Microchimica Acta

, 186:677 | Cite as

DNAzyme assisted recycling amplification method for ultrasensitive amperometric determination of lead(II) based on the use of a hairpin assembly on a composite prepared from nitrogen doped graphene, perylenetetracarboxylic anhydride, thionine and gold nanoparticles

  • Yidan Ma
  • Chao Yu
  • Yujie Yu
  • Jun Chen
  • Rufei Gao
  • Junlin HeEmail author
Original Paper


A Pb(II)-DNAzyme is used in an amperometric method for the determination of Pb(II). The method is based on two feedback processes. In the first, the Pb(II)-DNAzyme initiates a reaction in presence of Pb(II) in a micro-tube to release a linear DNA (S1). In the second, the S1 triggers the recycling amplification between two types of hairpin-shaped DNA templates (H1 and H2) which consist of a primer sequence and a Pb(II)-DNAzyme substrate sequence. The Pb(II)-DNAzyme has excellent cleavage specificity toward the substrate sequence in S1 that combined firstly with H1 and then is linked to H2. This process will connect H1 and H2. After hybridization with H1 and H2 to form two DNA complexes, S1 is released and initiates the next recycling process. This results in efficient amplification. A glassy carbon electrode (GCE) was immersed into solution of HAuCl4 to electrodeposit a layer of gold nanoparticles. This is followed by the assembly of the hairpin probe H1 on the GCE. In addition, a nanohybrid consisting of 3, 4, 9, 10-Perylenetetracarboxylic acid (PTCA) and nitrogen-doped graphene (NG) was loaded with electroactive thionine (Thi) and gold to form nanoparticles of type NG-PTCA-Thi-Au. This is responsible for generating the amperometric signal (best measured at around −0.30 V vs. SCE) and also acts as the reducing agent for synthesizing the NG-PTCA-Thi-Au nanohybrid. H2 is immobilized on NG-PTCA-Thi-Au to form a new tracer label. The concentration of Pb(II) in a solution can be quantified by determination of the amount of cleaved S1. The method has high sensitivity and selectivity for Pb(II). The detection limit is 0.42 pM (S/N = 3), and the detection range extends from 1 pM to 1000 nM.

Graphical abstract

Schematic representation of electrodes for the determination of the lead ions (Pb2+). The sensor is using Pb2+-DNAzyme assisted recycling amplification based on hairpin assembly on a composite prepared from nitrogen doped graphene, perylenetetracarboxylic anhydride, thionine and gold nanoparticles (NG-PTCA-Thi-Au). This versatile platform expands studies on the detection of heavy metal ions.


Pb2+ detection 8–17 DNAzyme Catalytic hairpin assembly Dual signal amplification Square wave voltammetry NG PTCA AuNPs 



We are grateful for the financial support from the National Natural Science Foundation of China (No. 31571554), and the Outstanding Graduate Student Cultivation Program of Chongqing Medical University (No. BJRC201915).

Compliance with ethical standards

Conflict of interest

The author(s) declare that they have no competing interests.

Ethical approval

This article does not contain any studies with human or animal subjects.

Supplementary material

604_2019_3790_MOESM1_ESM.doc (7 mb)
ESM 1 (DOC 7163 kb)


  1. 1.
    Roushani M, Baghelani YM, Mavaei M, Abbasi S, Mohammadi SZ (2018) Preparation of modified magnetic cobalt nanoparticles as a new magnetic sorbent for the Preconcentration and determination of trace amounts of Lead ions in environmental water and soil (air-dust) samples. Commun Soil Sci Plant Anal 49:645–657. CrossRefGoogle Scholar
  2. 2.
    Singh P, Mittal LS, Kumar K, Sharma P, Bhargava G, Kumar S (2018) Multifunctional metallo-supramolecular interlocked hexagonal microstructures for the detection of lead and thiols in water. Chem Commun (Camb) 54:9482–9485. CrossRefGoogle Scholar
  3. 3.
    Needleman H (2004) Lead poisoning. Annu Rev Med 55:209–222. CrossRefPubMedGoogle Scholar
  4. 4.
    Li J, Lu Y (2000) A highly sensitive and selective catalytic DNA biosensor for Lead ions. J Am Chem Soc 122:10466–10467. CrossRefGoogle Scholar
  5. 5.
    Lu Y, Li X, Wang G, Tang W (2013) A highly sensitive and selective optical sensor for Pb2+ by using conjugated polymers and label-free oligonucleotides. Biosens Bioelectron 39:231–235. CrossRefPubMedGoogle Scholar
  6. 6.
    Zhu D, Liu W, Zhao D, Hao Q, Li J, Huang J, Shi J, Chao J, Su S, Wang L (2017) Label-free electrochemical sensing platform for MicroRNA-21 detection using Thionine and gold nanoparticles co-functionalized MoS2 Nanosheet. ACS Appl Mater Interfaces 9:35597–35603. CrossRefPubMedGoogle Scholar
  7. 7.
    Li Y, Yu C, Yang B, Liu Z, Xia P, Wang Q (2018) Target-catalyzed hairpin assembly and metal-organic frameworks mediated nonenzymatic co-reaction for multiple signal amplification detection of miR-122 in human serum. Biosens Bioelectron 102:307–315. CrossRefPubMedGoogle Scholar
  8. 8.
    Xie H, Chai Y, Yuan Y, Yuan R (2017) Highly effective molecule converting strategy based on enzyme-free dual recycling amplification for ultrasensitive electrochemical detection of ATP. Chem Commun (Camb) 53:8368–8371. CrossRefGoogle Scholar
  9. 9.
    Liu S, Wang Y, Ming J, Lin Y, Cheng C, Li F (2013) Enzyme-free and ultrasensitive electrochemical detection of nucleic acids by target catalyzed hairpin assembly followed with hybridization chain reaction. Biosens Bioelectron 49:472–477. CrossRefPubMedGoogle Scholar
  10. 10.
    Huang J, Wu J, Li Z (2016) Molecular beacon-based enzyme-free strategy for amplified DNA detection. Biosens Bioelectron 79:758–762. CrossRefPubMedGoogle Scholar
  11. 11.
    Zang Y, Lei J, Ling P, Ju H (2015) Catalytic hairpin assembly-programmed porphyrin-DNA complex as Photoelectrochemical initiator for DNA biosensing. Anal Chem 87:5430–5436. CrossRefPubMedGoogle Scholar
  12. 12.
    Zhou Q, Lin Y, Lin Y, Wei Q, Chen G, Tang D (2016) Highly sensitive electrochemical sensing platform for lead ion based on synergetic catalysis of DNAzyme and au-Pd porous bimetallic nanostructures. Biosens Bioelectron 78:236–243. CrossRefPubMedGoogle Scholar
  13. 13.
    Roushani M, Shahdost-Fard F (2019) Applicability of AuNPs@N-GQDs nanocomposite in the modeling of the amplified electrochemical ibuprofen aptasensing assay by monitoring of riboflavin. Bioelectrochemistry 126:38–47. CrossRefPubMedGoogle Scholar
  14. 14.
    Chen M, Hou C, Huo D, Bao J, Fa H, Shen C (2016) An electrochemical DNA biosensor based on nitrogen-doped graphene/au nanoparticles for human multidrug resistance gene detection. Biosens Bioelectron 85:684–691. CrossRefPubMedGoogle Scholar
  15. 15.
    Wu J, He J, Zhang C, Chen J, Niu Y, Yuan Q, Yu C (2018) PdPt nanoparticles anchored on the N-G with the integration of PANI nanohybrids as novel redox probe and catalyst for the detection of rs1801177. Biosens Bioelectron 102:403–410. CrossRefPubMedGoogle Scholar
  16. 16.
    Rezaei B, Jamei HR, Ensafi AA (2018) An ultrasensitive and selective electrochemical aptasensor based on rGO-MWCNTs/chitosan/carbon quantum dot for the detection of lysozyme. Biosens Bioelectron 115:37–44. CrossRefPubMedGoogle Scholar
  17. 17.
    Gan S, Zhong L, Engelbrekt C, Zhang J, Han D, Ulstrup J, Chi Q, Niu L (2014) Graphene controlled H- and J-stacking of perylene dyes into highly stable supramolecular nanostructures for enhanced photocurrent generation. Nanoscale 6:10516–10523. CrossRefPubMedGoogle Scholar
  18. 18.
    Feng L, Chen Y, Ren J, Qu X (2011) A graphene functionalized electrochemical aptasensor for selective label-free detection of cancer cells. Biomaterials 32:2930–2937. CrossRefPubMedGoogle Scholar
  19. 19.
    Chang Y, Xie S, Chai Y, Yuan Y, Yuan R (2015) 3,4,9,10-Perylenetetracarboxylic acid/o-phenylenediamine nanomaterials as novel redox probes for electrochemical aptasensor systems based on an Fe3O4 magnetic bead as a nonenzymatic catalyst. Chem Commun (Camb) 51:7657–7660. CrossRefGoogle Scholar
  20. 20.
    Li F, Yang H, Shan C, Zhang Q, Han D, Ivaska A, Niu L (2009) The synthesis of perylene-coated graphene sheets decorated with au nanoparticles and its electrocatalysis toward oxygen reduction. J Mater Chem 19:4022. CrossRefGoogle Scholar
  21. 21.
    Han J, Ma J, Ma Z (2013) One-step synthesis of graphene oxide-thionine-au nanocomposites and its application for electrochemical immunosensing. Biosens Bioelectron 47:243–247. CrossRefPubMedGoogle Scholar
  22. 22.
    Su S, Zou M, Zhao H, Yuan C, Xu Y, Zhang C, Wang L, Fan C, Wang L (2015) Shape-controlled gold nanoparticles supported on MoS(2) nanosheets: synergistic effect of thionine and MoS(2) and their application for electrochemical label-free immunosensing. Nanoscale 7:19129–19135. CrossRefPubMedGoogle Scholar
  23. 23.
    Yun W, Cai D, Jiang J, Zhao P, Huang Y, Sang G (2016) Enzyme-free and label-free ultra-sensitive colorimetric detection of Pb(2+) using molecular beacon and DNAzyme based amplification strategy. Biosens Bioelectron 80:187–193. CrossRefPubMedGoogle Scholar
  24. 24.
    Zhang J, Tang Y, Teng L, Lu M, Tang D (2015) Low-cost and highly efficient DNA biosensor for heavy metal ion using specific DNAzyme-modified microplate and portable glucometer-based detection mode. Biosens Bioelectron 68:232–238. CrossRefPubMedGoogle Scholar
  25. 25.
    Xue S, Jing P, Xu W (2016) Hemin on graphene nanosheets functionalized with flower-like MnO2 and hollow AuPd for the electrochemical sensing lead ion based on the specific DNAzyme. Biosens Bioelectron 86:958–965. CrossRefPubMedGoogle Scholar
  26. 26.
    Yang Z, Jiang W, Liu F, Zhou Y, Yin H, Ai S (2015) A novel electrochemical immunosensor for the quantitative detection of 5-hydroxymethylcytosine in genomic DNA of breast cancer tissue. Chem Commun (Camb) 51:14671–14673. CrossRefGoogle Scholar
  27. 27.
    Lei YM, Wen RX, Zhou J, Chai YQ, Yuan R, Zhuo Y (2018) Silver ions as novel Coreaction accelerator for remarkably enhanced Electrochemiluminescence in a PTCA-S2O8(2-) system and its application in an ultrasensitive assay for mercury ions. Anal Chem 90:6851–6858. CrossRefPubMedGoogle Scholar
  28. 28.
    Xu LL, Zhang W, Shang L, Ma RN, Jia LP, Jia WL, Wang HS, Niu L (2018) Perylenetetracarboxylic acid and carbon quantum dots assembled synergistic electrochemiluminescence nanomaterial for ultra-sensitive carcinoembryonic antigen detection. Biosens Bioelectron 103:6–11. CrossRefPubMedGoogle Scholar
  29. 29.
    Hu C, Yang DP, Xu K, Cao H, Wu B, Cui D, Jia N (2012) Ag@BSA core/shell microspheres as an electrochemical interface for sensitive detection of urinary retinal-binding protein. Anal Chem 84:10324–10331. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  • Yidan Ma
    • 1
    • 2
  • Chao Yu
    • 3
  • Yujie Yu
    • 1
    • 2
  • Jun Chen
    • 3
  • Rufei Gao
    • 1
    • 2
  • Junlin He
    • 1
    • 2
    Email author
  1. 1.School of Public Health and ManagementChongqing Medical UniversityChongqingChina
  2. 2.Joint International Research Laboratory of Reproduction & DevelopmentChongqing Medical UniversityChongqingChina
  3. 3.College of PharmacyChongqing Medical UniversityChongqingChina

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