Nucleic Acid-Induced Tetraphenylethene Probe Noncovalent Self

Sep 9, 2014 - Wenying Li , Qingfeng Zhang , Huipeng Zhou , Jian Chen , Yongxin Li ... Yunyi Zhang , Yongxin Li , Na Yang , Xue Yu , Chunhua He , Niu N...
0 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/ac

Nucleic Acid-Induced Tetraphenylethene Probe Noncovalent SelfAssembly and the Superquenching of Aggregation-Induced Emission Jian Chen,† Yan Wang,†,‡ Wenying Li,†,‡ Huipeng Zhou,† Yongxin Li,† and Cong Yu*,†,‡ †

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China ‡ University of the Chinese Academy of Sciences, Beijing 100049, P. R. China S Supporting Information *

ABSTRACT: Superquenching of aggregation-induced emission (AIE) has been utilized in biosensing for the first time. A positively charged tetraphenylethene derivative (compound 1) showed no emission in an aqueous buffer solution. A single-stranded DNA (a polyanion) induced aggregation of compound 1, and strong compound 1 aggregate emission was observed. When the DNA was labeled with a quencher molecule, compound 1 aggregate emission was efficiently quenched. On the basis of this observation, a new, simple, sensitive and selective DNA methyltransferase (MTase) assay has been developed. A quencher-labeled double-stranded DNA could induce aggregation of compound 1, and superquenching of compound 1 AIE was observed. In the presence of MTase and an endonuclease, the DNA could be specifically methylated and cleaved into single-stranded DNA fragments. The quencher molecule was released, and a turn-on emission signal was detected.

F

number of novel biosensing techniques for the sensing of enzyme activities, nucleic acids, and other biomolecules.29−32 However, the synthesis of CPEs is rather complicated, timeconsuming, and expensive, and the water solubility of CPEs is relatively poor, which limits their potential applications. In this work, we report for the first time the superquenching of AIE. A positively charged TPE derivative (compound 1, Scheme 1) was synthesized. Compound 1 monomer showed no emission in an aqueous buffer solution. A single-stranded DNA (a polyanion) induced aggregation of compound 1 via electrostatic interactions, and strong compound 1 aggregate emission was observed. When the DNA was labeled with a small molecule quencher (dabcyl, Figure S1, Supporting Information), compound 1 aggregate emission was efficiently quenched due to the fluorescence resonance energy transfer (FRET) from compound 1 aggregate to the quencher (Scheme 1a). By using this type of superquenching, a new method for the sensitive and selective detection of DNA methyltransferase (MTase) activity has been developed. DNA methylation is the covalent attachment of a methyl group to the target cytosine or adenine base in a specific DNA sequence.33 It plays important roles in many biological processes, such as genomic expression, transcription, and

luorometric methods based on noncovalent self-assembly of small molecular probes have been widely used in bioanalysis and biosensing.1−9 Aggregation-induced emission (AIE) based fluorometric assay is a new technique developed in recent years.6−11 AIE is a unique photophysical phenomenon.12,13 The molecules with AIE characteristics are nonemissive in the monomeric form but are highly emissive in the aggregated form due to the restriction of intramolecular rotations, which reduces considerably the energy dissipation through the nonradiative means.14,15 Tetraphenylethene (TPE), a propeller-shaped compound, is a well-known AIE fluorogen.16 A single-stranded DNA (a polyanion) could induce strong aggregation of a cationic TPE derivative in an aqueous buffer solution. As a result, strong TPE emission could be observed.17−19 Fluorogen−quencher pairs have been widely used for the development of many novel biosensing techniques.20−22 For instance, covalent labeling of a fluorogen and a quencher at the two termini of a hairpin-shaped oligonucleotide has been used as a molecular beacon probe for the sensing of nucleic acids, proteins, enzyme activities, etc.23−25 Superquenching of the fluorescence of conjugated polyelectrolytes (CPEs) has been reported in the literature.26−28 It was observed that the fluorescence of CPEs could be quenched by very low amounts of oppositely charged small molecule quenchers. The quenching of excitation could pass through a large number of polymer repeating units via electron or energy transfer. This phenomenon has been utilized for the development of a © 2014 American Chemical Society

Received: July 8, 2014 Accepted: September 9, 2014 Published: September 9, 2014 9866

dx.doi.org/10.1021/ac502496h | Anal. Chem. 2014, 86, 9866−9872

Analytical Chemistry

Article

Reagent Database Inc. (Shanghai, China). All oligonucleotides, including the quencher-labeled oligonucleotides, were synthesized and Ultra-PAGE purified by Sangon Biological Engineering Technology & Service Co. Ltd. (Shanghai, China) (Table 1). Dam MTase, M.SssI MTase, and DpnI were purchased

Scheme 1. (a) Nucleic Acid-Induced TPE Probe Noncovalent Self-Assembly and the Superquenching of AIE. (b) Schematic Illustration of the Dam MTase Activity Assay Strategy

Table 1. Oligonucleotides Used in the Current Investigation (5′ → 3′)a

cellular differentiation.34,35 A number of human diseases have been found to be associated with abnormal gene methylations.36−38 The methylation process is catalyzed by DNA MTase using S-adenosyl-methionine (SAM) as the methyl donor. Sensing DNA MTase activity and its inhibitor screening are of great importance in many areas of basic research and clinical diagnosis. In recent years, DNA MTase has been detected by a number of electrochemical,39−42 chemiluminescent,43 electrochemical luminescent,44,45 colorimetric,46−48 and fluorometric49−54 methods. Fluorometric methods have drawn more attention because of the higher sensitivity and ease of operation compared with the other methods. In this work, based on the superquenching of AIE, a novel fluorescent turn-on DNA MTase sensing method has been developed (Scheme 1b). A quencher-labeled double-stranded DNA could induce aggregation of compound 1, and the superquenching of compound 1 AIE was observed. In the presence of MTase and an endonuclease, the DNA could be specifically methylated and then cleaved into single-stranded DNA fragments. The quencher molecule was released, and a turn-on emission signal was detected.

a

The complementary sequences are marked with underline, the Dam MTase recognition sites are given in red, and the DpnI cleavage point is marked with “↓”.

from New England Biolabs, Inc. (USA). All other chemicals were of analytical grade and used without further purification. All stock and buffer solutions were prepared with water purified with Milli-Q A10 (Millipore, Billerica, MA, USA). The nucleic acid and enzyme solutions were stored at 4 °C before use. Duplex DNAs (dsDNA1 = DNA-a + DNA20; dsDNA2 = DNA-b + DNA20; dsDNA3 = DNA-c + DNA-d) were obtained using a simple annealing procedure. Equimolar amounts (50 μM) of the complementary single-stranded nucleic acid strands were mixed in an aqueous buffer solution (5 mM Tris-HCl, 50 mM NaCl, pH 8.0). The mixtures were incubated in a 90 °C water bath for about 10 min and gradually cooled to ambient temperature. The obtained duplex DNA sample solutions were stored at 4 °C before use. Superquenching of AIE by Quencher-Labeled DNA. Different quencher-labeled DNAs were added to the solution of compound 1 (20 μM) in 5 mM Tris-HCl buffer at pH 8.0 (for double-stranded DNA, 50 mM NaCl was added). The samples were mixed briefly and stabilized at 10 °C for 3 min, and then the emission spectra were recorded (total sample volume: 400 μL). Sensing of Dam MTase Activity. A total volume of 50 μL of the sample mixture containing 2 μM dsDNA3-2Q, the reaction buffer [1 μL was added; buffer composition: 200 mM Tris-HAc, 500 mM KAc, 100 mM Mg(Ac)2, 10 mM DTT, pH 7.9], 80 μM SAM, 400 U/mL DpnI, and Dam MTase of various concentrations (0, 0.25, 0.5, 1, 2.5, 5, 10, 20, 40, 80, and 160 U/mL, respectively) was incubated at 37 °C for 5 h. The sample solutions were incubated at 90 °C for 10 min to

■

EXPERIMENTAL SECTION Instrumentation. Emission spectra were recorded with a Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon Inc., USA). Excitation wavelength was 350 nm, and the emission spectra were corrected against PMT response. Excitation and emission slit widths were both 5 nm. Quartz cuvettes with 10 mm path length and 2 mm window width were used for the emission measurements. Materials. Compound 1 was prepared following the literature procedures (Scheme S1, Figures S6, S7, and S8).17−19 SYBR Green I (10000× concentrate) was purchased from Generay Biotech Co. (Shanghai, China). 5-Fluorouracil, gentamycin, and benzylpenicillin were purchased from Aladdin 9867

dx.doi.org/10.1021/ac502496h | Anal. Chem. 2014, 86, 9866−9872

Analytical Chemistry

Article

inactivate Dam MTase and DpnI, and then they were allowed to cool to ambient temperature. The resulting solutions were added to a solution mixture of compound 1 (final concentration: 20 μM). The samples were mixed briefly and stabilized at 25 °C for 3 min, and then the emission spectra were recorded (total sample volume: 100 μL). Dam MTase Activity Assay in Biological Fluid. A total volume of 50 μL of the sample mixture containing 1% calf serum, 2 μM dsDNA3-2Q, the reaction buffer [1 μL was added; buffer composition: 200 mM Tris-HAc, 500 mM KAc, 100 mM Mg(Ac)2, 10 mM DTT, pH 7.9], 80 μM SAM, 400 U/mL DpnI, and Dam MTase of various concentrations (0, 5, 10, 20, and 40 U/mL, respectively) was incubated at 37 °C for 5 h. And the Dam MTase activity was determined as described in the “Sensing of Dam MTase Activity” section. Dam MTase Inhibitor Screening. A total volume of 50 μL of the sample mixture containing 2 μM dsDNA3-2Q, the reaction buffer [1 μL was added; buffer composition: 200 mM Tris-HAc, 500 mM KAc, 100 mM Mg(Ac)2, 10 mM DTT, pH 7.9], 80 μM SAM, 400 U/mL DpnI, 10 U/mL Dam MTase, and inhibitors of various concentrations (1 μM for 5fluorouracil and benzylpenicillin; 0, 0.25, 0.5, 0.75, and 1 μM for gentamycin, respectively) was incubated at 37 °C for 5 h. MTase activity was determined as described in the “Sensing of Dam MTase Activity” section. The relative activity (RA) of Dam MTase was defined as (RA) = [F(inhibitor) − F0]/[F(no inhibitor) − F0]

in which the F(inhibitor) and F(no inhibitor) referred to the emission intensity of compound 1 in the presence or absence of gentamycin, and F0 referred to the background emission intensity of compound 1.43

■

RESULTS AND DISCUSSION

Superquenching of AIE by Quencher-Labeled SingleStranded DNA. When 2 μM single-stranded DNA (DNA20, 20 base long) was added to a solution of 20 μM compound 1, a strong emission band (F0) with the band maximum at 470 nm was observed. By contrast, when 2 μM of the quencher-labeled DNA20 (DNA20-Q) was introduced, only a very small emission band (F Q ) was observed (Figure 1a). The corresponding maximum emission intensity ratio (the F0/FQ value) was 10.6 (Figure 1c). The results clearly suggest that the compound 1 AIE was efficiently quenched due to the FRET from the noncovalent TPE aggregates to the quencher. The influence of DNA chain length on the quenching efficiency was studied. Figure 1b shows that when longer DNA strands (1 μM DNA40-Q, 40 base long; 0.67 μM DNA59-Q, 59 base long) were mixed with 20 μM compound 1, less efficient quenching of compound 1 AIE was observed. The F0/FQ values were 3.3 for DNA40-Q and 2.3 for DNA59-Q. The results indicate that the use of a nucleic acid of longer chain length could cause part of the compound 1 aggregates to move further away from the quencher molecule, which were more difficult to quench. In addition, when both ends of a nucleic acid were labeled with quencher molecules, better quenching effect was obtained. For instance, upon the addition of 2 μM two-quencher-labeled DNA20 (DNA20-2Q), the F0/FQ value reached 23.7 (a quenching efficiency of about 95.8%), which is much higher than that of DNA20-Q. The fluorescence quenching could be easily observed by the naked eye, as indicated in the inset in Figure 1c.

Figure 1. (a, b) Quenching of compound 1 (20 μM) AIE by quencher-labeled single-stranded DNAs of different chain length (Q: with one labeled quencher; 2Q: with two labeled quenchers). The nucleic acid base concentration was kept constant at 40 μM in all cases. (c) The corresponding F0/FQ values. Inset: photograph of the sample solutions of compound 1 in the presence of DNA20 (left) and DNA20−2Q (right) under UV light illumination (365 nm). Buffer: 5 mM Tris-HCl, pH 8.0.

Superquenching of AIE by Quencher-Labeled Double-Stranded DNA. Quencher-labeled double-stranded DNA could also induce efficient quenching of compound 1 AIE. Four different double-stranded DNAs were selected (Figure 2). The complementary duplexes all consist of a short single strand (20 base long) labeled with one or two quenchers and a long single

Figure 2. Quencher-labeled double-stranded DNAs used in the current investigation. 9868

dx.doi.org/10.1021/ac502496h | Anal. Chem. 2014, 86, 9866−9872

Analytical Chemistry

Article

base long single strand labeled with two quenchers at both ends, and a 42 base long complementary strand. dsDNA3-2Q could induce aggregation of compound 1, and the superquenching of compound 1 AIE was observed. (2) dsDNA3-2Q contained the Dam recognition sequence (5′-G-A-T-C-3′) at the middle of the duplex DNA, which could be methylated (5′G-mA-T-C-3′) by Dam MTase in the presence of SAM. DpnI could specifically recognize and cleave the fully methylated GmA-T-C site. (3) In the presence of Dam MTase and DpnI, dsDNA3-2Q was methylated and cleaved into four singlestranded DNA fragments, which included two short quencherlabeled DNA fragments (7 base long) and two 21 base long DNA fragments. The 21 base long DNA fragments could induce aggregation of compound 1; thus, a turn-on aggregation-induced emission could be detected, and a fluorometric assay for DNA MTase activity was therefore established. Sensing of Dam MTase Activity. Control experiments were conducted to verify the principle of the assay. SYBR Green I is a widely used nucleic acid staining dye. It preferentially binds to double-stranded DNA and produces a significant fluorescence enhancement. Single-stranded nucleic acid could also be stained by SYBR Green I and gave a much weaker emission enhancement.54,55 Figure S2 shows that when SYBR Green I was added to the solution of dsDNA3 (with no quencher labeled), a strong emission band was observed. After the enzymatic reaction with Dam MTase and DpnI, significant decrease of emission intensity was observed. The results clearly indicate that dsDNA3 could be methylated and cleaved in the presence of Dam MTase and DpnI, and then dissociated into four single-stranded fragments (7, 7, 21, and 21 base long, respectively) under the experimental conditions (25 °C). The kinetics of the Dam MTase enzymatic reaction was studied. Figure 4 shows that, in the presence of 160 U/mL Dam MTase and 400 U/mL DpnI, the emission intensity of compound 1 increased gradually with prolonged enzymatic reaction time. After 5 h of the enzymatic reaction, the emission intensity reached its maximum. The results suggest that an increasing percentage of the substrate strand (dsDNA3-2Q) was gradually methylated by Dam MTase and then cleaved by DpnI with the increase of the enzymatic reaction time. And increasing percentage of the quencher molecule was released; therefore, increased intensity of compound 1 AIE was observed. Figure 5 shows that the emission intensity of compound 1 increased gradually with the addition of increasing concentrations of Dam MTase (0−160 U/mL). A linear relationship was obtained at a Dam MTase concentration range of 0−20 U/ mL. The linear regression equation is F = 7.29C + 65.84 (correlation coefficient R2 = 0.995), in which F is the emission intensity of compound 1 at 470 nm and C is the Dam MTase concentration in U/mL. Our assay is quite sensitive compared with the previously reported fluorometric methods.49,52,53 Without the use of any enzyme based signal amplification, the activity of 0.25 U/mL Dam MTase could be easily detected. Selectivity Study. The selectivity of the assay was also studied. M.SssI MTase, another DNA MTase, was selected as a potential interference enzyme. It can specifically methylate the cytosine residues within the double-stranded DNA recognition sequence of 5′-C-G-3′.43,54 Figure 6 shows that, under the same experimental conditions, 160 U/mL Dam MTase could induce a significant compound 1 emission increase. In contrast, M.SssI MTase of the same concentration could not induce an obvious

strand (59 base long) without any labeling. It was observed that when the quencher molecule was located relatively away from the center of the duplexes, the quenching of compound 1 AIE was less efficient (Figure 3a). The corresponding F0/FQ values

Figure 3. (a, b) Quenching of compound 1 (20 μM) AIE by different quencher-labeled double-stranded DNAs (500 nM, Q: with one labeled quencher, 2Q: with two labeled quenchers). (c) The corresponding F0/FQ values. Buffer: 5 mM Tris-HCl, 50 mM NaCl, pH 8.0.

were 1.31 and 1.91 for single (dsDNA1-Q) and double (dsDNA1-2Q) quencher-labeled duplexes, respectively (Figure 3c). In contrast, for the duplexes with the quencher located at the middle, more efficient quenching of compound 1 AIE was observed (Figure 3b). The corresponding F0/FQ values reached 2.68 and 10.54 for single (dsDNA2-Q) and double (dsDNA22Q) quencher-labeled duplexes, respectively. This observation formed the basis for the DNA MTase activity detection. MTase Activity Sensing Strategy. DNA adenine methylation (Dam) MTase and the restriction endonuclease DpnI were chosen as the model MTase and endonuclease, respectively. The overall sensing strategy is schematically illustrated in Scheme 1b. (1) A quencher-labeled doublestranded DNA (dsDNA3-2Q) was used. It consisted of a 14 9869

dx.doi.org/10.1021/ac502496h | Anal. Chem. 2014, 86, 9866−9872

Analytical Chemistry

Article

Figure 4. (a) Changes in the emission spectrum of compound 1 with enzymatic reaction time. (b) Plot of the changes in emission intensity of compound 1 at 470 nm against enzymatic reaction time.

Figure 5. (a) Changes in the emission spectrum of compound 1 with Dam MTase concentration (0, 0.25, 0.5, 1, 2.5, 5, 10, 20, 40, 80, and 160 U/mL). (b) Plot of the changes in the emission intensity of compound 1 at 470 nm against Dam MTase concentration. Inset: expanded linear region of the curve.

emission increase. The results clearly suggest that our assay is highly selective for Dam MTase. Dam MTase Activity Assay in Biological Fluid. The assay was also tested in complex sample mixtures. Sample mixtures containing 1% calf serum and Dam MTase of various concentrations were tested. The assay was conducted under the same experimental conditions. Figure S3 shows that the more Dam MTase was spiked, the larger the emission intensity increase of compound 1 was obtained. The results clearly indicate that our assay could be used in complex sample mixtures. Inhibitor Screening. The assay could also be used for the screening of potential DNA MTase inhibitors. A number of drugs, such as 5-fluorouracil, benzylpenicillin, and gentamycin, were tested. It was reported that all these drugs had no inhibition effect on the activity of DpnI when their concentrations were no more than 1 μM.49 Thus, the drugs at the concentration of no more than 1 μM were used in our study. While maintaining the concentration of each drug at 1 μM, the influence of the drugs on the activity of Dam MTase was investigated. Figure S4 shows that the emission intensity of compound 1 decreased significantly upon the addition of the drugs. These drugs inhibit the activity of Dam MTase differently, and Figure 7a shows that 5-fluorouracil is the most effective inhibitor. In addition, the emission intensity of compound 1 decreased gradually with the increase of 5fluorouracil concentration (Figure S5), which indicates that the inhibition was more effective at higher drug concentrations (Figure 7b). The results clearly suggest that our assay could be used for the screening of potential DNA MTase inhibitors.

Figure 6. Selectivity of the assay. The concentrations of Dam MTase and M.SssI MTase are both of 160 U/mL.

■

CONCLUSIONS In summary, we report for the first time the use of superquenching of AIE in biosensing. A single-stranded DNA induced aggregation of a positively charged TPE derivative (compound 1), and strong compound 1 aggregate emission was observed. When the DNA was labeled with a quencher molecule, compound 1 aggregate emission was efficiently quenched. On the basis of this observation, a new, simple, sensitive, and selective DNA MTase assay has been developed. Our assay has several important features. First, superquenching of AIE was utilized for the first time. AIE could be efficiently quenched by a simple quencher molecule. Second, it is based on the fluorescence “turn-on” mode, which could reduce 9870

dx.doi.org/10.1021/ac502496h | Anal. Chem. 2014, 86, 9866−9872

Analytical Chemistry

Article

(4) Liao, D.; Chen, J.; Zhou, H.; Wang, Y.; Li, Y.; Yu, C. Anal. Chem. 2013, 85, 2667−2672. (5) Chen, J.; Liao, D.; Wang, Y.; Zhou, H.; Li, W.; Yu, C. Org. Lett. 2013, 15, 2132−2135. (6) Liu, Y.; Deng, C.; Tang, L.; Qin, A.; Hu, R.; Sun, J. Z.; Tang, B. Z. J. Am. Chem. Soc. 2011, 133, 660−663. (7) Leung, C. W. T.; Hong, Y.; Hanske, J.; Zhao, E.; Chen, S.; Pletneva, E. V.; Tang, B. Z. Anal. Chem. 2014, 86, 1263−1268. (8) Wang, M.; Gu, X.; Zhang, G.; Zhang, D.; Zhu, D. Anal. Chem. 2009, 81, 4444−4449. (9) Xue, W.; Zhang, G.; Zhang, D.; Zhu, D. Org. Lett. 2010, 12, 2274−2277. (10) Shi, H.; Liu, J.; Geng, J.; Tang, B. Z.; Liu, B. J. Am. Chem. Soc. 2012, 134, 9596−9572. (11) Shi, H.; Kwok, R. T. K.; Liu, J.; Xing, B.; Tang, B. Z.; Liu, B. J. Am. Chem. Soc. 2012, 134, 17972−17981. (12) Luo, J.; Xie, Z.; Lam, J. W. Y.; Cheng, L.; Chen, H.; Qiu, C.; Kwok, H. S.; Zhan, X.; Liu, Y.; Zhu, D.; Tang, B. Z. Chem. Commun. 2001, 37, 1740−1741. (13) Tang, B. Z.; Zhan, X.; Yu, G.; Lee, P. P. S.; Liu, Y.; Zhu, D. J. Mater. Chem. 2001, 11, 2974−2978. (14) Li, Z.; Dong, Y.; Mi, B.; Tang, Y.; Häussler, M.; Tong, H.; Dong, Y.; Lam, J. W. Y.; Ren, Y.; Sung, H. H. Y.; Wong, K. S.; Gao, P.; Williams, I. D.; Kwok, H. S.; Tang, B. Z. J. Phys. Chem. B 2005, 109, 10061−10066. (15) Tong, H.; Dong, Y.; Hong, Y.; Häussler, M.; Lam, J. W. Y.; Sung, H. H.-Y.; Yu, X.; Sun, J.; Williams, I. D.; Kwok, H. S.; Tang, B. Z. J. Phys. Chem. C 2007, 111, 2287−2294. (16) Chen, Q.; Bian, N.; Cao, C.; Qiu, X.-L.; Qi, A.-D.; Han, B.-H. Chem. Commun. 2010, 46, 4067−4069. (17) Tong, H.; Hong, Y.; Dong, Y.; Häussler, M.; Lam, J. W. Y.; Li, Z.; Guo, Z.; Guo, Z.; Guo, Z.; Tang, B. Z. Chem. Commun. 2006, 42, 3705−3707. (18) Hong, Y.; Häussler, M.; Lam, J. W. Y.; Li, Z.; Sin, K. K.; Dong, Y.; Tong, H.; Liu, J.; Qin, A.; Renneberg, R.; Tang, B. Z. Chem.Eur. J. 2008, 14, 6428−6437. (19) Hong, Y.; Xiong, H.; Lam, J. W. Y.; Häussler, M.; Liu, J.; Yu, Y.; Zhong, Y.; Sung, H. H. Y.; Williams, I. D.; Wong, K. S.; Tang, B. Z. Chem.Eur. J. 2010, 16, 1232−1245. (20) Feng, F.; Tang, Y.; Wang, S.; Li, Y.; Zhu, D. Angew. Chem., Int. Ed. 2007, 46, 7882−7886. (21) Wang, B.; Jiao, H.; Li, W.; Liao, D.; Wang, F.; Yu, C. Chem. Commun. 2011, 47, 10269−10271. (22) Zhang, W.; Zhu, L.; Qin, J.; Yang, C. J. Phys. Chem. B 2011, 115, 12059−12064. (23) Yang, C. J.; Pinto, M.; Schanze, K.; Tan, W. Angew. Chem., Int. Ed. 2005, 44, 2572−2576. (24) Wang, K.; Tang, Z.; Yang, C. J.; Kim, Y.; Fang, X.; Li, W.; Wu, Y.; Medley, C. D.; Cao, Z.; Li, J.; Colon, P.; Lin, H.; Tan, W. Angew. Chem., Int. Ed. 2009, 48, 856−870. (25) Tang, D.; Liao, D.; Zhu, Q.; Wang, F.; Jiao, H.; Zhang, Y.; Yu, C. Chem. Commun. 2011, 47, 5485−5487. (26) Jones, R. M.; Bergstedt, T. S.; Buscher, C. T.; McBranch, D.; Whitten, D. Langmuir 2001, 17, 2568−2571. (27) Liu, Y.; Jiang, S.; Schanze, K. S. Chem. Commun. 2003, 39, 650− 651. (28) Achyuthan, K. E.; Bergstedt, T. S.; Chen, L.; Jones, R. M.; Kumaraswamy, S.; Kushon, S. A.; Ley, K. D.; Lu, L.; McBranch, D.; Mukundan, H.; Rininsland, F.; Shi, X.; Xia, W.; Whitten, D. G. J. Mater. Chem. 2005, 15, 2648−2656. (29) Kushon, S. A.; Ley, K. D.; Bradford, K.; Jones, R. M.; McBranch, D.; Whitten, D. Langmuir 2002, 18, 7245−7249. (30) Kushon, S. A.; Bradford, K.; Marin, V.; Suhrada, C.; Armitage, B. A.; McBranch, D.; Whitten, D. Langmuir 2003, 19, 6456−6464. (31) Kumaraswamy, S.; Bergstedt, T.; Shi, X.; Rininsland, F.; Kushon, S.; Xia, W.; Ley, K.; Achyuthan, K.; McBranch, D.; Whitten, D. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7511−7515.

Figure 7. (a) Influence of different drugs on the activity of Dam MTase. (1) No drug, (2) 5-fluorouracil, (3) benzylpenicillin, and (4) gentamycin. Drug concentration: 1 μM each. (b) Relative activity of Dam MTase against 5-fluorouracil concentration.

considerably the likelihood of false-positive signals. Third, compound 1 could be easily prepared, and the assay is fairly simple and cost-effective. Fourth, without the use of any enzyme based signal amplification, sensitive detection could be obtained. We envision that the principle of the assay could also be used for the development of novel sensing techniques for the detection of various biomolecules.

■

ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: (+86)-431-85262710. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (973 Program, No. 2011CB911002), the National Natural Science Foundation of China (21075119), and the Open Project of the State Key Laboratory of Supramolecular Structure and Materials (Grant No. SKLSSM201415).

■

REFERENCES

(1) González, D. C.; Savariar, E. N.; Thayumanavan, S. J. Am. Chem. Soc. 2009, 131, 7708−7716. (2) Wang, B.; Yu, C. Angew. Chem., Int. Ed. 2010, 49, 1485−1488. (3) Mizusawa, K.; Takaoka, Y.; Hamachi, I. J. Am. Chem. Soc. 2012, 134, 13386−13395. 9871

dx.doi.org/10.1021/ac502496h | Anal. Chem. 2014, 86, 9866−9872

Analytical Chemistry

Article

(32) Rininsland, F.; Xia, W.; Wittenburg, S.; Shi, X.; Stankewicz, C.; Achyuthan, K.; McBranch, D.; Whitten, D. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15295−15300. (33) Cheng, X.; Roberts, R. J. Nucleic Acids Res. 2001, 29, 3784− 3795. (34) Heithoff, D. M.; Sinsheimer, R. L.; Low, D. A.; Mahan, M. J. Science 1999, 284, 967−970. (35) Jaenisch, R.; Bird, A. Nat. Gene. Supp. 2003, 33, 245−254. (36) Heithoff, D. M.; Sinsheimer, R. L.; Low, D. A.; Mahan, M. J. Science 1999, 284, 967−970. (37) Jones, P. A.; Baylin, S. B. Nat. Rev. 2002, 3, 415−428. (38) Robertson, K. D. Nat. Rev. 2005, 6, 597−610. (39) Wu, H.; Liu, S.; Jiang, J.; Shen, G.; Yu, R. Chem. Commun. 2012, 48, 6280−6282. (40) Li, W.; Wu, P.; Zhang, H.; Cai, C. Anal. Chem. 2012, 84, 7583− 7590. (41) Wang, Y.; He, X.; Wang, K.; Su, J.; Chen, Z.; Yan, G.; Du, Y. Biosens. Bioelectron. 2013, 41, 238−243. (42) Yin, H.; Zhou, Y.; Xu, Z.; Chen, L.; Zhang, D.; Ai, S. Biosens. Bioelectron. 2013, 41, 492−497. (43) Zeng, Y.; Hu, J.; Long, Y.; Zhang, C. Anal. Chem. 2013, 85, 6143−6150. (44) Wang, M.; Xu, Z.; Chen, L.; Yin, H.; Ai, S. Anal. Chem. 2012, 84, 9072−9078. (45) Li, Y.; Luo, X.; Yan, Z.; Zheng, J.; Qi, H. Chem. Commun. 2013, 49, 3869−3871. (46) Song, G.; Chen, C.; Ren, J.; Qu, X. ACS Nano 2009, 3, 1183− 1189. (47) Li, W.; Liu, Z.; Lin, H.; Nie, Z.; Chen, J.; Xu, X.; Yao, S. Anal. Chem. 2010, 82, 1935−1941. (48) Zheng, X.; Qiu, J.; Zhang, L.; Wang, Z.; Liang, R. Chem. Commun. 2013, 49, 3546−3548. (49) Li, J.; Yan, H.; Wang, K.; Tan, W.; Zhou, X. Anal. Chem. 2007, 79, 1050−1056. (50) Feng, F.; Tang, Y.; He, F.; Yu, M.; Duan, X.; Wang, S.; Li, Y.; Zhu, D. Adv. Mater. 2007, 19, 3490−3495. (51) Lee, J.; Kim, Y.-K.; Min, D.-H. Anal. Chem. 2011, 83, 8906− 8912. (52) Tian, T.; Xiao, H.; Long, Y.; Zhang, X.; Wang, S.; Zhou, X.; Liu, S.; Zhou, X. Chem. Commun. 2012, 48, 10031−10033. (53) Zhao, X.-H.; Gong, L.; Zhang, X.-B.; Yang, B.; Fu, T.; Hu, R.; Tan, W.; Yu, R. Anal. Chem. 2013, 85, 3614−3620. (54) Wang, Y.; Chen, J.; Chen, Y.; Li, W.; Yu, C. Anal. Chem. 2014, 86, 4371−4378. (55) Liao, D.; Jiao, H.; Wang, B.; Lin, Q.; Yu, C. Analyst 2012, 137, 978−982.

9872

dx.doi.org/10.1021/ac502496h | Anal. Chem. 2014, 86, 9866−9872