靶标循环扩增电化学阻抗法检测DNA

Cite this:DOI:10.1039/c1cc14902d Target recycling amplification for sensitive and label-free impedimetric genosensing based on hairpin DNA and graphene/Au nanocomposites wYing Chen,a Bingying Jiang,b Yun Xiang,*a Yaqin Chai a and Ruo Yuan*aReceived 8th August 2011,Accepted 27th October 2011DOI:10.1039/c1cc14902dThe presence of exonuclease III leads to direct recycling and reuse of the target DNA,which in turn results in substantial signal amplification for highly sensitive,label-freeimpedimetric detection of specific DNA sequences.Graphene is a two-dimensional single layer of sp 2-hybridized carbon sheets.1Since its first isolation in 2004,graphene has attracted great research attention due to its unique properties,such as high surface area,2,3amazing intrinsic mobility,excellent thermal and electrical conductivity.4,5Recently,graphene and graphene-based materials have been increasingly used in biosensor applications.One of the major goals of the biosensor technology is to determine specific DNA sequences for various applications in drug discovery,diagnosis of diseases,forensic and food technology.Currently,specific and sensitive detection of DNA is mainly based on the transduction of hybridization of complementary DNA strands by fluorescence,6–8quartz microbalance,9,10surface plasmon resonance spectroscopy,11,12atomic force microscopy 13and electrochemical (EC)techniques.14–16Among these transduction methods,EC DNA sensors have shown promising potentials for point-of-care diagnostics and other important applications.Being highly sensitive and label-free,electrochemical impedance spectroscopy (EIS)has gained more and more attention as a powerful tool for probing molecular binding events such as DNA hybridizations.17–19The formation of the complexes upon bioaffinity bindings commonly changes the interfacial electron transfer kinetics between the redox probe and the electrode and generates quantitative signal outputs monitored by EIS for biosensing.However,one of the challenges in EIS-based detections is to amplify the signal mon routes for amplified EIS detection of a trace amount of target molecules require modifications of either the target or the probe molecules with labels,20–23which sacrifices the inherent advantages (e.g.simplicity and label-free capability)of this technique.In response,we reportherein a convenient signal amplification strategy for highly sensitive,label-free EIS determination of DNA sequences based on enzymatic target recycling and graphene/AuNP nanocompo-sites,as well as the hairpin DNA probes.The introduction of the catalytic enzyme,exonuclease III (Exo III),into the sensing system leads to direct recycling and reuse of the target DNA,which in turn amplifies the EIS signal.This enables amplified EIS detection of a trace level of specific DNA sequences in a convenient and truly label-free fashion.Our enzyme-assisted target recycling amplification strategy for sensitive and label-free EIS detection of DNA is illustrated in Scheme 1.This involves a one-step EC reduction of a mixture of graphene oxide and HAuCl 4to form graphene/AuNP nanocomposites on a screen printed carbon electrode (see ESI w for details),self-assembly of the hairpin DNA probes on the modified electrode and surface blocking with 6-mercapto-1-hexanol (MCH),addition of the target DNA together with Exo III and EIS measurement of the resulting electrode.The target DNA present hybridizes with the hairpin probe to form a double stranded DNA.At the same time,Exo III specifically cleaves the open,hybridized hairpin DNA and releases the target DNA because of the unique enzymatic property of Exo III,which catalyzes the stepwise removal of mononucleotides from the blunt or recessed 30-hydroxyl termini of a duplex DNA and is inactive to single stranded DNA.Upon enzymatic cleavage of the probe,the released target DNA again hybridizes with the remaining hairpin DNA probe and triggers another target recycling cycle.Therefore,a smallScheme 1Illustration of the enzyme-assisted target recycling for amplified EIS detection of DNA on a graphene/AuNP modified electrode.aKey Laboratory on Luminescence and Real-Time Analysis,Ministry of Education,School of Chemistry and ChemicalEngineering,Southwest University,Chongqing 400715,P.R.China.E-mail:yunatswu@,yuanruo@;Fax:+86-23-68252277;Tel:+86-23-68253172bSchool of Chemistry and Chemical Engineering,Chongqing University of Technology,Chongqing 400054,P.R.Chinaw Electronic supplementary information (ESI)available:Preparation of LBL assemblies and ECL detections.See DOI:10.1039/c1cc14902dChemCommDynamic Article Links/chemcommCOMMUNICATIOND o w n l o a d e d b y S H A N D O N G U N I VE R S I T Y o n 12 N o v e m b e r 2011P u b l i s h e d o n 11 N o v e m b e r 2011 o n h t t p ://p u b s .r s c .o r g | d o i :10.1039/C 1C C 14902DView Online / Journal Homepageamount of target DNA is expected to efficiently remove a large number of hairpin DNA probes from the electrode surface,leading to a substantial decrease in electron transfer resistance(R et )monitored by EIS.The decrease in R et can thus be related to the quantity of the target DNA in the testing samples.The stepwise sensor fabrication process is monitored by EIS and the resulting Nyquist plots are displayed in Fig.1.The EIS data were fitted to a Randles equivalent circuit (inset in Fig.1),which includes the solution resistance (R s ),R et ,the constant phase element (Q dl )and Warburg impedance (W ).In the Nyquist diagram,the diameter of the semicircle reflects the R et of redox conversion of the electroactive marker [Fe(CN)6]3À/4Àon the electrode at certain applied potential.This process is strongly dependent upon any modification to the electrode surface.As can be seen in Fig.1,the R et value decreases significantly after the deposition of the graphene/AuNPs on the electrode surface.This dramatic decrease in R et can be primarily attributed to the excellent electrical conductivity of both the electrochemically reduced graphene oxide 24and AuNPs,25which promotes the electron transfer from the electroactive markers to the electrode.However,after self-assembly of a mixed monolayer of the hairpin DNA probes and MCH on the graphene/AuNP modified electrode,a sub-stantial increase in R et is observed.Such an increase in R et is basically due to two facts.First,the negative charges on the phosphate backbone of the surface immobilized hairpin probes repel [Fe(CN)6]3À/4Àfrom the electrode,leading to an increase in R et .Second,the steric hinderance of the hairpin structure of the probe prevents [Fe(CN)6]3À/4Àfrom approaching the electrode and causes increase in R et .26The combination of these two effects thus results in a substantial increase in R et after formation of the mixed monolayer on the electrode.In order to demonstrate the dramatic signal amplification capability of the proposed label-free assay protocol,we compared our enzyme-assisted target recycling detection approach with the conventional method.From the Nyquist plots in Fig.2,we can see that the presence of 500pM target DNA (without Exo III)results in a small drop (20.7%)in R et (curve b vs.curve a),which is due to the decrease in the steric hinderance of thehairpin DNA probe upon hybridization and easy approaching of [Fe(CN)6]3À/4Àto the electrode.27While in the presence of both target DNA (500pM)and Exo III (5U),the target DNA can be used multiple times and more hairpin DNA probes are removed from the surface due to the catalytic target recycling by Exo III.A substantial decrease (60.0%)in R et is observed (curve c vs.curve a).When no target DNA is present in the testing sample,the addition of Exo III alone causes a negligible effect on R et (curve d).These results clearly suggest significant signal amplification by the incorporation of the enzyme-assisted target recycling in EIS detection of DNA.EIS measurements of the target DNA at various were used to determine the detection limit and dynamic range of the proposed sensor.Typical Nyquist plots of the sensor before and after incubation with the target DNA are displayed in Fig.3.It is obvious that the R et value shows a dependence upon the concentration of the target DNA (Fig.3A).As the concentration of the target DNA increases,the R et value decreases accordingly.The corresponding calibration plot (Fig.3B)of log c vs.D R et (the difference before and after incubation with the target DNA)exhibits a dynamic range from 50fM to 5nM and the limit of detection could be estimated to be 10fM based on the 3s rule.This detection limit is about 20–500-fold than other universal EIS detection strategies.26,28–33Such a low detection limit is primarily due to the involvement of the target recycling signal amplification in the assay protocol.The reproducibility of the proposed method was evaluated by performing a series of six repetitive experiments for the target sequence at the concentration of 500pM,Fig.1Nyquist diagrams for (a)bare SPCE electrode,(b)graphene/AuNP modified SPCE and (c)hairpin DNA and MCH self-assembled graphene/AuNPs/SPCE.Inset:equivalent circuit used to fit the EIS data.EIS measurements were carried out in 0.1M KCl solution containing 5mM (1:1)[Fe(CN)6]3À/4Àwith the range from 10kHz to 50MHz and an alternate voltage of 5mV.Fig.2Nyquist diagrams for sensors incubated with (a)buffer,(b)complementary DNA (500pM),(c)complementary DNA (500pM)with Exo III (5U)and (d)Exo III (5U).Fig.3(A)Nyquist diagrams for sensors incubated with different concentrations of target DNA:(a)5nM,(b)500pM,(c)50pM,(d)5pM,(e)500fM,(f)50fM,(g)blank (0fM target).(B)The resulting calibration plot of log c vs.D R et for the target DNA over the 50fM to 5nM range (error bars:SD,n =3)D o w n l o a d e d b y S H A N D O N G U N I VE R S I T Y o n 12 N o v e m b e r 2011P u b l i s h e d o n 11 N o v e m b e r 2011 o n h t t p ://p u b s .r s c .o r g | d o i :10.1039/C 1C C 14902Dwhich yielded a relative standarddeviation of 7.3%.This indicates that our assay protocol is coupled with good reproducibility.The selectivity of the sensor was examined by incubation with complementary,non-complementary and base mismatch DNAs and the EIS results are shown in Fig.4.The addition of 500pM non-complementary DNA (Fig.4b)to the sensor causes insignificant decrease in R et compared to the blank test (Fig.4a).Despite that the presence of 500pM one-base and three-base mismatch DNAs leads to decrease in R et ,it is not comparable with that of the presence of 500pM target DNA.These results indicate that the label-free impedimetric sensor could effectively distinguish target DNA from non-complementary and base mismatch DNA.Such discrimination between target and non-target DNA suggests the inherent high selectivity of the hairpin DNA probes 26as well as the minimized non-specific adsorption by the surface blocking 34,35and washing.In summary,we have demonstrated a convenient signal amplification strategy for sensitive and selective EIS detection of DNA target on a graphene/AuNP modified electrode.Our approach couples the high sensitivity of the EIS technique with the enzyme-assisted target recycling signal amplification to achieve low femtomolar detection of target gene sequences.Moreover,unlike other endonucleases such as nicking endo-nuclease and Fok I enzyme,which require a specific sequence for enzyme cleavage,Exo III is sequence independent.This unique property of Exo III makes the proposed sensor hold great potential for highly sensitive,selective and simple detection of a wide range of target DNA sequences.This work is supported by National Science Foundation of China (No.20905062and 20675064),Natural Science Foundation Project of Chongqing City (CSTC-2009BB5052),State Key Laboratory of Electroanalytical Chemistry (SKLEAC2010009),China Postdoctoral Science Foundation (20090460715and 201003305),Scientific Research Foundation for the Returned Overseas Chinese Scholars and research funds from 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