An electrochemical aptamer-based assay for femtomolar determination of insulin using a screen printed electrode modified with mesoporous carbon and 1,3,6,8-pyrenetetrasulfonate


The authors describe an electrochemical method for aptamer-based determination of insulin at femtomolar concentrations. The surface of a screen printed electrode was modified with ordered mesoporous carbon that was chemically modified with 1,3,6,8-pyrenetetrasulfonate (TPS). The amino-terminated aptamer was then covalently linked to TPS via reactive sulfonyl chloride groups. Subsequently, the redox probe Methylene Blue (MB) was interacted into the aptamer. The MB-modified binds to insulin and this results in the release of MB and a decreased signal as obtained by differential pulse voltammetry, best at a working voltage of −0.3 V (versus silver pseudo-reference electrode). Insulin can be quantified by this method in the 1.0 fM to 10.0 pM concentration range, with a 0.18 fM limit of detection (at 3σ/slope). The assay was applied to the determination of insulin in spiked human serum samples. The method is highly sensitive, selective, stable, and has a wide analytical range.

The surface of a screen printed electrode was modified with ordered mesoporous carbon-1,3,6,8-pyrenetetrasulfonate. The amino-terminated aptamer was then linked to the 1,3,6,8-pyrenetetrasulfonate. Then, the Methylene Blue was interacted into the aptamer. The modified electrode was applied to the determination of insulin.

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

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


  1. 1.

    Shabanpoor F, Separovic F, Wade JD (2009) Chapter 1 the human insulin superfamily of polypeptide hormones. In: Vitamins & Hormones, vol Volume 80. Academic Press, pp 1–31

  2. 2.

    Pagliuca FW, Millman JR, Gürtler M, Segel M, Van Dervort A, Ryu JH, Peterson QP, Greiner D, Melton DA Generation of functional human pancreatic β cells in vitro. Cell 159:428–439

  3. 3.

    Iwase H, Kobayashi M, Nakajima M, Takatori T The ratio of insulin to C-peptide can be used to make a forensic diagnosis of exogenous insulin overdosage. Forensic Sci Int 115:123–127

  4. 4.

    Khaksa G, Nalini K, Bhat M, Udupa N (1998) High-performance liquid chromatographic determination of insulin in rat and human plasma. Anal Biochem 260:92–95

    CAS  Article  Google Scholar 

  5. 5.

    Ho ENM, Wan TSM, Wong ASY, Lam KKH, Stewart BD (2011) Doping control analysis of insulin and its analogues in equine urine by liquid chromatography–tandem mass spectrometry. J Chromatogr A 1218:1139–1146

    CAS  Article  Google Scholar 

  6. 6.

    Ortner K, Buchberger W, Himmelsbach M (2009) Capillary electrokinetic chromatography of insulin and related synthetic analogues. J Chromatogr A 1216:2953–2957

    CAS  Article  Google Scholar 

  7. 7.

    Gobi KV, Iwasaka H, Miura N (2007) Self-assembled PEG monolayer based SPR immunosensor for label-free detection of insulin. Biosens Bioelectron 22:1382–1389

    CAS  Article  Google Scholar 

  8. 8.

    Wang Y, Gao D, Zhang P, Gong P, Chen C, Gao G, Cai L (2014) A near infrared fluorescence resonance energy transfer based aptamer biosensor for insulin detection in human plasma. Chem Commun 50:811–813

    CAS  Article  Google Scholar 

  9. 9.

    Salimi A, Hallaj R (2012) Cobalt oxide nanostructure-modified glassy carbon electrode as a highly sensitive flow injection amperometric sensor for the picomolar detection of insulin. J Solid State Chem 16:1239–1246

    CAS  Google Scholar 

  10. 10.

    Amouzadeh Tabrizi M, Shamsipur M, Mostafaie A (2016) A high sensitive label-free immunosensor for the determination of human serum IgG using overoxidized polypyrrole decorated with gold nanoparticle modified electrode. Mater Sci Eng C 59:965–969

    CAS  Article  Google Scholar 

  11. 11.

    Shao F, Jiao L, Wei Q, Li H (2017) Ternary Pt-co-cu nanodendrites for ultrasensitive voltammetric determination of insulin at very low working potential. Microchim Acta 184:2031–2038

    CAS  Article  Google Scholar 

  12. 12.

    Amouzadeh Tabrizi M, Shamsipur M, Saber R, Sarkar S, Ebrahimi V (2017) A high sensitive visible light-driven photoelectrochemical aptasensor for shrimp allergen tropomyosin detection using graphitic carbon nitride-TiO2 nanocomposite. Biosens Bioelectron 98:113–118

    CAS  Article  Google Scholar 

  13. 13.

    Jiang W, Tian D, Zhang L, Guo Q, Cui Y, Yang M (2017) Dual signal amplification strategy for amperometric aptasensing using hydroxyapatite nanoparticles. Application to the sensitive detection of the cancer biomarker platelet-derived growth factor BB. Microchim Acta 184:4375–4381

    CAS  Article  Google Scholar 

  14. 14.

    Huang J, Luo X, Lee I, Hu Y, Cui XT, Yun M (2011) Rapid real-time electrical detection of proteins using single conducting polymer nanowire-based microfluidic aptasensor. Biosens Bioelectron 30:306–309

    CAS  Article  Google Scholar 

  15. 15.

    Xin Y, Li Z, Zhang Z (2015) Photoelectrochemical aptasensor for the sensitive and selective detection of kanamycin based on au nanoparticle functionalized self-doped TiO2 nanotube arrays. Chem Commun 51:15498–15501

    CAS  Article  Google Scholar 

  16. 16.

    Shamsipur M, Farzin L, Amouzadeh Tabrizi M, Molaabasi F (2015) Highly sensitive label free electrochemical detection of VGEF165 tumor marker based on “signal off” and “signal on” strategies using an anti-VEGF165 aptamer immobilized BSA-gold nanoclusters/ionic liquid/glassy carbon electrode. Biosens Bioelectron 74:369–375

    CAS  Article  Google Scholar 

  17. 17.

    Amouzadeh Tabrizi M, Shamsipur M, Farzin L (2015) A high sensitive electrochemical aptasensor for the determination of VEGF165 in serum of lung cancer patient. Biosens Bioelectron 74:764–769

    CAS  Article  Google Scholar 

  18. 18.

    Yuan DS, Zeng J, Chen J, Liu Y (2009) Highly ordered mesoporous carbon synthesized via in situ template for supercapacitors. Int J Electrochem Sci 4:562–570

    CAS  Google Scholar 

  19. 19.

    Alsharaeh E, Ahmed F, Aldawsari Y, Khasawneh M, Abuhimd H, Alshahrani M (2016) Novel synthesis of holey reduced graphene oxide (HRGO) by microwave irradiation method for anode in lithium-ion batteries. Sci Rep 6:29854–29867

    CAS  Article  Google Scholar 

  20. 20.

    Yang Y, Ge L, Rudolph V, Zhu Z (2014) In situ synthesis of zeolitic imidazolate frameworks/carbon nanotube composites with enhanced CO2 adsorption. Dalton Trans 43:7028–7036

    CAS  Article  Google Scholar 

  21. 21.

    Amouzadeh Tabrizi M, Shamsipur M, Saber R, Sarkar S, Zolfaghari N (2017) An ultrasensitive sandwich-type electrochemical immunosensor for the determination of SKBR-3 breast cancer cell using rGO-TPS/FeHCFnano labeled anti-HCT as a signal tag. Sensors Actuators B Chem 243:823–830

    CAS  Article  Google Scholar 

  22. 22.

    Zhang F-T, Nie J, Zhang D-W, Chen J-T, Zhou Y-L, Zhang X-X (2014) Methylene blue as a G-Quadruplex binding probe for label-free homogeneous electrochemical biosensing. Anal Chem 86:9489–9495

    CAS  Article  Google Scholar 

  23. 23.

    Pan D, Zuo X, Wan Y, Wang L, Zhang J, Song S, Fan C (2007) Electrochemical interrogation of interactions between surface-confined DNA and methylene blue. Sensors 7:2671–2680

    CAS  Article  Google Scholar 

  24. 24.

    Jun S, Joo SH, Ryoo R, Kruk M, Jaroniec M, Liu Z, Ohsuna T, Terasaki O (2000) Synthesis of new, Nanoporous carbon with hexagonally ordered Mesostructure. J Am Chem Soc 122:10712–10713

    CAS  Article  Google Scholar 

  25. 25.

    Chen J, Zhang J, Wang K, Lin X, Huang L, Chen G (2008) Electrochemical biosensor for detection of BCR/ABL fusion gene using locked nucleic acids on 4-Aminobenzenesulfonic acid-modified glassy carbon electrode. Anal Chem 80:8028–8034

    CAS  Article  Google Scholar 

  26. 26.

    Zhuang H-S, Huang J-L, Chen G-N (2004) Synthesis of a new biacridine and its use as the chemiluminescent probe for immunoassay of carcinoembryonic antigen. Anal Chim Acta 512:347–353

    CAS  Article  Google Scholar 

  27. 27.

    Hu Y, Hua S, Li F, Jiang Y, Bai X, Li D, Niu L (2011) Green-synthesized gold nanoparticles decorated graphene sheets for label-free electrochemical impedance DNA hybridization biosensing. Biosens Bioelectron 26:4355–4361

    CAS  Article  Google Scholar 

  28. 28.

    Wang D-W, Li F, Fang H-T, Liu M, G-Q L, Cheng H-M (2006) Effect of pore packing defects in 2-D ordered mesoporous carbons on ionic transport. J Phys Chem B 110:8570–8575

    CAS  Article  Google Scholar 

  29. 29.

    Qiao Y, Bao S-J, Li CM (2010) Electrocatalysis in microbial fuel cells-from electrode material to direct electrochemistry. Energy Environ Sci 3:544–553

    CAS  Article  Google Scholar 

  30. 30.

    Wang L, Bai J, Bo X, Zhang X, Guo L (2011) A novel glucose sensor based on ordered mesoporous carbon–au nanoparticles nanocomposites. Talanta 83:1386–1391

    CAS  Article  Google Scholar 

  31. 31.

    Laviron E (1979) General expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systems. J Electroanal Chem Interfacial Electrochem 101:19–28

    CAS  Article  Google Scholar 

  32. 32.

    A S, VB G (2011) Methods for the determination of limit of detection and limit of quantitation of the analytical methods. Chron Young Sci 2:21–25

    Article  Google Scholar 

Download references

Author information



Corresponding authors

Correspondence to Mahmoud Amouzadeh Tabrizi or Mojtaba Shamsipur.

Ethics declarations

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

Electronic supplementary material


(DOCX 178 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Amouzadeh Tabrizi, M., Shamsipur, M., Saber, R. et al. An electrochemical aptamer-based assay for femtomolar determination of insulin using a screen printed electrode modified with mesoporous carbon and 1,3,6,8-pyrenetetrasulfonate. Microchim Acta 185, 59 (2018).

Download citation


  • Insulin
  • Screen printed electrode
  • Modified electrode
  • Ordered mesoporous carbon
  • 1,3,6,8-Pyrenetetrasulfonate
  • Differential pulse voltammetry
  • Methylene Blue