrate the formation of GO/AgNPs/luminol composites (Fig. 1D). GO (curve a) displayed a maximum absorption at 229 nm due to the π–πn transition of aromatic C ¼C bonds and a shoulder around 300 nm due to the n–πn transition of C ¼O bonds (Dong et al., 2010; Li et al., 2008). The characteristic absorption peaks of luminol appeared around at 219 nm, 300 nm, and 350 nm (curve b), respectively (He et al., 2011b). When the reduction of AgNO3 to AgNPs/luminol with luminol in the presence of ethanol, an obvious absorption peak occurred at about 427 nm (curve c) corresponding to the characteristic absorption peak of AgNPs, while the absorption peaks of luminol displayed a modest shift, indicated the successful formation of AgNPs/luminol. After decoration with AgNPs on the surface of GO nanosheets (curve d), in addition to the characteristic absorption peaks of GO, a new peak at about 425 nm was observed, which was the characteristic absorption peak of AgNPs due to their surface plasmon resonance (Li et al., 2007b). Meanwhile, the three peaks of luminol with a modest shift are also observed for the GO/ AgNPs/luminol dispersion, which strongly supports that luminol molecules are attached to the surface of the as-prepared GO/ AgNPs/luminol composites. In addition, the absorption peak of GO blue-shifted to 217 nm due to the exfoliation of layered GO sheets within the duration of the reaction (Chen et al., 2010), indicating
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Fig. 1. TEM images of (A) GO and (B) GO/AgNPs/luminol. (C) XRD patterns of (a) GO and (b) GO/AgNPs/luminol. (D) UV–vis absorption spectra of (a) GO, (b) luminol, (c) AgNPs/luminol and (d) GO/AgNPs/luminol in aqueous solution.
that GO was not reduced during the reaction process. These results supported the fact that AgNPs and luminol molecules were decorated on the surface of the GO surface through the one-pot synthetic method. 3.3. Characterization of the biosensor The stepwise reactions on the surface of the gold electrode were confirmed by electrochemical impedance spectroscopy (EIS). The impedance spectrum includes a semicircle portion and a linear portion. The semicircle portion observed at high frequencies corresponds to the charge-transfer limited process (Rct), a line portion observed at low frequencies by diffusion process (Park and Yoo, 2003). As shown in Fig. 2, the bare gold electrode showed a very small semicircle domain with the Rct of 367 Ω, the characteristics of a mass diffusional limiting electron-transfer process (curve a), indicating a very fast charge-transfer process of [Fe (CN)6]3 /4 . After self-assembly of thiol-terminated capture DNA (S1), the value of Rct increased to 1716 Ω (curve b), which indicated the successful assembling of S1, thus partly repelling the electron transfer of [Fe(CN)6]3 /4 to the gold electrode. Subsequent surface blocking with MCH also leaded to an increase
in Rct to 2269 Ω (curve c). After incubation of azide-terminated complementary ssDNA (S2), the Rct of 3871 Ω augmented gradually on account of the steric hindrance (curve d), indicating the successful formation of azide-terminated dsDNA. The further immobilization of alkynyl functionalized GO/AgNPs/luminol composites via click chemistry resulted in an further increased Rct to 5180 Ω (curve e), mainly due to the carboxyl and hydroxyl groups on the edges of the GO, in addition, luminol and its oxidation product 3-aminophthalate (AP2 ) were coexisted on the surface of GO/AgNPs/luminol. The stepwise increase of electron transfer resistance verified the successful fabrication of the ECL biosensor. However, after the DNA hybrid was methylated by Dam MTase and further cleaved by Dpn I, an obvious decrease of Rct to 2642 Ω emerged (curve f) corresponding to the cleavage of dsDNA probe, which could lead to the partial reducing of the GO/AgNPs/luminol, weakening the steric hindrance and accelerating the electron transfer between the redox probe and electrode surface. To give more detailed information about the impedance of the modified electrode, a modified Randles-equivalent circuit (inset in Fig. 2) was chosen to fit the measured results. All these experimental results demonstrated that the sensing interface has been successfully fabricated according to Scheme 1.
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Scheme 1. When GO/AgNPs/luminol/dsDNA electrode was methylated by Dam MTase and further cleaved by Dpn I at a specific site of dsDNA, the ECL response of luminol decreased significantly (curve g), revealing that some GO/AgNPs/luminol composites had been removed from the electrode surface due to the cleavage of the endonuclease. The possible ECL mechanisms are as follows (Miao, 2008; Richter, 2004). −H +
−e
−H +
H2 O2 ⇌+ HO−2 → HO2∙ ⇌+ O2∙−
(1)
LH− − e− → LH∙ → L∙− + H+
(2)
L∙− + O2∙− → LO22 −
(3)
LO22 − → AP2 −⁎ + N2
(4)
+H
+H
AP2 −⁎ → AP2 − + hv(425 nm)
Fig. 2. Nyquist plots of the different modified electrodes in 1.0 mol/L KCl solution containing 5 mM [Fe(CN)6]3 /4 . (a) Bare electrode, and (b) S1, (c) MCH/S1, (d) S2/ MCH/S1, and (e) GO/AgNPs/luminol/S2/MCH/S1 modified electrodes, and (f) electrode (e) reacted with 10 U/mL Dam MTase and 50 U/mL Dpn I.
3.4. ECL behavior of the biosensor To demonstrate the feasibility of the developed biosensor, ECL responses of different modified electrodes were investigated. As shown in Fig. 3, no ECL signals were observed on bare electrode (curve a), S1 modified electrode (curve b) and azide-terminated dsDNA modified electrode (curve c). After assembling alkynyl functionalized GO/AgNPs/luminol composites onto the azide-terminated dsDNA modified electrode via click chemistry, a strong ECL signal was observed (curve d), resulting from the ECL reaction of luminol. In comparison with the GO/luminol (curve e) and AgNPs/luminol (curve f), 49.4-fold and 3.3-fold enhancement in ECL signals was observed using GO/AgNPs/luminol composites as probe, indicating that the GO/AgNPs/luminol composites greatly amplified the ECL signal. These suggested the successful construction of the GO/AgNPs/luminol/dsDNA electrode according to
Fig. 3. ECL signals under step potential obtained on (a) bare electrode, and (b) S1, (c) S2/S1, (d) GO/AgNPs/luminol/S2/S1, (e) GO/luminol/S2/S1, (f) AgNPs/luminol/S2/ S1 modified electrodes, and (g) electrode (d) reacted with 20 U/mL Dam MTase and Dpn I 50 U/mL. Initial potential, 0.7 V; final potential, 0.1 V; pulse period, 30 s; pulse time, 1.0 s; working buffer, 1.0 mM H2O2 containing 0.02 mol/L CBS (pH 10.53).
(5) 2n
where LH is the deprotonated luminol and AP is the excited state product, it is noted that GO/AgNPs play important role to amplify the ECL signal of luminol. GO will increase the interface area of the modified electrode to capture more AgNPs and luminol through Ag–π interaction and π–π stacking; and AgNPs will facilitate the electron transfer at the electrode interface and catalyze the ECL process of luminol in both electrode and chemical reactions (He et al., 2011b; Wang et al., 2009). Thus, the GO/AgNPs/ luminol composites can mediate the ECL reaction even at a low content, implying highly efficient ECL signal amplification. At higher Dam MTase activity, there are more GO/AgNPs/luminol composites removed from the electrode surface due to the cleavage of Dpn I, resulting in a significant decrease of the ECL signal. Therefore, the as-designed ECL biosensor can be used for Dam MTase activity characterization. 3.5. ECL measurement of dam MTase activity In order to achieve the maximum sensitivity for the detection of Dam MTase, experimental parameters including the ECL detection mode (Figs. S1 and S2), pH of detection solution (Fig. S3A), methylation time (Fig. S3B), initial potential (Fig. S3C), pulse period (Fig. S3D) and the concentration of H2O2 (Fig. S3E) were optimized. Under the optimal conditions, quantitative detection of Dam MTase was performed. Fig. 4A shows the variance of ECL intensity with the concentration of Dam MTase. As the concentration of Dam MTase increased, more S1/S2 hybrids were methylated. After being treated with Dpn I, the methylated S1/S2 hybrids were cleaved and GO/AgNPs/luminol composites were released from the electrode surface, which resulted in the decrease of ECL signal. The ECL signal was inverse proportional to the concentration of Dam MTase, which could be used for Dam MTase detection. Fig. 4B shows the curve of ECL intensity with different Dam MTase concentrations. The ECL peak intensity decreased linearly with the logarithmic value of Dam MTase concentrations in the range of 0.1 to 20 U/mL, and the detection limit was calculated to be 0.03 U/mL at 3s. The detection range of the method was much wider than those of previously reported 1.0–10 U/mL based on gold nanoparticles coupled with enzyme-linkage reaction for the detection of DNA Methylation (Li et al., 2010) and 0.1–1.0 U/mL based on the controllable assembly of single wall carbon nanotubes using label-free electrochemical assay for methyltransferase activity detection (Wang et al., 2013). The detection limit was much lower than those of 0.14 U/mL on a label-free colorimetric assay based on unmodified Au nanorods as a signal sensing probe coupled with enzyme-linkage reaction (Zheng et al., 2013) and 0.12 U/mL using AuNPs amplification based on a sensitive signal-
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Fig. 4. (A) ECL signal obtained from GO/AgNPs/luminol/dsDNA modified electrode by different concentrations of Dam MTase. The curves from bottom to the top were obtained with different concentration: 0.1, 0.2, 0.5, 1.0, 5.0, 10.0, 20.0, 30.0 and 50.0 U/mL, respectively. (B) The response curve obtained with different concentrations of Dam MTase. Inset is the linear relationship between the ECL intensity and the logarithm of the Dam MTase concentration.
on electrochemical assay for DNA MTase activity (He et al., 2011a). Results indicated that employing the GO/AgNPs/luminol composites as the ECL signal probe is an effective strategy. Stable and high ECL signals (Fig. S4) were observed when the biosensor was successively scanned, which signified that the ECL biosensor possessed excellent potential cycling stability. The longterm stability of the biosensor was evaluated by measuring the ECL signal after storing at 4 °C for different time period. No obvious change of the ECL response was found within 4 weeks, supplying a high long-term stability and prolong lifetime of the ECL biosensor for Dam MTase activity detection. To evaluate the practicability of the biosensor, we applied this sensor in human serum samples for Dam MTase detection. As shown in Fig. S5, comparable responses were found for Dam MTase in both NEBuffer 4 and diluted human serum. Control experiment indicated that no Dam MTase was found in human serum. The ECL intensities from the human serum were well consistent with those
from NEBuffer 4, suggesting diluted human serum had no influence on detecting the activity of Dam MTase. The recoveries were in the range of 94.6–106.3% for measurement of 1, 5 and 10 U mL 1 Dam MTase spiked in serum with a maximum RSD of 5.8%, which were comparable to those reported in literatures (Zhao et al., 2014; Chen and Zhao, 2013). These results indicated that the proposed strategy holds a great promise in practical applications for DNA MTase detection with great accuracy and reliability. 3.6. Assay of the inhibition of dam MTase activity Since DNA methylation is closely related to our health and disease and inhibitors of DNA methylase have potential application in cancer therapy and antimicrobial infection, screening of inhibitors of DNA MTase receives more and more interest, and we have, thus, checked whether our method can be employed for screen of
Fig. 5. (A) Influence of different drugs on the activity of Dam MTase. (1) No drug, (2) mitomycin, (3) platinol, (4) benzylpenicillin, (5) 5-fluorouracil. (B) Inhibition of the activity of Dam MTase by 5-fluorouracil. Relationship between ECL intensity and the concentrations of 5-fluorouracil. Inset: ECL intensity-time behaviors at different concentrations of 5-fluorouracil. The concentration of 5-fluorouracil were 0, 1, 10, 20, 50 and 100 μM, respectively.
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Dam MTase inhibitors. The mechanism is that, if transferring of the methyl group to the DNA residue is blocked in the presence of inhibitor, the specific site of the DNA will not be recognized and cleaved; thus, the ECL intensity increased with the increase of inhibition of Dam MTase. To date, the reported small molecule Dam MTase inhibitors mainly contain two categories, antibiotic drugs (benzylpenicillin) and anticancer drugs (5-fluorouracil, platinol and mitomycin). As can be seen from Fig. 5A, benzylpenicillin and 5-fluorouracil could strongly inhibit the methylase. The inhibition efficiency of 5-fluorouracil was the most serious one, which may decrease the activity of Dam MTase by about 50%. However, mitomycin and platinol had almost no effect on the methylation, in good agreement with the order obtained in previous reports (Zheng et al., 2013). We also investigated how the concentration of 5-fluorouracil influenced the activity of methylase. The concentration of 5-fluorouracil was kept under 100 mM. As shown in Fig. 5B, the ECL intensity increased with increasing the concentration of 5-fluorouracil, clearly indicating significant dose-dependent inhibition. The IC50 value was the inhibitor concentration required to reduce enzyme activity by 50%. The relationship between ECL intensity and 5-fluorouracil concentrations is plotted in inset of Fig. 5B, the IC50 of 5-fluorouracil was calculated to be 18.2 μM, which was much lower than those of 100 μM based on the electrochemical assay (Wang et al., 2013; Jing et al., 2014). This indicated that the proposed electrochemiluminescence method was highly sensitive for screening inhibitor of Dam MTase. Therefore, the above results demonstrated that our method can be successfully used in the assay of Dam MTase inhibitors.
4. Conclusions In conclusion, we have successfully developed a novel, homogeneous, signal-off ECL DNA sensor for the sensitive and specific detection of Dam MTase using GO/AgNPs/luminol composites as the ECL signal probe. As a result, the as-proposed ECL biosensor offers a highly sensitive strategy for Dam MTase activity monitoring with a low detection limit of 0.03 U/mL, wide linear range, and good stability. More importantly, this assay is able to be performed in human serum with satisfying recovery, holding great potential for further application in complex biology samples. Moreover, the proof-of-concept method also shows excellent performance on screen Dam MTase inhibitor. The general and robust method can also be ready for other Dam MTase activities and inhibition assays. Given the important roles of Dam MTase in some disease related biological processes, this biosensor shows great potential for a high throughput assay in clinic diagnostics and drug discovery applications.
Acknowledgments We gratefully acknowledge the supports from the National Natural Science Foundation of China (Nos. 21163014 and 21265017) and the Program for New Century Excellent Talents in University (NCET-11-1002 and NCET-13-0848).
Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.07.079.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
One-pot synthesis of GO/AgNPs/luminol composites with electrochemiluminescence activity for sensitive detection of DNA methyltransferase activity Hui-Fang Zhao, Ru-Ping Liang, Jing-Wu Wang, Jian-Ding Qiu n Department of Chemistry, Nanchang University, Nanchang 330031, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 23 May 2014 Received in revised form 27 July 2014 Accepted 30 July 2014 Available online 12 August 2014
DNA methyltransferases catalyze the transfer of a methyl group from S-adenosylmethionine to the target adenine or cytosine, eventually inducing the DNA methylation in both prokaryotes and eukaryotes. Herein, we developed a novel electrochemiluminescence biosensor to quantify DNA adenine methylation (Dam) methyltransferase (MTase) employing signal amplification of GO/AgNPs/luminol composites to enhance the assay sensitivity. The method was developed by designing a capture probe DNA, which was immobilized on gold electrode surface, to hybridize with azide complementary DNA to form the azideterminated dsDNA. Then, alkynyl functionalized GO/AgNPs/luminol composites as the signal probe were immobilized to azide-terminated dsDNA modified electrode via click chemistry, resulting in a high electrochemiluminescence (ECL) signal. Once the DNA hybrid was methylated (under catalysis of Dam MTase) and further cleaved by Dpn I endonuclease (a site-specific endonuclease recognizing the duplex symmetrical sequence of 5′-G-Am-T-C-3′), GO/AgNPs/luminol composites release from the electrode surface to the solution, leading to significant reduction of the ECL signal. The change of the ECL intensity is related to the methylation status and MTase activity, which forms the basis of MTase activity assay and site-specific methylation determination. This novel strategy can be further used as a universal method for other transferase determination by designing various transferase-specific DNA sequences. In addition, this method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis. & 2014 Elsevier B.V. All rights reserved.
Keywords: ECL Dam MTase GO/AgNPs/luminol Click chemistry Biosensor
1. Introduction DNA methylation, which refers to methyltransferases (MTase)catalyzed covalent addition of a methyl group to adenine or cytosine residues in the specific DNA sequence (Lee et al., 2010), is a critical process existing in both prokaryotes and eukaryotes. DNA methylation occurs at the C-5/N-4 positions of cytosine and at the N-6 position of adenine and is catalyzed by DNA MTases (Okano et al., 1999; Palmer and Marinus, 1994). A number of human diseases have been found to be associated with aberrant gene methylation (Baylin et al., 2001; Costello et al., 2000). During this aberrant methylation process, the DNA MTase acts as a crucial participator to transfer a methyl group from S-adenosylmethionine (SAM) to the N-6 position of adenine in 5′-G-A-T-C-3′ (Schmitt et al., 1997). Recent studies have testified that aberrant DNA methylation influences the interaction between DNA and n
Corresponding author. Tel./fax: þ 86 791 83969518. E-mail address: [email protected] (J.-D. Qiu).
http://dx.doi.org/10.1016/j.bios.2014.07.079 0956-5663/& 2014 Elsevier B.V. All rights reserved.
protein, and alters gene expression, which may result in tumor occurrence and tumor growth (Shames et al., 2007). Notably, the DNA MTases have been treated as a potential target for anticancer drugs (Heithoff et al., 1999; Low et al., 2001). Therefore, the development of a sensitive method for the MTase assay is of significance for both fundamental biochemical research and drug discovery. In these years, compared to high-performance liquid chromatography (Reenilä et al., 1995), radioactive labeling (Som and Friedman, 1991), fluorescence (Feng et al., 2007; Li et al., 2007a), and colorimetric analysis systems (Li et al., 2010; Zheng et al., 2013), DNA methyltransferases activity assays based on electrochemical methods have been developed quickly because of the advantages of cheap instruments, simple operation, easy separation, and high sensitivity and selectivity. Electrochemical biosensors have been designed lately for the detection of DNA methyltransferases activity by measuring the current and charge responses of redox probes conjugated during the DNA methylation processes (He et al., 2011a; Li et al., 2012a; Liu et al., 2011). Liu
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et al. reported the quantitative measurements of methyltransferase activity based on oxidation current of ferrocene (Liu et al., 2011). To amplify the electrochemical response and improve the sensitivity, thionine/GO composites labeled dsDNA for the methyltransferases assay was also designed by measuring the redox currents of thionine, yet a low sensitivity was achieved (Li et al., 2012a). A sensitive and simple signal-on electrochemical assay for detection of Dam methyltransferase (Dam MTase) activity based on DNA-functionalized gold nanoparticles (AuNPs) amplification coupled with enzyme-linkage reactions was presented (He et al., 2011a). Despite the improvement of these methods, sophisticated procedures of electroactive labeling are needed, and sometimes, the performances such as linear range and sensitivity were also limited. Therefore, it is still a challenge in developing sensitive, rapid, accurate, and simple methods for the profiling of Dam MTase activity and inhibition. Electrogenerated chemiluminescence (ECL) is a light emission process in a redox reaction of electrogenerated reactants, which combines the electrochemical and luminescent techniques (Richter, 2004). Compared to the conventional electrochemical methods and luminescence techniques, the ECL technique not only shows high sensitivity and wide dynamic concentration response range but also is potential and spatial controlled. ECL biosensor is a powerful device for ultrasensitive biomolecule detection and quantification by combining the selectivity of the biological recognition elements and the sensitivity of ECL technique and is widely used in immunoassay (Jie et al., 2008; Liu and Ju, 2008), DNA analysis (Duan et al., 2010; Hu et al., 2009), environmental detection and clinic diagnostics (Miao and Bard, 2004; Wang et al., 2009). In spite of its excellent properties, few studies have been conducted concerning the detection of DNA methyltransferase activity and inhibition by ECL methods (Li et al., 2012b, 2013). Functional nanomaterials have attracted considerable interest motivated by their promising applications ranging from chemistry to life sciences, materials, nanosciences, engineering sciences, and environmental sciences (Daniel and Astruc, 2003; Klajn et al., 2010). Gold nanomaterials possess excellent stability, good biocompatibility, ease of self-assembly and unique optical, catalytic, redox-reactive, electrochemical, and surface properties (Daniel and Astruc, 2003). To date, a series of functionalized gold nanomaterials with novel magnetic, surface-enhance Raman scattering and catalytic properties have been successfully synthesized (Debouttière et al., 2006; Kisailus et al., 2005; Qian et al., 2009). However, few studies have been reported regarding nanomaterials with novel chemiluminescent (CL) property. Recently, Chai et al. synthetized luminol functionalized AuNPs as ECL tags for ultrasensitive DNA assay (Chai et al., 2010). He et al. employed luminol– AgNPs labeling to detect M. tuberculosis DNA, and the as-prepared luminol–AgNPs exhibited much better CL/ECL activity than previously reported luminol–AuNPs (He et al., 2011b). In addition, graphene oxide (GO), a two-dimensional derivative of graphene, which consists of an atomically thin sheet of graphite, has recently attracted much attention due to its unique optical, thermal, mechanical, and electrochemical properties (Chen et al., 2012b; Dreyer et al., 2010). To further expand their application fields, the preparation of nanocomposites based GO with unique properties is very significant. For example, Cui and coworkers synthesized lucigenin functionalized Pt nanoparticles/reduced graphene oxide nanocomposites with chemiluminescence activity (He and Cui, 2012a). Then, He et al. synthesized luminol functionalized sliver nanoparticles/graphene oxide composites for “signal on” chemiluminescent detection of glutathione activity, with good sensitivity and low detection limit (He and Cui, 2012b). In spite of their excellent properties, few studies have been conducted concerning the detection of MTase activity and inhibition base on luminol
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functionalized sliver nanoparticles/graphene oxide composites as ECL labeling. In the present work, the proof-of-concept of a novel ECL biosensor for the Dam MTase activity and inhibition analysis using highly chemiluminescent GO/AgNPs/luminol composites to assemble large amount of luminol for amplifying ECL signal was demonstrated. Firstly, azide-terminated complementary DNA was hybridized on the capture probe DNA modified gold electrode surface to form azide-terminated duplex DNA modified electrode. The alkynyl functionalized GO/AgNPs/luminol composites were then assembled on the modified electrode surface via click chemistry for ECL signal generation and amplification. Once the DNA adenine methylation methyltransferase and methylationresponsive restriction endonuclease Dpn I are presented, the ECL signal would be significantly reduced by releasing GO/AgNPs/ luminol from the electrode surface into solution due to the digestion of dsDNA at the site of 5′-G-Am-T-C-3′. The ECL signal change is related to the methylation status and MTase activity, which forms the basis of MTase activity assay and site-specific methylation determination.
2. Materials and methods 2.1. Materials, apparatus and synthesis of graphene oxide sheets Materials, apparatus and synthesis of graphene oxide sheets were presented in Supplementary material. 2.2. Synthesis of alkynyl functionalized GO/AgNPs/luminol composites In a typical synthesis (He and Cui, 2012b), ethanol/H2O solution containing 9 mL absolute ethanol and 5 mL ultrapure water, 2 mL of 5 mmol/L AgNO3 and 0.33 mL of 1 mg/mL GO aqueous solution were placed in a 50 mL beaker flask fitted with a magnetic stirrer, and then 0.5 mL of 0.01 mol/L luminol was quickly added to the mixed solution under stirring and was reacted for 4 h at room temperature. During this time, the color of the reaction solution was gradually changed from light yellow to deep yellow, indicated that the GO/AgNPs/luminol composites were successfully synthesized. In this strategy, luminol was not only used as reducing agent for the effective reduction of AgNO3 to AgNPs, the excess luminol was also be used as stabilizers directly coated onto the AgNPs surface through the Ag–N covalent interaction (He et al., 2011b) and in addition used as ECL indicator immobilized on the surface of GO by π-π stacking and hydrogen bonding (He and Cui, 2012b). The obtained GO/AgNPs/luminol composites were centrifuged (12 500 rpm) twice and redispersed with 0.1 mol/L NaOH solution. 1 mM propargylamine was added to 0.2 mL GO/AgNPs/luminol composites and incubated at room temperature for 3 h to form the alkynyl functionalized GO/AgNPs/luminol composites. Then, the obtained composites were centrifuged at 12500 rpm for 10 min and the precipitates were redispersed with 200 mL click chemistry solution containing CuSO4 5H2O (1.0 μM), TBTA (1.1 μM) and sodium L-ascorbate (4.0 μM) in H2O/MeOH/THF (2:2:1 v/v/v) (Krishna and Caruthers, 2012). 2.3. Modification of gold electrode with ECL reagent via click chemistry A gold electrode (diameter of 2 mm) was polished carefully with 1.0, 0.3, and 0.05 mm α-Al2O3 powder on fine abrasive paper and then thoroughly rinsed with ethanol and water. Prior to immobilizing DNA, the gold electrode was scanned in 0.5 M H2SO4 between 0.2 and 1.55 V (vs Ag/AgCl) at 100 mV/s until a
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reproducible cyclic voltammogram peak was obtained, followed by sonication again and drying with N2. 10 μL of probe immobilization buffer containing 2 μM thiol-terminated capture DNA was dripped onto the cleaned electrode surface and incubated overnight at room temperature in humidity. The capture DNA modified electrode was rinsed with ultrapure water, followed by immersion in 1 mM MCH solution for 1 h to block the nonspecific binding sites on the electrode. After washing with PBS, 10 μL of DNA hybridization buffer containing 2 μM of azide-terminated complementary DNA was dripped onto the electrode surface at 37 °C and incubated for 2 h to form azide-terminated duplex DNA modified electrode (Chen et al., 2012a; He et al., 2011a). After rinsing with WB and drying with N2, the prepared azide-terminated duplex DNA modified electrodes were finally immersed into a click chemistry solution containing alkynyl functionalized GO/AgNPs/ luminol composites for 7 h at room temperature to obtain the GO/ AgNPs/luminol modified electrode. 2.4. ECL characterization of dam MTase-catalyzed methylation reaction and dam MTase activity detection Dam MTase-catalyzed methylation was performed by incubating the GO/AgNPs/luminol modified electrode into an assay buffer solution (50 mM Tris–HCl and 50 mM KCl, pH 7.5) containing a desired amount of Dam MTase and SAM at 37 °C for 2 h. Subsequently, the modified electrode was immersed in extension solution (20 mM Tris–HCl, 10 mM Mg(Ac)2, 50 mM KAc, pH 7.9, and 50 U/mL Dpn I) and incubated at 37 °C for 2 h. After washing with PBS thoroughly, the resulting electrode was characterized by ECL technique in a 1.0 mM H2O2 working solution. In the inhibition experiment, a desired concentration of inhibitors was also contained in the assay buffer solutions, and the procedures were similar as above.
3. Results and discussion 3.1. ECL strategy for dam MTase activity detection The developed ECL strategy in profiling the methyltransferase activity is described in Scheme 1. The thiolated-terminated capture DNA was firstly immobilized onto the gold electrode surface through the Au-S bond and then hybridized with azide-terminated complementary DNA to form a rigid azide-terminated dsDNA. Subsequently, alkynyl functionalized GO/AgNPs/luminol as the ECL signal probe was immobilized to the azide-terminated dsDNA modified electrode via click chemistry, generating a strong ECL signal. In the presence of Dam MTase and SAM, the DNA hybrid was methylated. Dpn I restriction endonuclease, which is a widely used restriction enzyme in the gene-specific methylation assay, recognizes and cleaves on the 3′ side of the modified adenine within the methylated sequence to yield DNA fragments possessing fully base-paired termini. After being digested by Dpn I, the DNA hybrid was cleaved at a specific site of 5′-G-Am-T-C-3′ and GO/AgNPs/luminol composites were released from the electrode surface into solution, resulting in significant reduction of the ECL signal. At higher activity of Dam MTase, more DNA hybrid was methylated and cleaved, and more GO/AgNPs/luminol composites were released into solution, and thus, a weaker ECL response were obtained, offering a quantitative readout of Dam MTase activity. 3.2. Characterization of GO/AgNPs/luminol composites The morphology, structure, size and dispersion of GO/AgNPs/ luminol composites were examined by TEM, XRD and UV–vis absorption. The TEM image of the GO displayed a uniform
Scheme 1. Schematic illustration of the preparation of GO/AgNPs/luminol composites and the procedures of the MTase activity assay.
structure, which appeared transparent and was folded over on one edge with isolated small fragments of graphene on its surface (Fig. 1A). After the reduction of AgNO3 by luminol onto the surface of GO, dark particles with a uniform size of 18 nm are fairly well monodispersed on the surface of GO (Fig. 1B). Compared with GO (Fig. 1C, trace a), the XRD pattern of the GO/AgNPs/luminol composites (Fig. 1C, trace b) showed new reflection peaks at 38.2°, 44.3°, 64.5°, and 77.6°, corresponding to (111), (200), (220), and (311) crystallographic orientations of fcc Ag, respectively (He and Cui, 2012b), demonstrating the successful deposition of AgNPs on GO surface. Furthermore, UV–vis spectra were also performed to illustrate the formation of GO/AgNPs/luminol composites (Fig. 1D). GO (curve a) displayed a maximum absorption at 229 nm due to the π–πn transition of aromatic C ¼C bonds and a shoulder around 300 nm due to the n–πn transition of C ¼O bonds (Dong et al., 2010; Li et al., 2008). The characteristic absorption peaks of luminol appeared around at 219 nm, 300 nm, and 350 nm (curve b), respectively (He et al., 2011b). When the reduction of AgNO3 to AgNPs/luminol with luminol in the presence of ethanol, an obvious absorption peak occurred at about 427 nm (curve c) corresponding to the characteristic absorption peak of AgNPs, while the absorption peaks of luminol displayed a modest shift, indicated the successful formation of AgNPs/luminol. After decoration with AgNPs on the surface of GO nanosheets (curve d), in addition to the characteristic absorption peaks of GO, a new peak at about 425 nm was observed, which was the characteristic absorption peak of AgNPs due to their surface plasmon resonance (Li et al., 2007b). Meanwhile, the three peaks of luminol with a modest shift are also observed for the GO/ AgNPs/luminol dispersion, which strongly supports that luminol molecules are attached to the surface of the as-prepared GO/ AgNPs/luminol composites. In addition, the absorption peak of GO blue-shifted to 217 nm due to the exfoliation of layered GO sheets within the duration of the reaction (Chen et al., 2010), indicating
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Fig. 1. TEM images of (A) GO and (B) GO/AgNPs/luminol. (C) XRD patterns of (a) GO and (b) GO/AgNPs/luminol. (D) UV–vis absorption spectra of (a) GO, (b) luminol, (c) AgNPs/luminol and (d) GO/AgNPs/luminol in aqueous solution.
that GO was not reduced during the reaction process. These results supported the fact that AgNPs and luminol molecules were decorated on the surface of the GO surface through the one-pot synthetic method. 3.3. Characterization of the biosensor The stepwise reactions on the surface of the gold electrode were confirmed by electrochemical impedance spectroscopy (EIS). The impedance spectrum includes a semicircle portion and a linear portion. The semicircle portion observed at high frequencies corresponds to the charge-transfer limited process (Rct), a line portion observed at low frequencies by diffusion process (Park and Yoo, 2003). As shown in Fig. 2, the bare gold electrode showed a very small semicircle domain with the Rct of 367 Ω, the characteristics of a mass diffusional limiting electron-transfer process (curve a), indicating a very fast charge-transfer process of [Fe (CN)6]3 /4 . After self-assembly of thiol-terminated capture DNA (S1), the value of Rct increased to 1716 Ω (curve b), which indicated the successful assembling of S1, thus partly repelling the electron transfer of [Fe(CN)6]3 /4 to the gold electrode. Subsequent surface blocking with MCH also leaded to an increase
in Rct to 2269 Ω (curve c). After incubation of azide-terminated complementary ssDNA (S2), the Rct of 3871 Ω augmented gradually on account of the steric hindrance (curve d), indicating the successful formation of azide-terminated dsDNA. The further immobilization of alkynyl functionalized GO/AgNPs/luminol composites via click chemistry resulted in an further increased Rct to 5180 Ω (curve e), mainly due to the carboxyl and hydroxyl groups on the edges of the GO, in addition, luminol and its oxidation product 3-aminophthalate (AP2 ) were coexisted on the surface of GO/AgNPs/luminol. The stepwise increase of electron transfer resistance verified the successful fabrication of the ECL biosensor. However, after the DNA hybrid was methylated by Dam MTase and further cleaved by Dpn I, an obvious decrease of Rct to 2642 Ω emerged (curve f) corresponding to the cleavage of dsDNA probe, which could lead to the partial reducing of the GO/AgNPs/luminol, weakening the steric hindrance and accelerating the electron transfer between the redox probe and electrode surface. To give more detailed information about the impedance of the modified electrode, a modified Randles-equivalent circuit (inset in Fig. 2) was chosen to fit the measured results. All these experimental results demonstrated that the sensing interface has been successfully fabricated according to Scheme 1.
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Scheme 1. When GO/AgNPs/luminol/dsDNA electrode was methylated by Dam MTase and further cleaved by Dpn I at a specific site of dsDNA, the ECL response of luminol decreased significantly (curve g), revealing that some GO/AgNPs/luminol composites had been removed from the electrode surface due to the cleavage of the endonuclease. The possible ECL mechanisms are as follows (Miao, 2008; Richter, 2004). −H +
−e
−H +
H2 O2 ⇌+ HO−2 → HO2∙ ⇌+ O2∙−
(1)
LH− − e− → LH∙ → L∙− + H+
(2)
L∙− + O2∙− → LO22 −
(3)
LO22 − → AP2 −⁎ + N2
(4)
+H
+H
AP2 −⁎ → AP2 − + hv(425 nm)
Fig. 2. Nyquist plots of the different modified electrodes in 1.0 mol/L KCl solution containing 5 mM [Fe(CN)6]3 /4 . (a) Bare electrode, and (b) S1, (c) MCH/S1, (d) S2/ MCH/S1, and (e) GO/AgNPs/luminol/S2/MCH/S1 modified electrodes, and (f) electrode (e) reacted with 10 U/mL Dam MTase and 50 U/mL Dpn I.
3.4. ECL behavior of the biosensor To demonstrate the feasibility of the developed biosensor, ECL responses of different modified electrodes were investigated. As shown in Fig. 3, no ECL signals were observed on bare electrode (curve a), S1 modified electrode (curve b) and azide-terminated dsDNA modified electrode (curve c). After assembling alkynyl functionalized GO/AgNPs/luminol composites onto the azide-terminated dsDNA modified electrode via click chemistry, a strong ECL signal was observed (curve d), resulting from the ECL reaction of luminol. In comparison with the GO/luminol (curve e) and AgNPs/luminol (curve f), 49.4-fold and 3.3-fold enhancement in ECL signals was observed using GO/AgNPs/luminol composites as probe, indicating that the GO/AgNPs/luminol composites greatly amplified the ECL signal. These suggested the successful construction of the GO/AgNPs/luminol/dsDNA electrode according to
Fig. 3. ECL signals under step potential obtained on (a) bare electrode, and (b) S1, (c) S2/S1, (d) GO/AgNPs/luminol/S2/S1, (e) GO/luminol/S2/S1, (f) AgNPs/luminol/S2/ S1 modified electrodes, and (g) electrode (d) reacted with 20 U/mL Dam MTase and Dpn I 50 U/mL. Initial potential, 0.7 V; final potential, 0.1 V; pulse period, 30 s; pulse time, 1.0 s; working buffer, 1.0 mM H2O2 containing 0.02 mol/L CBS (pH 10.53).
(5) 2n
where LH is the deprotonated luminol and AP is the excited state product, it is noted that GO/AgNPs play important role to amplify the ECL signal of luminol. GO will increase the interface area of the modified electrode to capture more AgNPs and luminol through Ag–π interaction and π–π stacking; and AgNPs will facilitate the electron transfer at the electrode interface and catalyze the ECL process of luminol in both electrode and chemical reactions (He et al., 2011b; Wang et al., 2009). Thus, the GO/AgNPs/ luminol composites can mediate the ECL reaction even at a low content, implying highly efficient ECL signal amplification. At higher Dam MTase activity, there are more GO/AgNPs/luminol composites removed from the electrode surface due to the cleavage of Dpn I, resulting in a significant decrease of the ECL signal. Therefore, the as-designed ECL biosensor can be used for Dam MTase activity characterization. 3.5. ECL measurement of dam MTase activity In order to achieve the maximum sensitivity for the detection of Dam MTase, experimental parameters including the ECL detection mode (Figs. S1 and S2), pH of detection solution (Fig. S3A), methylation time (Fig. S3B), initial potential (Fig. S3C), pulse period (Fig. S3D) and the concentration of H2O2 (Fig. S3E) were optimized. Under the optimal conditions, quantitative detection of Dam MTase was performed. Fig. 4A shows the variance of ECL intensity with the concentration of Dam MTase. As the concentration of Dam MTase increased, more S1/S2 hybrids were methylated. After being treated with Dpn I, the methylated S1/S2 hybrids were cleaved and GO/AgNPs/luminol composites were released from the electrode surface, which resulted in the decrease of ECL signal. The ECL signal was inverse proportional to the concentration of Dam MTase, which could be used for Dam MTase detection. Fig. 4B shows the curve of ECL intensity with different Dam MTase concentrations. The ECL peak intensity decreased linearly with the logarithmic value of Dam MTase concentrations in the range of 0.1 to 20 U/mL, and the detection limit was calculated to be 0.03 U/mL at 3s. The detection range of the method was much wider than those of previously reported 1.0–10 U/mL based on gold nanoparticles coupled with enzyme-linkage reaction for the detection of DNA Methylation (Li et al., 2010) and 0.1–1.0 U/mL based on the controllable assembly of single wall carbon nanotubes using label-free electrochemical assay for methyltransferase activity detection (Wang et al., 2013). The detection limit was much lower than those of 0.14 U/mL on a label-free colorimetric assay based on unmodified Au nanorods as a signal sensing probe coupled with enzyme-linkage reaction (Zheng et al., 2013) and 0.12 U/mL using AuNPs amplification based on a sensitive signal-
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Fig. 4. (A) ECL signal obtained from GO/AgNPs/luminol/dsDNA modified electrode by different concentrations of Dam MTase. The curves from bottom to the top were obtained with different concentration: 0.1, 0.2, 0.5, 1.0, 5.0, 10.0, 20.0, 30.0 and 50.0 U/mL, respectively. (B) The response curve obtained with different concentrations of Dam MTase. Inset is the linear relationship between the ECL intensity and the logarithm of the Dam MTase concentration.
on electrochemical assay for DNA MTase activity (He et al., 2011a). Results indicated that employing the GO/AgNPs/luminol composites as the ECL signal probe is an effective strategy. Stable and high ECL signals (Fig. S4) were observed when the biosensor was successively scanned, which signified that the ECL biosensor possessed excellent potential cycling stability. The longterm stability of the biosensor was evaluated by measuring the ECL signal after storing at 4 °C for different time period. No obvious change of the ECL response was found within 4 weeks, supplying a high long-term stability and prolong lifetime of the ECL biosensor for Dam MTase activity detection. To evaluate the practicability of the biosensor, we applied this sensor in human serum samples for Dam MTase detection. As shown in Fig. S5, comparable responses were found for Dam MTase in both NEBuffer 4 and diluted human serum. Control experiment indicated that no Dam MTase was found in human serum. The ECL intensities from the human serum were well consistent with those
from NEBuffer 4, suggesting diluted human serum had no influence on detecting the activity of Dam MTase. The recoveries were in the range of 94.6–106.3% for measurement of 1, 5 and 10 U mL 1 Dam MTase spiked in serum with a maximum RSD of 5.8%, which were comparable to those reported in literatures (Zhao et al., 2014; Chen and Zhao, 2013). These results indicated that the proposed strategy holds a great promise in practical applications for DNA MTase detection with great accuracy and reliability. 3.6. Assay of the inhibition of dam MTase activity Since DNA methylation is closely related to our health and disease and inhibitors of DNA methylase have potential application in cancer therapy and antimicrobial infection, screening of inhibitors of DNA MTase receives more and more interest, and we have, thus, checked whether our method can be employed for screen of
Fig. 5. (A) Influence of different drugs on the activity of Dam MTase. (1) No drug, (2) mitomycin, (3) platinol, (4) benzylpenicillin, (5) 5-fluorouracil. (B) Inhibition of the activity of Dam MTase by 5-fluorouracil. Relationship between ECL intensity and the concentrations of 5-fluorouracil. Inset: ECL intensity-time behaviors at different concentrations of 5-fluorouracil. The concentration of 5-fluorouracil were 0, 1, 10, 20, 50 and 100 μM, respectively.
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Dam MTase inhibitors. The mechanism is that, if transferring of the methyl group to the DNA residue is blocked in the presence of inhibitor, the specific site of the DNA will not be recognized and cleaved; thus, the ECL intensity increased with the increase of inhibition of Dam MTase. To date, the reported small molecule Dam MTase inhibitors mainly contain two categories, antibiotic drugs (benzylpenicillin) and anticancer drugs (5-fluorouracil, platinol and mitomycin). As can be seen from Fig. 5A, benzylpenicillin and 5-fluorouracil could strongly inhibit the methylase. The inhibition efficiency of 5-fluorouracil was the most serious one, which may decrease the activity of Dam MTase by about 50%. However, mitomycin and platinol had almost no effect on the methylation, in good agreement with the order obtained in previous reports (Zheng et al., 2013). We also investigated how the concentration of 5-fluorouracil influenced the activity of methylase. The concentration of 5-fluorouracil was kept under 100 mM. As shown in Fig. 5B, the ECL intensity increased with increasing the concentration of 5-fluorouracil, clearly indicating significant dose-dependent inhibition. The IC50 value was the inhibitor concentration required to reduce enzyme activity by 50%. The relationship between ECL intensity and 5-fluorouracil concentrations is plotted in inset of Fig. 5B, the IC50 of 5-fluorouracil was calculated to be 18.2 μM, which was much lower than those of 100 μM based on the electrochemical assay (Wang et al., 2013; Jing et al., 2014). This indicated that the proposed electrochemiluminescence method was highly sensitive for screening inhibitor of Dam MTase. Therefore, the above results demonstrated that our method can be successfully used in the assay of Dam MTase inhibitors.
4. Conclusions In conclusion, we have successfully developed a novel, homogeneous, signal-off ECL DNA sensor for the sensitive and specific detection of Dam MTase using GO/AgNPs/luminol composites as the ECL signal probe. As a result, the as-proposed ECL biosensor offers a highly sensitive strategy for Dam MTase activity monitoring with a low detection limit of 0.03 U/mL, wide linear range, and good stability. More importantly, this assay is able to be performed in human serum with satisfying recovery, holding great potential for further application in complex biology samples. Moreover, the proof-of-concept method also shows excellent performance on screen Dam MTase inhibitor. The general and robust method can also be ready for other Dam MTase activities and inhibition assays. Given the important roles of Dam MTase in some disease related biological processes, this biosensor shows great potential for a high throughput assay in clinic diagnostics and drug discovery applications.
Acknowledgments We gratefully acknowledge the supports from the National Natural Science Foundation of China (Nos. 21163014 and 21265017) and the Program for New Century Excellent Talents in University (NCET-11-1002 and NCET-13-0848).
Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.07.079.
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