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A graphene oxide-based enzyme-free signal amplification platform for homogeneous DNA detection† Zhen Zhang, Yufei Liu, Xinghu Ji,* Xia Xiang and Zhike He* A graphene oxide (GO) based enzyme-free signal amplification platform for homogeneous DNA sensing is developed with simplicity and high sensitivity. In the absence of the target DNA, labeled hairpin probe 1 (H1) and probe 2 (H2) were adsorbed on the surface of GO, resulting in the fluorescence quenching of the dyes and minimizing the background fluorescence. The addition of the target DNA facilitated the formation of double-stranded DNA (dsDNA) between H1 and H2, causing the probes to separate from GO and release the target DNA through a strand displacement reaction. Meanwhile, the whole reaction started anew. This is an excellent isothermal signal amplification technique without the involvement of enzymes. By
Received 23rd May 2014 Accepted 1st July 2014
monitoring the change of the fluorescence intensity, the target DNA not only can be determined in buffer solution, but also can be detected in 1% serum solution spiked with a series of concentrations of
DOI: 10.1039/c4an00933a
the target DNA. In addition, the consumption amount of the probes in this method is lower than that in
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traditional molecular beacon methods.
1. Introduction Highly sensitive and selective DNA detection is of great demand in gene proling, drug screening, clinical diagnostics, environmental analysis and food safety.1–5 To improve the detection sensitivity, great efforts have been devoted to signal amplication such as polymerase chain reaction (PCR),6 ligase chain reaction (LCR),7 rolling circle amplication (RCA),8 exonucleaseassisted amplication9 and strand displacement amplication,10 etc. Despite high sensitivity and selectivity, many of these methods are enzyme-based DNA biosensors, which greatly increase the complexity, and restrict their universal application.11 Recently, some enzyme-free signal amplication methods have been reported for the development of low-cost, point-of-care diagnostics.12–14 One of these methods, catalytic hairpin assembly (CHA), which relies only on hybridization and strand-exchange reactions to achieve amplication, has been fabricated as a robust enzyme-free amplication platform with a variety of detection modalities.15–17 These methods do not need any enzymes or thermal-cycling procedures and can be easily achieved without complicated procedures. However, many fabricated DNA sensors based on target-catalyzed hairpin Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China. E-mail: [email protected]; Fax: +86-27-6875-4067; Tel: +8627-6875-6557 † Electronic supplementary information (ESI) available: Nondenaturing polyacrylamide gel (20%) analysis of the formation of the H1–H2 complex and uorescence emission spectra in the presence of different amounts of the target DNA in 1% serum. See DOI: 10.1039/c4an00933a
4806 | Analyst, 2014, 139, 4806–4809
assembly suffer from a low signal to noise ratio,18 tedious modifying process19 and easy digestion by nucleases, which conne their application.20 Graphene oxide (GO), a water-soluble derivative of graphene, has received rapidly increasing attention because of its extraordinary electronic, optical, and thermal properties.21 It has been reported that GO can interact with single-stranded DNA (ssDNA) by p–p stacking interaction between the nucleotide bases and GO, but hardly interacts with rigid dsDNA or aptamer–target complexes.22–27 Most notably, GO is also a very effective uorescence quencher for organic uorescent molecules.28–34 It has been reported that GO can signicantly decrease the background uorescence of molecular beacon (MB) to improve the sensitivity of MB-based DNA detection.35 The uorescence quenching property of GO also makes it an attractive and robust molecular signal modulator for an alternative homogeneous assay that does not require covalently linked uorescent probes. Moreover, GO can protect DNA against enzymatic cleavage and enhance the intracellular stability of DNA compared with the free DNA probe.36 Herein, we developed a facile, simple, cost-effective and enzyme-free method for homogeneous DNA detection based on the target-catalyzed hairpin DNA assembly and the superquenching property of GO. In the absence of the target DNA, dye labeled hairpin probes were adsorbed on the surface of GO, resulting in the uorescence quenching of the dyes and exhibiting minimal background uorescence. Upon the addition of the target DNA, many dsDNA between the two hairpin-shaped probes are formed which leads to the separation of dyes from GO and uorescence recovery. In the proposed method, we
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introduce the efficient uorescence quenching ability of GO to target-catalyzed hairpin DNA assembly.
2.
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2.1
Experimental section Apparatus and chemicals
Tris(hydroxymethyl)aminomethane hydrochloride (Tris) was purchased from Sigma-Aldrich (St. Louis, MO, USA). GO was purchased from Sinocarbon Materials Technology Co., Ltd. All oligonucleotides with different sequences were synthesized by Sangon Biotechnology Co., Ltd (Shanghai, China). Ultrapure water obtained from a Millipore water purication system (18.2 MU cm at 25 C water) and puried by using a Milli-Q Academic purication set (Millipore, Bedford, MA, USA) was used throughout. The sequences of the oligonucleotides used in this work are as follows: H1 probe: 50 -FAM-AAGTAGTGATTGAGCGTGATGAATGT CACTACTTCAACTCGCATTCATCACGCTCAATC-30 H2 probe: 50 -FAM-TGATGAATGCGAGTTGAAGTAGTGA CATTCATCACGCTCAATCACTACTTCAACTCGCA-30 Target DNA: 50 -GACATTCATCACGCTCAATCACTACTT-30 Single-base mismatched sequences (M1): 50 -GACATTCAT CACGCTCAATCAGTACTT-30 Double-base mismatched sequences (M2): 50 -GACATTCAT CACGCTCAATCAGTTCTT-30 Three-base mismatched sequences (M3): 50 -GACATTCAT CACGCTCAATCAGTTCAT-30 All other chemicals not mentioned here were of analyticalreagent grade or better. Fluorescence spectra were obtained in a RF-5301PC spectrophotometer (Shimadzu, Japan), with 10 nm band-pass spectrometer slits. 2.2
Procedure for sample preparation
The reaction was performed by mixing 3 nM H1, 3 nM H2, 4 mg mL1 GO, different concentrations of the target DNA and Tris buffer (10 mM Tris, 150 mM NaCl, pH 8.0) to a nal volume of 500 mL, followed by incubating at 37 C for 2 h and then cooling to room temperature. The uorescence intensity was monitored by exciting the sample at 480 nm and measuring the emission at 520 nm. The slits for excitation and emission were both set at 10 nm.
3. 3.1
Scheme 1 Schematic illustration of the GO-based sensor for amplification detection of DNA via target-catalyzed hairpin assembly.
process similar to DNA branch migration and trigger the formation of another H1–H2 complex. In this way, a single target can generate many H1–H2 duplexes, which can separate from the surface of GO resulting in the uorescence restore. Based on the variation of uorescence signals, the target is determined and we can detect the target with high sensitivity. 3.2
The feasibility of this method
First, we test the feasibility of this method. As shown in Fig. 1, the system containing only H1 exhibits very weak uorescence emission due to the uorescence quenching caused by GO (Fig. 1, curve a). Upon the addition of the target DNA, the uorescence increases due to the formation of the H1-target DNA duplex structure which has a weaker strength with GO (Fig. 1, curve b). In the absence of the target DNA, only a weak uorescence signal was observed (Fig. 1, curve c) for the reason that the stem-loop structures of H1 and H2 are quite stable so that little H1–H2 complex is formed. The uorescence has signicant enhancement upon simultaneous addition of H1, H2 and the target DNA (Fig. 1, curve d), and it becomes much higher than that by the addition of only H1, H1 and target or H1 and H2. Compared with the conventional method, which employs H1 only, the H2-aided amplication method leads to a 4-fold increase in the signal intensity. We also used nondenaturing polyacrylamide gel (PAGE gel) to investigate the viability of our strategy. (see Fig. S1 in the ESI†). When mixing
Results and discussion The principle of detection
Scheme 1 depicts the principle of the analytical process for amplied detection of DNA. Two hairpin probes (H1 and H2) are labeled with 6-carboxyuorescein (FAM) at their 50 ends. In the absence of the target DNA, H1 and H2, which are in the stem-closed state, interact slowly with each other so that they can be absorbed on the surface of GO and exhibit a low uorescence signal. When the target appears, the opening of the hairpin structure of H1 is facilitated, making the exposed sequence available for hybridization with the hairpin DNA H2. Then, the target can be displaced from H1 by H2 through a
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Fig. 1 Fluorescence spectra of (a) H1, (b) H1 and T, (c) H1 and H2, and (d) H1, H2 and T. The concentration of the target DNA is 1 nM, GO is 4 mg mL1, and H1 and H2 are 3 nM, respectively.
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the hairpin probes H1 and H2, there was almost no H1–H2 complex formation (Fig. S1,† lane 2). This phenomenon indicated that the two stable hairpins H1 and H2 can coexist in solution. Upon the addition of the target DNA, a new product band (H1–H2 complex) can be seen distinctly on the PAGE gel (Fig. S1,† lane 3–5). Meanwhile, the brightness of the band gradually increased as the concentration of the target increased. These results demonstrated that the target DNA can catalyze the assembly of hairpins H1 and H2. 3.3
Optimization of the reaction conditions
To obtain better performance of this DNA sensor, concentrations of GO and incubation time were optimized. As shown in Fig. 2a, the uorescence intensity decreased gradually with the increasing concentration of GO from 0 to 4 mg mL1 and then gave a slight decrease when it was above 4 mg mL1. The maximal quenching efficiencies were about 90%. 4 mg mL1 GO was selected for further experiments. In addition, incubation time is a major factor that inuences the hybridization of hairpin probes with the target DNA, therefore different incubation times in the range from 10 to 150 min were investigated in this study. The results in Fig. 2b indicated that the uorescence intensities of FAM increased with the increase of incubation time in the range from 10 to 120 min and then the uorescence signal remained constant. Therefore, the most suitable incubation time of 120 min was selected. 3.4
The linear correlation and the detection limit
The uorescence signals toward different concentrations of the target DNA were measured under the optimal conditions. Fig. 3 shows the uorescence spectra in the presence of different concentrations of the target DNA. The uorescence intensity showed linear increase with increasing concentrations of the target DNA. The inset of Fig. 3 depicts the emission intensity plotted against the concentrations of the target DNA, showing a good linear response toward the target DNA concentration over the range from 0.4 nM to 5 nM, and the tted regression equation is F ¼ 17.82CT + 32.86 (R2 ¼ 0.9887). The detection
Fig. 2 (a) Fluorescence quenching of H1 and H2 in the presence of different concentrations of GO: 0, 1, 2, 3, 4, 5, and 6 mg mL1 (from the top to the bottom). Inset: linear curve of fluorescence quenching for probes. The data shown in the figures represent the average of three independent experiments (n ¼ 3). (b) Time response of fluorescence intensity of samples containing 3 nM H1, 3 nM H2 and 5 nM target DNA.
4808 | Analyst, 2014, 139, 4806–4809
Fig. 3 Changes in the fluorescence spectra upon increasing the concentration of the target DNA: 0, 0.4, 0.8, 2, 3, 4, 5, 8, 10, 15 and 20 nM (from the bottom to the top). Inset: linear curve of target DNA detection. The data shown in the figures represent the average of three independent experiments (n ¼ 3).
limit of this method is 0.2 nM based on a linear tting and the noise level of 3s (where s is the standard deviation of a blank solution, n ¼ 11). Relative to other GO-DNA sensors, this enzyme-free biosensor provided better or comparable sensitivity for DNA detection.37,38 To authenticate the feasibility of this approach in complex biological matrices, we performed this DNA sensor in 1% serum. The uorescence intensity of the sensor increased with the increasing DNA concentration (see Fig. S2 in the ESI†). The calibration curve for DNA detection is shown in the inset of Fig. S2† and the linear equation is y ¼ 20.46x + 403.6 (R2 ¼ 0.9665), where y is the uorescence intensity and x is the concentration of the target DNA. It clearly shows that this GO-based DNA sensor could be used in practical samples sensitively.
3.5
Selectivity analysis
Fig. 4 displays the uorescence intensity observed upon the addition of the target DNA and the mismatched DNA. As shown, great uorescence enhancement was obtained upon the addition of perfectly matched DNA and the uorescence signal for the target DNA was approximately two times higher than that of the single-base mismatched target DNA under the same concentration. The results indicate that this proposed method
Fig. 4 Histogram of the change of fluorescence intensities of the sensing system including 5 nM of the complementary target, singlebase mismatched target (M1), double-base mismatched target (M2), three-base mismatched target (M3) and the absence of the target DNA (blank). The data shown in the figures represent the average of three independent experiments (n ¼ 3).
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can perfectly distinguish the complementary sequences from the single-base mismatched sequences. This high specicity was owing to the weak hybridization of mismatched DNA with probes.
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4. Conclusions In conclusion, a simple, selective, cost-efficient and enzyme-free signal amplication uorescent biosensor for DNA detection by integrating two single-labeled hairpin-shaped DNA and GO was developed. Compared with the reported DNA sensors based on the target catalyzed hairpin assembly, this present strategy has three advantages: rstly, there is no quench group labeled at the end of the hairpin DNA probes, which is simple and costeffective; secondly, the background is signicantly reduced, owing to the high quenching efficiency of GO; thirdly, the amount of probes (H1 and H2) in the total reaction system is reduced compared to other CHA-based sensor systems. Moreover, this GO-based DNA sensor could be used in 1% serum assays. In view of these advantages, this sensitive uorescent DNA biosensor has great potential in the area of DNA diagnostics and clinical analysis.
Acknowledgements This work was nancially supported by the National Key Scientic Program-Nanoscience and Nanotechnology (2011CB933600) and the National Science Foundation of China (21275109 and 21205089).
Notes and references 1 J. G. Hacia, J. B. Fan, O. Ryder, L. Jin, K. Edgemon, G. Ghandour, R. A. Mayer, B. Sun, L. Hsie, C. M. Robbins, L. C. Brody, D. Wang, E. S. Lander, R. Lipshutz, S. P. Fodor and F. S. Collins, Nat. Genet., 1999, 22, 164–167. 2 M. Smilkstein, N. Sriwilaijaroen, J. X. Kelly, P. Wilairat and M. Riscoe, Antimicrob. Agents Chemother., 2004, 48, 1803– 1806. 3 F. D. Bushman, T. Fujiwara and R. Craigie, Science, 1990, 249, 1555–1558. 4 Y. Wen, F. Xing, S. He, S. Song, L. Wang, Y. Long, D. Li and C. Fan, Chem. Commun., 2010, 46, 2596–2598. 5 Y. Liu Stratton, S. Roy and C. K. Sen, Toxicol. Lett., 2004, 150, 29–42. 6 K. Mullis, F. Faloona, S. Scharf, R. Saiki, G. Horn and H. Erlich, Biotechnology, 1992, 24, 17–27. 7 J. Luo, D. E. Bergstrom and F. Barany, Nucleic Acids Res., 1996, 24, 3071–3078. 8 Z. Cheglakov, Y. Weizmann, B. Basnar and I. Willner, Org. Biomol. Chem., 2007, 5, 223–225. 9 F. Xuan, X. Luo and I. M. Hsing, Anal. Chem., 2012, 84, 5216– 5220. 10 J. L. He, Z. S. Wu, H. Zhou, H. Q. Wang, J. H. Jiang, G. L. Shen and R. Q. Yu, Anal. Chem., 2010, 82, 1358–1364. 11 B. Fu, J. Cao, W. Jiang and L. Wang, Biosens. Bioelectron., 2013, 44, 52–56.
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12 H. M. Choi, J. Y. Chang, L. A. Trinh, J. E. Padilla, S. E. Fraser and N. A. Pierce, Nat. Biotechnol., 2010, 28, 1208–1212. 13 D. Y. Zhang, A. J. Turbereld, B. Yurke and E. Winfree, Science, 2007, 318, 1121–1125. 14 P. Yin, H. M. Choi, C. R. Calvert and N. A. Pierce, Nature, 2008, 451, 318–322. 15 S. Liu, Y. Wang, J. Ming, Y. Lin, C. Cheng and F. Li, Biosens. Bioelectron., 2013, 49, 472–477. 16 P. B. Allen, S. A. Arshad, B. Li, X. Chen and A. D. Ellington, Lab Chip, 2012, 12, 2951–2958. 17 A. X. Zheng, J. Li, J. R. Wang, X. R. Song, G. N. Chen and H. H. Yang, Chem. Commun., 2012, 48, 3112–3114. 18 H. Li, J. Ren, Y. Liu and E. Wang, Chem. Commun., 2014, 50, 704–706. 19 J. Huang, X. Su and Z. Li, Anal. Chem., 2012, 84, 5939–5943. 20 C. Liu, Z. Wang, H. Jia and Z. Li, Chem. Commun., 2011, 47, 4661–4663. 21 B. Feng, L. Guo, L. Wang, F. Li, J. Lu, J. Gao, C. Fan and H. Qing, Anal. Chem., 2013, 85, 7732–7737. 22 Y. Wang, Z. Li, D. Hu, C. T. Lin, J. Li and Y. Lin, J. Am. Chem. Soc., 2010, 132, 9274–9276. 23 X. H. Zhao, R. M. Kong, X. B. Zhang, H. M. Meng, W. N. Liu, W. Tan, G. L. Shen and R. Q. Yu, Anal. Chem., 2011, 83, 5062– 5066. 24 Y. Zhang, Y. Liu, S. J. Zhen and C. Z. Huang, Chem. Commun., 2011, 47, 11718–11720. 25 Y. Wen, C. Peng, D. Li, L. Zhuo, S. He, L. Wang, Q. Huang, Q. H. Xu and C. Fan, Chem. Commun., 2011, 47, 6278–6280. 26 L. Sheng, J. Ren, Y. Miao, J. Wang and E. Wang, Biosens. Bioelectron., 2011, 26, 3494–3499. 27 X. H. Zhao, Q. J. Ma, X. X. Wu and X. Zhu, Anal. Chim. Acta, 2012, 727, 67–70. 28 C. Zhang, Y. Yuan, S. Zhang, Y. Wang and Z. Liu, Angew. Chem., Int. Ed., 2011, 50, 6851–6854. 29 M. Luo, X. Chen, G. Zhou, X. Xiang, L. Chen, X. Ji and Z. He, Chem. Commun., 2012, 48, 1126–1128. 30 S. He, B. Song, D. Li, C. Zhu, W. Qi, Y. Wen, L. Wang, S. Song, H. Fang and C. Fan, Adv. Funct. Mater., 2010, 20, 453–459. 31 H. Wang, Q. Zhang, X. Chu, T. Chen, J. Ge and R. Yu, Angew. Chem., Int. Ed., 2011, 50, 7065–7069. 32 Z. Yue, P. Lv, H. Yue, Y. Gao, D. Ma, W. Wei and G. Ma, Chem. Commun., 2013, 49, 3902–3904. 33 B. Gulbakan, E. Yasun, M. I. Shukoor, Z. Zhu, M. You, X. Tan, H. Sanchez, D. H. Powell, H. Dai and W. Tan, J. Am. Chem. Soc., 2010, 132, 17408–17410. 34 W. Wu, H. Hu, F. Li, L. Wang, J. Gao, J. Lu and C. Fan, Chem. Commun., 2011, 47, 1201–1203. 35 F. Li, Y. Huang, Q. Yang, Z. Zhong, D. Li, L. Wang, S. Song and C. Fan, Nanoscale, 2010, 2, 1021–1026. 36 C. H. Lu, C. L. Zhu, J. Li, J. J. Liu, X. Chen and H. H. Yang, Chem. Commun., 2010, 46, 3116–3118. 37 M. Zhang, H. N. Le and B. C. Ye, ACS Appl. Mater. Interfaces, 2013, 5, 8278–8282. 38 C. H. Lu, H. H. Yang, C. L. Zhu, X. Chen and G. N. Chen, Angew. Chem., Int. Ed., 2009, 48, 4785–4787.
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A graphene oxide-based enzyme-free signal amplification platform for homogeneous DNA detection† Zhen Zhang, Yufei Liu, Xinghu Ji,* Xia Xiang and Zhike He* A graphene oxide (GO) based enzyme-free signal amplification platform for homogeneous DNA sensing is developed with simplicity and high sensitivity. In the absence of the target DNA, labeled hairpin probe 1 (H1) and probe 2 (H2) were adsorbed on the surface of GO, resulting in the fluorescence quenching of the dyes and minimizing the background fluorescence. The addition of the target DNA facilitated the formation of double-stranded DNA (dsDNA) between H1 and H2, causing the probes to separate from GO and release the target DNA through a strand displacement reaction. Meanwhile, the whole reaction started anew. This is an excellent isothermal signal amplification technique without the involvement of enzymes. By
Received 23rd May 2014 Accepted 1st July 2014
monitoring the change of the fluorescence intensity, the target DNA not only can be determined in buffer solution, but also can be detected in 1% serum solution spiked with a series of concentrations of
DOI: 10.1039/c4an00933a
the target DNA. In addition, the consumption amount of the probes in this method is lower than that in
www.rsc.org/analyst
traditional molecular beacon methods.
1. Introduction Highly sensitive and selective DNA detection is of great demand in gene proling, drug screening, clinical diagnostics, environmental analysis and food safety.1–5 To improve the detection sensitivity, great efforts have been devoted to signal amplication such as polymerase chain reaction (PCR),6 ligase chain reaction (LCR),7 rolling circle amplication (RCA),8 exonucleaseassisted amplication9 and strand displacement amplication,10 etc. Despite high sensitivity and selectivity, many of these methods are enzyme-based DNA biosensors, which greatly increase the complexity, and restrict their universal application.11 Recently, some enzyme-free signal amplication methods have been reported for the development of low-cost, point-of-care diagnostics.12–14 One of these methods, catalytic hairpin assembly (CHA), which relies only on hybridization and strand-exchange reactions to achieve amplication, has been fabricated as a robust enzyme-free amplication platform with a variety of detection modalities.15–17 These methods do not need any enzymes or thermal-cycling procedures and can be easily achieved without complicated procedures. However, many fabricated DNA sensors based on target-catalyzed hairpin Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China. E-mail: [email protected]; Fax: +86-27-6875-4067; Tel: +8627-6875-6557 † Electronic supplementary information (ESI) available: Nondenaturing polyacrylamide gel (20%) analysis of the formation of the H1–H2 complex and uorescence emission spectra in the presence of different amounts of the target DNA in 1% serum. See DOI: 10.1039/c4an00933a
4806 | Analyst, 2014, 139, 4806–4809
assembly suffer from a low signal to noise ratio,18 tedious modifying process19 and easy digestion by nucleases, which conne their application.20 Graphene oxide (GO), a water-soluble derivative of graphene, has received rapidly increasing attention because of its extraordinary electronic, optical, and thermal properties.21 It has been reported that GO can interact with single-stranded DNA (ssDNA) by p–p stacking interaction between the nucleotide bases and GO, but hardly interacts with rigid dsDNA or aptamer–target complexes.22–27 Most notably, GO is also a very effective uorescence quencher for organic uorescent molecules.28–34 It has been reported that GO can signicantly decrease the background uorescence of molecular beacon (MB) to improve the sensitivity of MB-based DNA detection.35 The uorescence quenching property of GO also makes it an attractive and robust molecular signal modulator for an alternative homogeneous assay that does not require covalently linked uorescent probes. Moreover, GO can protect DNA against enzymatic cleavage and enhance the intracellular stability of DNA compared with the free DNA probe.36 Herein, we developed a facile, simple, cost-effective and enzyme-free method for homogeneous DNA detection based on the target-catalyzed hairpin DNA assembly and the superquenching property of GO. In the absence of the target DNA, dye labeled hairpin probes were adsorbed on the surface of GO, resulting in the uorescence quenching of the dyes and exhibiting minimal background uorescence. Upon the addition of the target DNA, many dsDNA between the two hairpin-shaped probes are formed which leads to the separation of dyes from GO and uorescence recovery. In the proposed method, we
This journal is © The Royal Society of Chemistry 2014
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Paper
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introduce the efficient uorescence quenching ability of GO to target-catalyzed hairpin DNA assembly.
2.
Published on 01 July 2014. Downloaded by Northeastern University on 22/10/2014 21:57:36.
2.1
Experimental section Apparatus and chemicals
Tris(hydroxymethyl)aminomethane hydrochloride (Tris) was purchased from Sigma-Aldrich (St. Louis, MO, USA). GO was purchased from Sinocarbon Materials Technology Co., Ltd. All oligonucleotides with different sequences were synthesized by Sangon Biotechnology Co., Ltd (Shanghai, China). Ultrapure water obtained from a Millipore water purication system (18.2 MU cm at 25 C water) and puried by using a Milli-Q Academic purication set (Millipore, Bedford, MA, USA) was used throughout. The sequences of the oligonucleotides used in this work are as follows: H1 probe: 50 -FAM-AAGTAGTGATTGAGCGTGATGAATGT CACTACTTCAACTCGCATTCATCACGCTCAATC-30 H2 probe: 50 -FAM-TGATGAATGCGAGTTGAAGTAGTGA CATTCATCACGCTCAATCACTACTTCAACTCGCA-30 Target DNA: 50 -GACATTCATCACGCTCAATCACTACTT-30 Single-base mismatched sequences (M1): 50 -GACATTCAT CACGCTCAATCAGTACTT-30 Double-base mismatched sequences (M2): 50 -GACATTCAT CACGCTCAATCAGTTCTT-30 Three-base mismatched sequences (M3): 50 -GACATTCAT CACGCTCAATCAGTTCAT-30 All other chemicals not mentioned here were of analyticalreagent grade or better. Fluorescence spectra were obtained in a RF-5301PC spectrophotometer (Shimadzu, Japan), with 10 nm band-pass spectrometer slits. 2.2
Procedure for sample preparation
The reaction was performed by mixing 3 nM H1, 3 nM H2, 4 mg mL1 GO, different concentrations of the target DNA and Tris buffer (10 mM Tris, 150 mM NaCl, pH 8.0) to a nal volume of 500 mL, followed by incubating at 37 C for 2 h and then cooling to room temperature. The uorescence intensity was monitored by exciting the sample at 480 nm and measuring the emission at 520 nm. The slits for excitation and emission were both set at 10 nm.
3. 3.1
Scheme 1 Schematic illustration of the GO-based sensor for amplification detection of DNA via target-catalyzed hairpin assembly.
process similar to DNA branch migration and trigger the formation of another H1–H2 complex. In this way, a single target can generate many H1–H2 duplexes, which can separate from the surface of GO resulting in the uorescence restore. Based on the variation of uorescence signals, the target is determined and we can detect the target with high sensitivity. 3.2
The feasibility of this method
First, we test the feasibility of this method. As shown in Fig. 1, the system containing only H1 exhibits very weak uorescence emission due to the uorescence quenching caused by GO (Fig. 1, curve a). Upon the addition of the target DNA, the uorescence increases due to the formation of the H1-target DNA duplex structure which has a weaker strength with GO (Fig. 1, curve b). In the absence of the target DNA, only a weak uorescence signal was observed (Fig. 1, curve c) for the reason that the stem-loop structures of H1 and H2 are quite stable so that little H1–H2 complex is formed. The uorescence has signicant enhancement upon simultaneous addition of H1, H2 and the target DNA (Fig. 1, curve d), and it becomes much higher than that by the addition of only H1, H1 and target or H1 and H2. Compared with the conventional method, which employs H1 only, the H2-aided amplication method leads to a 4-fold increase in the signal intensity. We also used nondenaturing polyacrylamide gel (PAGE gel) to investigate the viability of our strategy. (see Fig. S1 in the ESI†). When mixing
Results and discussion The principle of detection
Scheme 1 depicts the principle of the analytical process for amplied detection of DNA. Two hairpin probes (H1 and H2) are labeled with 6-carboxyuorescein (FAM) at their 50 ends. In the absence of the target DNA, H1 and H2, which are in the stem-closed state, interact slowly with each other so that they can be absorbed on the surface of GO and exhibit a low uorescence signal. When the target appears, the opening of the hairpin structure of H1 is facilitated, making the exposed sequence available for hybridization with the hairpin DNA H2. Then, the target can be displaced from H1 by H2 through a
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Fig. 1 Fluorescence spectra of (a) H1, (b) H1 and T, (c) H1 and H2, and (d) H1, H2 and T. The concentration of the target DNA is 1 nM, GO is 4 mg mL1, and H1 and H2 are 3 nM, respectively.
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the hairpin probes H1 and H2, there was almost no H1–H2 complex formation (Fig. S1,† lane 2). This phenomenon indicated that the two stable hairpins H1 and H2 can coexist in solution. Upon the addition of the target DNA, a new product band (H1–H2 complex) can be seen distinctly on the PAGE gel (Fig. S1,† lane 3–5). Meanwhile, the brightness of the band gradually increased as the concentration of the target increased. These results demonstrated that the target DNA can catalyze the assembly of hairpins H1 and H2. 3.3
Optimization of the reaction conditions
To obtain better performance of this DNA sensor, concentrations of GO and incubation time were optimized. As shown in Fig. 2a, the uorescence intensity decreased gradually with the increasing concentration of GO from 0 to 4 mg mL1 and then gave a slight decrease when it was above 4 mg mL1. The maximal quenching efficiencies were about 90%. 4 mg mL1 GO was selected for further experiments. In addition, incubation time is a major factor that inuences the hybridization of hairpin probes with the target DNA, therefore different incubation times in the range from 10 to 150 min were investigated in this study. The results in Fig. 2b indicated that the uorescence intensities of FAM increased with the increase of incubation time in the range from 10 to 120 min and then the uorescence signal remained constant. Therefore, the most suitable incubation time of 120 min was selected. 3.4
The linear correlation and the detection limit
The uorescence signals toward different concentrations of the target DNA were measured under the optimal conditions. Fig. 3 shows the uorescence spectra in the presence of different concentrations of the target DNA. The uorescence intensity showed linear increase with increasing concentrations of the target DNA. The inset of Fig. 3 depicts the emission intensity plotted against the concentrations of the target DNA, showing a good linear response toward the target DNA concentration over the range from 0.4 nM to 5 nM, and the tted regression equation is F ¼ 17.82CT + 32.86 (R2 ¼ 0.9887). The detection
Fig. 2 (a) Fluorescence quenching of H1 and H2 in the presence of different concentrations of GO: 0, 1, 2, 3, 4, 5, and 6 mg mL1 (from the top to the bottom). Inset: linear curve of fluorescence quenching for probes. The data shown in the figures represent the average of three independent experiments (n ¼ 3). (b) Time response of fluorescence intensity of samples containing 3 nM H1, 3 nM H2 and 5 nM target DNA.
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Fig. 3 Changes in the fluorescence spectra upon increasing the concentration of the target DNA: 0, 0.4, 0.8, 2, 3, 4, 5, 8, 10, 15 and 20 nM (from the bottom to the top). Inset: linear curve of target DNA detection. The data shown in the figures represent the average of three independent experiments (n ¼ 3).
limit of this method is 0.2 nM based on a linear tting and the noise level of 3s (where s is the standard deviation of a blank solution, n ¼ 11). Relative to other GO-DNA sensors, this enzyme-free biosensor provided better or comparable sensitivity for DNA detection.37,38 To authenticate the feasibility of this approach in complex biological matrices, we performed this DNA sensor in 1% serum. The uorescence intensity of the sensor increased with the increasing DNA concentration (see Fig. S2 in the ESI†). The calibration curve for DNA detection is shown in the inset of Fig. S2† and the linear equation is y ¼ 20.46x + 403.6 (R2 ¼ 0.9665), where y is the uorescence intensity and x is the concentration of the target DNA. It clearly shows that this GO-based DNA sensor could be used in practical samples sensitively.
3.5
Selectivity analysis
Fig. 4 displays the uorescence intensity observed upon the addition of the target DNA and the mismatched DNA. As shown, great uorescence enhancement was obtained upon the addition of perfectly matched DNA and the uorescence signal for the target DNA was approximately two times higher than that of the single-base mismatched target DNA under the same concentration. The results indicate that this proposed method
Fig. 4 Histogram of the change of fluorescence intensities of the sensing system including 5 nM of the complementary target, singlebase mismatched target (M1), double-base mismatched target (M2), three-base mismatched target (M3) and the absence of the target DNA (blank). The data shown in the figures represent the average of three independent experiments (n ¼ 3).
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can perfectly distinguish the complementary sequences from the single-base mismatched sequences. This high specicity was owing to the weak hybridization of mismatched DNA with probes.
Published on 01 July 2014. Downloaded by Northeastern University on 22/10/2014 21:57:36.
4. Conclusions In conclusion, a simple, selective, cost-efficient and enzyme-free signal amplication uorescent biosensor for DNA detection by integrating two single-labeled hairpin-shaped DNA and GO was developed. Compared with the reported DNA sensors based on the target catalyzed hairpin assembly, this present strategy has three advantages: rstly, there is no quench group labeled at the end of the hairpin DNA probes, which is simple and costeffective; secondly, the background is signicantly reduced, owing to the high quenching efficiency of GO; thirdly, the amount of probes (H1 and H2) in the total reaction system is reduced compared to other CHA-based sensor systems. Moreover, this GO-based DNA sensor could be used in 1% serum assays. In view of these advantages, this sensitive uorescent DNA biosensor has great potential in the area of DNA diagnostics and clinical analysis.
Acknowledgements This work was nancially supported by the National Key Scientic Program-Nanoscience and Nanotechnology (2011CB933600) and the National Science Foundation of China (21275109 and 21205089).
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