Biosensors and Bioelectronics 54 (2014) 285–291
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Carbon nanotube signal amplification for ultrasensitive fluorescence polarization detection of DNA methyltransferase activity and inhibition Yong Huang, Ming Shi, Limin Zhao, Shulin Zhao n, Kun Hu, Zheng-Feng Chen, Jia Chen, Hong Liang n School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin 541004, China
art ic l e i nf o
a b s t r a c t
Article history: Received 25 July 2013 Received in revised form 2 October 2013 Accepted 21 October 2013 Available online 13 November 2013
A versatile sensing platform based on multiwalled carbon nanotube (MWCNT) signal amplification and fluorescence polarization (FP) is developed for the simple and ultrasensitive monitoring of DNA methyltransferase (MTase) activity and inhibition in homogeneous solution. This method uses a dyelabeled DNA probe that possess a doubled-stranded DNA (dsDNA) part for Mtase and its corresponding restriction endonuclease recognition, and a single-stranded DNA part for binding MWCNTs. In the absence of MTase, the dye-labeled DNA is cleaved by restriction endonuclease, and releases very short DNA carrying the dye that cannot bind to MWCNTs, which has relatively small FP value. However, in the presence of MTase, the specific recognition sequence in the dye-labeled DNA probe is methylated and not cleaved by restriction endonuclease. Thus, the dye-labeled methylated DNA product is adsorbed onto MWCNTs via strong π–π stacking interactions, which leads to a significant increase in the FP value due to the enlargement of the molecular volume of the dye-labeled methylated DNA/MWCNTs complex. This provides the basic of a quantitative measurement of MTase activity. By using the MWCNT signal amplification approach, the detection sensitivity can be significantly improved by two orders of magnitude over the previously reported methods. Moreover, this method also has high specificity and a wide dynamic range of over five orders of magnitude. Additionally, the suitability of this sensing platform for MTase inhibitor screening has also been demonstrated. This approach may serve as a general detection platform for sensitive assay of a variety of DNA MTases and screening potential drugs. & 2013 Elsevier B.V. All rights reserved.
Keywords: Multiwalled carbon nanotubes Fluorescence polarization Nanosensors DNA methyltransferase Inhibition
1. Introduction DNA methyltransferase (MTase)-mediated DNA methylation is an important epigenetic event that plays a pivotal role in DNA repair, gene transcription, and embryogenesis (Choy et al., 2010). Moreover, the aberrant methylation of CpG islands in promoter regions of genes is a new generation of cancer biomarkers, and can be regarded as a hallmark of various diseases (Jaenisch and Bird, 2003; Jeltsch et al., 2007). Additionally, DNA MTase is also a novel family of pharmacological targets for the treatment of tumors (Esteller et al., 2001; Szyf, 2005). Thus, evaluations of DNA MTase activity and inhibition are essential for clinical diagnostics and drug development. Traditional assay methods such as radioactive labeling, gel electrophoresis and high performance liquid chromatography (Som and Friedman, 1991; Rebeck and Samson, 1991; Reenila et al., 1995) have been established for DNA MTase detection. However, these methods are time-intensive, DNA-consuming, n
Corresponding authors. Tel.: þ 86 773 5856104; fax: þ86 773 5832294. E-mail addresses: [email protected] (S. Zhao), [email protected] (H. Liang). 0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.10.065
laborious, not sensitive, or require isotope labeling. Many of these limitations are now being addressed by the development of some new methods (Li et al., 2012, 2013; Liu et al., 2010; Tian et al., 2012). For example, fluorescence assays based on hairpin fluorescence molecular beacon based DNA probe, cationic conjugated polymer/ DNA complexes and graphene oxide-mediated DNA probe have been developed for assaying DNA MTases (Li et al., 2007; Feng et al., 2007). Colorimetric assays based on gold nanoparticle (AuNP) aggregation or horseradish peroxidase-mimicking DNAzyme were reported for detection of DNA MTase (Song et al., 2009; Li et al., 2010). In addition, electrochemical biosensors based on the use of ferrocene, alkaline phosphatase or AuNPs as labels have also been developed for assay of DNA MTase activity (He et al., 2011; Wang et al., 2010; Liu et al., 2011; Wu et al., 2012). Albeit substantial progress was accomplished, a method that is simpler and more sensitive still is necessitated. Carbon nanotubes (CNTs) attracts growing interest as a new, water-soluble material for biological studies due to their unique structural, mechanical and optoelectronic properties (Wang et al., 2013; Chen et al., 2011). Particularly interesting is the interaction of nucleic acids with CNTs. It is found that single-stranded nucleic
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acids adsorb strongly on CNTs, while duplex DNAs cannot bind to MWCNTs stably (Tang et al., 2006). This property has been employed to elaborately design sensors for various molecular targets in homogeneous solution (Zhang et al., 2010a; Wu et al., 2008). For example, the desorption of a fluorophorelabeled single-stranded DNA by the complementary nucleic acid was used for rapid and sensitive fluorescence detection of nucleic acids (Liu et al., 2009; Yang et al., 2008; Zhu et al., 2008). Similarly, the desorption of fluorophore-labeled nucleic acids from CNTs through the generation of aptamer-substrate complexes was implemented to develop CNT-based sensor systems for fluorescent detection of biomolecules (Zhen et al., 2010; Zhang et al., 2011a). In addition, the release of fluorophore-labeled metal-specific oligonucleotides from CNTs through the formation of metal ionmediated duplex DNA structures was also used for the fluorescence detection of metal ions (Zhao et al., 2010; Zhang et al., 2010b). Although these techniques can be quite powerful, greater sensitivity and specificity are often required, particularly when working with limited amounts of sample material or when target concentration is extremely low. Fluorescence polarization (FP) is a simple signaling approach that provides a quantitative measure for the rotational motion of a fluorescently labeled molecule. The FP value P is sensitive to changes in the rotational motion of fluorescently labeled molecules, which depends on the size of molecule. If a fluorescence molecule is free in solution, it rotates at a rate commensurate with its size and hence will have a relatively small P value. However, when the fluorescence molecule binds with another substance to form a complex, its rotational rate decreases and the P value will increase; the degree of variation depends on the strength of the binding interaction and the size of the formed complex (Zhang et al., 2011b; Deng et al., 2007). This technique has found widespread applications in monitoring the binding events of biomolecules and asssaying of various biomolecules or cells (Deng et al., 2010; Ruta et al., 2009; Zhang et al., 2011c, 2012; Perrier et al., 2010). Recently, to improve the detection sensitivity, several amplifying strategies that employ proteins (Cui et al., 2012; Zhu et al., 2012), AuNPs (Yin et al., 2010; Ye and Yin, 2008; Huang et al., 2011), silica nanoparticles (Huang et al., 2012), and graphene oxide (Liu et al., 2013; Yu et al., 2013) as FP enhancers for detection of small molecules and proteins have been proposed. Nonetheless, FP-based sensing platforms that implement CNTs as a signal amplifier are at present unknown. In the present work, we report a novel amplified sensing platform based on multiwalled carbon nanotube (MWCNT) signal amplification and fluorescence polarization (FP) for highly sensitive and selective detection of DNA MTase activity and inhibition. To the best of our knowledge, this is the first example of using CNTs as a signal amplifier for FP assay of biomolecules. Compared with traditional methods for DNA MTase assays, this proposed protocol does not require separation and troublesome procedures, which is very simple and fast. Most importantly, the introduction of MWCNTs causes a significant amplification of the detection signal, which substantially improves the detection sensitivity by two orders of magnitude over the previously reported methods. Moreover, the application of this MWCNTbased FP sensing platform for DNA MTase inhibitor screening has also been demonstrated.
2. Experimental 2.1. Materials and reagents All oligonucleotides were purchased from the Sangon Biotech Co. (Shanghai, China) and purified by HPLC. The sequences of the
involved oligonucleotides were as follow: 5′-CGA TCC CGC TGC CGG GCC CCG CTG CCC TGT GCC GAA TTT TTT-3′ (DNA-1) and 5′-GGC CCG GCA GCG GGA TCG-FAM-3′ (cDNA-1) were used for Dam MTase detection; and 5′-GTG AAT TCC ATC CGA CCC CCG CTG TCA TGT GCC GAA TTT AAA -3′ (DNA-2) and 5'-GGG TCG GAT GGA ATT CAC-FAM-3′ (cDNA-2) were used for assaying EcoRI MTase. The Dam MTase, AluI MTase, EcoRI MTase, DpnII endonuclease, EcoRI endonuclease and S-adenosyl-L-methiolnine (SAM) were purchased from New England Biolabs (NEB, UK). Standardized human serum was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Multiwalled carbon nanotubes (MWCNTs) were purchased from Shenzhen Nanotech Port Co. Ltd. (Shenzhen, China), and oxidized and purified as described previously (Zhen et al., 2010). Other chemicals were of analytical grade. Water was purified by using a Milli-Q plus 185 equip from Millipore (Bedford, MA).
2.2. Apparatus Fluorescence polarization (FP) measurements were carried out using an FL3-P-TCSPC system (Jobin Yvon, Inc., Edison, NJ, USA) with 300 μL cuvette. The FP of the sample solution was monitored by exciting the sample at 494 nm and measuring the emission at 520 nm (Fig. S1). And slits for both the excitation and the emission were set at 5 nm. HPLC analysis was performed by using an LC-10ATVP system equipped with RF-10AXL fluorescence detector (Shimadzu, Kyoto, Japan). The assays were carried out on a C18 column (250 4.6 mm i.d., 5 μm particle sizes, Elite, China). The HPLC assay used 494 nm and 520 nm as excitation and detection wavelengths, respectively.
2.3. DNA MTase activity assay To prepare duplex DNA substrate of Dam MTase (dsDNA-1), 2.5 μL of 100 μM FAM-labeled DNA-1 was mixed with a 1.2-fold excess of cDNA-1 in pH 7.5 buffer containing 50 mM Tris–HCl and 50 mM NaCl. Then, the mixture was annealed by heating to 90 1C for 5 min and followed by slow cooling at room temperature for 1 h. For Dam MTase assay, reaction mixture was prepared by mixing Dam MTase enzyme stock with 45 nM annealed dsDNA-1 in 200 μL reaction buffer (pH 7.5 Tris–HCl buffer containing 50 mM Tris, 50 mM NaCl, 10 mM dithiothreitol, 0.3 mM SAM) and incubated at 37 1C for 1 h. Then, 50 μL of DpnII reaction buffer containing 50 units of DpnII was added and incubated for 30 min. Finally, 50 μL of MWCNTs solution were added to the above reaction mixture to give a final concentration of 60 μg/mL, and incubated for another 30 min. The obtained sample solution was used for FP measurements. FP was measured by using the L-format configuration and FluorEssence™ software with constant wavelength analysis to achieve a FP value. The G factor was initially set to zero, to let the system measure G automatically. The FP value was also calculated automatically by the instrument. The integration time was set to 3 s for the FP measurements. Over five FP measurements were taken each time, and they were then averaged for further data processing. In case of EcoRI MTase, duplex DNA substrate of EcoRI MTase (dsDNA-2) was annealed by mixing 2.5 μL of 100 μM FAM-labeled DNA-2 and a 1.2-fold excess of cDNA-2 in pH 7.5 buffer containing 50 mM Tris–HCl and 50 mM NaCl. Then, the mixture was annealed by heating to 90 1C for 5 min, followed by slow cooling at room temperature for 1 h. Other procedure for EcoRI MTase activity assay was similar to detection of EcoRI MTase activity mentioned above, except that Dam MTase, dsDNA-1 and DpnII endonuclease were replace of EcoRI MTase, and dsDNA-2, and EcoRI endonuclease, respectively.
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2.4. DNA MTase inhibition assay Dam MTase was used as a model enzyme for inhibition assay. For Dam MTase inhibition assay, reaction mixture was prepared by mixing 5 units Dam MTase and different concentrations of inhibitors with 45 nM annealed dsDNA-1 in 200 μL reaction buffer (pH 7.5 Tris–HCl buffer containing 50 mM Tris, 50 mM NaCl, 10 mM dithiothreitol, 0.3 mM SAM) and incubated at 37 1C for 1 h. Other assay steps were the same as those of the detection of Dam MTase activity described above. 2.5. HPLC assay Samples for HPLC assays were prepared as follows: Dam MTase (100 units) complexed with 45 nM annealed dsDNA-1 was added into 200 μL reaction buffer (pH 7.5 Tris–HCl buffer containing 50 mM Tris, 50 mM NaCl, 10 mM dithiothreitol, 0.3 mM SAM) and incubated at 37 1C for 6 h. Then, 50 μL of DpnII reaction buffer containing 50 units of DpnII was added and incubated for 30 min. After that, 50 μL of MWCNTs solution was added to the above reaction mixture to give a final concentration of 60 μg/mL, and incubated for another 30 min. Finally, the reaction mixture was centrifuged to collect the supernatant and analyzed by HPLC. Blank samples were prepared similar to the procedure mentioned above except without the addition of Dam MTase. HPLC assay was carried out on a C18 column at a rate of 1.0 mL/min, with a 25 min gradient from 10 to 17% acetonitrile in 0.1 M triethylammonium acetate at pH 7.0.
3. Results and discussion 3.1. Principle of the MWCNT-based FP sensing platform Scheme 1 depicts the working principle of the MWCNT-based amplified FP assay for the detection of DNA MTase. This assay uses a fluorescein amidite (FAM)-labeled DNA probe, which contains a double-stranded DNA (dsDNA) part to serve as the specific recognition sequence of both DNA MTase and its corresponding restriction endonuclease, and a single-stranded DNA part for anchoring the DNA to the surface of MWCNTs. In the absence of DNA MTase, this FAM-labeled DNA probe is cleaved by restriction endonuclease, and generates the very short DNA fragments carrying the FAM dye that cannot bind to MWCNTs stably. In this situation, the FAM dye exhibits relatively small P value. However,
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in the presence of target DNA MTase, the specific recognition sequence within the FAM-labeled DNA probe is methylated and blocked the cleavage by its corresponding restriction endonuclease. Thus, the FAM-labeled methylated DNA product is adsorbed onto CNTs via strong π–π stacking interactions between the single-stranded DNA within the FAM-labeled methylated DNA product and the carbon nanotube sidewalls (Liu et al., 2009; Zhen et al., 2010; Zhu et al., 2008), which leads to a significant increase in the P value due to the enlargement of the molecular volume of the formed FAM-labeled methylated DNA/MWCNTs complexes with slow rotation. By monitoring the increase in the P value of the FAM dye, we could detect the target DNA MTase with very high sensitivity. In addition, this assay can be easily adapted to screening inhibitors for DNA MTase because DNA methylation by a DNA MTase is restrained in the presence of inhibitors. Of note, the use of MWCNTs in our new assay provides a novel means of signal amplification, and substantially improves the performance and sensitivity of the FP assay method for detecting DNA MTase in homogeneous solution (Fig. 1). 3.2. Feasibility study To demonstrate the proof-of-principle, the amplified assay was first tested by using Dam MTase and its substrate FAM-labeled dsDNA-1 as models. We investigated the FP responses of the FAMlabeled dsDNA-1 in the absence and presence of Dam MTase with subsequent incubation with MWCNTs. It was found that the P value of the solution with Dam MTase was increased significantly compared with that of the solution without Dam MTase (Fig. 1). The enhancement of the P value was attributed to the fact that the FAM-labeled dsDNA-1 was methylated by Dam MTase and inhibited the DpnII-catalyzed DNA cleavage, and thus the FAM-labeled methylated DNA product was adsorbed onto MWCNTs via noncovalent interactions (Liu et al., 2009; Zhen et al., 2010; Zhu et al., 2008). The FP responses of the FAM-labeled dsDNA-1 in the absence and presence of Dam MTase without MWCNTs were also investigated. It was found that the P value is approximately 19 times higher in the system employing the MWCNTs compared with the system without MWCNTs (Fig. 1). A substantial increase in the P value was due to the enlargement of the molecular volume of the formed FAM-labeled methylated DNA/MWCNTs complexes in solution with slow rotation. These results demonstrated that MWCNTs could be used as an effective signal amplifier for the FP detection of DNA MTase activity. 3.3. HPLC characterization The viability of the proposed strategy was further investigated by HPLC with fluorescence detection. The HPLC assay was carried out by the procedure as described in Experimental section, and the results obtained are shown in Fig. 2. In the presence of DpnII but without Dam MTase, a peak was observed in the electropherogram, indicating the cleavage of the FAM-labeled dsDNA-1 by DpnII (trace a). However, in the presence of both Dam MTase and DpnII, no peak was obtained in the electropherogram (trace b). This fact demonstrated that the DNA methylation by Dam MTase inhibited the DpnII-catalyzed DNA cleavage, and the FAM-labeled methylated DNA product was adsorbed onto MWCNTs, which provides a solid foundation for the present assay. 3.4. Optimization of the size and amount of MWCNTs
Fig. 1. Fluorescence polarization changes upon addition of Dam MTase at a concentration of 1 U/mL. A blank (FAM-labeled dsDNA-1 without Dam MTase) was used as a control reference.
In proposed FP amplifying design, the size and amount of MWCNTs were the critical factors because they significantly affected the reporting FP signal. Therefore, it was necessary to optimize the size and amount of MWCNTs. Fig. S2 shows the
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Scheme 1. The principle of the MWCNT-based FP sensing platform for the detection of DNA MTase
Fig. 2. HPLC chromatograms obtained from an incubation solution of the FAMlabeled dsDNA-1 in the absence (a) and presence (b) of Dam MTase with subsequent incubation with DpnII and MWCNTs. Fluorescence detection wavelength was 520 nm for all traces.
effects of the size of the MWCNTs on the P values. Increasing the diameter of the MWCNTs (average diameter: 16 nm, 28 nm, 47, and 65 nm) changed the P values more obviously when the same concentration of Dam MTase was used. Therefore, MWCNTs with an average diameter of about 65 nm was used in this study. In addition, the amount of MWCNTs was also optimized. As shown in Fig. 3, the P value increased gradually with an increase in the concentration of MWCNTs from 0 to 60 μg/mL. Further increase of MWCNTs concentration, the P value decreased slightly. This phenomenon might be attributed by following reasons: (1) the increase of MWCNTs concentration would result in the adsorption of more amount of FAM-labeled methylated DNA product onto MWCNTs, and led to an increase of the reporting FP signal; (2) Too high of MWCNTs concentration would inhibit the release of DpnIIinduced cleaved products from the surface of MWCNTs, and hence reduced the reporting FP signal. Based on the above results, a concentration of 60.0 μg/mL MWCNTs was used for the following experiments. 3.5. Dam MTase activity assay To confirm the ability of the proposed MWCNT-based amplified FP assay to detect target DNA MTase, a series of different
Fig. 3. The effects of MWCNTs concentration on the fluorescence polarization changes of FAM-labeled dsDNA-1 upon analyzing Dam MTase at a concentration of 0.05 U/mL.
concentrations of Dam MTase were measured. Fig. 4 shows the FP responses of the sensing system for different Dam MTase concentrations. As the concentration of Dam MTase increased, the P value increased. This was consistent with the fact that the enhanced inhibition of the DpnII-catalyzed DNA cleavage by Dam MTase-catalyzed more DNA methylation, and the higher amount of FAM-labeled methylated DNA were adsorbed onto MWCNTs. This method allowed the detection of Dam MTase as low as 1.0 10 4 U/mL, which was two orders of magnitude lower than that of the reported fluorescence (Li et al., 2007), colorimetric (Song et al., 2009; Li et al., 2010) and electrochemical (He et al., 2011; Wang et al., 2010; Liu et al., 2011; Wu et al., 2012) methods. In contrast, when the FP assay method without MWCNT signal amplification was used, a sensitivity that corresponds to a Dam MTase concentration of 1 U/mL was obtained (Fig. 4). The substantial sensitivity improvement of the MWCNT-based amplified FP assay method was attributed to the slower rotation of fluorescent unit when DpnII-catalyzed DNA cleavage was inhibited by Dam MTase-catalyzed DNA methylation and the FAM-labeled methylated DNA product was adsorbed onto MWCNTs. The MWCNT-based amplified FP assay for Dam MTase was also specific. To evaluate this property, we challenged the system with two non-targeted DNA MTases: AluI MTase and EcoRI MTase.
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had no influence on the activity of DpnII when the concentrations of the compounds were no more than 1 μM. Afterwards, we tested the effects of the compounds mentioned above at 1 μM on Dam MTase activity to comparison of the inhibition abilities of test compounds. It was found that all of the drugs and the metal complexes inhibited the Dam MTase activity to a certain degree (Fig. 6A). Interestingly, the two metal complexes showed a stronger inhibition on the Dam MTase activity than the anticancer drugs. In addition, we also investigated how the concentration of [OGH] [AuCl4] DMSO influenced the activity of Dam MTase, and the results are shown in Fig. 6B. As the concentration of [OGH] [AuCl4] DMSO increased, inhibition was enhanced. These results demonstrated that the proposed MWCNT-based amplified FP assay has potential application in screening DNA MTase targeted drugs, which has significant value to antibiotics and anticancer therapeutics.
Fig. 4. Plots of fluorescence polarization changes as a function of Dam MTase concentrations for the sensing systems with or without MWCNTs.
3.7. Detection of dam MTase activity in Human serum A significant challenge for enzyme activity analysis is the ability to realize the detection of target in complex biological matrixes. To investigate if the MWCNT-based amplified FP assay
Fig. 5. Specificity of the MWCNT-based amplified FP assay. The concentration of the target Dam MTase was 0.05 U/mL, and other DNA MTases were 50 U/mL each.
Significantly higher P value was observed with the target Dam MTase than with non-targeted DNA MTases (Fig. 5). These results clearly demonstrated the high specificity of our MWCNT-based amplified FP assay. In addition, assay reproducibility was also investigated by analyzing Dam MTase standard solutions (at 0.001, 0.01, and 0.1 U/mL Dam MTase, respectively) five times. The results showed that the relative standard deviations (RSDs) were found to be r4.9%. 3.6. Dam MTase activity inhibition evaluation The MWCNT-based amplified FP assay has the potential for highthroughput screening of inhibitors of Dam MTase. We investigated the effects of two anticancer drugs (5-fluorouracil and cisplatin) and two metal complexes of 5-chloro-7-iodo-8-hydroxylquinoline (ClIQ) and oxoglaucine (OG) ([Sn(ClIQ)2Cl2] and [OGH][AuCl4] DMSO) synthesized in our laboratory (Chen et al., 2012, 2013), on the activity of Dam MTase. Before evaluating the inhibitory effects of the compounds on the activity of Dam MTase, we first investigated whether these compounds had influence on the DpnII activity. The experimental results found that the test compounds
Fig. 6. (A) Influence of different drugs and metal complexes on the activity of Dam MTase. The numbers of the horizontal bar represented different compounds tested: (1) no drug; (2) cisplatin; (3) 5-fluorouracil; (4) [Sn(ClIQ)2Cl2]; (5) [OGH][AuCl4] DMSO. All compounds were tested at 1 μM. (B) The effects of [OGH][AuCl4] DMSO on the activity of Dam MTase at different concentrations.
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method developed here was applicable to complex biological matrixes, standardized human serum sample without Dam MTase (diluted 10 times with 50 mM Tris–HCl buffer) samples and the spiked human serum samples, at three different concentrations of Dam MTase (0.005, 0.05 and 0.5 U/mL), were analyzed using the proposed method. The responses of the MWCNT-based amplified FP assay for Dam MTase in diluted human serum samples were compared with those obtained from Dam MTase in buffer solution. As shown in Fig. 7, comparable responses were found for Dam MTase in both buffer solution and diluted human serum. Then, three independent spiked serum samples were tested by the MWCNT-based amplified FP assay, and the activity of Dam MTase in the spiked serum was calculated by using the calibration curve of standard Dam MTase. The assay results of the spiked serum samples are shown in Table S1. The recoveries were found to be in the range of 92.8–97.6%. The precision of the method was evaluated by repeatedly analyzing each spiked serum sample for five times within one working day. The RSDs were between 2.9% and 4.8%. These results indicated the potentiality of the proposed method for detection of DNA MTase in real biological samples.
Fig. 8. Plots of fluorescence polarization changes as a function of EcoRI MTase concentrations for the sensing systems with or without MWCNTs.
MTase standard solutions (at 0.001, 0.01, and 0.1 U/mL Dam MTase, respectively) five times. 3.8. EcoRI MTase activity assay To illustrate the generality of our design, we applied this strategy to detect another DNA MTase, EcoRI MTase. By replacing the duplex DNA substrate of Dam MTase with the duplex DNA substrate (dsDNA-2) for EcoRI MTase, we similarly made the MWCNT-based amplified FP assay for detecting EcoRI MTase. As shown in Fig. 8, the MWCNT-based amplified FP assay could sensitively detect EcoRI MTase with a detection limit of 1.0 10 4 U/mL, which was 104 times lower than that of the FP assay strategy without MWCNT signal amplification. Meanwhile, this sensitivity was also two orders of magnitude higher than that of the reported fluorescence method (Feng et al., 2007). The MWCNT-based amplified FP assay also showed high specificity toward EcoRI MTase over other non-targeted DNA MTases, such as Dam MTase and AluI MTase. In addition, this assay also exhibited good reproducibility with RSDs less than 4.7% for assaying EcoRI
4. Conclusions In summary, we have developed a novel amplified sensing platform based on MWCNT signal amplification and FP detection for the assay of DNA MTase activity and inhibition in homogeneous solution. As a proof-of-concept, we demonstrate that this amplified assay can respectively detect Dam MTase and HaeIII MTase with high sensitivity and specificity. Furthermore, the application of this MWCNT-based sensing platform for DNA MTase inhibitor screening has also been demonstrated. This assay technique has several important features. First, by using MWCNT signal amplification approach, the sensitivity of this new type of assay can be significantly improved by two orders of magnitude over the previously reported methods (see Table S2). Second, compared with traditional methods for DNA MTase detection, this assay is done in homogeneous solution, and not requiring separation and troublesome procedures, which is quite simple and convenient. Third, the assay can be easily adapted to high throughput applications in an array format. Finally, this method may also be extended to detect other DNA MTases and screen their inhibitors by simply switching the corresponding DNA substrates. Also, given that endonucleases can cleave their specific DNA substrates, we expect that this new methodology may also find potential application in the detection of endonucleases through FP reduction. These quanlities endow the proposed MWCNT-based amplified FP sensing platform with great promise in DNA MTase-related basic research and drug development.
Acknowledgments
Fig. 7. Results obtained from the testing of human serum samples spiked with Dam MTase (red column) and Dam MTase in Tris–HCl buffer (black column). The numbers of the horizontal bar represented different concentrations of Dam MTase in human serum and Tris–HCl buffer: (1) 0 U/mL; (2) 0.005 U/mL; (3) 0.05 U/mL; (4) 0.5 U/mL. Human serum was diluted in 1:10 ratio with Tris–HCl buffer. (For interpretation of the references to color in this figure caption, the reader is referred to the web version of this article.)
This work was supported by the National Natural Science Foundations of China (No. 21175030), the National Basic Research Program of China (No. 2012CB723501), and the Natural Science Foundations of Guangxi Province (No. 2012GXNSFDA385001, 2013GXNSFBA019038, 2013GXNSFBA019044) as well as BAGUI Scholar Program and the project of Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Education of China (CMEMR2012-A19, CMEMR2013C06).
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Carbon nanotube signal amplification for ultrasensitive fluorescence polarization detection of DNA methyltransferase activity and inhibition Yong Huang, Ming Shi, Limin Zhao, Shulin Zhao n, Kun Hu, Zheng-Feng Chen, Jia Chen, Hong Liang n School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin 541004, China
art ic l e i nf o
a b s t r a c t
Article history: Received 25 July 2013 Received in revised form 2 October 2013 Accepted 21 October 2013 Available online 13 November 2013
A versatile sensing platform based on multiwalled carbon nanotube (MWCNT) signal amplification and fluorescence polarization (FP) is developed for the simple and ultrasensitive monitoring of DNA methyltransferase (MTase) activity and inhibition in homogeneous solution. This method uses a dyelabeled DNA probe that possess a doubled-stranded DNA (dsDNA) part for Mtase and its corresponding restriction endonuclease recognition, and a single-stranded DNA part for binding MWCNTs. In the absence of MTase, the dye-labeled DNA is cleaved by restriction endonuclease, and releases very short DNA carrying the dye that cannot bind to MWCNTs, which has relatively small FP value. However, in the presence of MTase, the specific recognition sequence in the dye-labeled DNA probe is methylated and not cleaved by restriction endonuclease. Thus, the dye-labeled methylated DNA product is adsorbed onto MWCNTs via strong π–π stacking interactions, which leads to a significant increase in the FP value due to the enlargement of the molecular volume of the dye-labeled methylated DNA/MWCNTs complex. This provides the basic of a quantitative measurement of MTase activity. By using the MWCNT signal amplification approach, the detection sensitivity can be significantly improved by two orders of magnitude over the previously reported methods. Moreover, this method also has high specificity and a wide dynamic range of over five orders of magnitude. Additionally, the suitability of this sensing platform for MTase inhibitor screening has also been demonstrated. This approach may serve as a general detection platform for sensitive assay of a variety of DNA MTases and screening potential drugs. & 2013 Elsevier B.V. All rights reserved.
Keywords: Multiwalled carbon nanotubes Fluorescence polarization Nanosensors DNA methyltransferase Inhibition
1. Introduction DNA methyltransferase (MTase)-mediated DNA methylation is an important epigenetic event that plays a pivotal role in DNA repair, gene transcription, and embryogenesis (Choy et al., 2010). Moreover, the aberrant methylation of CpG islands in promoter regions of genes is a new generation of cancer biomarkers, and can be regarded as a hallmark of various diseases (Jaenisch and Bird, 2003; Jeltsch et al., 2007). Additionally, DNA MTase is also a novel family of pharmacological targets for the treatment of tumors (Esteller et al., 2001; Szyf, 2005). Thus, evaluations of DNA MTase activity and inhibition are essential for clinical diagnostics and drug development. Traditional assay methods such as radioactive labeling, gel electrophoresis and high performance liquid chromatography (Som and Friedman, 1991; Rebeck and Samson, 1991; Reenila et al., 1995) have been established for DNA MTase detection. However, these methods are time-intensive, DNA-consuming, n
Corresponding authors. Tel.: þ 86 773 5856104; fax: þ86 773 5832294. E-mail addresses: [email protected] (S. Zhao), [email protected] (H. Liang). 0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.10.065
laborious, not sensitive, or require isotope labeling. Many of these limitations are now being addressed by the development of some new methods (Li et al., 2012, 2013; Liu et al., 2010; Tian et al., 2012). For example, fluorescence assays based on hairpin fluorescence molecular beacon based DNA probe, cationic conjugated polymer/ DNA complexes and graphene oxide-mediated DNA probe have been developed for assaying DNA MTases (Li et al., 2007; Feng et al., 2007). Colorimetric assays based on gold nanoparticle (AuNP) aggregation or horseradish peroxidase-mimicking DNAzyme were reported for detection of DNA MTase (Song et al., 2009; Li et al., 2010). In addition, electrochemical biosensors based on the use of ferrocene, alkaline phosphatase or AuNPs as labels have also been developed for assay of DNA MTase activity (He et al., 2011; Wang et al., 2010; Liu et al., 2011; Wu et al., 2012). Albeit substantial progress was accomplished, a method that is simpler and more sensitive still is necessitated. Carbon nanotubes (CNTs) attracts growing interest as a new, water-soluble material for biological studies due to their unique structural, mechanical and optoelectronic properties (Wang et al., 2013; Chen et al., 2011). Particularly interesting is the interaction of nucleic acids with CNTs. It is found that single-stranded nucleic
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acids adsorb strongly on CNTs, while duplex DNAs cannot bind to MWCNTs stably (Tang et al., 2006). This property has been employed to elaborately design sensors for various molecular targets in homogeneous solution (Zhang et al., 2010a; Wu et al., 2008). For example, the desorption of a fluorophorelabeled single-stranded DNA by the complementary nucleic acid was used for rapid and sensitive fluorescence detection of nucleic acids (Liu et al., 2009; Yang et al., 2008; Zhu et al., 2008). Similarly, the desorption of fluorophore-labeled nucleic acids from CNTs through the generation of aptamer-substrate complexes was implemented to develop CNT-based sensor systems for fluorescent detection of biomolecules (Zhen et al., 2010; Zhang et al., 2011a). In addition, the release of fluorophore-labeled metal-specific oligonucleotides from CNTs through the formation of metal ionmediated duplex DNA structures was also used for the fluorescence detection of metal ions (Zhao et al., 2010; Zhang et al., 2010b). Although these techniques can be quite powerful, greater sensitivity and specificity are often required, particularly when working with limited amounts of sample material or when target concentration is extremely low. Fluorescence polarization (FP) is a simple signaling approach that provides a quantitative measure for the rotational motion of a fluorescently labeled molecule. The FP value P is sensitive to changes in the rotational motion of fluorescently labeled molecules, which depends on the size of molecule. If a fluorescence molecule is free in solution, it rotates at a rate commensurate with its size and hence will have a relatively small P value. However, when the fluorescence molecule binds with another substance to form a complex, its rotational rate decreases and the P value will increase; the degree of variation depends on the strength of the binding interaction and the size of the formed complex (Zhang et al., 2011b; Deng et al., 2007). This technique has found widespread applications in monitoring the binding events of biomolecules and asssaying of various biomolecules or cells (Deng et al., 2010; Ruta et al., 2009; Zhang et al., 2011c, 2012; Perrier et al., 2010). Recently, to improve the detection sensitivity, several amplifying strategies that employ proteins (Cui et al., 2012; Zhu et al., 2012), AuNPs (Yin et al., 2010; Ye and Yin, 2008; Huang et al., 2011), silica nanoparticles (Huang et al., 2012), and graphene oxide (Liu et al., 2013; Yu et al., 2013) as FP enhancers for detection of small molecules and proteins have been proposed. Nonetheless, FP-based sensing platforms that implement CNTs as a signal amplifier are at present unknown. In the present work, we report a novel amplified sensing platform based on multiwalled carbon nanotube (MWCNT) signal amplification and fluorescence polarization (FP) for highly sensitive and selective detection of DNA MTase activity and inhibition. To the best of our knowledge, this is the first example of using CNTs as a signal amplifier for FP assay of biomolecules. Compared with traditional methods for DNA MTase assays, this proposed protocol does not require separation and troublesome procedures, which is very simple and fast. Most importantly, the introduction of MWCNTs causes a significant amplification of the detection signal, which substantially improves the detection sensitivity by two orders of magnitude over the previously reported methods. Moreover, the application of this MWCNTbased FP sensing platform for DNA MTase inhibitor screening has also been demonstrated.
2. Experimental 2.1. Materials and reagents All oligonucleotides were purchased from the Sangon Biotech Co. (Shanghai, China) and purified by HPLC. The sequences of the
involved oligonucleotides were as follow: 5′-CGA TCC CGC TGC CGG GCC CCG CTG CCC TGT GCC GAA TTT TTT-3′ (DNA-1) and 5′-GGC CCG GCA GCG GGA TCG-FAM-3′ (cDNA-1) were used for Dam MTase detection; and 5′-GTG AAT TCC ATC CGA CCC CCG CTG TCA TGT GCC GAA TTT AAA -3′ (DNA-2) and 5'-GGG TCG GAT GGA ATT CAC-FAM-3′ (cDNA-2) were used for assaying EcoRI MTase. The Dam MTase, AluI MTase, EcoRI MTase, DpnII endonuclease, EcoRI endonuclease and S-adenosyl-L-methiolnine (SAM) were purchased from New England Biolabs (NEB, UK). Standardized human serum was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Multiwalled carbon nanotubes (MWCNTs) were purchased from Shenzhen Nanotech Port Co. Ltd. (Shenzhen, China), and oxidized and purified as described previously (Zhen et al., 2010). Other chemicals were of analytical grade. Water was purified by using a Milli-Q plus 185 equip from Millipore (Bedford, MA).
2.2. Apparatus Fluorescence polarization (FP) measurements were carried out using an FL3-P-TCSPC system (Jobin Yvon, Inc., Edison, NJ, USA) with 300 μL cuvette. The FP of the sample solution was monitored by exciting the sample at 494 nm and measuring the emission at 520 nm (Fig. S1). And slits for both the excitation and the emission were set at 5 nm. HPLC analysis was performed by using an LC-10ATVP system equipped with RF-10AXL fluorescence detector (Shimadzu, Kyoto, Japan). The assays were carried out on a C18 column (250 4.6 mm i.d., 5 μm particle sizes, Elite, China). The HPLC assay used 494 nm and 520 nm as excitation and detection wavelengths, respectively.
2.3. DNA MTase activity assay To prepare duplex DNA substrate of Dam MTase (dsDNA-1), 2.5 μL of 100 μM FAM-labeled DNA-1 was mixed with a 1.2-fold excess of cDNA-1 in pH 7.5 buffer containing 50 mM Tris–HCl and 50 mM NaCl. Then, the mixture was annealed by heating to 90 1C for 5 min and followed by slow cooling at room temperature for 1 h. For Dam MTase assay, reaction mixture was prepared by mixing Dam MTase enzyme stock with 45 nM annealed dsDNA-1 in 200 μL reaction buffer (pH 7.5 Tris–HCl buffer containing 50 mM Tris, 50 mM NaCl, 10 mM dithiothreitol, 0.3 mM SAM) and incubated at 37 1C for 1 h. Then, 50 μL of DpnII reaction buffer containing 50 units of DpnII was added and incubated for 30 min. Finally, 50 μL of MWCNTs solution were added to the above reaction mixture to give a final concentration of 60 μg/mL, and incubated for another 30 min. The obtained sample solution was used for FP measurements. FP was measured by using the L-format configuration and FluorEssence™ software with constant wavelength analysis to achieve a FP value. The G factor was initially set to zero, to let the system measure G automatically. The FP value was also calculated automatically by the instrument. The integration time was set to 3 s for the FP measurements. Over five FP measurements were taken each time, and they were then averaged for further data processing. In case of EcoRI MTase, duplex DNA substrate of EcoRI MTase (dsDNA-2) was annealed by mixing 2.5 μL of 100 μM FAM-labeled DNA-2 and a 1.2-fold excess of cDNA-2 in pH 7.5 buffer containing 50 mM Tris–HCl and 50 mM NaCl. Then, the mixture was annealed by heating to 90 1C for 5 min, followed by slow cooling at room temperature for 1 h. Other procedure for EcoRI MTase activity assay was similar to detection of EcoRI MTase activity mentioned above, except that Dam MTase, dsDNA-1 and DpnII endonuclease were replace of EcoRI MTase, and dsDNA-2, and EcoRI endonuclease, respectively.
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2.4. DNA MTase inhibition assay Dam MTase was used as a model enzyme for inhibition assay. For Dam MTase inhibition assay, reaction mixture was prepared by mixing 5 units Dam MTase and different concentrations of inhibitors with 45 nM annealed dsDNA-1 in 200 μL reaction buffer (pH 7.5 Tris–HCl buffer containing 50 mM Tris, 50 mM NaCl, 10 mM dithiothreitol, 0.3 mM SAM) and incubated at 37 1C for 1 h. Other assay steps were the same as those of the detection of Dam MTase activity described above. 2.5. HPLC assay Samples for HPLC assays were prepared as follows: Dam MTase (100 units) complexed with 45 nM annealed dsDNA-1 was added into 200 μL reaction buffer (pH 7.5 Tris–HCl buffer containing 50 mM Tris, 50 mM NaCl, 10 mM dithiothreitol, 0.3 mM SAM) and incubated at 37 1C for 6 h. Then, 50 μL of DpnII reaction buffer containing 50 units of DpnII was added and incubated for 30 min. After that, 50 μL of MWCNTs solution was added to the above reaction mixture to give a final concentration of 60 μg/mL, and incubated for another 30 min. Finally, the reaction mixture was centrifuged to collect the supernatant and analyzed by HPLC. Blank samples were prepared similar to the procedure mentioned above except without the addition of Dam MTase. HPLC assay was carried out on a C18 column at a rate of 1.0 mL/min, with a 25 min gradient from 10 to 17% acetonitrile in 0.1 M triethylammonium acetate at pH 7.0.
3. Results and discussion 3.1. Principle of the MWCNT-based FP sensing platform Scheme 1 depicts the working principle of the MWCNT-based amplified FP assay for the detection of DNA MTase. This assay uses a fluorescein amidite (FAM)-labeled DNA probe, which contains a double-stranded DNA (dsDNA) part to serve as the specific recognition sequence of both DNA MTase and its corresponding restriction endonuclease, and a single-stranded DNA part for anchoring the DNA to the surface of MWCNTs. In the absence of DNA MTase, this FAM-labeled DNA probe is cleaved by restriction endonuclease, and generates the very short DNA fragments carrying the FAM dye that cannot bind to MWCNTs stably. In this situation, the FAM dye exhibits relatively small P value. However,
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in the presence of target DNA MTase, the specific recognition sequence within the FAM-labeled DNA probe is methylated and blocked the cleavage by its corresponding restriction endonuclease. Thus, the FAM-labeled methylated DNA product is adsorbed onto CNTs via strong π–π stacking interactions between the single-stranded DNA within the FAM-labeled methylated DNA product and the carbon nanotube sidewalls (Liu et al., 2009; Zhen et al., 2010; Zhu et al., 2008), which leads to a significant increase in the P value due to the enlargement of the molecular volume of the formed FAM-labeled methylated DNA/MWCNTs complexes with slow rotation. By monitoring the increase in the P value of the FAM dye, we could detect the target DNA MTase with very high sensitivity. In addition, this assay can be easily adapted to screening inhibitors for DNA MTase because DNA methylation by a DNA MTase is restrained in the presence of inhibitors. Of note, the use of MWCNTs in our new assay provides a novel means of signal amplification, and substantially improves the performance and sensitivity of the FP assay method for detecting DNA MTase in homogeneous solution (Fig. 1). 3.2. Feasibility study To demonstrate the proof-of-principle, the amplified assay was first tested by using Dam MTase and its substrate FAM-labeled dsDNA-1 as models. We investigated the FP responses of the FAMlabeled dsDNA-1 in the absence and presence of Dam MTase with subsequent incubation with MWCNTs. It was found that the P value of the solution with Dam MTase was increased significantly compared with that of the solution without Dam MTase (Fig. 1). The enhancement of the P value was attributed to the fact that the FAM-labeled dsDNA-1 was methylated by Dam MTase and inhibited the DpnII-catalyzed DNA cleavage, and thus the FAM-labeled methylated DNA product was adsorbed onto MWCNTs via noncovalent interactions (Liu et al., 2009; Zhen et al., 2010; Zhu et al., 2008). The FP responses of the FAM-labeled dsDNA-1 in the absence and presence of Dam MTase without MWCNTs were also investigated. It was found that the P value is approximately 19 times higher in the system employing the MWCNTs compared with the system without MWCNTs (Fig. 1). A substantial increase in the P value was due to the enlargement of the molecular volume of the formed FAM-labeled methylated DNA/MWCNTs complexes in solution with slow rotation. These results demonstrated that MWCNTs could be used as an effective signal amplifier for the FP detection of DNA MTase activity. 3.3. HPLC characterization The viability of the proposed strategy was further investigated by HPLC with fluorescence detection. The HPLC assay was carried out by the procedure as described in Experimental section, and the results obtained are shown in Fig. 2. In the presence of DpnII but without Dam MTase, a peak was observed in the electropherogram, indicating the cleavage of the FAM-labeled dsDNA-1 by DpnII (trace a). However, in the presence of both Dam MTase and DpnII, no peak was obtained in the electropherogram (trace b). This fact demonstrated that the DNA methylation by Dam MTase inhibited the DpnII-catalyzed DNA cleavage, and the FAM-labeled methylated DNA product was adsorbed onto MWCNTs, which provides a solid foundation for the present assay. 3.4. Optimization of the size and amount of MWCNTs
Fig. 1. Fluorescence polarization changes upon addition of Dam MTase at a concentration of 1 U/mL. A blank (FAM-labeled dsDNA-1 without Dam MTase) was used as a control reference.
In proposed FP amplifying design, the size and amount of MWCNTs were the critical factors because they significantly affected the reporting FP signal. Therefore, it was necessary to optimize the size and amount of MWCNTs. Fig. S2 shows the
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Scheme 1. The principle of the MWCNT-based FP sensing platform for the detection of DNA MTase
Fig. 2. HPLC chromatograms obtained from an incubation solution of the FAMlabeled dsDNA-1 in the absence (a) and presence (b) of Dam MTase with subsequent incubation with DpnII and MWCNTs. Fluorescence detection wavelength was 520 nm for all traces.
effects of the size of the MWCNTs on the P values. Increasing the diameter of the MWCNTs (average diameter: 16 nm, 28 nm, 47, and 65 nm) changed the P values more obviously when the same concentration of Dam MTase was used. Therefore, MWCNTs with an average diameter of about 65 nm was used in this study. In addition, the amount of MWCNTs was also optimized. As shown in Fig. 3, the P value increased gradually with an increase in the concentration of MWCNTs from 0 to 60 μg/mL. Further increase of MWCNTs concentration, the P value decreased slightly. This phenomenon might be attributed by following reasons: (1) the increase of MWCNTs concentration would result in the adsorption of more amount of FAM-labeled methylated DNA product onto MWCNTs, and led to an increase of the reporting FP signal; (2) Too high of MWCNTs concentration would inhibit the release of DpnIIinduced cleaved products from the surface of MWCNTs, and hence reduced the reporting FP signal. Based on the above results, a concentration of 60.0 μg/mL MWCNTs was used for the following experiments. 3.5. Dam MTase activity assay To confirm the ability of the proposed MWCNT-based amplified FP assay to detect target DNA MTase, a series of different
Fig. 3. The effects of MWCNTs concentration on the fluorescence polarization changes of FAM-labeled dsDNA-1 upon analyzing Dam MTase at a concentration of 0.05 U/mL.
concentrations of Dam MTase were measured. Fig. 4 shows the FP responses of the sensing system for different Dam MTase concentrations. As the concentration of Dam MTase increased, the P value increased. This was consistent with the fact that the enhanced inhibition of the DpnII-catalyzed DNA cleavage by Dam MTase-catalyzed more DNA methylation, and the higher amount of FAM-labeled methylated DNA were adsorbed onto MWCNTs. This method allowed the detection of Dam MTase as low as 1.0 10 4 U/mL, which was two orders of magnitude lower than that of the reported fluorescence (Li et al., 2007), colorimetric (Song et al., 2009; Li et al., 2010) and electrochemical (He et al., 2011; Wang et al., 2010; Liu et al., 2011; Wu et al., 2012) methods. In contrast, when the FP assay method without MWCNT signal amplification was used, a sensitivity that corresponds to a Dam MTase concentration of 1 U/mL was obtained (Fig. 4). The substantial sensitivity improvement of the MWCNT-based amplified FP assay method was attributed to the slower rotation of fluorescent unit when DpnII-catalyzed DNA cleavage was inhibited by Dam MTase-catalyzed DNA methylation and the FAM-labeled methylated DNA product was adsorbed onto MWCNTs. The MWCNT-based amplified FP assay for Dam MTase was also specific. To evaluate this property, we challenged the system with two non-targeted DNA MTases: AluI MTase and EcoRI MTase.
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had no influence on the activity of DpnII when the concentrations of the compounds were no more than 1 μM. Afterwards, we tested the effects of the compounds mentioned above at 1 μM on Dam MTase activity to comparison of the inhibition abilities of test compounds. It was found that all of the drugs and the metal complexes inhibited the Dam MTase activity to a certain degree (Fig. 6A). Interestingly, the two metal complexes showed a stronger inhibition on the Dam MTase activity than the anticancer drugs. In addition, we also investigated how the concentration of [OGH] [AuCl4] DMSO influenced the activity of Dam MTase, and the results are shown in Fig. 6B. As the concentration of [OGH] [AuCl4] DMSO increased, inhibition was enhanced. These results demonstrated that the proposed MWCNT-based amplified FP assay has potential application in screening DNA MTase targeted drugs, which has significant value to antibiotics and anticancer therapeutics.
Fig. 4. Plots of fluorescence polarization changes as a function of Dam MTase concentrations for the sensing systems with or without MWCNTs.
3.7. Detection of dam MTase activity in Human serum A significant challenge for enzyme activity analysis is the ability to realize the detection of target in complex biological matrixes. To investigate if the MWCNT-based amplified FP assay
Fig. 5. Specificity of the MWCNT-based amplified FP assay. The concentration of the target Dam MTase was 0.05 U/mL, and other DNA MTases were 50 U/mL each.
Significantly higher P value was observed with the target Dam MTase than with non-targeted DNA MTases (Fig. 5). These results clearly demonstrated the high specificity of our MWCNT-based amplified FP assay. In addition, assay reproducibility was also investigated by analyzing Dam MTase standard solutions (at 0.001, 0.01, and 0.1 U/mL Dam MTase, respectively) five times. The results showed that the relative standard deviations (RSDs) were found to be r4.9%. 3.6. Dam MTase activity inhibition evaluation The MWCNT-based amplified FP assay has the potential for highthroughput screening of inhibitors of Dam MTase. We investigated the effects of two anticancer drugs (5-fluorouracil and cisplatin) and two metal complexes of 5-chloro-7-iodo-8-hydroxylquinoline (ClIQ) and oxoglaucine (OG) ([Sn(ClIQ)2Cl2] and [OGH][AuCl4] DMSO) synthesized in our laboratory (Chen et al., 2012, 2013), on the activity of Dam MTase. Before evaluating the inhibitory effects of the compounds on the activity of Dam MTase, we first investigated whether these compounds had influence on the DpnII activity. The experimental results found that the test compounds
Fig. 6. (A) Influence of different drugs and metal complexes on the activity of Dam MTase. The numbers of the horizontal bar represented different compounds tested: (1) no drug; (2) cisplatin; (3) 5-fluorouracil; (4) [Sn(ClIQ)2Cl2]; (5) [OGH][AuCl4] DMSO. All compounds were tested at 1 μM. (B) The effects of [OGH][AuCl4] DMSO on the activity of Dam MTase at different concentrations.
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method developed here was applicable to complex biological matrixes, standardized human serum sample without Dam MTase (diluted 10 times with 50 mM Tris–HCl buffer) samples and the spiked human serum samples, at three different concentrations of Dam MTase (0.005, 0.05 and 0.5 U/mL), were analyzed using the proposed method. The responses of the MWCNT-based amplified FP assay for Dam MTase in diluted human serum samples were compared with those obtained from Dam MTase in buffer solution. As shown in Fig. 7, comparable responses were found for Dam MTase in both buffer solution and diluted human serum. Then, three independent spiked serum samples were tested by the MWCNT-based amplified FP assay, and the activity of Dam MTase in the spiked serum was calculated by using the calibration curve of standard Dam MTase. The assay results of the spiked serum samples are shown in Table S1. The recoveries were found to be in the range of 92.8–97.6%. The precision of the method was evaluated by repeatedly analyzing each spiked serum sample for five times within one working day. The RSDs were between 2.9% and 4.8%. These results indicated the potentiality of the proposed method for detection of DNA MTase in real biological samples.
Fig. 8. Plots of fluorescence polarization changes as a function of EcoRI MTase concentrations for the sensing systems with or without MWCNTs.
MTase standard solutions (at 0.001, 0.01, and 0.1 U/mL Dam MTase, respectively) five times. 3.8. EcoRI MTase activity assay To illustrate the generality of our design, we applied this strategy to detect another DNA MTase, EcoRI MTase. By replacing the duplex DNA substrate of Dam MTase with the duplex DNA substrate (dsDNA-2) for EcoRI MTase, we similarly made the MWCNT-based amplified FP assay for detecting EcoRI MTase. As shown in Fig. 8, the MWCNT-based amplified FP assay could sensitively detect EcoRI MTase with a detection limit of 1.0 10 4 U/mL, which was 104 times lower than that of the FP assay strategy without MWCNT signal amplification. Meanwhile, this sensitivity was also two orders of magnitude higher than that of the reported fluorescence method (Feng et al., 2007). The MWCNT-based amplified FP assay also showed high specificity toward EcoRI MTase over other non-targeted DNA MTases, such as Dam MTase and AluI MTase. In addition, this assay also exhibited good reproducibility with RSDs less than 4.7% for assaying EcoRI
4. Conclusions In summary, we have developed a novel amplified sensing platform based on MWCNT signal amplification and FP detection for the assay of DNA MTase activity and inhibition in homogeneous solution. As a proof-of-concept, we demonstrate that this amplified assay can respectively detect Dam MTase and HaeIII MTase with high sensitivity and specificity. Furthermore, the application of this MWCNT-based sensing platform for DNA MTase inhibitor screening has also been demonstrated. This assay technique has several important features. First, by using MWCNT signal amplification approach, the sensitivity of this new type of assay can be significantly improved by two orders of magnitude over the previously reported methods (see Table S2). Second, compared with traditional methods for DNA MTase detection, this assay is done in homogeneous solution, and not requiring separation and troublesome procedures, which is quite simple and convenient. Third, the assay can be easily adapted to high throughput applications in an array format. Finally, this method may also be extended to detect other DNA MTases and screen their inhibitors by simply switching the corresponding DNA substrates. Also, given that endonucleases can cleave their specific DNA substrates, we expect that this new methodology may also find potential application in the detection of endonucleases through FP reduction. These quanlities endow the proposed MWCNT-based amplified FP sensing platform with great promise in DNA MTase-related basic research and drug development.
Acknowledgments
Fig. 7. Results obtained from the testing of human serum samples spiked with Dam MTase (red column) and Dam MTase in Tris–HCl buffer (black column). The numbers of the horizontal bar represented different concentrations of Dam MTase in human serum and Tris–HCl buffer: (1) 0 U/mL; (2) 0.005 U/mL; (3) 0.05 U/mL; (4) 0.5 U/mL. Human serum was diluted in 1:10 ratio with Tris–HCl buffer. (For interpretation of the references to color in this figure caption, the reader is referred to the web version of this article.)
This work was supported by the National Natural Science Foundations of China (No. 21175030), the National Basic Research Program of China (No. 2012CB723501), and the Natural Science Foundations of Guangxi Province (No. 2012GXNSFDA385001, 2013GXNSFBA019038, 2013GXNSFBA019044) as well as BAGUI Scholar Program and the project of Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Education of China (CMEMR2012-A19, CMEMR2013C06).
Y. Huang et al. / Biosensors and Bioelectronics 54 (2014) 285–291
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