Skip to main content

Advertisement

SpringerLink
Log in

A Simple QD–FRET Bioprobe for Sensitive and Specific Detection of Hepatitis B Virus DNA

  • ORIGINAL PAPER
  • Published:
Journal of Fluorescence Aims and scope Submit manuscript

Abstract

We report here a simple quantum dot-FRET (QD-FRET) bioprobe based on fluorescence resonance energy transfer (FRET) for the sensitive and specific detection of hepatitis B virus DNA (HBV DNA). The proposed one-pot HBV DNA detection method is very simple, rapid and convenient due to the elimination of the washing and separation steps. In this study, the water-soluble CdSe/ZnS QDs were prepared by replacing the trioctylphosphine oxide on the surface of QDs with mercaptoacetic acid (MAA). Subsequently, DNA was attached to QDs surface to form the functional QD-DNA bioconjugates by simple surface ligand exchange. After adding 6-carboxy-X-rhodamine (ROX)-modified HBV DNA (ROX-DNA) into the QD-DNA bioconjugates solution, DNA hybridization between QD-DNA bioconjugates and ROX-DNA was formed. The resulting hybridization brought the ROX fluorophore, the acceptor, and the QDs, the donor, into proximity, leading to energy transfer from QDs to ROX. When ROX-DNA was displaced by the unlabeled HBV DNA, the efficiency of FRET was dramatically decreased. Based on the changes of both fluorescence intensities of QDs and ROX, HBV DNA could be detected with high sensitivity and specificity. Under the optimized conditions, the linear range of HBV DNA determination was 2.5 – 30 nmol L−1, with a correlation coefficient (R) of 0.9929 and a limit of detection (3σ black) of 1.5 nmol L−1. The relative standard deviation (R.S.D.) for 12 nmol L−1 HBV DNA was 0.9 % (n = 5). There was no interference to non-complementary DNA. Time-resolved fluorescence spectra and fluorescence images were performed to verify the validity of this method and the results were satisfying.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Bruchez M, Moronne M Jr, Gin P, Weiss S, Alivisatos AP (1998) Semiconductor nanocrystals as fluorescent biological labels. Science 281:2013–2016

    Article  PubMed  CAS  Google Scholar 

  2. Medintz IL, Uyeda HT, Goldman ER, Mattoussi H (2005) Quantum dot bioconjugates for imaging,labelling and sensing. Nat Mater 4:435–446

    Article  PubMed  CAS  Google Scholar 

  3. Huang S, Xiao Q, Li R, Guan HL, Liu J, Liu XR, He ZK, Liu Y (2009) A simple and sensitive method for L-Cysteine detection based on the fluorescence intensity increment of quantum dots. Anal Chim Acta 645:73–78

    Article  PubMed  CAS  Google Scholar 

  4. Huang S, Xiao Q, He ZK, Liu Y, Tinnefeld P, Su XR, Peng XN (2008) A high sensitive and specific QDs FRET bioprobe for MNase. Chem Commun 44:5990–5992

    Google Scholar 

  5. Qiu T, Zhao D, Zhou GH, Liang Y, He ZK, Liu ZH, Peng XN, Zhou L (2010) A positively charged QDs-based FRET probe for micrococcal nuclease detection. Analyst 135:2394–2399

    Article  PubMed  CAS  Google Scholar 

  6. Chen CY, Cheng CT, Lai CW, Wu PW, Wu KC, Chou PT, Chou YH, Chiu HT (2006) Potassium ion recognition by 15-crown-5 functionalized CdSe/ZnS quantum dots in H2O. Chem Commun 42:263–265

    Google Scholar 

  7. Medintz IL, Clapp AR, Mattoussi H, Goldman ER, Fisher B, Mauro JM (2003) Self-assembled nanoscale biosensors based on quantum dot FRET donors. Nat Mater 2:630–638

    Article  PubMed  CAS  Google Scholar 

  8. Tang B, Cao LH, Xu KH, Zhou LH, Ge JC, Li QL, Yu LJ (2008) A New Nanobiosensor for Glucose with High Sensitivity and Selectivity in Serum Based on Fluorescence Resonance Energy Transfer (FRET) between CdTe Quantum Dots and Au Nanoparticles. Chem Eur J 14:3637–3644

    Google Scholar 

  9. Freeman R, Gill R, Shweky I, Kotler M, Banin U, Willner I (2009) Biosensing and Probing of Intracellular Metabolic Pathways by NADH-Sensitive Quantum Dots. Angew Chem Int Ed 48:309–313

    Article  CAS  Google Scholar 

  10. Tomasulo M, Yildiz I, Kaanumalle SL, Raymo FM (2006) pH-Sensitive Ligand for Luminescent Quantum Dots. Langmuir 22:10284–10290

    Article  PubMed  CAS  Google Scholar 

  11. Snee PT, Somers RC, Nair G, Zimmer JP, Bawendi MG, Nocera DG (2006) A Ratiometric CdSe/ZnS Nanocrystal pH Sensor. J Am Chem Soc 128:13320–13321

    Article  PubMed  CAS  Google Scholar 

  12. Levy M, Cater SF, Ellington AD (2005) Quantum-Dot Aptamer Beacons for the Detection of Proteins. ChemBioChem 6:2163–2166

    Article  PubMed  CAS  Google Scholar 

  13. Oh E, Hong MY, Lee D, Nam SH, Yoon HC, Kim HS (2005) Inhibition Assay of Biomolecules based on Fluorescence Resonance Energy Transfer (FRET) between Quantum Dots and Gold Nanoparticles. J Am Chem Soc 127:3270–3271

    Article  PubMed  CAS  Google Scholar 

  14. Xu CJ, Xing BG, Rao JH (2006) A self-assembled quantum dot probe for detecting β-lactamase activity. Biochem Biophys Res Commun 344:931–935

    Article  PubMed  CAS  Google Scholar 

  15. Chang E, Miller JS, Sun JT, Yu WW, Colvin VL, Drezek R, West JL (2005) Protease-activated quantum dot probes. Biochem Biophys Res Commun 334:1317–1321

    Google Scholar 

  16. Medintz IL, Clapp AR, Brunel FM, Tiefenbrunn T, Uyeda HT, Chang EL, Deschamps JR, Dawson PE, Mattoussi H (2006) Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot-peptide conjugates. Nat Mater 5:581–589

    Article  PubMed  CAS  Google Scholar 

  17. Shi LF, Paoli VD, Rosenzweig N, Rosenzweig Z (2006) Synthesis and Application of Quantum Dots FRET-Based Protease Sensors. J Am Chem Soc 128:10378–10379

    Article  PubMed  CAS  Google Scholar 

  18. Zhang CY, Yeh HC, Kuroki MT, Wang TH (2005) Single-quantum-dot-based DNA nanosensor. Nat Mater 4:826–831

    Article  PubMed  CAS  Google Scholar 

  19. Zhang CY, Hu J (2010) Single Quantum Dot-Based Nanosensor for Multiple DNA Detection. Anal Chem 82:1921–1927

    Article  PubMed  CAS  Google Scholar 

  20. Peng H, Zhang LJ, Kjllman THM, Soeller C, Travas-Sejdic J (2007) DNA Hybridization Detection with Blue Luminescent Quantum Dots and Dye-Labeled Single-Stranded DNA. J Am Chem Soc 129:3048–3049

    Article  PubMed  CAS  Google Scholar 

  21. Jiang GX, Susha AS, Lutich AA, Stefani FD, Feldman J, Rogach AL (2010) Cascaded FRET in Conjugated Polymer/Quantum Dot/Dye-Labeled DNA Complexes for DNA Hybridization Detection (出版年为 : 2009年). ACSNANO 3:4127–4131

    Google Scholar 

  22. Algar WR, Krull UJ (2007) Towards multi-colour strategies for the detection of oligonucleotide hybridization using quantum dots as energy donors in fluorescence resonance energy transfer (FRET). Anal Chim Acta 581:193–201

    Article  PubMed  CAS  Google Scholar 

  23. Wang X, Lou XH, Wang Y, Guo QC, Fang Z, Zhong XH, Mao HJ, Jin QH, Wu L, Zhao H, Zhao JL (2010) QDs-DNA nanosensor for the detection of hepatitis B virus DNA and the single-base mutants. Biosens Bioelectron 25:1934–1940

    Article  PubMed  CAS  Google Scholar 

  24. Medintz IL, Berti L, Pons T, Grimes AF, English DS, Alessandrini A, Facci P, Mattoussi H (2007) A Reactive Peptidic Linker for Self-Assembling Hybrid Quantum Dot?DNA Bioconjugates. Nano Lett 7:1741–1748

    Article  PubMed  CAS  Google Scholar 

  25. Wu SM, Tian ZQ, Zhang ZL, Huang BH, Jiang P, Xie ZX, Pang DW (2010) Direct fluorescence in situ hybridization (FISH) in Escherichia coli with a target-specific quantum dot-based molecular beacon. Biosens Bioelectron 26:491–496

    Article  PubMed  CAS  Google Scholar 

  26. Mast EE, Alter MJ, Margolis HS (1999) Strategies to prevent and control hepatitis B and C virus infections: a global perspective. Vaccine 17:1730–1733

    Article  PubMed  CAS  Google Scholar 

  27. Malik AH, Lee WM (2000) Chronic hepatitis B virus infection: Treatment strategies for the next millennium. Ann Intern Med 132:723–731

    Article  PubMed  CAS  Google Scholar 

  28. Selabe SG, Song E, Burnett RJ, Mphahlele MJ (2009) Frequent detection of hepatitis B virus variants associated with lamivudine resistance in treated South African patients infected chronically with different HBV genotypes. J Med Virol 81:996–1001

    Article  PubMed  CAS  Google Scholar 

  29. Ntziora F, Parakevis D, Haida C, Magiorkinis E, Manesis E, Papatheodoridis G, Manolakopoulos S, Beloukas A, Chryssoy S, Magiorkinis G, Sypsa V, Hatzakis A (2009) Quantitative Detection of the M204V Hepatitis B Virus Minor Variants by Amplification Refractory Mutation System Real-Time PCR Combined with Molecular Beacon Technology. J Clin Microbiol 47:2544–2550

    Article  PubMed  CAS  Google Scholar 

  30. Hige S, Yamamoto Y, Yoshida S, Kobayashi T, Horimoto H, Yamamoto K, Sho T, Natsuizaka M, Nakanishi M, Chuma M, Asaka M (2010) Sensitive Assay for Quantification of Hepatitis B Virus Mutants by Use of a Minor Groove Binder Probe and Peptide Nucleic Acids. J Clin Microbiol 48:4487–4494

    Article  PubMed  CAS  Google Scholar 

  31. Xu GL, You QM, Pickerill S, Zhong HY, Wang HY, Shi J, Luo Y, You P, Kong HM, Lu FM, Hu L (2010) Application of PCR-LDR-nucleic acid detection strip in detection of YMDD mutation in hepatitis B patients treated with lamivudine. J Med Virol 82:1143–1149

    Article  PubMed  CAS  Google Scholar 

  32. Ye YK, Zhao JH, Yan F, Zhu YL, Ju XH (2003) Electrochemical behavior and detection of hepatitis B virus DNA PCR production at gold electrode. Biosens Bioelectron 18:1501–1508

    Article  PubMed  CAS  Google Scholar 

  33. Ariksoysal DO, Karadeniz H, Erdem A, Sengonul A, Sayiner AA, Ozsoz M (2005) Label-Free Electrochemical Hybridization Genosensor for the Detection of Hepatitis B Virus Genotype on the Development of Lamivudine Resistance. Anal Chem 77:4908–4917

    Article  PubMed  CAS  Google Scholar 

  34. Hong SP, Kim NK, Hwang SG, Chung HJ, Kim S, Han JH, Kim HT, Rim KS, Kang MS, Yoo W, Kim SO (2004) Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments. J Hepatol 40:837–844

    Article  PubMed  CAS  Google Scholar 

  35. Guan HL, Cai M, Wang Y, He ZK (2010) Label‐free DNA sensor based on fluorescent cationic polythiophene for the sensitive detection of hepatitis B virus oligonucleotides. Luminescence 25:311–316

    Article  PubMed  CAS  Google Scholar 

  36. Peng ZA, Peng XG (2001) Formation of high-quality CdTe, CdSe, and CdS nanocrystals using CdO as precursor. J Am Chem Soc 123:183–184

    Article  PubMed  CAS  Google Scholar 

  37. Zhou DJ, Bruckbauer A, Abell C, Klenerman D, Kang DJ (2005) Fabrication of Three-Dimensional Surface Structures with Highly Fluorescent Quantum Dots by Surface-Templated Layer-by-Layer Assembly. Adv Mater 17:1243–1248

    Article  CAS  Google Scholar 

  38. Xiao Q, Huang S, Qi ZD, Zhou B, He ZK, Liu Y (2008) Conformation, thermodynamics and stoichiometry of HSA adsorbed to colloidal CdSe/ZnS quantum dots. BBA-Proteins Proteom 1784:1020–1027

    Article  CAS  Google Scholar 

  39. Lei Y, Xiao Q, Huang S, Xu WS, Zhang Z, He ZK, Liu Y, Deng FJ (2011) Impact of CdSe/ZnS quantum dots on the development of zebrafish embryos. J Nanopart Res 13:6895–6906

    Article  CAS  Google Scholar 

  40. Xiao Q, Zhou B, Huang S, Tian FF, Guan HL, Ge YS, Liu XR, He ZK, Liu Y (2009) Direct observation of the binding process between protein and quantum dots by in situ surface plasmon resonance measurements. Nanotechnology 20:325101

    Article  PubMed  Google Scholar 

  41. Yu WW, Qu LH, Guo WZ, Peng XG (2003) Experimental determination of the extinction coefficient of CdTe, CdSe, and CdS nanocrystals. Chem Mater 15:2854–2860

    Article  CAS  Google Scholar 

  42. Crosby GA, Demas JN (1971) The measurement of photo-luminescence quantum yields. J Phys Chem 75:991–1024

    Article  CAS  Google Scholar 

  43. Parak WJ, Gerion D, Zanchet D, Woerz AS, Pellegrino T, Micheel C, Williams SC, Seitz M, Bruehl RE, Bryant Z, Bustamante C, Bertozzi CR, Alivisatos AP (2002) Conjugation of DNA to Silanized Colloidal Semiconductor Nanocrystalline Quantum Dots. Chem Mater 14:2113–2119

    Article  CAS  Google Scholar 

  44. Sklar LA, Hudson BS, Simoni RD (1977) Conjugated polyene fatty acids as fluorescent probes: synthetic phospholipid membrane studies. Biochemistry 16:819–828

    Article  PubMed  CAS  Google Scholar 

  45. Stryer L (1978) Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem 47:819–846

    Article  PubMed  CAS  Google Scholar 

  46. Lee SF, Osborne M (2007) Photodynamics of a Single Quantum Dot:? Fluorescence Activation, Enhancement, Intermittency, and Decay J Am Chem Soc 129:8936–8937

    Article  PubMed  CAS  Google Scholar 

  47. Pons T, Medintz IL, Wang X, English DS, Mattoussi H (2006) Solution-Phase Single Quantum Dot Fluorescence Resonance Energy Transfer. J Am Chem Soc 128:15324–15331

    Article  PubMed  CAS  Google Scholar 

  48. Valeur B, Brochon JC (1999) New trends in fluorescence spectroscopy. Springer Press, Berlin, p 25

    Google Scholar 

  49. Zhang CY, Johnson LW (2006) Quantum Dot-Based Fluorescence Resonance Energy Transfer with Improved FRET Efficiency in Capillary Flows. Anal Chem 78:5532–5537

    Article  PubMed  CAS  Google Scholar 

  50. Zhang CY, Johnson LW (2007) Microfluidic Control of Fluorescence Resonance Energy Transfer: Breaking the FRET Limit. Angew Chem Int Ed 46:3482–3485

    Article  CAS  Google Scholar 

  51. Lakowicz JR (1999) Principles of fluorescence spectroscopy, 2nd edn. Kluwer Academic/Plenum Publishers, New York

    Book  Google Scholar 

  52. Haldar KK, Sen T, Patra A (2010) Metal Conjugated Semiconductor Hybrid Nanoparticle-Based Fluorescence Resonance Energy Transfer. J Phys Chem C 114:4869–4874

    Article  CAS  Google Scholar 

  53. Saini S, Srinivas G, Bagchi B (2009) Distance and Orientation Dependence of Excitation Energy Transfer: From Molecular Systems to Metal Nanoparticles. J Phys Chem B 113:1817–1832

    Article  PubMed  CAS  Google Scholar 

  54. Lee A, Coombs NA, Gourevich I, Kumacheva E, Scholes GD (2009) Lamellar Envelopes of Semiconductor Nanocrystals. J Am Chem Soc 131:10182–10188

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (Grant No. 21203035, 21273065, 21261005) and Guangxi Natural Science Foundation (2013GXNSFCA019005, 2013GXNSFBA019029).

Author information

Authors and Affiliations

  1. College of Chemistry and Life Science, Guangxi Teachers Education University, Nanning, 530001, People’s Republic of China

    Shan Huang, Hangna Qiu, Qi Xiao, Chusheng Huang & Wei Su

  2. Key Laboratory of Beibu Gulf Environment Change and Resources Utilization (Guangxi Teachers Education University), Ministry of Education, Nanning, People’s Republic of China

    Qi Xiao & Baoqing Hu

Authors
  1. Shan Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hangna Qiu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qi Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chusheng Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wei Su
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Baoqing Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Qi Xiao.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Huang, S., Qiu, H., Xiao, Q. et al. A Simple QD–FRET Bioprobe for Sensitive and Specific Detection of Hepatitis B Virus DNA. J Fluoresc 23, 1089–1098 (2013). https://doi.org/10.1007/s10895-013-1238-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10895-013-1238-2

Keywords

Search

Navigation