Probing DNA Hybridization by Impedance Measurement Based on CdS-Oligonucleotide Nanoconjugates

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Probing DNA Hybridization by Impedance Measurement Based onCdS-Oligonucleotide NanoconjugatesYing Xu, Hong Cai, Pin-Gang He, Yu-Zhi Fang*

Department of Chemistry, East China Normal University, Shanghai 200062, P.R. China*e-mail: [email protected]

Received: June 30, 2003Final version: August 12, 2003

AbstractA novel, sensitive DNA hybridization detection protocol, based on DNA-quantum dots nanoconjugates coupled withelectrochemical impedance spectroscopy (EIS) detection, is described. The amino-linked ss-DNA probe wascovalently immobilized onto a self-assembled mercaptoacetic acid monolayer modified gold electrode; afterhybridization with the target ssDNA-CdS nanoconjugates, EIS was used to detect the change of interfacial electron-transfer resistance (Ret) of the redox marker, [Fe(CN)6]4�/3�, from solution to transducer surface. The results showedthat when target ssDNA-CdS nanoconjugates hybridized with probe oligonucleotide, a double helix film formed onthe electrode, a remarkably increased Ret value was observed. Only complementary DNA sequence had an obvioussignal compared with three-base mismatched or non-completely matched sequences under the optimizedexperimental conditions. Due to having more negative charges, space resistance and the semiconductor property,CdS nanoparticle labels on target DNA could improve the sensitivity to two orders of magnitude when compared withnon-CdS tagged DNA sequences.

Keywords: DNA hybridization, Impedance detection, Self-assembled monolayer (SAM), CdS nanoparticles

1. Introduction

Interest in the DNA hybridization biosensors has beenincreasing due to the major developments in humangenetics. An effective DNA-sensing system to detectspecific nucleic acid sequences is playing an importantrole in many areas, such as clinical diagnosis, medicine,epidemic prevention, environmental protection and bioen-gineering. Various techniques including radiochemical [1],electrochemical [2], colorimetric [3] and chemiluminescent[4] methods have been developed for DNA analysis.Among them, electrochemical techniques are a novel anddeveloping tool which combines biochemical, electrochem-ical, medical and electronic techniques with the advantagesof being simple, reliable, cheap, sensitive and selective forgenetic detection, as well as being compatible with DNAbiochip. Such devices rely on conversion of the DNA base-pair recognition event into a useful electrical signal. It candetect the increased current signal of the redox indicator [5,6], including cationic metal complexes [7], organic inter-calating compounds [8, 9]; metal nanoparticles labels [10,11] or enzyme labels [12, 13] (the latter are accomplished byan electrochemical measurement of the product of theenzymatic reaction) [14]; it also can monitor the changes inthe instinctive redox activity of DNA that occurred from thehybridization event [15, 16], exploit different rates ofelectron transfer [17, 18] and detect changes in theconductivity [19, 20], capacitance [21] or Faradic impedance[22 ± 24] accompanying the hybridization event.

The unique optical, electrical and bio-compatible proper-ties offer nanometer sized materials excellent prospects in aDNA sensor [25]. Besides the application of gold [10] andsilver nanoparticles [11] in electrochemical DNA hybrid-ization detection protocol, semiconductor quantum dots(such as CdS, PbS, ZnS) as oligonucleotide labeling tags forstripping potentiometric detection of DNA hybridizationhave been addressed as a developing approach to DNAhybridization analysis [26, 27]. The reported steps conven-tionally involved hybridization of the target DNA with thesemiconductor nanoparticle-DNA probe, followed by thedissolution of the nanoparticles anchored on the hybrids andthe stripping determination of the dissolved metal ions. Thedissolution step was a requisite to the potentiometricdetection, for the impossibility of direct detection thevoltammetric signal of Cd grafted on the electrode surfacedue to the semiconductor property of CdS. However, the useof semiconductor quantum dots-oligonucleotide nanocon-jugates for DNA hybridization sensing by EIS (electro-chemical impedance spectroscopy) was rarely reported. Inthis article, a type of cadmium sulfide (CdS) nanoparticlescoated with free carboxyl groups on its surface was directlysynthesized in aqueous solution, which could covalentlybind with the amine groups modified target ssDNA [28],which produced DNA-CdS nanoconjugates (shown inFig. 1). EIS technique, as well as cyclic voltammetry, provedto be a sensitive and selective approach to detect DNAhybridization occurrence as double helix carrying nano-conjugates film on the electrode resulting in the increase ofinterfacial electron-transfer resistance. Compared with the

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Electroanalysis 2004, 16, No. 1-2 ¹ 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/elan.200302923

reported stripping potentiometric detection method, thisprotocol eliminated the complex electrochemical detectionprocess. Moreover, because of the negative charges, spaceresistance and semiconductor characteristics of CdS tags,the use of CdS nanoparticle greatly improved DNA hybrid-ization detection sensitivity.

2. Experimental

2.1. Reagents

Thioglycollic acid (SH�CH2�COOH) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) were pur-chased from Sigma. The various oligonucleotides werepurchased from Shenggong Bioengineering Ltd. Company(Shanghai, China) having the following sequences:

Probe:5�-NH2-GAGCGCGCAACATTTCAGGTGGA-3�Complementary target:5�-NH2-TCG ACCTGAAATGTTGCGCCACTC-3�Three-mismatched oligonucleotides:5�-NH2-TCGTCCT GAAACGTTGCGCCTCTC-3�Non-complementary sequence:5�-NH2-GAGCGCGCAACATT TCAGGTGGA-3�

The following buffers were used: 0.1 M PBS (0.1 MNaCl� 0.01 M sodium phosphate buffer, pH 7.0), 0.75 MPBS (0.75 M NaCl� 0.01 M sodium phosphate buffer,pH 7.0). Other reagents were commercially available andwere all of analytical reagent grade. All of the solutions wereprepared with ultrapure water from an Aquapro system.

2.2. Apparatus

AC impendence (ACI) measurements and cycle voltamme-try (CV) scans were all performed with a CH InstrumentsModel 660 electrochemical analyzer (CH Instruments Inc.,US). The three-electrode system consisted of a gold workingelectrode (ca. 1.77 mm2 sensing area), an Ag/AgCl refer-ence electrode with KCl saturated, and a platinum wirecounter electrode. All electrochemical measurements werecarried out in a 10 mL cell.

2.3. Construction of a ssDNA Monolayer-FunctionedGold Electrode Transducer

To remove any previous organic layer, the gold electrode(ca. 1.5 mm diameter) was treated in boiling 2.0 M KOH for1 h, following ultrasound in Piranha solution (3 :1 H2SO4/H2

O2 v/v) for 10 min, then sonicated in water for 10 min. Afterthat, the electrode was voltammetrically cycled and charac-terized in 0.2 M H2SO4 from�0.5 V to�1.4 V (vs. Ag/AgCl )with scan rate of 0.10 V/s until a stable cyclic voltammogramwas obtained.

The fresh electrode was immersed in a 1.0 mM SH�CH2

�COOH (diluted in water) thiol solution for 2 h to obtain aself-assembled mercaptoacetic acid monolayer modifiedgold electrode. The free polar carboxylic acid groups wereactivated 1 h by 0.01 M EDAC solution (0.01 M sodiumphosphate buffer, pH 5.7), which then reacted with theamino-group on the ssDNA probe by dipping the electrodeinto 10�6 M NH2-ssDNA probe solution (0.01 M sodiumphosphate buffer, pH 7.0) with 6 h stirring.

2.4. Preparation of CdS-DNA Nanoconjugates

Cadmium sulfide nanoclusters were prepared according tothe literature [29] by using mercaptoacetic acid as thestabilizer. Briefly, 19.2 �L mercaptoacetic acid (RSH) wasadded to 100 mL 1 mM CdCl2 solution, pH was adjusted toca. 11 with 0.5 M NaOH and the solution was bubbled withnitrogen for 30 min, then 50 mL 1.34 mM Na2S was addeddropwise to the mixture solution. The reaction was carriedout for 24 h under bubbling nitrogen, and gradually ayellow-green colloid was formed. The colloid was stable formonths at room temperature. The CdS nanoclusters have anaverage diameter of 5 nm as measured by TEM (trans-mission electron microscope).

The nanoconjugates reaction was operated at roomtemperature with stirring 2 mL CdS nanoparticles(pH 3.5) and 2 OD target ssDNA with 0.01 M EDAC for18 h. Finally the nanoconjugates were isolated by centrifu-gation for 30 min at 10000 rpm. The obtained DNA-CdSnanoparticles precipitate was washed with water, re-centri-fuged and re-dispersed in water, stored at �5 �C forhybridization.

2.5. Hybridization of Target CdS-DNA Nanoconjugateswith Probe Oligonucleotide Functionalized AuElectrode

Hybridization reaction was carried out by incubating theprobe ssDNA functionalized Au electrode into a stirred,40 �C hybridization solution (0.75 M PBS buffer, pH 7.0)containing a certain concentration of target DNA-CdSnanocojugates for 1 h to yield the double DNA helix, theelectrode then was washed with the same PBS buffersolution for 10 min.

Fig. 1. The skeleton of CdS-DNA nanocojugates.

151CdS-Oligonucleotide Nanoconjugates

Electroanalysis 2004, 16, No. 1-2 ¹ 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2.6. Electrochemical Detection

Cyclic voltammogram and Faradic impedance measure-ments were performed in a 0.1 M PBS buffer solution(pH 7.0) containing a 0.01 M K3[Fe(CN)6]/K4[Fe(CN)6](1 : 1) mixture as a redox marker. Impedance measurementswere performed at a bias potential of 0.17 V (vs. Ag/AgCl)in the frequency range from 105 Hz to 1.0 Hz at a samplingrate of 12 points per decade (AC amplitude: 5 mV). Theimpedance spectra were plotted in the form of complexplane diagrams (Nyquist plots, Zim vs. Zre). The CVmeasurements were operated from �0.1 V to �0.7 V (vs.Ag/AgCl) with scan rate 100 mV/s to obtain a goodreproducibility.

3. Results and Discussion

Impedance spectroscopy, specifically Faradaic impedancespectroscopy (Fig. 2), is an effective technique to probe thefeatures of surface-modified electrodes [30, 31]. For the EISdetection, if a redox marker like Fe(CN)3�

6 /Fe(CN)4�6

existed, the equivalent circuit is outlined in Figure 2 (inset).Among the elements listed in the equivalent circuit, Ret

corresponded to the interfacial electron-transfer resistancefor redox marker, Fe(CN)3��4�

6 , Cdl was the double-layercapacitance, Rs was the ohmic resistance and Zw resultedfrom Warburg impedance. According to the circuit, a typicalshape of a Faradic impedance spectrum (presented in theform of a Nyquist plot,Zim vs.Zre) is a semicircle region lyingon the Zre axis followed by a straight line. The semicircleportion, observed at higher frequencies, corresponded tothe electron-transfer limited process, and theRet equaled therespective semicircle diameters. However, the linear partwas characteristic of the lower frequencies range andrepresented the diffusion-limited electron-transfer process.Here, we utilized the semicircle diameters (Ret) to observethe change of electronic transfer resistance.

3.1. Characterization of NH2-ssDNA Functioned Film onElectrode Surface

Figure 3 illustrates the Faradic impedance changes accom-panying the stepwise electrode modification process. Shownin this spectrum, after SH�CH2�COOH monolayer self-assembled on Au electrode, the interfacial electron-transferresistance Ret corresponding to the respective semicirclediameters increased from 546.9 � (curve A) to 1 760 �

(curve B). The non-conductivity characteristic ofSH�CH2�COOH film and a large quantity of negativecharges from �COO� groups perturbed the interfacialelectron-transfer rates between the electrode and theelectrolyte solution. After activated by EDAC, the negativecharges of�COO� groups were sealed, and the net positivecharges adduct carrying under experimental condition(pH 5.7) would attract negative redox marker, thus Ret

decreased to curve C (192.7 �), even lower than bare Auelectrode. After the ssDNA immobilized on the electrodesurface, the electron-transfer probing by negatively chargedredox marker, [Fe(CN)6]3�/4�, was hindered again, so Ret

increased to curve D (978.9 �).To evaluate the immobilized probe, differential pulse

voltammetry (DPV) were recorded in a blank phosphatebuffer (pH 7.0) solution before and after immobilization ofthe probe. In the voltammogram recorded for the modifiedelectrode after probe immobilization, two peaks appearedat �0.90 Vand � 1.35 V, which correspond to the oxidationof guanine and adenine on the probe oligonucleotides. Thisis similar to other reports [32] regarding the oxidationpotential of guanine (�0.87 V) and adenine (�1.12 V) of

Fig. 2. Faradic impedance spectra presented in the form ofNyquist plot, Zim vs. Zre, in the presence of redox marker. Inset:Equivalent circuit corresponding to the impedance features.

Fig. 3. Nyquist diagram (Zim versus Zre) for the Faradic impe-dance measured operated in the 0.1 M PBS buffer solutioncontaining a 10 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1 :1) mixture at apotential of 0.17 V (vs.Ag/AgCl) in the frequency for 105 Hz to1 Hz, a sampling rate of 12 points per decade, AC amplitude 5 mV.A) bare gold electrode, B) carboxyl-terminate SAM functionedAu electrode formed by SH�CH2�COOH self-assembly, C)EDAC activated RSH functioned electrode, D) NH2-ssDNAmodified gold electrode formed by condensation reaction be-tween 5�-amino group of ssDNA and the terminal group on Au.

152 Y. Xu et al.


DNA [33], which were modified on a substrate differentfrom ours.

3.2. Electrochemical Impedance Detection of DNAHybridization Involving a CdS-DNA Nanoconjugate

The principle of electrochemical impedance detection ofDNA hybridization with a CdS tag, depicted in Figure 4,consisted of the following main steps: a) immobilizationprobe ssDNA on self-assembly RSH monolayer modifiedAu electrode; b) EIS detection the Ret of ssDNA/Auelectrode; c) hybridization with target ssDNA-CdS nano-cojugates; d) EIS detection the Ret of dsDNA/Au. Figure 5

illustrates a typicalRet enhancement from ssDNA to dsDNAafter hybridization with 10�9 M DNA-CdS nanocojugatestarget. We can see that double-stranded helix formation onthe electrode surface greatly retarded the redox markerfrom penetrating dsDNA film to the Au bare active sites.

The sensitivity of the CdS nanoparticle-based electro-chemical impedance hybridization assay was investigated byvarying the target oligonucleotide-CdS nanoconjugatesconcentration. Experiments showed that a double helixformed on the electrode would be expected to retard theinterfacial electron-transfer kinetics and to increase theelectron-transfer resistance. The analytical signal, �Ret

(�Ret �RdsDNA �RssDNA), had a linear relationship with thelogarithmic value of the target DNA-CdS concentrationranging from 10�11 M to 10�7 M (shown in Fig. 6A). Theregression equation was Y� 3731.02� 319.42 X (X: Lg[target DNA], Y: �Ret) with a correlation coefficient (R)0.97911. Detection limit was 4.5� 10�12 M using 3 s (where sis the standard deviation of blank solution, n� 11). Controlexperiment showed that if hybridization with non-CdSlabeled target ssDNA (shown in Fig. 6B), Y� 3 409.2�360.64 X (X: Lg [target ssDNA],Y:�Ret) and the correlationcoefficient of the linear curve was 0.99931. Detection limitwas 1.43� 10�10 M. The results illustrated that doublestranded helix with CdS tags which having more negativecharges, space resistance and the semiconductor character-istics yielded more resistance for electron transfer fromsolution to electrode surface and increased 2 orders ofmagnitude for detection sensitivity.

When captured DNA probe hybridized with target DNAhaving or not-having CdS labeling tags, effect of thehybridization time on EIS signal is shown in Figure 7.When DNA probe hybridized with oligonucleotide-CdSnanoconjugates target, EIS signal increased from 15 min to120 min until a plateau at 150 min (Fig. 7, signal �), itshowed that due to lots of space resistance and negativecharges existence, hybridization with nanoconjugates need-ed more time than conventional practice with bare targetDNA (shown in Fig. 7, � ).

Fig. 4. Schematic representation of this analytical protocol. A)bare gold electrode; B) carboxy1-terminate SAM functioned Auelectrode formed by SH�CH2�COOH self-assembly; C) NH2-ssDNA modified gold electrode formed by condensation reactionbetween 5�-amino-linked ssDNA and the terminal group on Au inthe presence of activator EDAC; D) double helix formation onelectrode surface formed by hybridization with ssDNA-CdS targetnanocojugates.

Fig. 5. Nyquist diagram (Zim versus Zre) for the Faradic impe-dance of ssDNA/Au and dsDNA after hybridization with 10�9 Mtarget DNA-CdS nanoparticles 50 min at 40 �C measured in the0.1 M PBS buffer solution containing a 10 mM K3[Fe(CN)6]/K4

[Fe(CN)6] (1 : 1) mixture at a potential of 0.17 V (vs.Ag/AgCl) inthe frequency for 105 Hz to 1 Hz, a sampling rate of 12 points perdecade, AC amplitude 5 mV.

Fig. 6. The plots of �Ret vs. complementary oligonucleotideconcentration. A) with CdS-ssDNA hybridized at 40 �C with60 min stirring; B) with non-CdS ssDNA hybridized at 40 �C with30 min stirring.



3.3. Optimization of DNA Hybridization Conditions

The influence of relevant experimental parameters, includ-ing the RSH self-assembly extent, the redox markerconcentration and the solution ionic strength for EISdetection were all investigated here. In order to ensure amoderate monolayer film for an ideal signal change and tolimit the nonspecific absorption of CdS-oligonucleotideDNA targets, 2 h was applied as the assembly time.

Concentration of redox marker also had a great influenceto the EIS signal detected. According to the equilibrium:Ret �RT(nFIo)�1

Io � nFAket[S]

whereR is the gas constant,T is the temperature (K),A is theelectrode area (cm2), [S] corresponds to the concentration ofthe redox marker (mol cm�3), n is the number of transferredelectrons per molecule of the redox probe and ket isheterogeneous electron-transfer rate constant [34, 35], wecould deduce:

Ret-dsDNA �RT(n2F2Aket-dsDNA[S])�1 (1)

Ret-ssDNA �RT(n2F2Aket-ssDNA[S])�1 (2)

�Ret �RdsDNA �RssDNA �RT(n2F2A[S])�1 Ket (3)

(where Ket �ket-dsDNA�1 � ket-ssDNA

�1).

If we fixed the other elements and only changed the redoxmarker concentration from [S] to [S] �, according toEquation 3 we gained �Ret: �Ret�� [S]� : [S]. The exper-imental results obtained (shown in Table 1) indicated thatour experiment coincided with theory well.

Ionic strength of the background electrolyte would alsoaffect the signal change (shown in Table 2). With the lowerionic strength a higher �Ret-to-Ret-ssDNA was obtained.Concentration of NaCl had a more clear effect thanphosphate buffer. Therefore, if we lower the backgroundelectrolyte ionic strength, we could get an increased signal-to-background. However, in consideration of the stability ofdsDNA, 0.1 M PBS was chose as the detection electrolyte.

The selectivity of the CdS nanocluster electrochemicalhybridization assay was investigated by using the CdS-oligonucleotide DNA target (complementary, non-comple-mentary and three-base mismatched sequences) to hybrid-ize with the probe ssDNA immobilized on the electrode.Under the optimized conditions, only the complementarysequence gave a significant response (shown in Fig. 8).

4. Conclusions

The construction of bio-material assemblies on electrodesurfaces for recognition or catalysis are of substantialfundamental and practical importance for DNA sensingand other bioelectronics devices such as a tailored biosensorand immunosensor. Several methods for the electrochem-ical transduction of the formation of double helix on theelectrode surface were reported. Here we have demon-strated a simple method for the sensitive analysis of DNAusing Faradic impedance spectroscopy. The use of oligonu-cleotide-CdS nanoconjugates provides a means to confirmand amplify the analysis of the target DNA. Compared with

Fig. 7. Diagrams (Zim vs. Zre) of �Ret (�Ret�RdsDNA �RssDNA)corresponding to the hybridization time with 10�9 M complemen-tary ssDNA-CdS nanoconjugates (�) and 10�7 M complementarynon-CdS tagged ssDNA (� ) at 40 �C, stirring.

Table 1. The effect of concentration of redox marker K3[Fe(CN)6]/K4[Fe(CN)6] to the EIS signal.

Fe(CN)3��4�6 Concentration (mM) �Ret (�) [S] � : [S] �Ret :�Ret�

10/10 978.7 1.60 (10 : 6.25) 1.69 (1 654.0 : 978.7)6.25/6.25 1654.0 1.38(6.25 : 4.5) 1.47 (2 436.9 : 1654.0)4.5/4.5 2436.9 2.22 (10 : 4.5) 2.49 (2 436.9 : 978.7)

Table 2. Background electrolyte ionic strength-dependent �Ret (�Ret �RdsDNA �RssDNA ).

Background electrolyte �Ret (�) Signal-to-background(�Ret :�Ret-ssDNA )

100 mM phosphate buffer� 0.1 M NaCl (pH 7.0) 473.6 0.76310 mM phosphate buffer� 0.1 M NaCl (pH 7.0) 978.7 1.0010 mM phosphate buffer� 0.01 M NaCl (pH 7.0) 1739.5 1.35

154 Y. Xu et al.


the reported stripping potentiometric detection method,this protocol eliminated the complex electrochemical de-tection process. And in theory, the nanoparticles such asPbS, ZnS, Au, and silica which can label DNA and are coatedwith negative charge group have the possibility to beemployed in this protocol for enhanced hybridizationdetection sensitivity. Development of quantum dot bio-conjugates coupled with impedance measurement for bio-logical assay are opening a new possibility in recognizingspecific analytes such as protein, DNA or viruses.

5. Acknowledgement

We thank the National Nature Science Foundation of China(NSFC), which financially supported this work. (No.29875008).

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Fig. 8. The �Ret corresponding to hybridization with 10�9 Mdifferent ssDNA sequences at 40 �C with 1 h stirring.



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Probing DNA Hybridization by Impedance Measurement Based on CdS-Oligonucleotide Nanoconjugates