A sensitive and specific electrochemiluminescent sensor for lead based

on DNAzymew

Xi Zhu, Zhenyu Lin,* Lifeng Chen, Bin Qiu and Guonan Chen*

Received (in Cambridge, UK) 8th June 2009, Accepted 6th August 2009

First published as an Advance Article on the web 21st August 2009

DOI: 10.1039/b911191c

A specific ECL sensor for Pb2+ based on DNAzyme has

been developed for the first time; the detection limit of 1.1 �10�11 mol l�1 is much lower than those of fluorescent,

colorimetric or electrical biosensors.

DNAzymes can recognize target analytes or catalyze specific

chemical and biological reactions. Many cofactors, such as

amino acids, nucleic acids, metal ions and small organic

molecules can affect the catalytic activity of DNAzyme, which

makes it a novel platform for developing highly selective

sensors. To date, several DNAzyme sensor studies have been

reported,1–6 and among them, lead-dependent DNAzyme has

been paid much attention, since lead is a common environ-

mental contaminant. Many optical methods have been

developed for Pb2+ sensing based on the behaviour of the

DNAzyme,7–10 but they suffer from possible drawbacks

including the potential for false signals arising from contami-

nating colorants, fluorophores and quenchers. In addition,

cumbersome optical equipment was often needed. In order to

overcome these drawbacks, electrochemical detection methods

combined with DNAzyme have been applied to lead detection.

Xiao et al. developed a Pb2+ electrochemical sensor by

tethering a redox-active group to the DNAzyme and cleaving

the enzyme substrate from the electrode surface.11 Shen et al.

also developed a sensitive electrochemical sensor based on

DNA-Au bio-barcode amplification.12 These initial studies

indicated that electrochemical detection had many advantages

over optical detection, but this research was a first step, and

much more optimization was still needed.

Electrochemiluminescence (ECL) sensors possess the

advantages of both electrochemical and chemiluminescent

sensors, such as high sensitivity, ease of control and the use

of simple equipment.13 Many highly sensitive and selective

ECL DNA sensors have been developed by using high quantum

efficiency ECL labels.14,15 Specially, tris(2,20-bipyridine)-

ruthenium(II) (TBR) and its derivatives have high quantum

efficiencies and their ECL intensities can be further enhanced

by using co-reactors. For example, Miao and Bard employed

TBR as an ECL label and developed an ECL sensor for an

anthrax-related specific DNA sequence with a detection limit

of 30 pM.16 The results showed that the sensitivity of ECL is

much higher than that of electrochemical detection.

In the present work, we describe a DNAzyme-based ECL

sensor for lead, which combines the high selectivity of

DNAzyme with the high sensitivity of ECL. The Pb2+-specific

DNAzyme employed in this study is ‘‘8–17’’ DNAzyme.17,18

Compared with other lead detection methods, this proposed

method has the merits of simpler equipment, higher selectivity

and sensitivity—the sensor has a lower detection limit than

colormetric or electrochemical sensors. In addition, the

proposed methods can be further applied to establish a range

of DNAzyme-based ECL sensors.

The principle of the ECL biosensor for lead is shown in

Fig. 1 (the sequences of the DNA are shown in the ESIw). Thesensor consists of a 50-thiol modified DNA (DNAzyme)

attached to a gold electrode, a 50-amino modified DNA

(DNA substrate strand), and Ru(bpy)32+ N-hydroxyl-

succinimide (TBR-NHS) ester. The DNA substrate strand

can extend on both the 30 and 50 ends as long as the enzyme

recognition portion is reserved. A linker (TTTTT) is inserted

between the thiol group and the DNA-enzyme sequence. The

TTTTT linker extends the DNA above the 6-mercaptohexanol

(MCH) layer for complete hybridization whilst maintaining

DNAzyme activity.7 Thiol modified DNAzymes are

immobilized onto the surface of the gold electrode via

thiol–Au interactions, and the surface density of the

DNAzyme on the gold electrode was about 4.5 �1011 molecules cm�2, as calculated by the method reported

previously.19 The DNA substrate modified with the ECL label

TBR-NHS ester can hybridize with DNAzyme to make a

double-stranded DNA (ds-DNA).

Fig. 1 Principle of the ECL Pb2+ sensor based on DNAzyme.

Ministry of Education Key Laboratory of Analysis and DetectionTechnology for Food Safety, Department of Chemistry,Fuzhou University, Fuzhou, Fujian 350002, China.E-mail: gnchen@fzu.edu.cn, zylin@fzu.edu.cn;Fax: +86 591-83713866w Electronic supplementary information (ESI) available: Experimentaldetails, sensor preparation, ECL measurements, optimization ofself-assembly time and the stability of the modified electrode. SeeDOI: 10.1039/b911191c

6050 | Chem. Commun., 2009, 6050–6052 This journal is �c The Royal Society of Chemistry 2009

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When the modified electrode is immersed into a solution

containing Pb2+, the DNAzyme catalyzes the hydrolytic

cleavage of the ds-DNA into two pieces. In this case, the

TBR-NHS ester is removed, which results in the reduction of

the ECL intensity. The ECL intensity decreases with the

increase of Pb2+ concentration. Accordingly, the concentration

of Pb2+ in solution can be obtained indirectly.

The TBR labels were incorporated onto the 50 end of the

DNA substrate using the phosphoramidite method as

previously shown.20 1 OD of DNA substrate was dissolved

in 250 ml of TAE buffer, and then 200 ml of 6.0 � 10�4 mol l�1

Ru(bpy)2(dcbpy)NHS and 10 ml of 0.10 mol l�1 TAE buffer

were added, followed by shaking at low speed overnight at

room temperature. Then, 100 ml of 3 mol l�1 NaAc and 2 ml of

ethanol were added into the mixture, and precipitation was

carried out in a refrigerator over 12 h. The mixture was

centrifuged for 30 min. The precipitate was preserved

and rinsed with cold 70% ethanol solution twice and dried

in air. The dried precipitate was re-dissolved in TAE buffer

(pH 8.2) to obtain the target DNA-TBR solution and stored

at �16 1C.

The incorporation procedure is a relatively reliable method,

its success rate is over 80%.21 We used different concentrations

of standard TBR solutions to get the evolution of the ECL

intensities and the TBR concentrations. Then we detected the

ECL intensities from the modified electrode and calculated the

amount of TBR modified on the electrode. By this method, we

estimate that the amount of Ru(II) labels on the modified

electrode was about 10�13 mole.

The immobilization of single-stranded DNA (ss-DNA) on

the gold electrodes and the subsequent hybridization to form

ds-DNA can be characterized by faradic electrochemical

impedance spectroscopy (EIS).22,23 The impedance spectra

consist of a semicircular portion at high frequencies and a

linear part at low frequencies. The semicircle relates to

an electron transfer-limited process, while the linear part

corresponds to a diffusion-limited process. The change in the

diameter of the semicircle reflects the change in the interfacial

charge-transfer resistance (Rct). The change of the Rct value

reflects the extent of the immobilization and hybridization of

DNA on the gold electrode surface. Therefore, we can know

the properties of DNA immobilization and hybridization

clearly by the EIS measurement.

Optimization of the DNA self-assembly process is described

in the ESI.w Fig. 2 shows Nyquist plots of [Fe(CN)6]3�/4� at

the bare (a), ss-DNA modified (b), and ds-DNA modified gold

electrodes (c). For the bare Au electrode, the value of Rct was

about 78 O, displaying a very small semicircular domain. After

immobilization of DNAzyme, the value of Rct increased from

78 O to 246 O. The negatively charged phosphate backbone of

the DNA immobilized on the bare Au electrode prevented the

negatively charged redox probe [Fe(CN)6]3�/4� from reaching

the gold electrode, thus inhibiting interfacial charge transfer,

resulting in a larger Rct value than that of the bare gold

electrode.24 When the DNAzyme was hybridized with the

target DNA substrate strand on the electrode surface, the

Rct was enhanced to a much larger value (476 O).After hybridization, the negative charge on the gold electrode

surface will again increase and thereby prevent [Fe(CN)6]3�/4�

from reaching the gold electrode more effectively, thus

raising the Rct value. Therefore, the immobilization and the

hybridization of DNA are clearly shown to happen on the

bare gold electrode according to the change of the Rct value

by EIS.

When Pb2+ is introduced into the sensing system, the

substrate strand hybridized to the DNAzyme is broken into

two pieces, and TBR-NHS is released from the electrode

surface, which causes a decrease in the ECL intensity. It is

clear that the Pb2+ concentration can influence the amount of

TBR-NHS ester bound with the DNA on the modified

electrode. Fig. 3(A) shows ECL intensities from the modified

electrodes in the presence of different Pb2+ concentrations in

TAE solution (pH = 8.2) containing 10�5 mol l�1 TPA. The

ECL intensity is found to decrease with increasing Pb2+

concentration. Fig. 3(B) shows the relationship between

concentration of Pb2+ and ECL intensity. The ECL intensity

decreases linearly with Pb2+ concentration in the range

of 2.5 � 10�10 to 1.0 � 10�9 mol l�1. The regression

equation is

I/a.u. = �15.76CPb/(10�10 mol l�1) + 198.6, R = �0.9922.

where I is the ECL intensity, CPb is the Pb2+ concentration

and R is the regression coefficient. The detection limit for Pb2+

is 1.1 � 10�11 mol l�1 (defined as S/N = 3), which is lower

Fig. 2 Nyquist plots corresponding to the different electrodes.

(a) Bare gold electrode; (b) ss-DNA modified gold electrode and

(c) ds-DNA modified gold electrode. The data were recorded in the

presence of 5.0 mmol l�1 [Fe(CN)6]3�/4�, containing 0.1 mol l�1 KCl,

upon application of a biasing potential of 0.218 V, and a 5 mV

alternating voltage in the frequency range of 1–10 000 Hz.

Fig. 3 (A) ECL intensity curves from the modified ECL sensors with

different Pb2+ concentrations under CV scanning. (a) 0; (b) 2.5 �10�10; (c) 5.0� 10�10; (d) 7.5� 10�10; (e) 1.0 � 10�9 mol l�1 in 10 mM

TAE (pH 8.2) containing 10�5 mol l�1 TPA at the scan rate of

50 mV s�1, potential range: 0.8–1.6 V; (B) the calibration curves.

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than those of fluorescent,7 electrical12 and colorimetric25,26

biosensors for Pb2+.

The DNAzyme-based sensor is clearly sensitive to its

target ion. We also studied the specificity of the ECL sensor

by making measurements using several other divalent metal

ions. Fig. 4 shows the changes in the ECL intensity for

DNAzyme-modified gold electrodes after reaction with

0.5 nmol l�1 Pb2+, Ba2+, Cu2+, Ni2+, Mn2+, Zn2+ and

when using a blank buffer solution. Apart from Pb2+, the

reduction in the ECL intensity is very small for all the divalent

metal ions, with only Ba2+ causing a little interference.

However, in the presence of Pb2+, the reduction of the ECL

intensity is obvious. The response to lead ions is easily

distinguishable and therefore this ECL biosensor has a good

selectivity for discriminating Pb2+ from other contaminating

divalent ions.

In summary, an ECL sensor for the detection of Pb2+ based

on DNAzyme as a recognition element using Ru(bpy)32+ for

ECL signal readout has been designed. The sensor exhibits

excellent sensitivity and selectivity. The limit of detection is as

low as 0.1 nmol l�1, which is better than those of fluorescent,

colorimetric and electrical biosensors for Pb2+ detection.

The main advantages of the present sensor come from the

combination of the selectivity of DNAzyme and the sensitivity

of ECL, which may provide a platform for the fabrication

of ECL sensors for analysis of many small molecules or

metal ions. For example, DNAzymes specific for Cu2+,

Zn2+, Co2+ and Hg2+ have also been obtained. Hence,

one can use the same signal discrimination method to design

ECL sensors to detect and quantify many metal ions

conveniently.

This project was financially supported by the National

Basic Research Program of China (No. 2010CB732403), the

National Nature Sciences Funding of China (20735002, 20877019)

and the Special Foundation for Young Scientists of Fujian

Province, China (2008F3057).

Notes and references

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Fig. 4 ECL intensity changes for modified gold electrodes after

reaction with various divalent metal ions (all at 0.5 nmol l�1).

6052 | Chem. Commun., 2009, 6050–6052 This journal is �c The Royal Society of Chemistry 2009

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