Elsevier

Sensors and Actuators B: Chemical

Volume 222, January 2016, Pages 1083-1089
Sensors and Actuators B: Chemical

A Pb2+-ion electrochemical biosensor based on single-stranded DNAzyme catalytic beacon

https://doi.org/10.1016/j.snb.2015.08.046Get rights and content

Highlights

  • A selective and sensitive electrochemical lead ion biosensor was developed based on the single-stranded DNAzyme with ferrocene beacon.

  • The receptor was a single stranded oligonucleotide, which contained the DNAzyme and the enzyme substrate as well.

  • The sensor employs a stem-loop structure dually labeled with thiol and ferrocene at either end of a hairpin shaped DNA.

  • The assay represents a versatile detection method and can be easily extended for use with other metal ions with various metal ions-specific DNAzymes.

Abstract

A novel strategy for selective and sensitive electrochemical lead ion (Pb2+) biosensor was developed based on the single-stranded DNAzyme catalytic beacon. A DNAzyme that requires Pb2+ for activation was selected and labeled with redox-active ferrocene (Fc) for signal transducer. The Fc-labeled single-stranded DNAzyme (Fc-ssDNAzyme) was self-assembled through Ssingle bondAu bonding on a gold electrode surface. In the presence of Pb2+, the ssDNAzyme was activated and catalyzed the hydrolytic cleavage of the substrate strand, resulting in the removal of the substrate strand along with the Fc from the Au electrode surface. The dissociation of Fc caused a decrease of electrochemical signal (“signal-off”). Under the optimal conditions, the electrochemical signal of Fc decreased directly with the increasing Pb2+ concentration, exhibiting a linear response in the range of 0.5 nM to 5 μM with a detection limit of 0.25 nM. This strategy is simple, sensitive and selective with the minimal reagents and working steps, thereby holds great potential for Pb2+ detection in real environmental sample analysis.

Introduction

Heavy metals contamination, especially lead ion (Pb2+), has gained increasing attention now as they can induce serious threat to human health and environment. Because of its potential bioaccumulation and toxicity, Pb2+ has been considered as a major environmental pollutant [1]. Lead poisoning is related to various neurotoxin effects, including anemia, memory loss, irritability, and mental retardation, especially in children [2], [3]. Therefore, it is highly desirable to develop a sensitive, selective, economical and on-site method for Pb2+ detection in environmental and biological samples.

Various analytical techniques for Pb2+ detection have been developed, such as atomic absorption spectrometry (AAS) [4], [5], atomic emission spectrometry (AES), inductively coupled plasma atomic emission spectrometry (ICP-AES) [6], inductively coupled plasma mass spectrometry (ICP-MS) [7], and X-ray fluorescence spectrometry [8]. However, these traditional analytical methods usually require sophisticated instruments, complicated operation and sample preparation/pretreatment procedures, which limit their wide-spread applications. On the contrary, electrochemical methods have overcome those limitations as they show several important advantages including low-cost, simplicity, selectivity and sensitivity [9].

DNAzymes, artificial deoxyribozymes, are specific single strand DNA sequences that can catalyze the cleavage of their substrates in the presence of a particular metal ion, showing extra-high metal-binding affinity and specificity. The catalytic activities of the DNAzymes can be regulated by specific metal ion co-factors. Such unique metal ion-dependant activity makes DNAzymes an attractive and general platform to design metal ion sensors [10], [11], [12]. Aroused by this, DNAzyme have been converted into biosensors for lead detection by colorimetric [13], [14], fluorescence [15], [16], electrochemiluminescent [17], [18], or electrochemical methods [19], [20], [21].

It has been reported that single-stranded DNAzyme (ssDNAzyme) can be used as a versatile construction material for self-assemble into complex structure with the introduction of nanomaterials and amplification strategies. In the present work, we describe a novel sensitive and selective electrochemical DNAzyme biosensor for lead ions. This design takes advantage of ferrocene moiety, which can be labeled with single-stranded DNAzyme (Fc-ssDNAzyme), as signal transducer [22], [23], [24]. To decrease the complexity of the sensing system, the Pb2+-specific DNAzyme strand and its substrate strand with Fc terminus were combined into a DNAzyme strand. In the presence of Pb2+, the ssDNAzyme was activated and catalyzed the hydrolytic cleavage of the substrate strand, resulting in the removal of the substrate strand along with the Fc from the electrode surface. The dissociation of Fc caused a decrease of electrochemical signal (“signal-off”), which was directly related to the concentration of Pb2+ in the assay solution. It is different from the previous electrochemical sensors for lead ions in which the complex structure and signal amplification was introduced, whereas simple molecule of Fc was used as signal transducer and without signal amplification process in the present study. Therefore, the proposed electrochemical biosensor is very simple and shows excellent sensitivity and selectivity compared with other sensors.

Section snippets

Materials and apparatus

The Pb2+-specific DNAzyme (5′-NH2(CH2)6-GTAGAGAAGGrATATCACTCATTTTTT TTTTTGAGTGATAAAGCTGGCCGAGCCTCTTCTCTAC-SH-3′, rA represents adenosine ribonucleotide) was obtained from TaKaRa Biotech. Co., Ltd. (Dalian, China). Ferrocene-carboxylic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide sodium salt (Sulfo-NHS), 2-mercaptoethanol and 6-mercaptohexanol (MCH) were purchased from Aladdin. All other chemicals were of analytical grade, and used without

Sensor design

The design strategy of the DNAzyme based sensing system was depicted in Scheme 1. The Pb2+-specific DNAzyme we have employed, the “8–17” DNAzyme, is a sequence-specific nuclease acting on a single stranded DNA substrate containing a single, sessile ribo-adenine (rA). The electrochemical DNA sensor employs a stem-loop structure dually labeled with thiol and ferrocene (Fc) at either end of a hairpin shaped DNA, which is self-assembled at the Au electrode surface. In the initial state, the DNA

Conclusions

In conclusion, a novel strategy for sensitive and selective electrochemical Pb2+ biosensor was developed based on the Fc-labeled single-stranded DNAzyme catalytic beacon. Compared with other electrochemical sensors using the redox-labeled DNA as a signal transducer, our new system has two main advantages. First, the design of previously reported electrochemical DNAzyme sensors is complex since the sensors rely on the unique structures of different DNA-target pairs, such as stem-loop, hairpin,

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (No. 21205104 and 21463028), International Education Cooperation Base of Yunnan Minzu University (YMU 218-02001001002129), Graduate Student Project (2014J075) of Department of Education of Yunnan Province, and partially supported by the Open Funding Project of the State Key Laboratory of Chemo/Biosensing and Chemometrics (No. 2013014), Hunan University, PR China.

Yanli Zhang received her BSc (2002) degree in chemistry from Yan’an University, MSc and PhD degrees in analytical chemistry from Yunnan University and Hunan University in 2005 and 2008, respectively. Presently, she is an associate professor at Yunnan Minzu University. Her research interests include electrochemical analysis, electrochemical chemical and biological sensors.

References (34)

  • H. Needleman

    Lead poisoning

    Annu. Rev. Med.

    (2004)
  • H.L. Needleman et al.

    The health effects of low level exposure to lead

    Annu. Rev. Public Health

    (1991)
  • N.H. Bings et al.

    Atomic spectroscopy

    Anal. Chem.

    (2006)
  • R. Kunkel et al.

    Atomic absorption analysis of strong heavy metal chelating agents in water and waste water

    Anal. Chem.

    (1973)
  • H. Elfering et al.

    Determination of organic lead in soils and waters by hydride generation inductively coupled plasma atomic emission spectrometry

    Analyst

    (1998)
  • J.F. Wu et al.

    Low blank preconcentration technique for the determination of lead, copper, and cadmium in small-volume seawater samples by isotope dilution ICPMS

    Anal. Chem.

    (1997)
  • S. Arzhantsev et al.

    Rapid limit tests for metal impurities in pharmaceutical materials by X-ray fluorescence spectroscopy using wavelet transform filtering

    Anal. Chem.

    (2011)
  • Cited by (35)

    • Recent advances in the construction of functional nucleic acids with isothermal amplification for heavy metal ions sensor

      2022, Microchemical Journal
      Citation Excerpt :

      With the introduction of Pb2+, the DNAzyme could be cleavage, resulting in the removal of the enzyme-strand labeled by Fc from the surface of Au electrode, producing a decreased electrochemical signal. The assay exhibited a linear response in the range of 0.5 nM to 5 µM with a limit of detection (LOD) of 0.25 nM[59]. Zhang et al. reported an unusual fluorescence sensing device sensor utilizing the Pb2+-specific DNAzyme and the double-strand-chelating dye Picogreen (PG) as a fluorescent indicator of duplex DNAs.

    • Fundamentals of sensor technology

      2022, Advanced Sensor Technology: Biomedical, Environmental, and Construction Applications
    • Review of recent progress on DNA-based biosensors for Pb<sup>2+</sup> detection

      2021, Analytica Chimica Acta
      Citation Excerpt :

      AuNP-free colorimetric sensors have also been reported, and those are discussed in Section 6.3. Using RNA-cleaving DNAzymes as recognition elements, electrochemical sensors achieved outstanding performance in selectivity, sensitivity, convenience, and cost for Pb2+ detection [78–89]. A classic example was by Pb2+-induced cleavage of the substrate strand to make the label on the enzyme strand closer to the electrode surface to enhance the electrochemical signal (Fig. 8A) [44].

    View all citing articles on Scopus

    Yanli Zhang received her BSc (2002) degree in chemistry from Yan’an University, MSc and PhD degrees in analytical chemistry from Yunnan University and Hunan University in 2005 and 2008, respectively. Presently, she is an associate professor at Yunnan Minzu University. Her research interests include electrochemical analysis, electrochemical chemical and biological sensors.

    Shixiu Xiao received her BSc (2011) degree in chemical engineering and technology from Qinzhou University. She is a graduate student at Yunnan Minzu University. Her current research focuses on electrochemical biosensors.

    Haizhen Li received her BSc (2013) degree in chemistry from Yunnan Minzu University. She is a graduate student at Yunnan Minzu University. Her current researches include electrochemical sensors and biosensors.

    Hongjun Liu received his BSc (2013) degree in chemistry from Yunnan Minzu University. He is a graduate student at Yunnan Minzu University. His research is focused on electrochemical sensors and biosensors.

    Pengfei Pang received his BSc (2002) degree in chemistry from Yan’an University, MSc and PhD degrees in analytical chemistry from Hunan University in 2005 and 2008, respectively. He is currently working as an associate professor at Yunnan Minzu University. His research interests include electrochemical chemical and biological sensors.

    Hongbin Wang received his BSc (1989) degree in chemistry from Sichuan University and MSc degree in 2003 from Kunming University of Science and Technology. Presently, he is a professor at Yunnan Minzu University. His research interests include functional materials and application, environmental chemistry and separation and analysis technology.

    Zhan Wu received her PhD degree in chemistry in 2010 from Hunan University. She is currently an assistant professor at Hunan University. Her research is focused on electrochemical biosensors.

    Wenrong Yang received his PhD degree in chemistry in 2002 from the University of New South Wales. He is currently a senior lecture at Deakin University, working on biological and biomedical applications of CNTs and graphene. He also is exploring single-molecule conductivity by scanning probe microscopy.

    View full text