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Cite this: Chem. Commun., 2014, 50, 6211 Received 20th January 2014, Accepted 12th March 2014
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Reporter-triggered isothermal exponential amplification strategy in ultrasensitive homogeneous label-free electrochemical nucleic acid biosensing† Ji Nie, De-Wen Zhang, Fang-Ting Zhang, Fang Yuan, Ying-Lin Zhou* and Xin-Xiang Zhang*
DOI: 10.1039/c4cc00475b www.rsc.org/chemcomm
A simple and novel reporter-triggered isothermal exponential amplification reaction (R-EXPAR) integrated with a miniaturized electrochemical device was developed, which achieved excellent improvement (five orders of magnitude) of sensitivity toward reporter, G-quadruplex. This R-EXPAR strategy has been successfully implemented to construct a homogeneous label-free electrochemical sensor for ultrasensitive DNA detection.
Sequence-specific nucleic acid detection is essential for detecting clinical pathogens, bio-threat agents and the early diagnosis of cancer or genetic disease in a post-genomic era. An ideal point-ofcare (POC) detection should fulfil the ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free and Deliverable to end users) criteria represented by the World Health Organization.1 Towards these goals, electrochemical detections show compelling advantages, such as simplicity, low cost, less power requirement and miniaturized platform,2–4 and they perform well with cloudy or colored samples, which might cause interference in optimal assays. Most nucleic acid electrochemical sensors are based on heterogeneous format.5–8 The immobilization of a probe DNA onto the electrode is necessary to perform a relevant recognition event with target DNA. Although high sensitivity is provided, the heterogeneous format shows some intrinsic drawbacks: complex and timeconsuming immobilization process; expensive modification of linking group; lower hybridization efficiency due to steric hindrance. As the complement to heterogeneous assay, homogeneous electrochemical sensor seems more suitable to achieve a desired simple, rapid, robust and easy-to-operate POC nucleic acid detection. Compared with labelled assays,9–11 label-free ones are more attractive due to low cost. Until now, label-free electrochemical methods are mainly achieved via double-stranded DNA-intercalating redox probes.4 While taking Beijing National Laboratory for Molecular Sciences (BNLMS), MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry, Peking University, Beijing 100871, China. E-mail: [email protected], [email protected]; Fax: +86-10-62754112; Tel: +86-10-62754112 † Electronic supplementary information (ESI) available: Experimental details and additional figures. See DOI: 10.1039/c4cc00475b
This journal is © The Royal Society of Chemistry 2014
advantages of design simplicity and operation convenience, they often suffer from background interference of non-selectivity intercalation and loss of sensitivity.12 A more direct, specific and efficient signal read-out mode is in great need. G-quadruplex, with repetitive G-rich structural motifs, can act as DNAzyme possessing peroxidase-mimic activity when binding with hemin.13 Since it can be flexibly encoded into nucleic acid based strategy, the generation of G-quadruplex sequence through target DNA recognition process might become a novel signal read-out mode to meet the demands of constructing label-free and immobilization-free electrochemical nucleic acid sensors. However, the sensitivity, which is correlated directly to the detectable concentration of G-quadruplex sequence, might be limited due to the much lower catalytic activity comparing with horseradish peroxidase (HRP). A powerful reporter-amplification strategy should be introduced to offer sufficiently sensitive signal read-out for an ideal electrochemical POC assay. Herein, we first developed reporter-triggered isothermal exponential amplification reaction (R-EXPAR) strategy, integrating with a miniaturized electrochemical device,14 for the construction of ultrasensitive homogeneous label-free nucleic acid biosensor. EXPAR is an isothermal molecular chain reaction, which can synthesize short oligonucleotides with high amplification efficiency.15 The R-EXPAR circuits can realize the exponential multiplication of reporter element (G-quadruplex) within a short time, which will achieve the tremendous signal read-out growth. This simple and rapid amplification for reporter molecules is anticipated to remarkably improve the sensitivity of assays that treat G-quadruplex generation as signal read-out. Our R-EXPAR strategy is illustrated in Scheme 1. EAD2, an intramolecular parallel G-quadruplex sequence, which showed high peroxidase-mimic activity, was chosen as reporter (denoted as ‘‘Y’’). The template Y0 –Y0 was designed with two repeated complementary sequences of Y. The two regions were separated by the complementary of nicking recognition and cleavage site (insert legend of Scheme 1). Once the Y priming the Y 0 –Y0 template, the primertemplate could be polymerized by vent (exo-) polymerase and cleaved by Nt.BstNBI nicking enzyme. The created oligonucleotide
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Fig. 2 Calibration curves of signal responses Di vs. EAD2 concentrations (a) with R-EXPAR, and (b) without R-EXPAR. Error bars represent SD of three independent experiments. RSD o 13.2%. Scheme 1 The principle of R-EXPAR strategy integrated with an ultramicroelectrode/micropipette-tip based miniaturized electrochemical device.
was then released via melting or strand displacement. Since it was specifically designed the same to trigger Y, an exponential amplification circuit could be achieved while the new Y triggered another Y0 –Y0 . Subsequently, EAD2 can form EAD2–hemin DNAzyme and be detected in hydroquinone (HQ)–H2O2 system by the ultramicroelectrode/micropipette-tip based miniaturized electrochemical device in a small volume. To validate the feasibility of R-EXPAR for EAD2 G-quadruplex sequence, we first measured the CVs of the amplification production triggered by 10 nM EAD2 and without triggering (the negative control) using HQ as an electron mediator. The oxidation/reduction quasi-steady state currents were proportional to the concentrations of HQ/BQ (benzoquinone) individually in solution. In the presence of DNAzyme and H2O2, HQ was chemically oxidized to BQ. Therefore, the corresponding oxidation current was decreased accompanying the increase of reduction current.16 As shown in Fig. 1A, the R-EXPAR production triggered by 10 nM EAD2 (curve b) shows significant decrease of oxidation peak current and increase of reduction peak current compared with the negative control (curve a). In fact, 10 nM EAD2–hemin DNAzyme without amplification showed no obvious difference to the negative control (not provided here). It indicated that the R-EXPAR strategy realized the generation of EAD2. The generated EAD2 was successfully detected via G-quadruplex/ hemin catalyzed HQ–H2O2 system based on our miniaturized electrochemical device. The corresponding photograph analyzed
Fig. 1 (A) The CVs of 1 mM HQ and 1 mM H2O2 in 0.1 M phosphate buffer (pH 7.4) with 12.5 mM hemin and 5 mL R-EXPAR production: (a) without trigger sequence, and (b) triggered by 10 nM EAD2. (B) The amperometric response curves of (a) negative control, (b) R-EXPAR production triggered by 1 nM EAD2, (c) 1 nM HRP, and (d) 1 nM EAD2 at 0.1 V.
6212 | Chem. Commun., 2014, 50, 6211--6213
by agarose gel electrophoresis (Fig. S1, ESI†) can further verify the amplification of EAD2 in the strategy. In Fig. 1B, we compared the peroxidase catalytic activity of EAD2–hemin DNAzyme with and without R-EXPAR, and HRP via amperometric response curves. R-EXPAR production triggered by 1 nM EAD2 (curve b) caused 5.61 nA reduction current increase after 6 min catalytic reaction, while 1 nM HRP (curve c) showed only about 1.54 nA change. The HRP-mimicking DNAzyme amplified through EXPAR showed significantly higher peroxidase activity than that of the natural enzyme with the same concentration. Without amplification, the Di–t curve of ideally formed 1 nM EAD2–hemin DNAzyme (curve d) did not show obvious difference to that of the negative control (curve a). It is apparent that the simple and rapid R-EXPAR exhibited excellent performance in magnifying the quantity of reporter molecules to a dramatic level. To quantitatively evaluate the magnification level of R-EXPAR towards reporter, the sensitivity of EAD2 detection was tested (Fig. 2, curve a). The variations of reduction currents at different EAD2 concentrations (potential fixed at 0.1 V) along 6 min catalytic reaction were recorded for quantification. With the increase of the EAD2 concentration, a corresponding sharp change in current response was observed (curve a). It is consistent with the mechanism that more reporters Y triggered more exponential circuits and generated more reporters during the same extension/nicking time. With a dynamic range from 1 pM to 10 nM, as low as 1 pM EAD2 can be magnified into experimentally detectable concentrations based on a signal-to-noise ratio (S/N) of 3. Also, we detected the EAD2 without the proposed strategy (curve b) under the same experimental conditions. A 100 nM EAD2 with excess hemin which was supposed to form ideally 100 nM DNAzyme only gained 0.63 nA current response change. 10 nM or lower than 10 nM EAD2 formed DNAzymes could not be detected. The comparison demonstrated that our R-EXPAR was able to achieve conspicuous five orders of magnitude amplification (from 100 nM down to 1 pM) for G-quadruplex/hemin DNAzyme system. More attractively, only one simply designed Y 0 –Y 0 DNA template was required to perform the strategy in an extremely short time (15 min). Considering the cost-saving and laboursaving characteristics of the miniaturized homogeneous electrochemical device, the strategy shows great potential to be universally applied for G-quadruplex sequence based sensing modes with satisfactory sensitivity.
This journal is © The Royal Society of Chemistry 2014
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Fig. 3 (A) The principle of two-stage X 0 –Y 0 /Y 0 –Y 0 strategy. (B) The feasibility of two-stage X 0 –Y 0 /Y 0 –Y 0 strategy without target X (negative control), 100 pM target X and 1 nM random, individually. (C) Calibration curves of signal responses Di vs. target X concentrations, (a) via X 0 –Y 0 /Y 0 –Y 0 strategy; (b) via X 0 –Y 0 strategy. The leftmost dot of each curve means the negative control without target X. Error bars represent standard deviations of three independent experiments. RSD o 11.1%.
A simple model which included a two-stage X0 –Y0 /Y0 –Y0 circuit was designed to certify the application of our R-EXPAR strategy for target DNA detection (Fig. 3A). The target DNA X could hybridize with the recognition template X0 –Y0 . Through the polymerization and nicking, released Y sequence (designed to be EAD2) was obtained in a linear amplification manner. Following the conversion of X to reporter Y, the second stage R-EXPAR was performed. The undetectable slight amount of reporter EAD2 generated from X0 –Y0 target recognition could be amplified into a detectable amount via the tandem Y0 –Y0 protocol. As shown in Fig. 3B, 100 pM target X triggered 98% larger current variation than that performed without target X. To ensure the triggering event was due to the hybridization of target X with the relevant complementary region of recognition template X0 –Y0 , ten times of random sequence instead of target X was added. The Di–t curves illustrate that 1 nM random showed negligible difference to the negative control. It validated the high specificity of X0 –Y0 /Y0 –Y0 strategy to target DNA. The quantitative performance of R-EXPAR based X0 –Y0 /Y0 –Y0 strategy for target X was evaluated, which was compared with that of X0 –Y0 strategy (Fig. 3C). For the X0 –Y0 /Y0 –Y0 mode (curve a), the current response increased with the concentration of target X from 0 to 4 nM. The lowest detectable concentration for target X was 1 pM (10 amol in 10 mL) with a dynamic range from 1 pM to 1 nM. However, in the X0 –Y0 mode (curve b), without downstream amplification for reporter element, EAD2 was generated directly via linear conversion from recognition element to signal read-out element. 15 nM of target X was the lowest detectable concentration in X0 –Y0 mode. Thus nearly ten thousand times improvement of sensitivity was achieved via the additional R-EXPAR following a simple DNA recognition process. Comparing with other homogeneous electrochemical sensors (Table S2, ESI†), our assay shows excellent performance. The selectivity of X0 –Y0 /Y0 –Y0 strategy was tested by comparative exploration of the perfect-matched target DNA, two-bases mismatched
This journal is © The Royal Society of Chemistry 2014
Communication
DNA (M2) and three-bases mismatched DNA (M3) (Fig. S2, ESI†). The current changes for the M2 and M3 were 32.8% and 8.84% of that of the perfect-matched target DNA, respectively. To improve the selectivity for better discrimination among mismatches, more elaborated recognition event (e.g., adopting sensitive structure switches in response to the specific target triggering) should be involved. The results here indicate the potential that R-EXPAR coupled DNA recognition strategy could show good signal discrimination for mismatch among trace DNA samples. We also successfully evaluated the feasibility of the strategy spiked with 20% human serum (Table S3, ESI†). In conclusion, a simple and novel R-EXPAR strategy integrating an ultramicroelectrode/micropipette-tip based miniaturized electrochemical device was developed for label-free homogeneous nucleic acid biosensing. The reporter molecules (EAD2) via the rapid R-EXPAR can be detected down to 1 pM (five orders of magnitude improvement). Excellent sensitivity for target DNA detection was achieved in X0 –Y0 recognition strategy with a tandem R-EXPAR protocol. Simultaneously, the label-free and immobilization-free miniaturized electrochemical measurements exhibited outstanding performances like easy operation, low cost, high reliability and repeatability. Not only for sequence-specific nucleic acid detection, the R-EXPAR strategy showed great potential to be implemented to suitable nucleic acid based POC sensors towards other targets (e.g., small molecules or biomacromolecules with relevant aptamers) which were well-designed to create a G-quadruplex sequence as reporter. This work was supported by the National Natural Science Foundation of China (Nos. 21275009 and 20805002) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, MOE, China.
Notes and references 1 D. Mabey, R. W. Peeling, A. Ustianowski and M. D. Perkins, Nat. Rev. Microbiol., 2004, 2, 231–240. 2 M. R. Hartman, R. C. H. Ruiz, S. Hamada, C. Y. Xu, K. G. Yancey, Y. Yu, W. Han and D. Luo, Nanoscale, 2013, 5, 10141–10154. 3 F. Xuan, X. T. Luo and I. M. Hsing, Biosens. Bioelectron., 2012, 35, 230–234. 4 R. Miranda-Castro, D. Marchal, B. Limoges and F. Mavre, Chem. Commun., 2012, 48, 8772–8774. 5 R. Ren, C. Leng and S. Zhang, Chem. Commun., 2010, 46, 5758–5760. 6 Q. Wang, L. J. Yang, X. H. Yang, K. M. Wang, L. L. He, J. Q. Zhu and T. Y. Su, Chem. Commun., 2012, 48, 2982–2984. 7 S. F. Liu, C. F. Wang, C. X. Zhang, Y. Wang and B. Tang, Anal. Chem., 2013, 85, 2282–2288. 8 Z. Zhu, J. P. Lei, L. Liu and H. X. Ju, Analyst, 2013, 138, 5995–6000. 9 F. Xuan, X. T. Luo and I. M. Hsing, Anal. Chem., 2012, 84, 5216–5220. 10 J. K. Wu, C. H. Huang, G. F. Cheng, F. Zhang, P. G. He and Y. Z. Fang, Electrochem. Commun., 2009, 11, 177–180. 11 X. Luo, T. M.-H. Lee and I. M. Hsing, Anal. Chem., 2008, 80, 7341–7346. 12 G. Liu, Y. Wan, V. Gau, J. Zhang, L. Wang, S. Song and C. Fan, J. Am. Chem. Soc., 2008, 130, 6820–6825. 13 X. Cheng, X. Liu, T. Bing, Z. Cao and D. Shangguan, Biochemistry, 2009, 48, 7817–7823. 14 D. W. Zhang, J. X. Liu, J. Nie, Y. L. Zhou and X. X. Zhang, Anal. Chem., 2013, 85, 2032–2036. 15 J. Van Ness, L. K. Van Ness and D. J. Galas, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 4504–4509. 16 D.-W. Zhang, J. Nie, F.-T. Zhang, L. Xu, Y.-L. Zhou and X.-X. Zhang, Anal. Chem., 2013, 85, 9378–9382.
Chem. Commun., 2014, 50, 6211--6213 | 6213
Published on 13 March 2014. Downloaded by Dalhousie University on 04/07/2014 06:18:35.
COMMUNICATION
Cite this: Chem. Commun., 2014, 50, 6211 Received 20th January 2014, Accepted 12th March 2014
View Article Online View Journal | View Issue
Reporter-triggered isothermal exponential amplification strategy in ultrasensitive homogeneous label-free electrochemical nucleic acid biosensing† Ji Nie, De-Wen Zhang, Fang-Ting Zhang, Fang Yuan, Ying-Lin Zhou* and Xin-Xiang Zhang*
DOI: 10.1039/c4cc00475b www.rsc.org/chemcomm
A simple and novel reporter-triggered isothermal exponential amplification reaction (R-EXPAR) integrated with a miniaturized electrochemical device was developed, which achieved excellent improvement (five orders of magnitude) of sensitivity toward reporter, G-quadruplex. This R-EXPAR strategy has been successfully implemented to construct a homogeneous label-free electrochemical sensor for ultrasensitive DNA detection.
Sequence-specific nucleic acid detection is essential for detecting clinical pathogens, bio-threat agents and the early diagnosis of cancer or genetic disease in a post-genomic era. An ideal point-ofcare (POC) detection should fulfil the ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free and Deliverable to end users) criteria represented by the World Health Organization.1 Towards these goals, electrochemical detections show compelling advantages, such as simplicity, low cost, less power requirement and miniaturized platform,2–4 and they perform well with cloudy or colored samples, which might cause interference in optimal assays. Most nucleic acid electrochemical sensors are based on heterogeneous format.5–8 The immobilization of a probe DNA onto the electrode is necessary to perform a relevant recognition event with target DNA. Although high sensitivity is provided, the heterogeneous format shows some intrinsic drawbacks: complex and timeconsuming immobilization process; expensive modification of linking group; lower hybridization efficiency due to steric hindrance. As the complement to heterogeneous assay, homogeneous electrochemical sensor seems more suitable to achieve a desired simple, rapid, robust and easy-to-operate POC nucleic acid detection. Compared with labelled assays,9–11 label-free ones are more attractive due to low cost. Until now, label-free electrochemical methods are mainly achieved via double-stranded DNA-intercalating redox probes.4 While taking Beijing National Laboratory for Molecular Sciences (BNLMS), MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry, Peking University, Beijing 100871, China. E-mail: [email protected], [email protected]; Fax: +86-10-62754112; Tel: +86-10-62754112 † Electronic supplementary information (ESI) available: Experimental details and additional figures. See DOI: 10.1039/c4cc00475b
This journal is © The Royal Society of Chemistry 2014
advantages of design simplicity and operation convenience, they often suffer from background interference of non-selectivity intercalation and loss of sensitivity.12 A more direct, specific and efficient signal read-out mode is in great need. G-quadruplex, with repetitive G-rich structural motifs, can act as DNAzyme possessing peroxidase-mimic activity when binding with hemin.13 Since it can be flexibly encoded into nucleic acid based strategy, the generation of G-quadruplex sequence through target DNA recognition process might become a novel signal read-out mode to meet the demands of constructing label-free and immobilization-free electrochemical nucleic acid sensors. However, the sensitivity, which is correlated directly to the detectable concentration of G-quadruplex sequence, might be limited due to the much lower catalytic activity comparing with horseradish peroxidase (HRP). A powerful reporter-amplification strategy should be introduced to offer sufficiently sensitive signal read-out for an ideal electrochemical POC assay. Herein, we first developed reporter-triggered isothermal exponential amplification reaction (R-EXPAR) strategy, integrating with a miniaturized electrochemical device,14 for the construction of ultrasensitive homogeneous label-free nucleic acid biosensor. EXPAR is an isothermal molecular chain reaction, which can synthesize short oligonucleotides with high amplification efficiency.15 The R-EXPAR circuits can realize the exponential multiplication of reporter element (G-quadruplex) within a short time, which will achieve the tremendous signal read-out growth. This simple and rapid amplification for reporter molecules is anticipated to remarkably improve the sensitivity of assays that treat G-quadruplex generation as signal read-out. Our R-EXPAR strategy is illustrated in Scheme 1. EAD2, an intramolecular parallel G-quadruplex sequence, which showed high peroxidase-mimic activity, was chosen as reporter (denoted as ‘‘Y’’). The template Y0 –Y0 was designed with two repeated complementary sequences of Y. The two regions were separated by the complementary of nicking recognition and cleavage site (insert legend of Scheme 1). Once the Y priming the Y 0 –Y0 template, the primertemplate could be polymerized by vent (exo-) polymerase and cleaved by Nt.BstNBI nicking enzyme. The created oligonucleotide
Chem. Commun., 2014, 50, 6211--6213 | 6211
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Fig. 2 Calibration curves of signal responses Di vs. EAD2 concentrations (a) with R-EXPAR, and (b) without R-EXPAR. Error bars represent SD of three independent experiments. RSD o 13.2%. Scheme 1 The principle of R-EXPAR strategy integrated with an ultramicroelectrode/micropipette-tip based miniaturized electrochemical device.
was then released via melting or strand displacement. Since it was specifically designed the same to trigger Y, an exponential amplification circuit could be achieved while the new Y triggered another Y0 –Y0 . Subsequently, EAD2 can form EAD2–hemin DNAzyme and be detected in hydroquinone (HQ)–H2O2 system by the ultramicroelectrode/micropipette-tip based miniaturized electrochemical device in a small volume. To validate the feasibility of R-EXPAR for EAD2 G-quadruplex sequence, we first measured the CVs of the amplification production triggered by 10 nM EAD2 and without triggering (the negative control) using HQ as an electron mediator. The oxidation/reduction quasi-steady state currents were proportional to the concentrations of HQ/BQ (benzoquinone) individually in solution. In the presence of DNAzyme and H2O2, HQ was chemically oxidized to BQ. Therefore, the corresponding oxidation current was decreased accompanying the increase of reduction current.16 As shown in Fig. 1A, the R-EXPAR production triggered by 10 nM EAD2 (curve b) shows significant decrease of oxidation peak current and increase of reduction peak current compared with the negative control (curve a). In fact, 10 nM EAD2–hemin DNAzyme without amplification showed no obvious difference to the negative control (not provided here). It indicated that the R-EXPAR strategy realized the generation of EAD2. The generated EAD2 was successfully detected via G-quadruplex/ hemin catalyzed HQ–H2O2 system based on our miniaturized electrochemical device. The corresponding photograph analyzed
Fig. 1 (A) The CVs of 1 mM HQ and 1 mM H2O2 in 0.1 M phosphate buffer (pH 7.4) with 12.5 mM hemin and 5 mL R-EXPAR production: (a) without trigger sequence, and (b) triggered by 10 nM EAD2. (B) The amperometric response curves of (a) negative control, (b) R-EXPAR production triggered by 1 nM EAD2, (c) 1 nM HRP, and (d) 1 nM EAD2 at 0.1 V.
6212 | Chem. Commun., 2014, 50, 6211--6213
by agarose gel electrophoresis (Fig. S1, ESI†) can further verify the amplification of EAD2 in the strategy. In Fig. 1B, we compared the peroxidase catalytic activity of EAD2–hemin DNAzyme with and without R-EXPAR, and HRP via amperometric response curves. R-EXPAR production triggered by 1 nM EAD2 (curve b) caused 5.61 nA reduction current increase after 6 min catalytic reaction, while 1 nM HRP (curve c) showed only about 1.54 nA change. The HRP-mimicking DNAzyme amplified through EXPAR showed significantly higher peroxidase activity than that of the natural enzyme with the same concentration. Without amplification, the Di–t curve of ideally formed 1 nM EAD2–hemin DNAzyme (curve d) did not show obvious difference to that of the negative control (curve a). It is apparent that the simple and rapid R-EXPAR exhibited excellent performance in magnifying the quantity of reporter molecules to a dramatic level. To quantitatively evaluate the magnification level of R-EXPAR towards reporter, the sensitivity of EAD2 detection was tested (Fig. 2, curve a). The variations of reduction currents at different EAD2 concentrations (potential fixed at 0.1 V) along 6 min catalytic reaction were recorded for quantification. With the increase of the EAD2 concentration, a corresponding sharp change in current response was observed (curve a). It is consistent with the mechanism that more reporters Y triggered more exponential circuits and generated more reporters during the same extension/nicking time. With a dynamic range from 1 pM to 10 nM, as low as 1 pM EAD2 can be magnified into experimentally detectable concentrations based on a signal-to-noise ratio (S/N) of 3. Also, we detected the EAD2 without the proposed strategy (curve b) under the same experimental conditions. A 100 nM EAD2 with excess hemin which was supposed to form ideally 100 nM DNAzyme only gained 0.63 nA current response change. 10 nM or lower than 10 nM EAD2 formed DNAzymes could not be detected. The comparison demonstrated that our R-EXPAR was able to achieve conspicuous five orders of magnitude amplification (from 100 nM down to 1 pM) for G-quadruplex/hemin DNAzyme system. More attractively, only one simply designed Y 0 –Y 0 DNA template was required to perform the strategy in an extremely short time (15 min). Considering the cost-saving and laboursaving characteristics of the miniaturized homogeneous electrochemical device, the strategy shows great potential to be universally applied for G-quadruplex sequence based sensing modes with satisfactory sensitivity.
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 13 March 2014. Downloaded by Dalhousie University on 04/07/2014 06:18:35.
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Fig. 3 (A) The principle of two-stage X 0 –Y 0 /Y 0 –Y 0 strategy. (B) The feasibility of two-stage X 0 –Y 0 /Y 0 –Y 0 strategy without target X (negative control), 100 pM target X and 1 nM random, individually. (C) Calibration curves of signal responses Di vs. target X concentrations, (a) via X 0 –Y 0 /Y 0 –Y 0 strategy; (b) via X 0 –Y 0 strategy. The leftmost dot of each curve means the negative control without target X. Error bars represent standard deviations of three independent experiments. RSD o 11.1%.
A simple model which included a two-stage X0 –Y0 /Y0 –Y0 circuit was designed to certify the application of our R-EXPAR strategy for target DNA detection (Fig. 3A). The target DNA X could hybridize with the recognition template X0 –Y0 . Through the polymerization and nicking, released Y sequence (designed to be EAD2) was obtained in a linear amplification manner. Following the conversion of X to reporter Y, the second stage R-EXPAR was performed. The undetectable slight amount of reporter EAD2 generated from X0 –Y0 target recognition could be amplified into a detectable amount via the tandem Y0 –Y0 protocol. As shown in Fig. 3B, 100 pM target X triggered 98% larger current variation than that performed without target X. To ensure the triggering event was due to the hybridization of target X with the relevant complementary region of recognition template X0 –Y0 , ten times of random sequence instead of target X was added. The Di–t curves illustrate that 1 nM random showed negligible difference to the negative control. It validated the high specificity of X0 –Y0 /Y0 –Y0 strategy to target DNA. The quantitative performance of R-EXPAR based X0 –Y0 /Y0 –Y0 strategy for target X was evaluated, which was compared with that of X0 –Y0 strategy (Fig. 3C). For the X0 –Y0 /Y0 –Y0 mode (curve a), the current response increased with the concentration of target X from 0 to 4 nM. The lowest detectable concentration for target X was 1 pM (10 amol in 10 mL) with a dynamic range from 1 pM to 1 nM. However, in the X0 –Y0 mode (curve b), without downstream amplification for reporter element, EAD2 was generated directly via linear conversion from recognition element to signal read-out element. 15 nM of target X was the lowest detectable concentration in X0 –Y0 mode. Thus nearly ten thousand times improvement of sensitivity was achieved via the additional R-EXPAR following a simple DNA recognition process. Comparing with other homogeneous electrochemical sensors (Table S2, ESI†), our assay shows excellent performance. The selectivity of X0 –Y0 /Y0 –Y0 strategy was tested by comparative exploration of the perfect-matched target DNA, two-bases mismatched
This journal is © The Royal Society of Chemistry 2014
Communication
DNA (M2) and three-bases mismatched DNA (M3) (Fig. S2, ESI†). The current changes for the M2 and M3 were 32.8% and 8.84% of that of the perfect-matched target DNA, respectively. To improve the selectivity for better discrimination among mismatches, more elaborated recognition event (e.g., adopting sensitive structure switches in response to the specific target triggering) should be involved. The results here indicate the potential that R-EXPAR coupled DNA recognition strategy could show good signal discrimination for mismatch among trace DNA samples. We also successfully evaluated the feasibility of the strategy spiked with 20% human serum (Table S3, ESI†). In conclusion, a simple and novel R-EXPAR strategy integrating an ultramicroelectrode/micropipette-tip based miniaturized electrochemical device was developed for label-free homogeneous nucleic acid biosensing. The reporter molecules (EAD2) via the rapid R-EXPAR can be detected down to 1 pM (five orders of magnitude improvement). Excellent sensitivity for target DNA detection was achieved in X0 –Y0 recognition strategy with a tandem R-EXPAR protocol. Simultaneously, the label-free and immobilization-free miniaturized electrochemical measurements exhibited outstanding performances like easy operation, low cost, high reliability and repeatability. Not only for sequence-specific nucleic acid detection, the R-EXPAR strategy showed great potential to be implemented to suitable nucleic acid based POC sensors towards other targets (e.g., small molecules or biomacromolecules with relevant aptamers) which were well-designed to create a G-quadruplex sequence as reporter. This work was supported by the National Natural Science Foundation of China (Nos. 21275009 and 20805002) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, MOE, China.
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