A sensitive biosensor with a DNAzyme for lead( ii ) detection based on fluorescence turn-onThe Analyst


Yang Guo, Junting Li, Xiaoqian Zhang, Yanli Tang
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Cite this: Analyst, 2015, 140, 4642

Received 8th April 2015,

Accepted 29th April 2015

DOI: 10.1039/c5an00677e www.rsc.org/analyst

A sensitive biosensor with a DNAzyme for lead(II) detection based on fluorescence turn-on†

Yang Guo, Junting Li, Xiaoqian Zhang and Yanli Tang*

In this paper, we described a new DNAzyme-based fluorescent biosensor for the detection of Pb2+. In the biosensor, the bulged structure is formed between the substrate labeled with fluorescein amidite (FAM) and DNAzyme after being annealed. Ethidium bromide (EB), the DNA intercalator, then intercalates into the double-stranded DNA section. Once FAM is excited, the FRET takes place from FAM to EB, which leads to the fluorescence of FAM decreasing greatly. In the presence of Pb2+, the substrate is cleaved by

DNAzyme, which breaks the bulged structure. Then EB is released and the FRET from FAM to EB is inhibited. In this case, the fluorescence of FAM increases dramatically. Thus, the Pb2+ ions can be detected by measuring the fluorescence enhancement of FAM. Under optimal conditions, the increased fluorescence intensity ratio of FAM is dependent on the lead level in the sample, and exhibits a linear response over a

Pb2+ concentration range of 0–100 nM with a detection limit of 530 pM. The sensor showed high selectivity in the presence of a number of interference ions. The river water samples were also tested with satisfying results by using the new method. This sensor is highly sensitive and simple without any additional treatments, which provides a platform for other biosensors based on DNAzyme.


Heavy metals are widespread environmental pollutants, and most of them are toxic to human health such as lead ions (Pb2+).1 Pb2+ could cause serious damage to the brain and central nervous system even at low concentrations, leading to fatigue, headaches, irreversible learning difficulties, lung tumors, and illnesses.2,3 According to the Environmental Protection Agency (EPA), 15 ppb (0.07 μM) of lead is the safety limit in drinking water,4 while the International Agency for

Research on Cancer (IARC) has a lower threshold of 10 ppb (48.26 nM) in food and water. Therefore, the development of simple, rapid and sensitive sensors plays an important role in detection of lead ions in environmental and biological samples.

Traditional methods for detection of heavy metals have been developed for many years, including atomic adsorption spectrometry, inductively-coupled plasma mass spectrometry, inductively-coupled plasma atomic emission spectrometry, voltammetric detection and UV vis spectrometry.5–7 These methods present good sensitivity for lead, however most of them require time-consuming pretreatment procedures and expensive instruments, which limit their on-site and real-time detection. A lot of methods thus have been developed for metal assay in order to provide on-site analysis.8–13 Notably, fluorescent platforms for lead detection have attracted much attention because they have facile operation and high sensitivity, cause less cell-damage, and have the ability to provide real-time information.14–16

Very recently, Zhang and Zhu published a critical review on the development of functional nucleic acid-based sensors for the detection of heavy metal ions.17 DNAzyme as a sensor platform for detection of lead ions has been studied extensively for several years17,18 and many of the DNAzyme-based sensors required using advanced materials including quantum dots,19 gold nanoparticles,20–24 graphene,25,26 etc. In addition, some methods based on phosphorothioate modification,27 strand displacement amplification reaction,28 rolling circle amplification or exonuclease aided recycling amplification24,29,30 provide high sensitivity, however limitations still exist, such as needing complicated and long-time operation.

Herein, we developed a fluorescence turn-on method for sensitive and selective detection of Pb2+ based on 17E

DNAzyme and the cleavage substrate 17S labeled with FAM.31

In this method, ethidium bromide (EB), a double-stranded

DNA intercalator, is used as an energy acceptor in the fluorescence resonance energy transfer (FRET) process, leading to the fluorescence of FAM decreasing. First, 17E and 17S-FAM †Electronic supplementary information (ESI) available. See DOI: 10.1039/ c5an00677e

Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education,

Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of

Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062,

P. R. China. E-mail: yltang@snnu.edu.cn; Fax: (+086) 029-81530727;

Tel: (+086) 029-81530844 4642 | Analyst, 2015, 140, 4642–4647 This journal is © The Royal Society of Chemistry 2015

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View Journal | View Issue form a bulged structure after hybridization. EB then intercalates into the double-stranded DNA section. When the solution is excited at 490 nm, the FRET occurs from FAM to EB, which leads to the fluorescence intensity of FAM decreasing greatly.32–34 In the presence of lead ions, the substance 17S-FAM is cleaved by 17E DNAzyme, resulting in the FRET being broken. The fluorescence intensity of FAM thus increases dramatically. This fluorescence turn-on method exhibits excellent selectivity and sensitivity. Furthermore, this simple and rapid technique could provide a new platform for

DNAzyme-based sensors.


Reagents and instruments

Materials and measurements. The oligonucleotides were purchased from Takara. HEPES-Na, ethidium bromide (EB) and other chemicals were purchased from Shanghai Sangon

Biological Engineering Technology Co. Ltd. Lead(II) acetate trihydrate was purchased from Sinopharm Chemical Reagent Co.

Ltd. The oligonucleotide sequences used in our experiments are as follows: 17S-FAM, 5′-FAM-(CH2)6-ACT CAC TAT rAG GAA