Skip to main content
SpringerLink
Log in

Ultrasensitive aptamer-based thrombin assay based on metal enhanced fluorescence resonance energy transfer

  • Original Paper
  • Published:
Microchimica Acta Aims and scope Submit manuscript

Abstract

A sensitive “on-off” fluorescent protocol for thrombin detection is demonstrated. Firstly, thrombin aptamers which hybridize with labeled help DNA were immobilized on the surface of Ag@SiO2 nanoparticles (NPs). The silver core causes the label Cy5 to display strong metal-enhanced fluorescence. On addition of thrombin and graphene oxide (GO), thrombin (with its high affinity for the aptamers) displaces the Cy5-labeled help DNA which then binds to the surface of GO via π-stacking, causing fluorescence quenching of Cy5. The findings were used to design a thrombin assay that has a 0.05 nM detection limit and excellent selectivity. It was applied to the quantification of thrombin in spiked serum samples where is showed recoveries ranging from 97 % to 107 %, with relative standard deviations between 2.2 and 4.5 %.

A sandwich architecture of type Ag@SiO2-DNA-Cy5 was fabricated. The fluorescence of Cy5 is enhanced by Ag@SiO2, and quenched by GO. Upon the introduction of thrombin and GO, (a) the quadruplex-thrombin complex is formed, (b) the DNA duplexes are dismissed, and (c) helper DNA is released, which then is absorbed by GO, this leading to quenching.

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. Lakowicz JR (2006) Principles of Fluorescence Spectroscopy, Springer; 3rd edition, ISBN: 0387312781

  2. Resch-Genger U, Grabolle M, Cavaliere-Jaricot S, Nitschke R, Nann T (2008) Quantum dots versus organic dyes as fluorescent labels. Nat Meth 5:763–775

    Article  CAS  Google Scholar 

  3. Yang YM, Zhao Q, Feng W, Li FY (2012) Luminescent chemodosimeters for bioimaging. Chem Rev 113:192–270

    Article  Google Scholar 

  4. Cheng Z, Li G, Liu M (2015) A metal-enhanced fluorescence sensing platform based on new mercapto rhodamine derivatives for reversible Hg2+ detection. J Hazard Mater 287:402–411

    Article  CAS  Google Scholar 

  5. Li M, Wang QY, Shi XD, Hornak LA, Wu NQ (2011) Detection of mercury(II) by Quantum dot/DNA/Gold Nanoparticle Ensemble based nanosensor via nanometal surface energy transfer. Anal Chem 83:7061–7065

    Article  CAS  Google Scholar 

  6. Liu XQ, Wang F, Aizen R, Yehezkeli O, Willner I (2013) Graphene oxide/nucleic-acid-stabilized Silver Nanoclusters: functional hybrid materials for optical aptamer sensing and multiplexed analysis of pathogenic DNAs. J Am Chem Soc 135:11832–11839

    Article  CAS  Google Scholar 

  7. Wang YH, Wu ZJ, Liu ZH (2012) Upconversion fluorescence resonance energy transfer biosensor with aromatic polymer nanospheres as the lable-free energy acceptor. Anal Chem 85:258–264

    Article  Google Scholar 

  8. Ma K, Lu L, Qi Z, Feng J, Zhuo C, Zhang Y (2015) In situ induced metal-enhanced fluorescence: A new strategy for biosensing the total acetylcholinesterase activity in sub-microliter human whole blood. Biosen & Bioelectr 68:648–653

    Article  CAS  Google Scholar 

  9. Li H, Chen C-Y, Wei X, Qiang W, Li Z, Cheng Q, et al. (2012) Highly sensitive detection of proteins based on metal-enhanced fluorescence with novel Silver Nanostructures. Anal Chem 84:8656–8662

    Article  CAS  Google Scholar 

  10. Lu L, Qian Y, Wang L, Ma K, Zhang Y (2014) Metal-enhanced fluorescence-based Core shell Ag@SiO2 nanoflares for affinity biosensing via Target-induced structure switching of aptamer. ACS Appl Mat Inter 6:1944–1950

    Article  CAS  Google Scholar 

  11. Mujumdar RB, Ernst LA, Mujumdar SR, Lewis CJ, Waggoner AS (1993) Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjug Chem 4:105–111

    Article  CAS  Google Scholar 

  12. Onoda M, Uchiyama S, Santa T, Imai K (2002) The effects of spacer length on the fluorescence Quantum yields of the benzofurazan compounds bearing a donor acceptor system. Luminescence 17:11–14

    Article  CAS  Google Scholar 

  13. Lakowicz JR (2001) Radiative decay engineering: biophysical and biomedical applications. Anal Biochem 298:1–24

    Article  CAS  Google Scholar 

  14. Lakowicz JR (2006) Plasmonics in biology and Plasmon-Controlled Fluorescence. Plasmonics 1:5–33

    Article  CAS  Google Scholar 

  15. Lakowicz JR, Fu Y (2009) Modification of single molecule fluorescence near metallic nanostructures. Laser Photonics Rev 3:221–232

    Article  Google Scholar 

  16. Ji BT, Giovanelli E, Habert B, Spinicelli P, Nasilowski M, Xu XZ, Lequeux N, Hugonin JP, Marquier F, Greffet JJ, Dubertret B (2015) Non-blinking Quantum dot with a plasmonic nanoshell resonator. Nat Nano 10:170–175

    Article  CAS  Google Scholar 

  17. Zhang HF, Shuang SM, Sun LL, Chen AJ, Qin Y, Dong C (2014) Label-free aptasensor for thrombin using a glassy carbon electrode modified with a graphene-porphyrin composite. Microchim Acta 181:189–196

    Article  CAS  Google Scholar 

  18. Xu ZC, Huang XY, Dong CQ, Ren JC (2014) Fluorescence correlation spectroscopy of gold nanoparticles, and its application to an aptamer-based homogeneous thrombin assay. Microchim Acta 181:723–730

    Article  CAS  Google Scholar 

  19. Zhang LP, Li L (2015) Colorimetric thrombin assay using aptamer-functionalized gold nanoparticles acting as a peroxidase mimetic. Microchim Acta:1–6

  20. Li YB, Ling LS (2015) Aptamer-based fluorescent solid-phase thrombin assay using a silver-coated glass substrate and signal amplification by glucose oxidase. Microchim Acta 182:1849–1854

    Article  CAS  Google Scholar 

  21. Lin ZH, Pan D, Hu TY, Liu ZP, Su XG (2015) A near-infrared fluorescent bioassay for thrombin using aptamer-modified CuInS2 Quantum dots. Microchim Acta 182:1933–1939

    Article  CAS  Google Scholar 

  22. Lu CH, Li J, Lin MH, Wang YW, Yang HH, Chen X, Chen GN (2010) Amplified aptamer based assay through catalytic recycling of the analyte. Angew Chem 122:8632–8635

    Article  Google Scholar 

  23. Xue LY, Zhou XM, Xing D (2012) Sensitive and homogeneous protein detection based on Target-triggered aptamer hairpin switch and nicking enzyme assisted fluorescence signal amplification. Anal Chem 84:3507–3513

    Article  CAS  Google Scholar 

  24. Pérez-López B, Merkoçi A (2012) Carbon nanotubes and graphene in analytical sciences. Microchim Acta 179:1–16

    Article  Google Scholar 

  25. Duan Y, Ning Y, Song Y, Deng L (2014) Fluorescent aptasensor for the determination of salmonella typhimurium based on a graphene oxide platform. Microchim Acta 181:647–653

    Article  CAS  Google Scholar 

  26. Deng W, Jin DY, Drozdowicz-Tomsia K, Yuan JL, Wu J, Goldys EM (2011) Ultrabright Eu-Doped Plasmonic Ag@SiO2 nanostructures: time-gated bioprobes with single particle sensitivity and negligible background. Adv Mater 23:4649–4654

    Article  CAS  Google Scholar 

  27. Sui N, Wang LN, Yan TF, Liu FY, Sui J, Jiang YJ, Xiao HL, Liu MH, Yu WW (2014) Selective and sensitive biosensors based on metal-enhanced fluorescence. Sensors and Actuators B: Chem 202:1148–1153

    Article  CAS  Google Scholar 

  28. Yang LT, Ellington AD (2008) Real-time PCR detection of protein analytes with conformation-switching aptamers. Anal Biochem 380:164–173

    Article  CAS  Google Scholar 

  29. Lu CH, Yang HH, Zhu CL, Chen X, Chen GN (2009) A graphene platform for sensing biomolecules. Angew Chem 121:4785–4787

    Article  Google Scholar 

  30. 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 

  31. Sui N, Monnier V, Zakharko Y, Chevolot Y, Alekseev S, Bluet J-M, Lysenko V, Souteyrand E (2012) Fluorescent (Au@SiO2)SiC nanohybrids: influence of Gold nanoparticle diameter and SiC nanoparticle surface density. Plasmonics 8:85–92

    Article  Google Scholar 

  32. Sui N, Monnier V, Zakharko Y, Chevolot Y, Alekseev S, Bluet J-M, Lysenko V, Souteyrand E (2012) Plasmon-controlled narrower and blue-shifted fluorescence emission in (Au@SiO2)SiC nanohybrids. J Nanoparticle Res 14:1–10

    Google Scholar 

  33. Li F, Chao J, Li ZH, Xing S, Su S, Li XX, Song SP, Zuo XL, Fan CH, Liu B, Huang W, Wang LH, Wang LH (2015) Graphene oxide-assisted nucleic acids assays using conjugated polyelectrolytes-based fluorescent signal transduction. Anal Chem 87:3877–3883

    Article  CAS  Google Scholar 

  34. Bharadwaj P, Novotny L (2007) Spectral dependence of single molecule fluorescence enhancement. Opt Express 15:14266–14274

    Article  CAS  Google Scholar 

  35. Pang YF, Rong Z, Xiao R, Wang SQ (2015) "Turn on" and label-free core shell Ag@SiO2 nanoparticles-based metal-enhanced fluorescent (MEF) aptasensor for Hg2+, Sci Rep 5. 9541:1–5

  36. Wang Y, Bao L, Liu Z, Pang D-W (2011) Aptamer biosensor based on fluorescence resonance energy transfer from upconverting phosphors to carbon nanoparticles for thrombin detection in human plasma. Anal Chem 83:8130–8137

    Article  CAS  Google Scholar 

  37. Yu JM, Yang LR, Liang XF, Dong TT, Liu HZ (2015) Bare magnetic nanoparticles as fluorescence quenchers for detection of thrombin. Analyst 140:4114–4120

    Article  CAS  Google Scholar 

  38. Wang C, Zhai W, Wang Y, Yu P, Mao L (2015) MnO2 nanosheets based fluorescent sensing platform with organic dyes as a probe with excellent analytical properties. Analyst 140:4021–4029

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (21301103, 21501106), the 47th Scientific Research Foundation for the returned overseas Chinese scholars, the taishan scholarship, the Shandong Natural Science Foundation (ZR2012FZ007), the Shandong Province High Education Research And Development program (J13LA08).

Author information

Authors and Affiliations

  1. College of Materials Science and Engineering, Qingdao University of Science and Technology, Qingdao, 266042, China

    Ning Sui, Fengxia Xie, Fengya Liu, Hailian Xiao, Manhong Liu & William W. Yu

  2. Department of Chemistry and Physics, Louisiana State University, Shreveport, LA, 71115, USA

    William W. Yu

  3. College of Environment and Safety Engineering, Key Laboratory of Eco-chemical Engineering, Ministry of Education, Qingdao University of Science and Technology, Qingdao, 266042, China

    Lina Wang

Authors
  1. Ning Sui
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lina Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fengxia Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fengya Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hailian Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Manhong Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. William W. Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Lina Wang or William W. Yu.

Ethics declarations

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

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sui, N., Wang, L., Xie, F. et al. Ultrasensitive aptamer-based thrombin assay based on metal enhanced fluorescence resonance energy transfer. Microchim Acta 183, 1563–1570 (2016). https://doi.org/10.1007/s00604-016-1774-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00604-016-1774-y

Keywords

Search

Navigation