Skip to main content
Log in

Electrochemiluminescent aptasensor for thrombin using nitrogen-doped graphene quantum dots

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

Abstract

An electrochemiluminescent (ECL) aptamer based method is described for the determination of thrombin. Three-dimensional nitrogen-doped graphene oxide (3D-NGO) was placed on a glassy carbon electrode (GCE) to provide an electrode surface that displays excellent electrical conductivity and acts as a strong emitter of ECL. The modified electrode was further coated with chitosan via electrodeposition. Finally, the amino-modified aptamer was immobilized on the modified GCE. The interaction between thrombin and aptamer results in a decrease in ECL. The assay has a linear response in the 1 fM to 1 nM thrombin concentration range and a 0.25 fM lower detection limit (at an S/N ratio of 3). The method was applied to the determination of thrombin in spiked human plasma samples, and recoveries ranged between 94 and 105% (with RSDs of <3.6%). The calibration plot was recorded at potential and wavelength of fluorescence emission (wavelength: 445 nm; potential: 0 to -2 V).

A bare glassy carbon electrode (GCE) does not display electrochemiluminescence (ECL). If, however, nitrogen-doped graphene quantum dots, chitosan, and three-dimensional nitrogen-doped graphene oxide (NGQD-chitosan/3D-NGO) are electrodeposited on the GCE, strong ECL can be observed. The ECL intensity decreased after aptamer and bovine serum albumin (BSA) were dropped onto the electrode (curve a). However, the ECL further decreases after addition of thrombin (TB; curve b).

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

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

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

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Tsopanoglou NE, Maragoudakis ME (2009) The role of thrombin in angiogenesis. Springer:93–113

  2. Nishino A, Suzuki M, Ohtani H, Motohashi O, Mezawa K, Nagura H, Yoshimoto T (1993) Thrombin may contribute to the pathophysiology of central nervous system injury. PubMed J Neurotrauma 10:167–179

    Article  CAS  Google Scholar 

  3. Zhao J, Zhang Y, Li H, Wen Y, Fan X, Lin F, Tan L, Yao S (2011) Ultrasensitive electrochemical aptasensor for thrombin based on the amplification of aptamer-AuNPs-HRP conjugates. Biosens Bioelectron 26:2297–2303

    Article  CAS  PubMed  Google Scholar 

  4. Cai H, Lee TM, Hsing IM (2006) Label-free protein recognition using an aptamer-based impedance measurement assay. Sensors Actuators B Chem 114:433–437

    Article  CAS  Google Scholar 

  5. Bafrooei EH, Amini M, Ardakani MH (2016) An electrochemical aptasensor based on TiO2/MWCNT and a novel synthesized schiff base nanocomposite for the ultrasensitive detection of thrombin. Biosens Bioelectron 85:828–836

    Article  CAS  Google Scholar 

  6. Zhang H, Shuang S, Sun L, Chen A, 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 

  7. Wang X, Sun D, Tong Y, Zhong Y, Chen Z (2017) A voltammetric aptamer-based thrombin biosensor exploiting signal amplification via synergetic catalysis by DNAzyme and enzyme decorated AuPd nanoparticles on a poly (o-phenylenediamine) support. Microchim Acta 184:1791–1799

    Article  CAS  Google Scholar 

  8. Su Z, Xu X, Xu H, Zhang Y, Li C, Ma Y, Song D, Xie Q (2017) Amperometric thrombin aptasensor using a glassy carbon electrode modified with polyaniline and multiwalled carbon nanotubes tethered with a thiolated aptamer. Microchim Acta 184:1677–1682

    Article  CAS  Google Scholar 

  9. Cheng W, Pan J, Yang J, Zheng Z, Lu F, Chen Y, Gao W (2018) A photoelectrochemical aptasensor for thrombin based on the use of carbon quantum dot-sensitized TiO2 and visible-light photoelectrochemical activity. Microchim Acta 185:263

    Article  CAS  Google Scholar 

  10. Gao F, Du L, Tang D, Lu Y, Zhang Y, Zhang L (2015) A cascade signal amplification strategy for surface enhanced Raman spectroscopy detection of thrombin based on DNAzyme assistant DNA recycling and rolling circle amplification. Biosens Bioelectron 66:423–430

    Article  CAS  PubMed  Google Scholar 

  11. Li XM, Wang LL, Li CX (2015) Rolling-circle amplification detection of thrombin using surface-enhanced Raman spectroscopy with Core-Shell nanoparticle probe. Chem Europ J 21:6817–6822

    Article  CAS  Google Scholar 

  12. Bai Y, Feng F, Zhao L, Wang C, Wang H, Tian M, Qin J, Duan Y, He X (2013) Aptamer/thrombin/aptamer-AuNPs sandwich enhanced surface plasmon resonance sensor for the detection of subnanomolar thrombin. Biosens Bioelectron 47:265–270

    Article  CAS  PubMed  Google Scholar 

  13. Baek SH, Wark AW, Lee HJ (2014) Dual nanoparticle amplified surface plasmon resonance detection of thrombin at subattomolar concentrations. Anal Chem 86:9824–9829

    Article  CAS  PubMed  Google Scholar 

  14. Chang H, Tang L, Wang Y, Jiang J, Li J (2010) Graphene fluorescence resonance energy transfer aptasensor for the thrombin detection. Anal Chem 82:2341–2346

    Article  CAS  PubMed  Google Scholar 

  15. Na W, Liu X, Wang L, Su X (2015) Label-free aptamer biosensor for selective detection of thrombin. Anal Chim Acta 899:85–90

    Article  CAS  PubMed  Google Scholar 

  16. Xu Z, Huang X, Dong C, Ren J (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 

  17. Wen C-Y, Bi J-H, Wu L-L, Zeng J-B (2017) Aptamer-functionalized magnetic and fluorescent nanospheres for one-step sensitive detection of thrombin. Microchim Acta 185:77

    Article  CAS  Google Scholar 

  18. Wang G-L, Hu X-L, Wu X-M, Dong Y-M, Li Z-J (2016) Fluorescent aptamer-based assay for thrombin with large signal amplification using peroxidase mimetics. Microchim Acta 183:765–771

    Article  CAS  Google Scholar 

  19. Sui N, Wang L, Xie F, Liu F, Xiao H, Liu M, Yu W-W (2016) Ultrasensitive aptamer-based thrombin assay based on metal enhanced fluorescence resonance energy transfer. Microchim Acta 183:1563–1570

    Article  CAS  Google Scholar 

  20. Ma M, Zheng X (2015) Preparation of brightly fluorescent silica nanoparticles modified with lucigenin and chitosan, and their application to an aptamer-based sandwich assay for thrombin. Microchim Acta 182:2193–2199

    Article  CAS  Google Scholar 

  21. Li Y, Ling L (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 

  22. He J, Li G, Hu Y (2017) Aptamer-involved fluorescence amplification strategy facilitated by directional enzymatic hydrolysis for bioassays based on a metal-organic framework platform: highly selective and sensitive determination of thrombin and oxytetracycline. Microchim Acta 184:2365–2373

    Article  CAS  Google Scholar 

  23. Hao L, Zhao Q (2016) Microplate based assay for thrombin detection using an RNA aptamer as affinity ligand and cleavage of a chromogenic or a fluorogenic peptide substrate. Microchim Acta 183:1891–1898

    Article  CAS  Google Scholar 

  24. Guo L, Zhao Q (2016) Determination of the platelet-derived growth factor BB by a competitive thrombin-linked aptamer-based Fluorometric assay. Microchim Acta 183:3229–3235

    Article  CAS  Google Scholar 

  25. Zhuo B, Li Y, Huang X, Lin Y, Chen Y, Gao W (2015) An electrochemiluminescence aptasensing platform based on ferrocene-graphene nanosheets for simple and rapid detection of thrombin. Sensors Actuators B Chem 208:518–524

    Article  CAS  Google Scholar 

  26. Hong LR, Chai YQ, Zhao M, Liao N, Yuan R, Zhuo Y (2015) Highly efficient electrogenerated chemiluminescence quenching of PEI enhanced Ru(bpy)3 2+ nanocomposite by hemin and au@CeO2 nanoparticles. Biosens Bioelectron 63:392–398

    Article  CAS  PubMed  Google Scholar 

  27. Liu Y, Zhao Y, Fan Q, Khan MS, Li X, Zhang Y, Ma H, Wei Q (2018) Aptamer based electrochemiluminescent thrombin assay using carbon dots anchored onto silver-decorated polydopamine nanospheres. Microchim Acta 185:85

    Article  CAS  Google Scholar 

  28. Wen Q, Lu P, Yang P (2016) A glassy carbon electrode modified with in-situ generated chromium-loaded CdS nanoprobes and heparin for ultrasensitive electrochemiluminescent determination of thrombin. Microchim Acta 183:123–132

    Article  CAS  Google Scholar 

  29. Zhang L, Li L (2016) Colorimetric thrombin assay using aptamer-functionalized gold nanoparticles acting as a peroxidase mimetic. Microchim Acta 183:485–490

    Article  CAS  Google Scholar 

  30. Famulok M, Harting JS, Mayer G (2007) Functional aptamers and aptazymes in biotechnology, diagnostics and therapy. Chem Rev 107:3715–3743

    Article  CAS  PubMed  Google Scholar 

  31. Tombelli S, Minunni M, Mascini M (2005) Analytical applications of aptamers. Biosens Bioelectron 20:2424–2434

    Article  CAS  PubMed  Google Scholar 

  32. Bard AJ (2004) Electrogenerated chemiluminescence. CRC Press, 552

  33. Hu L, Xu G (2010) Applications and trends in electrochemiluminescence. Chem Soc Rev 39:3275–3304

    Article  CAS  PubMed  Google Scholar 

  34. Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, Sundaresan G, Wu AM, Gambhir SS, Weiss S (2005) Quantum dots for live cells in vivo imaging and diagnostics. Science 307:538–544

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Jie G, Chen K, Wang X, Lu Z (2016) Dual-stabilizer-capped CdSe quantum dots for “off–on” electrochemiluminescence biosensing of thrombin by target-triggered multiple amplification. RSC Adv 6:2065–2071

    Article  CAS  Google Scholar 

  36. Dong YP, Gao TT, Zhou Y, Zhu JJ (2014) Electrogenerated chemiluminescence resonance energy transfer between luminol and CdSe@ ZnS quantum dots and its sensing application in the determination of thrombin. Anal Chem 86:11373–11379

    Article  CAS  PubMed  Google Scholar 

  37. Lin L, Rong M, Luo F, Wang Y, Chen X (2014) Luminescent graphene quantum dots as new fluorescent materials for environmental and biological applications. TrAC Trends Anal Chem 54:83–102

    Article  CAS  Google Scholar 

  38. Martínez SB, Valcárcel M (2015) Graphene quantum dots in analytical science. TrAC Trends Anal Chem 72:93–113

    Article  CAS  Google Scholar 

  39. Li Y, Zhao Y, Cheng H, Hu Y, Shi G, Dai L, Qu L (2012) Nitrogen-doped graphene quantum dots with oxygen-rich functional groups. J Am Chem Soc Commun 134:15–18

    Article  CAS  Google Scholar 

  40. Novoselov KS, Geim AK, Morozov SV, Jiang D, Zhang Y, Dubonos SV, Grigorieva IV, Firsov AA (2004) Electric field effect in atomically thin carbon films. Science 306:666–669

    Article  CAS  PubMed  Google Scholar 

  41. Pumera M (2010) Graphene-based nanomaterials and their electrochemistry. Chem Soc Rev 39:4146–4157

    Article  CAS  PubMed  Google Scholar 

  42. Xu J, Wang Y, Hu S (2017) Nanocomposites of graphene and graphene oxides: synthesis, molecular functionalization and application in electrochemical sensors and biosensors. A review. Microchim Acta 184:1–44

    Article  CAS  Google Scholar 

  43. Dong S, Dao AQ, Zheng B, Tan Z, Fu C, Liu H, Xiao F (2015) One-step electrochemical synthesis of three-dimensional graphene foam loaded nickel-cobalt hydroxides nanoflakes and its electrochemical properties. Electrochim Acta 152:195–201

    Article  CAS  Google Scholar 

  44. Li J, Jiang J, Feng H, Xu Z, Tang S, Deng P, Qian D (2016) Facile synthesis of 3D porous nitrogen-doped graphene as an efficient electrocatalyst for adenine sensing. RSC Adv 6:31565–31573

    Article  CAS  Google Scholar 

  45. Chen S, Chen X, Xia T, Ma Q (2016) A novel electrochemiluminescence sensor for the detection of nitroaniline based on the nitrogen-doped graphene quantum dots. Biosens Bioelectron 85:903–908

    Article  CAS  PubMed  Google Scholar 

  46. Pur MRK, Hosseini M, Faridbod F, Dezfuli AS, Ganjali MR (2016) A novel solid-state electrochemiluminescence sensor for detection of cytochrome c based on ceria nanoparticles decorated with reduced graphene oxide nanocomposite. Anal Bioanal Chem 408:7193–7202

    Article  CAS  PubMed  Google Scholar 

  47. Ju J, Zhang R, He S, Chen W (2014) Nitrogen-doped graphene quantum dots-based fluorescent probe for the sensitive turn-on detection of glutathione and its cellular imaging. RSC Adv 4:52583–52589

    Article  CAS  Google Scholar 

  48. Lin L, Song X, Chen Y, Rong M, Zhao T, Jiang Y, Wang Y, Chen X (2015) One-pot synthesis of highly greenish-yellow fluorescent nitrogen-doped graphene quantum dots for pyrophosphate sensing via competitive coordination with Eu 3+ ions. Nanoscale 7:15427–15433

    Article  CAS  PubMed  Google Scholar 

  49. Du X, Jiang D, Liu Q, Zhu G, Mao H, Wang K (2015) Fabrication of graphene oxide decorated with nitrogen-doped graphene quantum dots and its enhanced electrochemiluminescence for ultrasensitive detection of pentachlorophenol. Analyst 140:1253–1259

    Article  CAS  PubMed  Google Scholar 

  50. Antonello S, Musumeci M, Wayner DDM, Maran F (1997) Electroreduction of Dialkyl peroxides. Activation−driving force relationships and bond dissociation free energies. J Am Chem Soc 119:9541–9549

    Article  CAS  Google Scholar 

  51. Meneses AB, Antonello S, Arévalo MC (2006) Maran F double-layer correction for Electron-transfer kinetics at glassy carbon and mercury electrodes in N, N-Dimethylformami. Electroanalysis 18:363–370

    Article  CAS  Google Scholar 

  52. Chen M, Hou C, Huo D, Fa H, Zhao Y, Shen C (2017) Sensitive electrochemical DNA biosensor based on three-dimensional nitrogen-doped graphene and Fe3O4 nanoparticles. Sensors Actuators B Chem 239:421–429

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was financially supported by State Key Laboratory of Fine Chemicals, the National Natural Science Foundation of China (No. 21272030, 21472016, 21306019, 21576042).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Shiguo Sun.

Ethics declarations

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

Electronic supplementary material

ESM 1

(DOCX 546 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Khonsari, Y.N., Sun, S. Electrochemiluminescent aptasensor for thrombin using nitrogen-doped graphene quantum dots. Microchim Acta 185, 430 (2018). https://doi.org/10.1007/s00604-018-2942-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s00604-018-2942-z

Keywords

Navigation