Microchimica Acta

, 186:158 | Cite as

Determination of Alzheimer biomarker DNA by using an electrode modified with in-situ precipitated molybdophosphate catalyzed by alkaline phosphatase-encapsulated DNA hydrogel and target recycling amplification

  • Xiaoyu Hua
  • Xingxing Zhou
  • Shijing Guo
  • Ting Zheng
  • Ruo Yuan
  • Wenju XuEmail author
Original Paper


An electrochemical biosensor is described for highly sensitive determination of tDNA, an Alzheimer’s disease (AD)-related biomarker. Electroactive molybdophosphate anions were precipitated in-situ on a glassy carbon electrode (GCE) via catalytic hydrolysis by alkaline phosphatase (ALP). This is followed by recycling amplification of tDNA. Four DNA strands (referred to as S1, S2, S3 and S4) were designed to assemble X-shape DNA (X-DNA) building blocks. These were further extended into four directions under the action of DNA polymerase. The resultant two X-DNA motifs were polymerize. Simultaneously, ALP is encapsulated into a hydrogels network to obtain a porous material of type ALP@DNAhg. The GCE was modified with reduced graphene oxide functionalized with gold nanoparticles (Au@rGO). If ALP@DNAhg are captured via strand displacement, tDNA recycling assembly for signal amplification is initiated. This results in the immobilization of large amounts of ALP. On introduction of pyrophosphate and molybdate (MoO42−), ALP will catalyze the hydrolysis of pyrophosphate to produce phosphate. It will react with molybdate to form redox active phosphomolybdate anions (PMo12O403−). Its amperometrical signal depends on the concentration of tDNA in the 1.0 × 10−2 to 1.0 × 104 pM concentration range, and the detection limit is 3.4 × 10−3 pM.

Graphical abstract

Schematic presentation of (a) preparation of alkaline phosphatase-encapsulated DNA hydrogel (ALP@DNAhg). (b) fabrication of the biosensor for target DNA (tDNA) based on ALP@DNAhg to catalyze in situ precipitation of electroactive molybdophosphate anion (PMo12O403−) and tDNA recycling amplification, achieving tDNA-dependent electrochemical signal readout (X-DNA: X-shape DNA building block. TdT: terminal deoxynucleotidyl transferase. dATP: deoxyadenosine triphosphate. dTTP: deoxythymidine triphosphate. X-DNA-pAn and X-DNA-pTn: X-DNA motifs with poly-A and poly-T tails. ALP: alkaline phosphatase. ALP@DNAhg: ALP-encapsulated DNA hydrogels. Au@rGO: gold nanoparticles-functionalized reduced graphene oxide. GCE: glass carbon electrode. HP1, 2: hairpin DNA 1, 2. MCH: 6-mercaptohexanol. tDNA: target DNA. CV: cyclic voltammetry).


Label-free detection Electrochemical biosensor AuNP-functionalized reduced graphene oxide X-shape DNA building block Pyrophosphate Molybdate In-situ precipitation Networked DNA structure Enzymatic catalysis Redox active phosphomolybdate anions 



The financial support by the National Natural Science Foundation (NNSF) of China (21775123) and the Natural Science Foundation Project of Chongqing (cstc2018jcyjAX0214) to this work was deeply appreciated.

Compliance with ethical standards

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

Supplementary material

604_2019_3283_MOESM1_ESM.doc (540 kb)
ESM 1 (DOC 540 kb)


  1. 1.
    Su S, Sun HF, Cao WF, Chao J, Peng HZ, Zuo XL, Yuwen LH, Fan CH, Wang LH (2016) Dual-target electrochemical biosensing based on DNA structural switching on gold nanoparticle-decorated MoS2 nanosheets. ACS Appl Mater Interfaces 8:6826–6833CrossRefGoogle Scholar
  2. 2.
    Cui L, Lu MF, Li Y, Tang B, Zhang CY (2018) A reusable ratiometric electrochemical biosensor on the basis of the binding of methylene blue to DNA with alternating AT base sequence for sensitive detection of adenosine. Biosens Bioelectron 102:87–93CrossRefGoogle Scholar
  3. 3.
    Zhao J, Gao J, Zheng T, Yang Z, Chai Y, Chen S, Yuan R, Xu W (2018) Highly sensitive electrochemical assay for Nosema bombycis gene DNA PTP1 via conformational switch of DNA nanostructures regulated by H+ from LAMP. Biosens Bioelectron 106:186–192CrossRefGoogle Scholar
  4. 4.
    Zheng TT, Zhang QF, Feng S, Zhu JJ, Wang Q, Wang H (2014) Robust nonenzymatic hybrid nanoelectrocatalysts for signal amplification toward ultrasensitive electrochemical cytosensing. J Am Chem Soc 136:2288–2291CrossRefGoogle Scholar
  5. 5.
    Ngo TA, Nakata E, Saimura M, Morii T (2016) Spatially organized enzymes drive cofactor-coupled cascade reactions. J Am Chem Soc 138:3012–3021CrossRefGoogle Scholar
  6. 6.
    Lv YQ, Chen SY, Shen YF, Ji JJ, Zhou Q, Liu SQ, Zhang YJ (2018) Competitive multiple-mechanism-driven electrochemiluminescent detection of 8-hydroxy-2′-deoxyguanosine. J Am Chem Soc 140:2801–2804CrossRefGoogle Scholar
  7. 7.
    Wu YM, Xu WJ, Bai LJ, Yuan YL, Yi HY, Chai YQ, Yuan R (2013) Ultrasensitive thrombin detection based on direct electrochemistry of highly loaded hemoglobin spheres-encapsulated platinum nanoparticles as labels and electrocatalysts. Biosens Bioelectron 50:50–56CrossRefGoogle Scholar
  8. 8.
    Xu MD, Zhuang JY, Chen X, Chen GN, Tang DP (2013) A difunctional DNA–AuNP dendrimer coupling DNAzyme with intercalators for femtomolar detection of nucleic acids. Chem Commun 49:7304–7306CrossRefGoogle Scholar
  9. 9.
    Liu TZ, Hu R, Zhang X, Zhang KL, Liu Y, Zhang XB, Bai RY, Li DL, Yang YH (2016) Metal–organic framework nanomaterials as novel signal probes for electron transfer mediated ultrasensitive electrochemical immunoassay. Anal Chem 88:12516–12523CrossRefGoogle Scholar
  10. 10.
    Li JY, Si L, Bao JC, Wang ZY, Dai ZH (2017) Fluorescence regulation of poly(thymine)-templated copper nanoparticles via an enzyme-triggered reaction toward sensitive and selective detection of alkaline phosphatase. Anal Chem 89:3681–3686CrossRefGoogle Scholar
  11. 11.
    Si Z, Xie B, Cahen Z, Tang C, Li T, Yang M (2017) Electrochemical aptasensor for the cancer biomarker CEA based on aptamer induced current due to formation of molybdophosphate. Microchim Acta 184:3215–3221CrossRefGoogle Scholar
  12. 12.
    Sadakane M, Steckhan E (1998) Electrochemical properties of polyoxometalates as electrocatalysts. Chem Rev 98:219–237CrossRefGoogle Scholar
  13. 13.
    Huang YX, Tang C, Liu J, Cheng J, Si ZZ, Li T, Yang MH (2017) Signal amplification strategy for electrochemical immunosensing based on a molybdophosphate induced enhanced redox current on the surface of hydroxyapatite nanoparticles. Microchim Acta 184:855–861CrossRefGoogle Scholar
  14. 14.
    Qu FL, Yang MH, Rasooly A (2016) Dual signal amplification electrochemical biosensor for monitoring the activity and inhibition of the Alzheimer’s related protease β-secretase. Anal Chem 88:10559–10565CrossRefGoogle Scholar
  15. 15.
    Hu LS, Hu SQ, Guo LY, Shen CC, Yang MH, Rasooly A (2017) DNA generated electric current biosensor. Anal Chem 89:2547–2552CrossRefGoogle Scholar
  16. 16.
    Feng KJ, Liu J, Deng L, Yu HJ, Yang MH (2018) Amperometric detection of microRNA based on DNA-controlled current of a molybdophosphate redox probe and amplification via hybridization chain reaction. Microchim Acta 185:28CrossRefGoogle Scholar
  17. 17.
    Xie SB, Yuan YL, Chai YQ, Yuan R (2015) Tracing phosphate ions generated during loop-mediated isothermal amplification for electrochemical detection of Nosema bombycis genomic DNA PTP1. Anal Chem 87:10268–10274CrossRefGoogle Scholar
  18. 18.
    Cai W, Xie SB, Tang Y, Chai YQ, Yuan R, Zhang J (2017) A label-free electrochemical biosensor for microRNA detection based on catalytic hairpin assembly and in situ formation of molybdophosphate. Talanta 163:65–71CrossRefGoogle Scholar
  19. 19.
    Shen CC, Li XZ, Rasooly A, Guo LY, Zhang K, Yang MH (2016) A single electrochemical biosensor for detecting the activity and inhibition of both protein kinase and alkaline phosphatase based on phosphate ions induced deposition of redox precipitates. Biosens Bioelectron 85:220–225CrossRefGoogle Scholar
  20. 20.
    He Y, Yang X, Yuan R, Chai YQ (2017) Switchable target-responsive 3D DNA hydrogels as a signal amplification strategy combining with SERS technique for ultrasensitive detection of miRNA 155. Anal Chem 89:8538–8544CrossRefGoogle Scholar
  21. 21.
    Lilienthal SV, Shpilt ZH, Wang F, Orbach R, Willner I (2015) Programmed DNAzyme-triggered dissolution of DNA-based hydrogels: means for controlled release of biocatalysts and for the activation of enzyme cascades. ACS Appl Mater Interfaces 7:8923–8931CrossRefGoogle Scholar
  22. 22.
    Li J, Zheng C, Cansiz S, Wu CC, Xu JH, Cui C, Liu Y, Hou WJ, Wang YY, Zhang LQ, Teng IT, Yang HH, Tan WH (2015) Self-assembly of DNA nanohydrogels with controllable size and stimuli-responsive property for targeted gene regulation therapy. J Am Chem Soc 137:1412–1415CrossRefGoogle Scholar
  23. 23.
    Li J, Mo LT, Lu CH, Fu T, Yang HH, Tan WH (2016) Functional nucleic acid-based hydrogels for bioanalytical and biomedical applications. Chem Soc Rev 45:1410CrossRefGoogle Scholar
  24. 24.
    Zhou L, Sun N, Xu LJ, Chen X, Cheng H, Wang J, Pei RJ (2016) Dual signal amplification by an “on-command” pure DNA hydrogel encapsulating HRP for colorimetric detection of ochratoxin A. RSC Adv 6:114500–114504CrossRefGoogle Scholar
  25. 25.
    Zhu XL, Mao XX, Wang ZH, Feng C, Chen GF, Li GX (2017) Fabrication of nanozyme@ DNA hydrogel and its application in biomedical analysis. Nano Res 10:959–970CrossRefGoogle Scholar
  26. 26.
    Mao XX, Chen GF, Wang ZH, Zhang YG, Zhu XL, Li GX (2018) Surface-immobilized and self-shaped DNA hydrogels and their application in biosensing. Chem Sci 9:811–818CrossRefGoogle Scholar
  27. 27.
    Zhu Z, Guan Z, Jia S, Lei Z, Lin S, Zhang H, Ma Y, Tian ZQ, Yang CJ (2014) Au@ Pt nanoparticle encapsulated target-responsive hydrogel with volumetric bar-chart chip readout for quantitative point-of-care testing. Angew Chem Int Ed 53:12503–12507Google Scholar
  28. 28.
    Liu SF, Wang CF, Zhang CX, Wang Y, Tang B (2013) Label-free and ultrasensitive electrochemical detection of nucleic acids based on autocatalytic and exonuclease III-assisted target recycling strategy. Anal Chem 85:2282–2288CrossRefGoogle Scholar
  29. 29.
    Wu XY, Chai YQ, Zhang P, Yuan R (2015) An electrochemical biosensor for sensitive detection of microRNA-155: combining target recycling with cascade catalysis for signal amplification. ACS Appl Mater Interfaces 7:713–720CrossRefGoogle Scholar
  30. 30.
    Shen CC, Zeng K, Luo JJ, Li XQ, Yang MH, Rasooly A (2017) Self-assembled DNA generated electric current biosensor for HER2 analysis. Anal Chem 89:10264–10269CrossRefGoogle Scholar
  31. 31.
    Long GL, Winefordner JD (1983) Limit of detection. A closer look at the IUPAC definition. Anal Chem 55:712A–724ACrossRefGoogle Scholar
  32. 32.
    Radi AE, Sanchez GLA, Baldrich E, O’Sullivan CK (2006) Reagentless, reusable, ultrasensitive electrochemical molecular beacon aptasensor. J Am Chem Soc 28:117–124CrossRefGoogle Scholar
  33. 33.
    Zhou ZX, Wei W, Zhang YJ, Liu SQ (2013) DNA-responsive disassembly of AuNP aggregates: influence of nonbase-paired regions and colorimetric DNA detection by exonuclease III aided amplification. J Mater Chem B 1:2851–2858CrossRefGoogle Scholar
  34. 34.
    Gao Y, Li BX (2014) Exonuclease III-assisted cascade signal amplification strategy for label-free and ultrasensitive chemiluminescence detection of DNA. Anal Chem 86:8881–8887CrossRefGoogle Scholar
  35. 35.
    Huang YL, Gao ZF, Luo HQ, Li NB (2017) Sensitive detection of HIV gene by coupling exonuclease III-assisted target recycling and guanine nanowire amplification. Sens Actuators B Chem 238:1017–1023CrossRefGoogle Scholar
  36. 36.
    Liu CC, Kanekiyo T, Xu HX, Bu GJ (2013) Apolipoprotein E and Alzheimer disease: risk, mechanisms and therapy. Nat Rev Neurol 9:106–118CrossRefGoogle Scholar
  37. 37.
    Yang ZH, Zhuo Y, Yuan R, Chai YQ (2015) Amplified thrombin aptasensor based on alkaline phosphatase and hemin/G-quadruplex-catalyzed oxidation of 1-naphthol. ACS Appl Mater Interfaces 7:10308–10315CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, School of Chemistry and Chemical EngineeringSouthwest UniversityChongqingPeople’s Republic of China

Personalised recommendations