Electrochemistry Communications 12 (2010) 985–988

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Electrochemistry Communications

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Ultrasensitive electrochemical detection of nucleic acid based on the isothermalstrand-displacement polymerase reaction and enzyme dual amplification

Yuqing He a,b,c, Kang Zeng a,⁎, Xibao Zhang b, Anant S. Gurung c, Meenu Baloda c, Hui Xu c, Guodong Liu c,⁎a Department of Dermatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Chinab Department of Dermatology, Guangzhou Institute of Dermatology, Guangzhou 510095, Chinac Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, 58105, United States

⁎ Corresponding authors. Tel.: +1 701 231 8697; faxE-mail addresses: npfk@fimmu.com (K. Zeng), guod

1388-2481/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.elecom.2010.05.007

a b s t r a c t

a r t i c l e i n f o

Article history:Received 25 March 2010Received in revised form 28 April 2010Accepted 12 May 2010Available online 20 May 2010

Keywords:ElectrochemicalDNAStrand-displacementEnzymeAmplification

We describe an ultrasensitive electrochemical detection of DNA protocol based on the isothermal strand-displacement polymerase reaction (ISDPR) and enzyme dual amplifications. Target DNA triggered an ISDPRto produce numerous bi-functionalized duplex DNA complexes. Following an immuno-magnetic collectionvia an immunoreaction between the attached digoxin on the duplex DNA and the anti-digoxin antibody onthe magnetic bead, horseradish (HRP) tracers were bound to the duplex DNA through a biotin–streptavidininteraction. The quantification of DNA was realized by square wave voltammetric detection of the enzymaticproducts with a screen-printed gold electrode. The voltammetric response was proportional to theconcentration of DNA in the range of 0.1 fM–0.5 pM, and the limit of detection was estimated to be 0.06 fM.The new protocol showed great promise for simple, cost-effective, and quantitative gene analysis.

: +1 701 231 8831.ong.liu@ndsu.edu (G. Liu).

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© 2010 Elsevier B.V. All rights reserved.

1. Introduction

The detection of specific sequences of DNA is of central importancefor the diagnosis and treatment of genetic diseases, for the detectionof infectious agents, and for reliable forensic analysis [1]. Recently,electrochemical (EC) biosensing and bioassay of nucleic acids haveattracted considerable interest because of their inherent simplicity,low cost, high sensitivity, and miniaturization [2]. Among the manymethods devised, amplification is one of the most important conceptsbecause it permits the highest analytical sensitivity. Recent activityhas focused on the development of magnetic bead (MB)-based ECDNA hybridization sensors [3,4], and nanomaterials-enhanced ECDNA bioassays [5]. Particularly, combination of nanomaterials withMB-based protocols provides sensitive methods to electrochemicallydetect several thousand DNA molecules [5]. However, preparation ofsuch DNA-conjugated nano-biolabels is complex and time consuming,therefore, design of alternative approaches for the sensitive EC detec-tion of DNA is in continuous demand.

Combination of EC detection and polymerase chain reaction (PCR)amplification offers an alternative approach to detect nucleic acidsamples [6]. However PCR requires precise control of temperaturecycling for successful DNA amplification, and the resultant instru-

mental restraint has been hampering its wider and more versatileapplications [7]. Recently, an amplified DNA detection method basedon isothermal strand-displacement polymerase reactions (ISDPRs)has been developed [8]. ISDPR is based on an isothermal amplificationprocess, which is free from the problems raised in PCR, and yieldslarge amounts of DNA products to enhance the signal and thesensitivity of DNA detection [8]. Although the ISDPR-based fluores-cence DNA assay offers a high specificity, there are still manychallenges. For example, the detection still needs expensive, dual-labeled probes; meanwhile, fluorophores result in a high fluores-cence background and, thus, a decrease in the detection sensitivity.Here, we presented a protocol for ultrasensitive detection of DNAbased on ISDPR, enzyme catalytic amplification and EC detection. Thepromising properties of the approach are reported in the followingsections.

2. Experimental

2.1. Apparatus

EC experiments were performed using a CHI 660A EC work station(CH Instruments, Austin, TX). A disposable, screen-printed gold elec-trode (SPGE) consisting of a gold working electrode, a gold counterelectrode, and an Ag/AgCl reference electrode was purchased fromPalm Instruments BV (Houten, The Netherlands). The MB assaysand separations were performed on a MCB 1200 Biomagnetic BeadProcessing Platform (Dexter, CA).

986 Y. He et al. / Electrochemistry Communications 12 (2010) 985–988

2.2. Reagents

The polymerase Klenow fragment exo– and streptavidin-modifiedMBs (1-μm diameter) were purchased from Biolabs, Inc. (NewEngland). Deoxynucleoside triphosphates (dNTP), dithiothreitol(DTT), Dimethyl sulfoxide (DMSO), biotinylated anti-digoxin anti-body, bovine serum albumin (BSA), o-aminophenol (o-AP), hydrogenperoxide, streptavidin-horseradish peroxidase (HRP), and phosphatebuffered saline (PBS, pH 7.4, 0.01 M), were purchased from Sigma-Aldrich (ST. LOUIS). The stock substrate solution of o-AP was prepareddaily or prior to use in the Britton–Robinson (BR) buffer at afinal concentration of 0.01 M. The hairpin and oligonucleotideprobes used in this study were obtained from Integrated DNATechnologies, Inc. (Coralville, IA). The oligonucleotide sequences wereas follows:

Biotinylated hairpin probe: 5′-/5BioTEG/TCTTGGACACAGTCTGTTT-GTGCATACCCTGTGTCCAAGA-3′Digoxin-modified primer: 5′ -DigN/TCTTGGACTarget DNA: 5′ -GTG GGT ATG CAC A AA CAG ACT GTG T- 3′Noncomplementary DNA: 5′-TGCAAGGTGTCAGTATAATCCGACG-TTTT-3′

Fig. 1. (A) Schematic illustration of the production of functionalized duplex DNA based on isoduplex DNA and electrochemical detection of enzymatic product.

2.3. Preparation of digoxin- and biotin-functionalized duplex DNAcomplexes with ISDPRs

Briefly, ISDPR was performed in a 100-μL solution consisting of5.0×10−8 M biotinylated hairpin probe, 5.0×10−8 M digoxin-primer,3 U polymerase Klenow fragment exo–, 50 μM dNTPs, 6% DMSO, 0.1%BSA, 1 mMDTT, and 5 mMMgCl2 in a 50-mMTris–HCl (pH 8.0) buffer.Target DNA at different concentrations was then added to everymixture sample solution and incubated at 42 °C for 1 h.

2.4. Immuno-magnetic collection of the formed duplex DNA complexes

Immuno-magnetic collection of the formed duplex DNA com-plexes was performed with a modified procedure [3]. Briefly, 5 μLstreptavidin-coated MBs were transferred into a 1.5-mL centrifugetube. The beads were then washed with 95 μL of PBS containing 0.1%Tween (PBST) and were suspended in 45 μL of PBS containing 1% BSA(PBSB, pH 7.4). Subsequently, 5 μL of the biotinylated anti-digoxinantibody (1 mg mL−1) were added, and the mixture was incubatedfor 20 min with gentle mixing. The antibody-coated MBs werewashed twice with 100 μL of PBST and suspended in the ISDPRproduct solution. The mixture was incubated for 30 min with gentle

thermal strand-displacement polymerase reactions; (B) immuno-magnetic collection of

Fig. 2. (A) Typical cyclic voltammograms of SPGE in the presence of 0.2 mM o-AP+H2O2 substrate (curve a) and corresponding enzymatic product (curve b), the enzymecatalytic reaction preceded for 20 min after addition of 0.1 U/mL HRP. Inset is thecorresponding SWV responses; (B) typical SWV responses in the presence 10 fM targetDNA (a), 10 nM noncomplementary DNA (b), and 0 nM target DNA.

987Y. He et al. / Electrochemistry Communications 12 (2010) 985–988

mixing. After magnetic separation, the duplex DNA-MBswere washedtwice with 100 μL of PBST and suspended in 45 μL of PBSB.Subsequently, 5.0 μL of streptavidin–HRP (1/100 dilution) wereadded and mixed for 20 min. The resulting microspheres werewashed 3 times and resuspended in 40 μL of 0.2-mM o-AP+H2O2

substrate solution. After a 5-min mixing and a magnetic separation,the suspensionwas transferred to a SPGE surface for ECmeasurement.

2.5. EC measurements

The enzymatic product was dropped to a SPGE surface to form an ECmicrocell. Square wave voltammetric (SWV) measurements wereperformed by applying a potential scanning at the range of −0.3 to0.1 V with a step of 4 mV, an amplitude of 25 mV, and a frequency of10 Hz. Baseline correctionof the resultingvoltammogramwasperformedusing the “linear baseline correction”mode of the CHI 660 software.

3. Results and discussion

3.1. Principle of the EC bioassay of DNA

The new protocol combines the amplification features of ISDPRsand enzymes with the sensitive electrochemical detection of enzy-matic products. The principle is shown in Fig. 1. The target DNA is firstused to generate numerous bi-functionalized, duplex DNA complexesthrough the ISDPRs (Fig. 1A). The ISDPR solution consists of abiotinylated hairpin probe, a digoxin-modified primer, polymerase,and buffer solution. In the presence of target DNA, the hairpin proberecognizes and hybridizes with it and undergoes a conformationalchange, leading to stem separation (Step 1). Following this step, thedigoxin-modified primer anneals with the open stem (Step 2) andtriggers a polymerization reaction in the presence of dNTPs/polymer-ase (Step 3). In the process of primer extension, the target is displacedby the polymerase with strand-displacement activity, after which abiotin- and digoxin-modified, duplex DNA complex is formed. Thedisplaced target hybridizes with another biotinylated hairpin probe,which triggers a new polymerization reaction (Step 4). Throughoutthis cyclical process, numerous biotin- and digoxin-modified, duplexDNA are produced. The biotinylated hairpin probe used in this studyacts as a template of polymerase reaction and biotin carrier. The biotinin the hairpin probe is used to capture streptavidin conjugated HRPtracer. The hairpin probe has stem long enough to ensure that stemhybridization affinity will be stronger than hybridization affinity withthe primer. In the absence of a target, the stem-loop conformationalprobe is unable to anneal with the primer to induce a polymerizationreaction. The resulting duplex DNA complexes are then captured byanti-digoxin antibody-conjugated MB (Fig. 1B). The reaction mixtureis subjected to a magnetic field to separate the excess reagents.Subsequently, streptavidin–HRP is introduced to react with the biotingroups of the bound, duplex DNA on theMB surface. The capturedHRPlabels are determined by SWVmeasurement of the enzymatic productin the presence of substrate.

In the current study, o-AP was selected as the substrate of HRP forenzymatic reaction to produce electroactive products, 3-aminophe-noxazone (3-APZ, Fig. 1B) [9]. The concentration of target DNA wasdetermined by measuring the SWV response of 3-APZ on a SPGE.Fig. 2A presents the typical cyclic voltammograms of the substrate(curve a) and the enzymatic product (curve b) on the SPGE. A coupleof well-defined redox peaks (Epa=−0.12 V, Epc=−0.18 V) wasobserved, and no redox peak was observed in the selected potentialrange for the substrate. The inset also presents the correspondingSWV responses on the SPGE. Such favorable responses offer a sensitiveapproach to quantify the activity of HRP, thus the DNA concentration.Fig. 2B displays the typical SWV responses of the samples containing10-fM target DNA (a), and 10-nM noncomplementary DNA (b) and 0-nM DNA (c) after the complete assays. A well-defined voltammetric

peak is observed for 10-fM DNA, while negligible signals are obtainedfor 10 nM noncomplementary DNA and 0 nM DNA, indicating ex-cellent specificity of the assay and negligible environmental contam-ination in ISDPRs.

3.2. Optimization of experimental parameters

To obtain the best sensitivity and reproducibility of the assay,analytical parameters, including the temperature of the ISDPR, theamount of anti-digoxin-conjugated MBs and substrate concentration,was optimized. It was found that an ISDPR temperature of 42 °C gavethe best signal-to-noise (S/N) ratio. A higher temperature leads to ahigh background signal and a low S/N ratio, which may be caused bythe conformational change of the hairpin probe at a high temperatureinducing the polymerase reactions even in the absence of target DNA.The amount of anti-digoxin-conjugated MB influences the amount ofduplex DNA bound to the surface of MBs, and the amount of thecaptured HRP labels. It was found that the response currents increasedupon raising the volume of anti-digoxin antibody-conjugated MBfrom 2.0 µL to 5.0 µL, and then, it started to level off (data not shown).Therefore, a volume of 5.0 µL anti-digoxin antibody-conjugated MB

Fig. 3. Typical SWV responses of DNA with increasing concentrations (from bottom totop (a to i): 0, 0.1, 0.5, 1, 5, 10, 50, 100, and 500 fM). Inset is the SWV responses of 0 fM(curve a) and 0.1 fM (curve b) target DNA.

988 Y. He et al. / Electrochemistry Communications 12 (2010) 985–988

was employed for the magnetic collection of the duplex DNAcomplexes. We studied the effect of the substrate concentration onthe electrochemical response of the assay. The results indicated thatthe S/N ratio would increase with an increasing substrate concentra-tion in the range of 0.01 mM to 0.2 mM (data not shown). A higherconcentration of o-AP leads to a decreased S/N ratio because of theincreased background signal, whichmay be caused by the oxidation ofo-AP at a high concentration level. Therefore, a 0.2-mM substrateconcentration was used throughout our experiments.

3.3. Assay performances

Under optimal experimental conditions, we examined the perfor-mance of the protocal with different concentrations of DNA. Fig. 3displays typical SWV responses for different concentrations of DNA.Well-defined voltammetric peaks are observed with such lowconcentrations of DNA. The peak-current increase with increasedDNA concentrations. The resulting plot of response vs. DNAconcentration is linear and suitable for quantitative work. Thefavorable response (see inset in Fig. 3) of the 0.1 fM target solutionindicates a detection limit (DL) of 0.06 fM based on the S/Ncharacteristics (S/N=3). Such a DL is comparable with that of the

carbon nanotube-enzyme based electrical protocol [10], and isconsiderably lower than the DLs reported for the fluorescence-basedISDPR protocol [8]. The amplified electrical signal is coupled with agood reproducibility. A series of eight repetitive measurements of 1-fMDNA yielded reproducible signals with relative standard deviationsof 6.5%.

4. Conclusions

We have demonstrated an ultrasensitive method for electrochem-ical detection of DNA based on the ISDPR and enzyme dualamplification events with high specificity. The detection limit of thisprotocol is 6.0×10−17 M. The new protocol provides a simple, fast andcost-effective approach for the detection of nucleic acid samples andcould readily find wide applications in molecular diagnosis laborato-ries. Further work will aim for the direct detection of DNA extractedfrom pathogens and viruses.

Acknowledgments

This research was supported by a grant from the North DakotaExperimental Program to Stimulate Competitive Research (EPSCoR)and new faculty startup funds at North Dakota State University. Y. Heacknowledges financial support from the Guangdong ProvincialNatural Science Foundation of China (no. 06021655; no. 07001961;and no. 07300648), the Key Research Plan of Guangzhou HealthDepartment, China (no. 2006-ZDi-07), and the Guangdong ProvinceHealth Department Research Fund of China (no. A2007543).

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