Microchimica Acta

, 186:286 | Cite as

Fluorometric determination of HIV DNA using molybdenum disulfide nanosheets and exonuclease III-assisted amplification

  • Lele Wang
  • Lianhua Dong
  • Gang Liu
  • Xizhong Shen
  • Jing Wang
  • Changfeng ZhuEmail author
  • Min Ding
  • Yanli WenEmail author
Original Paper


A convenient and ultrasensitive fluorometric method is described for the determination of HIV DNA. It exploits the strong difference in the affinities of MoS2 nanosheets for long ssDNA versus short oligonucleotide fragments. In addition, efficient signal amplification is accomplished by exonuclease III-assisted target recycling. When absorbed on the MoS2 nanosheets, the fluorescence of the FAM-labeled ssDNA probe (FP) is quenched. However, in the presence of HIV DNA, the FP hybridizes with target to form a duplex. As a result, the FP in the duplex will be stepwise hydrolyzed into short fragments by Exo III, and the fluorescence signal thus is retained because short fragments have low affinity for the MoS2 nanosheets. By using the Exo III-assisted target recycling amplification, the detection sensitivity is strongly improved. The sensor can detect DNA in a concentration as low as 5.3 pM (at an S/N ratio of 3), and the analytical range extends from 0.01 nM to 10 nM. The assay is simple, sensitive and specific, and conceivably represents a valuable tool in clinical studies related to the HIV.

Graphical abstract

Schematic presentation of fluorometric determination of HIV DNA based on molybdenum disulfide nanosheets and Exo III. When the fluorescence-tagged ssDNA probe hybridized with target to form a duplex, the Exo III-assisted target recycling amplification is generated. The method can detect as low as 5.3 pM HIV DNA.


Layered transition metal dichalcogenide nanosheets Enzyme amplification Fluorometric assay DNA biosensor 



This work was financially supported by National Natural Science Foundation of China (No. 21775104, 21605026, 81672720), the National Quality Infrastructure Program of China (2017YFF0204605), and Shanghai Rising-Star Program (16QB1403100).

Author contributions

L.W., Y.W., G.L., and C.Z. designed the experiments and wrote the main manuscript ;L. W.,L.D., M. D. and Y.W. performed experiments; X.S. and J.W. contributed to datacuration and formal analysis. All authors reviewed the manuscript.

Compliance with ethical standards

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

Supplementary material

604_2019_3368_MOESM1_ESM.doc (9.7 mb)
ESM 1 (DOC 9.73 mb)


  1. 1.
    Debouck C, Goodfellow PN (1999) DNA microarrays in drug discovery and development. Nat Genet 21:48–50CrossRefGoogle Scholar
  2. 2.
    Velusamy V, Arshak K, Korostynska O, Oliwa KAdley C (2010) An overview of foodborne pathogen detection: in the perspective of biosensors. Biotechnol Adv 28:232–254CrossRefGoogle Scholar
  3. 3.
    Paddle BM (1996) Biosensors for chemical and biological agents of defence interest. Biosens Bioelectron 11:1079–1113CrossRefGoogle Scholar
  4. 4.
    Rodriguez-Mozaz S, de Alda MJL, Barcelo D (2006) Biosensors as useful tools for environmental analysis and monitoring. Anal Bioanal Chem 386:1025–1041CrossRefGoogle Scholar
  5. 5.
    Wang KM, Tang ZW, Yang CYJ, Kim YM, Fang XH, Li W, Wu YR, Medley CD, Cao ZH, Li J, Colon P, Lin H, Tan WH (2009) Molecular engineering of DNA: molecular beacons. Angew Chem, Int Ed Engl 48:856–870CrossRefGoogle Scholar
  6. 6.
    Cao YWC, Jin RC, Mirkin CA (2002) Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection. Science 297:1536–1540CrossRefGoogle Scholar
  7. 7.
    Patolsky F, Lichtenstein A, Willner I (2000) Amplified microgravimetric quartz-crystal-microbalance assay of DNA using oligonucleotide-functionalized liposomes or biotinylated liposomes. J Am Chem Soc 122:418–419CrossRefGoogle Scholar
  8. 8.
    Larsson C, Rodahl M, Hook F (2003) Characterization of DNA immobilization and subsequent hybridization on a 2D arrangement of streptavidin on a biotin-modified lipid bilayer supported on SiO2. Anal Chem 75:5080–5087CrossRefGoogle Scholar
  9. 9.
    Patolsky F, Lichtenstein A, Willner I (2001) Detection of single-base DNA mutations by enzyme-amplified electronic transduction. Nat Biotechnol 19:253–257CrossRefGoogle Scholar
  10. 10.
    Fan CH, Plaxco KW, Heeger AJ (2003) Electrochemical interrogation of conformational changes as a reagentless method for the sequence-specific detection of DNA. Proc Natl Acad Sci U S A 100:9134–9137CrossRefGoogle Scholar
  11. 11.
    Yang RH, Jin JY, Chen Y, Shao N, Kang HZ, Xiao Z, Tang ZW, Wu YR, Zhu Z, Tan WH (2008) Carbon nanotube-quenched fluorescent oligonucleotides: probes that fluoresce upon hybridization. J Am Chem Soc 130:8351–8358CrossRefGoogle Scholar
  12. 12.
    Xu JJ, Zhao WW, Song S, Fan C, Chen HY (2014) Functional nanoprobes for ultrasensitive detection of biomolecules: an update. Chem Soc Rev 43:1601–1611CrossRefGoogle Scholar
  13. 13.
    Qi L, Xiao M, Wang X, Wang C, Wang L, Song S, Qu X, Li L, Shi J, Pei H (2017) DNA-encoded Raman-active anisotropic nanoparticles for microRNA detection. Anal Chem 89:9850–9856CrossRefGoogle Scholar
  14. 14.
    Zhang YA, Ning XP, Mao GB, Ji XH, He ZK (2018) Fluorescence turn-on detection of target sequence DNA based on silicon nanodot-mediated quenching. Anal Bioanal Chem 410:3209–3216CrossRefGoogle Scholar
  15. 15.
    He S, Song B, Li D, Zhu C, Qi W, Wen Y, Wang L, Song S, Fang H, Fan C (2010) A graphene nanoprobe for rapid, sensitive, and multicolor fluorescent DNA analysis. Adv Funct Mater 20:453–459CrossRefGoogle Scholar
  16. 16.
    Chen Y, Tan CL, Zhang H, Wang LZ (2015) Two-dimensional graphene analogues for biomedical applications. Chem Soc Rev 44:2681–2701CrossRefGoogle Scholar
  17. 17.
    Tan CL, Cao XH, Wu XJ, He QY, Yang J, Zhang X, Chen JZ, Zhao W, Han SK, Nam GH, Sindoro M, Zhang H (2017) Recent advances in ultrathin two-dimensional nanomaterials. Chem Rev 117:6225–6331CrossRefGoogle Scholar
  18. 18.
    Su S, Cao W, Liu W, Lu Z, Zhu D, Chao J, Weng L, Wang L, Fan C, Wang L (2017) Dual-mode electrochemical analysis of microRNA-21 using gold nanoparticle-decorated MoS2 nanosheet. Biosens Bioelectron 94:552–559CrossRefGoogle Scholar
  19. 19.
    Zhu C, Zeng Z, Li H, Li F, Fan C, Zhang H (2013) Single-layer MoS2-based nanoprobes for homogeneous detection of biomolecules. J Am Chem Soc 135:5998–6001CrossRefGoogle Scholar
  20. 20.
    Pumera M, Loo AH (2014) Layered transition-metal dichalcogenides (MoS2 and WS2) for sensing and biosensing. TrAC Trends Anal Chem 61:49–53CrossRefGoogle Scholar
  21. 21.
    Ping JF, Zhou YB, Wu YY, Papper V, Boujday S, Marks RS, Steele TWJ (2015) Recent advances in aptasensors based on graphene and graphene-like nanomaterials. Biosens Bioelectron 64:373–385CrossRefGoogle Scholar
  22. 22.
    Tan CL, Yu P, Hu YL, Chen JZ, Huang Y, Cai YQ, Luo ZM, Li B, Lu QP, Wang LH, Liu Z, Zhang H (2015) High-yield exfoliation of ultrathin two-dimensional ternary chalcogenide nanosheets for highly sensitive and selective fluorescence DNA sensors. J Am Chem Soc 137:10430–10436CrossRefGoogle Scholar
  23. 23.
    Zhu D, Liu W, Zhao DX, Hao Q, Li J, Huang JX, Shi JY, Chao J, Su S, Wang LH (2017) Label-free electrochemical sensing platform for MicroRNA-21 detection using thionine and gold nanoparticles co-functionalized MoS2 nanosheet. ACS Appl Mater Interfaces 9:35597–35603CrossRefGoogle Scholar
  24. 24.
    Deng HM, Yang XJ, Gao ZQ (2015) MoS2 nanosheets as an effective fluorescence quencher for DNA methyltransferase activity detection. Analyst 140:3210–3215CrossRefGoogle Scholar
  25. 25.
    Tang J, Huang YP, Cheng Y, Huang LL, Zhuang JY, Tang DP (2018) Two-dimensional MoS2 as a nano-binder for ssDNA: ultrasensitive aptamer based amperometric detection of Ochratoxin A. Microchim Acta 185:162CrossRefGoogle Scholar
  26. 26.
    Zuo XW, Zhang HG, Zhu Q, Wang WF, Feng J, Chen XG (2016) A dual-color fluorescent biosensing platform based on WS2 nanosheet for detection of Hg2+ and Ag+. Biosens Bioelectron 85:464–470CrossRefGoogle Scholar
  27. 27.
    Southern E (2006) Southern blotting. Nat Protoc 1:518–525CrossRefGoogle Scholar
  28. 28.
    Brown PO, Botstein D (1999) Exploring the new world of the genome with DNA microarrays. Nat Genet 21:33–37CrossRefGoogle Scholar
  29. 29.
    Li JWJ, Chu YZ, Lee BYH, Xie XLS (2008) Enzymatic signal amplification of molecular beacons for sensitive DNA detection. Nucleic Acids Res 36:e36CrossRefGoogle Scholar
  30. 30.
    Wang L, Huang ZC, Wang R, Liu YB, Qian C, Wu J, Liu JW (2018) Transition metal dichalcogenide nanosheets for visual monitoring PCR rivaling a real-time PCR instrument. ACS Appl Mater Interfaces 10:4409–4418CrossRefGoogle Scholar
  31. 31.
    Xiao MS, Man TT, Zhu CF, Pei H, Shi JY, Li L, Qu XM, Shen XZ, Li J (2018) MoS2 nanoprobe for MicroRNA quantification based on duplex-specific nuclease signal amplification. ACS Appl Mater Interfaces 10:7852–7858CrossRefGoogle Scholar
  32. 32.
    Hao LL, Gu HJ, Duan N, Wu SJ, Ma XY, Xia Y, Tao Z, Wang ZP (2017) An enhanced chemiluminescence resonance energy transfer aptasensor based on rolling circle amplification and WS2 nanosheet for Staphylococcus aureus detection. Anal Chim Acta 959:83–90CrossRefGoogle Scholar
  33. 33.
    Ge J, Xin G, Du YH, Chen JJ, Zhang L, Bai DM, Ji DY, Hu YL, Li ZH (2017) Highly sensitive fluorescence detection of mercury (II) ions based on WS2 nanosheets and T7 exonuclease assisted cyclic enzymatic amplification. Sensors Actuators B Chem 249:189–194CrossRefGoogle Scholar
  34. 34.
    Wang XZ, Hou T, Lu TT, Li F (2014) Autonomous exonuclease III-assisted isothermal cycling signal amplification: a facile and highly sensitive fluorescence DNA glycosylase activity assay. Anal Chem 86:9626–9631CrossRefGoogle Scholar
  35. 35.
    Henikoff S (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351–359CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Lele Wang
    • 1
  • Lianhua Dong
    • 2
  • Gang Liu
    • 1
  • Xizhong Shen
    • 3
    • 4
  • Jing Wang
    • 2
  • Changfeng Zhu
    • 3
    Email author
  • Min Ding
    • 1
  • Yanli Wen
    • 1
    Email author
  1. 1.Laboratory of BiometrologyShanghai Institute of Measurement and Testing TechnologyShanghaiPeople’s Republic of China
  2. 2.Division of Medical and Biological MeasurementNational Institute of MetrologyBeijingPeople’s Republic of China
  3. 3.Department of Gastroenterology and Hepatology, Zhongshan HospitalFudan UniversityShanghaiPeople’s Republic of China
  4. 4.Shanghai Institute of Liver Diseases, Zhongshan HospitalFudan UniversityShanghaiPeople’s Republic of China

Personalised recommendations