Biosensors and Bioelectronics 56 (2014) 237–242

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G-quadruplex based two-stage isothermal exponential amplification reaction for label-free DNA colorimetric detection Ji Nie, De-Wen Zhang, Cai Tie, Ying-Lin Zhou n, Xin-Xiang Zhang n Beijing National Laboratory for Molecular Sciences (BNLMS), MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry, Peking University, Beijing 100871, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 12 December 2013 Accepted 17 January 2014 Available online 24 January 2014

A novel G-quadruplex based two-stage isothermal exponential amplification reaction (GQ-EXPAR) was developed for label-free DNA colorimetric detection in this work. The exponential amplified trigger DNA in the first stage can convert into G-quadruplex sequence EAD2 by a linear amplification circuit in the second stage. Created EAD2 can form G-quadruplex/hemin DNAzyme to act as a direct signal readout element. The GQ-EXPAR combines the exponential amplification of DNA sequence and the peroxidasemimicking DNAzyme induced signal amplification, which achieves tandem dual-amplification. Taking advantages of isothermal incubation, this label-free homogeneous assay obviates the need of thermal cycling . As no complex synthesis or extra downstream operation is needed, the whole easy handling procedure can be finished in no more than 1 h. This assay allows the sensing of the model DNA with the limit of detection to be 2.5 pM. Moreover, it demonstrates good discrimination of mismatched sequences. The strategy has also been successfully implemented to sensitively detect Tay–Sachs genetic disorder mutant. & 2014 Elsevier B.V. All rights reserved.

Keywords: G-quadruplex DNA detection Isothermal exponential amplification Label-free Colorimetry

1. Introduction DNA detection is critical for accurate examination of clinical pathogen and the early diagnosis of cancer or genetic disease (Sassolas et al., 2007). An ideal assay should be rapid, real-time, simple and sensitive for quantification. It is ordinarily performed by amplifying trace amounts of target oligonucleotide to detectable levels. The most famous nucleic acid amplification is the polymerase chain reaction (PCR) (Saiki et al., 1988), which relies on temperature cycling protocol and polymerase activity. However, precision thermal cycling among three temperatures imposes instrument constraints. Many isothermal amplification techniques have emerged as alternatives with excellent performance, such as rolling circle amplification (RCA) (Lizardi et al., 1998), loopmediated amplification (LAMP) (Hsieh et al., 2012; Notomi et al., 2000), strand displacement amplification (SDA) (Connolly and Trau, 2010; Walker et al., 1992), helicase-dependent amplification reaction (HDA) (Huang et al., 2011; Vincent et al., 2004), hybridization chain reaction (HCR) (Dirks and Pierce, 2004; Dong et al., 2012) and nucleic acid sequence-based amplification (NASBA) (Compton, 1991). While proceeding at constant temperature,


Corresponding authors. Tel./fax: þ86 1062754112. E-mail addresses: [email protected] (Y.-L. Zhou), [email protected] (X.-X. Zhang). 0956-5663/$ - see front matter & 2014 Elsevier B.V. All rights reserved.

isothermal nucleic acid amplification shows more potential in realizing low-cost point of care molecular diagnosis. EXPAR (isothermal exponential amplification reaction) first reported by Galas' group is an isothermal molecular chain reaction combining polymerase strand extension and single strand nicking (Van Ness et al., 2003). It can synthesize short oligonucleotides with high amplification efficiency and rapid amplification kinetics due to the DNA biological circuit with feedback design. According to the number of templates and circuits involved, EXPAR in nucleic acid detection contains two main modes, one-stage and twostage: (1) in the one-stage mode, the most basic and original EXPAR, real-time fluorescence PCR machine is used for monitoring the generation of partially or completely dsDNA (double-stranded DNA) (Jia et al., 2010). As the fluorescent intensity is correlated to the amount of double-stranded regions, the point of inflection is used for quantification. Also, a kind of single-stranded long template is developed, which can create trigger and reporter oligonucleotides simultaneously. Extra time is needed to record the fluorescence signal via growing nanoclusters (Liu et al., 2012; Wang et al., 2013b) or achieve satisfactory detection limit (Wang et al., 2013a); (2) in the two-stage mode, DNA sequence amplification and conversion are two divided stages. The created singlestranded reporter oligonucleotides converted in the second stage can bridge DNA functionalized gold nanoparticles into aggregation (Tan et al., 2005) or hybridize with labeled DNA capture and reporter probes for quantum dot based fluorescence resonance energy transfer (Zhang and Zhang, 2012). The signal is directly


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correlated to the single-stranded reporter DNA produced in EXPAR circuit. Sophisticated equipment, time-consuming synthesis of nano-material or multistep operation after amplification reaction to achieve signal readout limits their further application in the simple and rapid genetic diagnosis. Treating color changes as signal readout might make elaborated DNA biosensors more intuitionistic and convenient without losing reliability and accuracy. Herein, we first developed a novel G-quadruplex based two-stage isothermal exponential amplification strategy for label-free DNA colorimetric detection. G-quadruplex is a higher-order nucleic acid nano-structure generated from repetitive G-rich sequence motifs. It can form G-quadruplex/hemin complex in the presence of hemin and mimic horseradish peroxidase activity (Cheng et al., 2009). Acting as a tag with catalytic activity for signal transduction, G-quadruplex can be flexibly designed into nucleic acid biosensors for label-free homogeneous amplification (Zhao et al., 2013). In our strategy, the exponential amplified target oligonucleotides in the first stage can convert into G-quadruplex sequences EAD2 for DNAzyme-based colorimetry by a linear amplification circuit in the second stage. Two kinds of amplification procedures combined into the system, the exponential amplification of DNA sequence via EXPAR and the G-quadruplex/hemin mimicperoxidase induced signal amplification, provide highly amplified efficiency and sensitive quantification. As no complex synthesis or extra downstream operation is needed, the developed assay achieves simple, rapid, sensitive, label-free and low-cost detection of DNA.

2. Experimental section 2.1. Reagents and apparatus All HPLC-purified DNA oligonucleotides (listed in Table S1) were synthesized by Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). The vent (exo-) DNA polymerase and nicking endonuclease Nt.BstNBI were purchased from New England Biolabs (Beverly, MA). TMB  2HCl (TMB: 4,40 -diamino-3,30 ,5,50 -tetramethylbiphenyl) was purchased from Ameresco (USA). Hemin was obtained from Sigma-Aldrich (St. Louis, MO, USA). It was prepared in dimethyl sulfoxide (DMSO) and stored at  20 1C as 5 mM stock solution. All other reagents were at least of analytical reagent grade. Human serum samples were kindly provided by Peking University Hospital (Beijing, China). Absorption signals were recorded on a Thermo Scientific Multiskan FC Microplate Photometer (USA).

Temperature gradient optimization was carried out by a TC-512 Gradient PCR (TECHNE, UK). 2.2. GQ-EXPAR assay The reaction mixtures for GQ-EXPAR were prepared separately as part A and part B. Part A consisted of X0 –X0 template, target DNA X, Nt.BstNBI buffer and dNTP. Part B consisted of X0 –Y0 template, the nicking endonuclease Nt.BstNBI, vent (exo-) DNA polymerase and ThermoPol buffer. Part A and Part B were mixed immediately containing X0 –X0 template (200 nM), X0 –Y0 template (300 nM), dNTP (375 μM), Nt.BstNBI (0.8 U/μL), vent (exo-) DNA polymerase (0.1 U/μL), different concentrations of target DNA X, 1  ThermoPol buffer (20 mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgCl2, 0.1% Triton X-100) and 0.5  Nt.BstNBI buffer (25 mM Tris–HCl, pH 7.9, 50 mM NaCl, 5 mM MgCl2, 0.5 mM DTT). The mixture at a volume of 10 μL was incubated at 55 1C for 30 min and quenched at 4 1C. In spiked bio-sample analysis, different concentrations of target oligonucleotides were spiked with 20% human serum and detected following the above procedure. In TS-mutant detection, the reaction was performed at 58 1C for 1 h. After the amplification reaction, 4 μL of 50 μM hemin and 14 μL 1  ThermoPol buffer were added. A 5 μL resulting solution dropped into 96-well plate was mixed with 100 μL TMB–H2O2 solution (0.12 mg/mL TMB  2HCl, 18 mM H2O2, 0.1 M NaH2PO4– Na2HPO4, pH 6.0) to start the chromogenic reaction. G-quadruplex/hemin DNAzyme can catalyze H2O2-mediated oxidation of TMB. After 3 min incubation at room temperature (avoiding direct light exposure), the reaction was terminated by addition of 50 μL 2 M H2SO4. Then photographs were taken or the absorbance was collected as (A450–A620) a dual wavelength mode by a Microplate Photometer.

3. Results and discussion 3.1. The principle of GQ-EXPAR This GQ-EXPAR integrates two stages (Scheme 1): the first stage is an exponential amplification with template X0 –X0 for target X. The second stage is a linear amplification via template X0 –Y0 which enables the conversion of amplified trigger X to a reporter Y for further colorimetry. The template X0 –X0 consists of two repeated complementary sequences of X separated by the complementary of nicking enzyme recognition site (50 -GAGTC-30 )

Scheme 1. Schematic principle of GQ-EXPAR. (Inset) The detail of nicking enzyme recognition site.

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and cleavage site (four bases downstream). After trigger X priming X0 –X0 template, the primer-template can be extended by vent (exo-) polymerase at 55 1C. Then, the key to achieve circuit circulating, nicking enzyme Nt.BstNBI cleaves a phosphodiester bond to create an oligonucleotide that either melts off or is displaced from template X0 –X0 . After dissociation, the regenerated primer-template can continue to produce oligonucleotide X in a manner of linear amplification. Created trigger X then primes other template X0 –X0 leading to exponential amplification circuit. X can also prime a second amplification template X0 –Y0 to trigger the second stage, the conversion of amplified X to signal reporter Y, EAD2 (a G-quadruplex sequence) in a linear amplification. EAD2 can self-assemble G-quadruplex sequence into active DNAzyme with hemin. Subsequently, it catalyzes colorimetric reaction of TMB–H2O2, which provides amplified optical signals. Exponential and linear amplification combined two-stage EXPAR and G-quadruplex/hemin DNAzyme amplification are the core of GQ-EXPAR, which actually achieves tandem dual-amplification. Rapid and direct monitoring of converted reporter EAD2 gets rid of tedious downstream operations for signal readout. Also, it avoids signal interference from non-specific background amplification induced by spontaneous extended dsDNA, which may cause significant background interference in assays based on dsDNA intercalative fluorescent dye. 3.2. Feasibility of GQ-EXPAR for DNA detection To evaluate the feasibility of GQ-EXPAR, a 22nt DNA sequence was used as a model to perform several controls. No polymerase means no extension to the transient primer/template duplex. Nicking enzyme makes no sense to the partly complementary duplex without dsDNA recognition and cleaving site. In contrast, with polymerase only, an ideally 1:1 stoichiometry of target X to elongated dsDNA is obtained. No nicking means no created new trigger X or reporter Y and no continuous circulation. As shown in Fig. 1(A), it was clear that the strategy was dependent on both the vent (exo-) polymerase and nicking enzyme Nt.BstNBI, and no signal increase was observed when either of them was omitted.


We also tested the system with and without template X0 –X0 , which was the key to develop a feedback circuit for exponential increasing. There only exists an X0 –Y0 linear amplification without template X0 –X0 . In the presence of 100 pM trigger X and X0 –Y0 , color change was not obvious (Fig. 1(B)). (We had tested the performance of X0 –Y0 linear amplification under the same condition, and above 10 nM target X was detectable, data not shown.) However, with both X0 –X0 and X0 –Y0 templates forming a complete GQ-EXPAR circuit, 100 pM target X triggered a remarkable colorimetric signal. To investigate the specificity, one hundred times of random sequences were added instead of trigger X and no triggering event had happened (Fig. S1). The corresponding photographs analyzed by agarose gel electrophoresis are illustrated in Fig. S2.

3.3. Optimization of the experimental conditions As target X can competitively hybridize with both X0 –X0 and X –Y0 , the ratio of templates X0 –X0 and X0 –Y0 is a fatal factor to influence the production of reporter Y. With higher molar ratios of X0 –X0 , the trigger X was sufficiently amplified by an exponential circuit in the first stage but not converted to reporter Y effectively; with lower molar ratios of X0 –X0 the exponential feedback circuit was hindered, which subsequently affected the quantity of reporter Y created in the second stage. Keeping the total concentration of templates X0 –X0 and X0 –Y0 to be 500 nM, the ratios of 1:4, 2:3, 3:2, and 4:1 were quantitatively evaluated (Fig. 2A). The levels of signal increasing were calculated to be 11.8%, 185.8%, 156.4% and 12.0%, successively. Therefore, the ratio of 2:3 was selected for template X0 –X0 :X0 –Y0 as the optimal condition. Amplification time is another essential factor to determine the performance of GQ-EXPAR. To avoid the signal increase induced by nonspecific background amplification in the absence of target X, such as the generation of reporter Y from spurious triggering event, an optimal 30 min was selected for GQ-EXPAR incubation under the optimal ratio of templates (Fig. 2B). Within the same given time, more initial target X can trigger more amplification 0

Fig. 1. (A) The influences of Nt.BstNBI and Vent (exo-) DNA polymerase on GQ-EXPAR performance. All the solutions contained template X0 –X0 , X0 –Y0 and 10 nM target X. (B) The control experiments of GQ-EXPAR performed without target X, without amplification template X0 –X0 and with 100 pM target X, individually.


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160 120 2.0


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Molar ratio of X'-X':X'-Y'


level of signal increase %


1.2 1.0 0.8 0.6 0.4

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Negative 1E-13







target X concentration / M 60

Fig. 3. (A) The photograph for the colorimetric detection of target X by naked eyes. (Negative control: detection reaction without target X) (B) Calibration curve of signal response (A450–A620) vs. target X concentrations. Signal responses were obtained via monitoring dual wavelengths after termination reaction. The experiment was performed under optimal conditions.

40 20 0 30




time / min

template and lead to more exponential circuit, which indicates more conversion to reporter EAD2. 3.4. The performance of GQ-EXPAR for DNA detection The quantification performance of GQ-EXPAR for target X detection was investigated under the above optimal conditions. The absorption values A450–A620 after termination of the TMB– H2O2 coloration were recorded in the presence of different target DNA concentrations. As shown in Fig. 3(B), the colorimetric responses increased when the target was raised from 0 to 10 nM. This indicated that the amount of created reporter EAD2 was highly dependent on the concentration of trigger X. Even 10 pM target X triggered remarkable color change that could be easily distinguished by naked eyes (Fig. 3A). Target X could be quantified ranging from 10 pM to 10 nM with the limit of detection (LOD) estimated to be 2.5 pM (25 amol in 10 μL) based on a signal-to-noise ratio (S/N) of 3. The specificity of the proposed strategy was further investigated by perfect-matched target X, single-base mismatched, twobases mismatched and three-bases mismatched DNAs (Fig. 4). The signal responses of two-bases and three-bases mismatched DNAs


level of signal increase %

Fig. 2. (A) Variation of the level of signal increase as a function of the molar ratio of X0 –X0 :X0 –Y0 . The total concentration of X0 –X0 and X0 –Y0 was kept at 500 nM. Level of signal increase¼(Y  N)/N  100%, where Y is the signal triggered by 1 nM target X and N is the signal of corresponding negative control in the absence of target X. (B) Variation of the level of background increase at different incubation times in the absence of target X. Experimental conditions: template X0 –X0 and X0 –Y0 concentrations were fixed to 200 nM and 300 nM, respectively.

100 80 60 40 20 0 control


single-base two-bases three-bases mismatch mismatch mismatch

Fig. 4. (A) The photograph of specificity evaluation for perfect-matched target X, single-base, two-bases and three-bases mismatched sequences. (B) The level of signal increase of the GQ-EXPAR assay for analyzing 100 pM perfect-matched target X, single-base, two-bases and three-bases mismatched sequences under the same experimental condition, individually. Level of signal increase¼(Y  N)/N  100%, where Y is the signal triggered by 100 pM DNA sample and N is the signal of corresponding negative control.

show no difference to that of the negative control (in the absence of target X). The single-base mismatched DNA shows 19.3% signal increase, which is about one-fifth of that triggered by the perfectmatched target X (Fig. 4B). We found that mismatched DNAs were

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more difficult to initialize the GQ-EXPAR. Only perfect-matched target X can trigger remarkable colorimetric response (Fig. 4A). Thus, our strategy exhibited good performance in the discrimination of mismatched sequences. To evaluate the feasibility of the GQ-EXPAR assay for more complex sample, we carried out the quantification performance by spiking target DNA into human serum. Also, 10 pM target X can trigger notable color change. In Fig. S3, a calibration curve is achieved with LOD calculated to be 4.6 pM (S/N ¼ 3). This indicated the potential of applying our strategy for DNA detection in a complicated biological matrix. 3.5. The generalization of GQ-EXPAR In order to test the general applicability of GQ-EXPAR we designed, the platform was further implemented to the detection of Tay–Sachs genetic disorder mutant. Tay–Sachs disease is an inherited disorder that leaves the body unable to produce hexosaminidase A for ganglioside GM2 degradation (Lu et al., 2012; Wang et al., 2012). Since the target sequence was increased to 25 nt length, several factors of GQ-EXPAR were adjusted. Thermal properties of DNA oligonucleotide, diffusion and annealing of trigger X to template, activity of thermophilic DNA polymerase and thermal denaturation of nicking enzyme were four important factors that should be balanced for seeking an optimal incubation temperature. The optimization of incubation temperature (Fig. S4) shows that 1 nM TS-mutant can trigger significant signal at 58 1C that is much better than those performed at 55 1C, 60 1C and 63 1C. We prefer to suppose that although the activity of Nt.BstNBI might be slightly restricted compared with that at 55 1C (the optimum temperature for Nt.BstNBI activity), higher temperature probably assisted the dissociation of created X (here the TS-mutant sequence) from an X0 –X0 template, which immediately took part in another exponential circuit rapidly. The temperature adjustment offered us a potential way to improve performance towards other target oligonucleotide with different length and specific


sequence. At 58 1C, we further optimized the incubation time of GQ-EXPAR. As a better sensitivity toward TS-mutant was achieved with longer incubation time (Fig. S5), 1 h was selected as a suitable condition. The quantitative performance of GQ-EXPAR for Tay–Sachs mutant sequence was investigated under the optimum conditions mentioned above. As shown in the corresponding calibration curve (Fig. 5), the signal response increased with the concentration of TS-mutant ranging from 5 pM to 1 nM. And 5 pM TS-mutant was easily detectable by naked eye. The LOD was estimated to be 1.7 pM (17 amol in 10 μL) based on an S/N of 3, which is comparable or even better than the recently reported assays for Tay–Sachs mutant detection (Lu et al., 2012; Wang et al., 2012). For comparison, both 100 pM mutant gene and normal gene were analyzed, individually. A five-fold colorimetric signal for mutant gene was observed, implying that the mutant can be discriminated from the normal gene (Fig. S6).

4. Conclusion In summary, we developed a novel GQ-EXPAR for simple, rapid, and label-free DNA colorimetry. It was achieved by flexibly engineering exponential and linear amplification circuits to bring out the continuous creation of trigger DNA and their conversion to signal readout element, a G-quadruplex sequence EAD2. EXPAR integrated with G-quadruplex/hemin DNAzyme contains following advantages: (1) the tandem dual-amplification, exponential circuit for trigger DNA and mimic-HRP DNAzyme, is involved to guarantee good quantification performance; (2) the label-free and onepot homogeneous DNA detection can be simply finished in an hour without any extra synthesis or separation process; (3) reporter oligonucleotide is directly utilized to produce the signal instead of complicated downstream operations. The strategy can detect as low as 2.5 pM model DNA with high specificity towards mismatched/perfect-matched sequences. The successful detection of Tay–Sachs mutant (LOD 1.7 pM) illustrated the feasibility of the GQ-EXPAR for DNA detection to be implemented to many more oligonucleotides. We envision the GQ-EXPAR to be a potential tool for point-of-care molecular diagnostics.

Acknowledgments 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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Appendix A. Supplementary material


Supplementary data associated with this article can be found in the online version at

1.0 0.8


0.6 0.4 0.2 Negative 1E-13






TS-mutant concentration / M Fig. 5. (A) The photograph for the colorimetric detection of TS-mutant by naked eyes. (Negative control: detection reaction without TS-mutant.) (B) Calibration curve of signal response (A450–A620) vs. TS-mutant concentrations. Signal responses were obtained via monitoring dual wavelengths after termination reaction. The experiment was performed under optimal conditions.

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G-quadruplex based two-stage isothermal exponential amplification reaction for label-free DNA colorimetric detection.

A novel G-quadruplex based two-stage isothermal exponential amplification reaction (GQ-EXPAR) was developed for label-free DNA colorimetric detection ...
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