This journal is©The Royal Society of Chemistry 2014 Chem. Commun., 2014, 50, 6211--6213 | 6211
Cite this:Chem. Commun., 2014,
50, 6211
Reporter-triggered isothermal exponentialamplification strategy in ultrasensitive homogeneouslabel-free electrochemical nucleic acid biosensing†
Ji Nie, De-Wen Zhang, Fang-Ting Zhang, Fang Yuan, Ying-Lin Zhou* andXin-Xiang Zhang*
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 repor-
ter, G-quadruplex. This R-EXPAR strategy has been successfully
implemented to construct a homogeneous label-free electro-
chemical sensor for ultrasensitive DNA detection.
Sequence-specific nucleic acid detection is essential for detectingclinical pathogens, bio-threat agents and the early diagnosis ofcancer or genetic disease in a post-genomic era. An ideal point-of-care (POC) detection should fulfil the ASSURED (Affordable, Sensi-tive, Specific, User-friendly, Rapid and Robust, Equipment-free andDeliverable to end users) criteria represented by the World HealthOrganization.1 Towards these goals, electrochemical detectionsshow compelling advantages, such as simplicity, low cost, lesspower requirement and miniaturized platform,2–4 and they performwell with cloudy or colored samples, which might cause interferencein optimal assays.
Most nucleic acid electrochemical sensors are based on hetero-geneous format.5–8 The immobilization of a probe DNA onto theelectrode is necessary to perform a relevant recognition event withtarget DNA. Although high sensitivity is provided, the heterogeneousformat shows some intrinsic drawbacks: complex and time-consuming immobilization process; expensive modification of linkinggroup; lower hybridization efficiency due to steric hindrance. As thecomplement to heterogeneous assay, homogeneous electrochemicalsensor seems more suitable to achieve a desired simple, rapid, robustand easy-to-operate POC nucleic acid detection. Compared withlabelled assays,9–11 label-free ones are more attractive due to low cost.Until now, label-free electrochemical methods are mainly achievedvia double-stranded DNA-intercalating redox probes.4 While taking
advantages of design simplicity and operation convenience, they oftensuffer from background interference of non-selectivity intercalationand loss of sensitivity.12 A more direct, specific and efficient signalread-out mode is in great need.
G-quadruplex, with repetitive G-rich structural motifs, can act asDNAzyme possessing peroxidase-mimic activity when binding withhemin.13 Since it can be flexibly encoded into nucleic acid basedstrategy, the generation of G-quadruplex sequence through targetDNA recognition process might become a novel signal read-outmode to meet the demands of constructing label-free andimmobilization-free electrochemical nucleic acid sensors. However,the sensitivity, which is correlated directly to the detectable concen-tration of G-quadruplex sequence, might be limited due to themuch lower catalytic activity comparing with horseradish peroxidase(HRP). A powerful reporter-amplification strategy should be intro-duced to offer sufficiently sensitive signal read-out for an idealelectrochemical POC assay.
Herein, we first developed reporter-triggered isothermal expo-nential amplification reaction (R-EXPAR) strategy, integrating with aminiaturized electrochemical device,14 for the construction of ultra-sensitive homogeneous label-free nucleic acid biosensor. EXPAR isan isothermal molecular chain reaction, which can synthesize shortoligonucleotides with high amplification efficiency.15 The R-EXPARcircuits can realize the exponential multiplication of reporter ele-ment (G-quadruplex) within a short time, which will achieve thetremendous signal read-out growth. This simple and rapid ampli-fication for reporter molecules is anticipated to remarkably improvethe sensitivity of assays that treat G-quadruplex generation as signalread-out.
Our R-EXPAR strategy is illustrated in Scheme 1. EAD2, an intra-molecular parallel G-quadruplex sequence, which showed highperoxidase-mimic activity, was chosen as reporter (denoted as ‘‘Y’’).The template Y0–Y0 was designed with two repeated complementarysequences of Y. The two regions were separated by the comple-mentary of nicking recognition and cleavage site (insert legendof Scheme 1). Once the Y priming the Y0–Y0 template, the primer-template could be polymerized by vent (exo-) polymerase andcleaved by Nt.BstNBI nicking enzyme. The created oligonucleotide
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 andadditional figures. See DOI: 10.1039/c4cc00475b
Received 20th January 2014,Accepted 12th March 2014
DOI: 10.1039/c4cc00475b
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6212 | Chem. Commun., 2014, 50, 6211--6213 This journal is©The Royal Society of Chemistry 2014
was then released via melting or strand displacement. Since it wasspecifically designed the same to trigger Y, an exponential amplifica-tion circuit could be achieved while the new Y triggered anotherY0–Y0. Subsequently, EAD2 can form EAD2–hemin DNAzyme andbe detected in hydroquinone (HQ)–H2O2 system by the ultramicro-electrode/micropipette-tip based miniaturized electrochemical devicein a small volume.
To validate the feasibility of R-EXPAR for EAD2 G-quadruplexsequence, we first measured the CVs of the amplification produc-tion triggered by 10 nM EAD2 and without triggering (the negativecontrol) using HQ as an electron mediator. The oxidation/reductionquasi-steady state currents were proportional to the concentrationsof HQ/BQ (benzoquinone) individually in solution. In the presenceof DNAzyme and H2O2, HQ was chemically oxidized to BQ. There-fore, the corresponding oxidation current was decreased accompa-nying the increase of reduction current.16 As shown in Fig. 1A, theR-EXPAR production triggered by 10 nM EAD2 (curve b) showssignificant decrease of oxidation peak current and increase ofreduction peak current compared with the negative control (curve a).In fact, 10 nM EAD2–hemin DNAzyme without amplification showedno obvious difference to the negative control (not provided here). Itindicated 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 miniaturizedelectrochemical device. The corresponding photograph analyzed
by agarose gel electrophoresis (Fig. S1, ESI†) can further verifythe amplification of EAD2 in the strategy.
In Fig. 1B, we compared the peroxidase catalytic activity ofEAD2–hemin DNAzyme with and without R-EXPAR, and HRP viaamperometric response curves. R-EXPAR production triggered by1 nM EAD2 (curve b) caused 5.61 nA reduction current increase after6 min catalytic reaction, while 1 nM HRP (curve c) showed onlyabout 1.54 nA change. The HRP-mimicking DNAzyme amplifiedthrough EXPAR showed significantly higher peroxidase activity thanthat of the natural enzyme with the same concentration. Withoutamplification, the Di–t curve of ideally formed 1 nM EAD2–heminDNAzyme (curve d) did not show obvious difference to that of thenegative control (curve a). It is apparent that the simple and rapidR-EXPAR exhibited excellent performance in magnifying the quan-tity of reporter molecules to a dramatic level.
To quantitatively evaluate the magnification level of R-EXPARtowards reporter, the sensitivity of EAD2 detection was tested (Fig. 2,curve a). The variations of reduction currents at different EAD2concentrations (potential fixed at �0.1 V) along 6 min catalyticreaction were recorded for quantification. With the increase of theEAD2 concentration, a corresponding sharp change in currentresponse was observed (curve a). It is consistent with the mecha-nism that more reporters Y triggered more exponential circuits andgenerated more reporters during the same extension/nicking time.With a dynamic range from 1 pM to 10 nM, as low as 1 pM EAD2can be magnified into experimentally detectable concentrationsbased on a signal-to-noise ratio (S/N) of 3. Also, we detected theEAD2 without the proposed strategy (curve b) under the sameexperimental conditions. A 100 nM EAD2 with excess heminwhich was supposed to form ideally 100 nM DNAzyme onlygained 0.63 nA current response change. 10 nM or lower than10 nM EAD2 formed DNAzymes could not be detected. Thecomparison demonstrated that our R-EXPAR was able to achieveconspicuous five orders of magnitude amplification (from100 nM down to 1 pM) for G-quadruplex/hemin DNAzymesystem. More attractively, only one simply designed Y0–Y0 DNAtemplate was required to perform the strategy in an extremelyshort time (15 min). Considering the cost-saving and labour-saving characteristics of the miniaturized homogeneous electro-chemical device, the strategy shows great potential to be universallyapplied for G-quadruplex sequence based sensing modes withsatisfactory sensitivity.
Scheme 1 The principle of R-EXPAR strategy integrated with an ultra-microelectrode/micropipette-tip based miniaturized electrochemicaldevice.
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) withouttrigger sequence, and (b) triggered by 10 nM EAD2. (B) The amperometricresponse curves of (a) negative control, (b) R-EXPAR production triggeredby 1 nM EAD2, (c) 1 nM HRP, and (d) 1 nM EAD2 at �0.1 V.
Fig. 2 Calibration curves of signal responses Di vs. EAD2 concentrations(a) with R-EXPAR, and (b) without R-EXPAR. Error bars represent SD ofthree independent experiments. RSD o 13.2%.
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A simple model which included a two-stage X0–Y0/Y0–Y0 circuitwas designed to certify the application of our R-EXPAR strategy fortarget DNA detection (Fig. 3A). The target DNA X could hybridizewith the recognition template X0–Y0. Through the polymerizationand nicking, released Y sequence (designed to be EAD2) wasobtained in a linear amplification manner. Following the conversionof X to reporter Y, the second stage R-EXPAR was performed. Theundetectable slight amount of reporter EAD2 generated from X0–Y0
target recognition could be amplified into a detectable amount viathe tandem Y0–Y0 protocol. As shown in Fig. 3B, 100 pM target Xtriggered 98% larger current variation than that performed withouttarget X. To ensure the triggering event was due to the hybridizationof target X with the relevant complementary region of recognitiontemplate X0–Y0, ten times of random sequence instead of target Xwas added. The Di–t curves illustrate that 1 nM random showednegligible difference to the negative control. It validated the highspecificity 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 thatof X0–Y0 strategy (Fig. 3C). For the X0–Y0/Y0–Y0 mode (curve a), thecurrent response increased with the concentration of target X from0 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 forreporter element, EAD2 was generated directly via linear conversionfrom recognition element to signal read-out element. 15 nM of targetX was the lowest detectable concentration in X0–Y0 mode. Thus nearlyten thousand times improvement of sensitivity was achieved via theadditional 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 comparativeexploration of the perfect-matched target DNA, two-bases mismatched
DNA (M2) and three-bases mismatched DNA (M3) (Fig. S2, ESI†). Thecurrent changes for the M2 and M3 were 32.8% and 8.84% of that ofthe perfect-matched target DNA, respectively. To improve the selectivityfor better discrimination among mismatches, more elaborated recog-nition event (e.g., adopting sensitive structure switches in response tothe specific target triggering) should be involved. The results hereindicate the potential that R-EXPAR coupled DNA recognition strategycould show good signal discrimination for mismatch among traceDNA samples. We also successfully evaluated the feasibility of thestrategy spiked with 20% human serum (Table S3, ESI†).
In conclusion, a simple and novel R-EXPAR strategy integratingan ultramicroelectrode/micropipette-tip based miniaturized electro-chemical device was developed for label-free homogeneous nucleicacid biosensing. The reporter molecules (EAD2) via the rapidR-EXPAR can be detected down to 1 pM (five orders of magnitudeimprovement). Excellent sensitivity for target DNA detection wasachieved in X0–Y0 recognition strategy with a tandem R-EXPARprotocol. Simultaneously, the label-free and immobilization-freeminiaturized electrochemical measurements exhibited outstandingperformances like easy operation, low cost, high reliability andrepeatability. Not only for sequence-specific nucleic acid detection,the R-EXPAR strategy showed great potential to be implemented tosuitable 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 asreporter.
This work was supported by the National Natural ScienceFoundation of China (Nos. 21275009 and 20805002) and theScientific Research Foundation for the Returned OverseasChinese Scholars, MOE, China.
Notes and references1 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 andY. 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.
Fig. 3 (A) The principle of two-stage X0–Y0/Y0–Y0 strategy. (B) The feasi-bility of two-stage X0–Y0/Y0–Y0 strategy without target X (negative control),100 pM target X and 1 nM random, individually. (C) Calibration curves ofsignal responses Di vs. target X concentrations, (a) via X0–Y0/Y0–Y0 strategy;(b) via X0–Y 0 strategy. The leftmost dot of each curve means the negativecontrol without target X. Error bars represent standard deviations of threeindependent experiments. RSD o 11.1%.
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