Microchimica Acta

, 186:771 | Cite as

A ratiometric electrochemiluminescent immunoassay for calcitonin by using N-(aminobutyl)-N-(ethylisoluminol) and graphite-like carbon nitride

  • Cong Zhang
  • Di Liu
  • Han Zhang
  • Xingrong Tan
  • Shihong ChenEmail author
Original Paper


A ratiometric electrochemiluminescent (ECL) assay is described for the determination of the calcium(II) regulator calcitonin (CT). The method is making use of (a) graphite-like carbon nitride (g-C3N4) as the cathodic luminophore, (b) N-(aminobutyl)-N-(ethylisoluminol) (ABEI) as the anodic luminophore, and (c) peroxodisulfate and dissolved oxygen as coreactants. The luminous potential of g-C3N4 and ABEI can be well distinguished because of their different luminescent properties. Energy transfer between g-C3N4 and ABEI is not observed, and the coreactants peroxodisulate and oxygen do not interfere with each other. Au nanoparticles were functionalized with g-C3N4 and placed on the electrode to serve as a matrix for immobilization of primary antibody (Ab1). In the presence of CT, it will bind to the electrode. Then secondary antibody (Ab2) modified with polyaniline (PANI) and ABEI is incubated onto the electrode. With the increase in the concentration of CT, the blue ECL of g-C3N4 is quenched by PANI, while the blue luminescence of ABEI is enhanced. This enables ratiometric detection of calcitonin by ratioing the internsities at 460 and 475 nm. Response is linear in the 0.1~40 pg·mL−1 CT concentration range, and the limit of detection is 23 fg·mL−1. The method breaks the limitation of common ECL ratiometric strategy, namely, two luminophores often share the common coreactant.

Graphical abstract

Schematic representation of an immunoassay where polyaniline (PANI) in a BSA-Ab2-ABEI-Au@PANI composite quenches the cathodic signal of a graphitic carbon nitride (Au-g-C3N4) modified with gold nanoparticles (Au), while N-(aminobutyl)-N-(ethylisolumino) (ABEI) in the BSA-Ab2-ABEI-Au@PANI composit produces an anodic signal that enables quantitation of calcitonin.


Electrochemiluminescence G-C3N4 Polyaniline Au nanoparticles Peroxodisulfate Dissolved oxygen Glassy carbon electrode Medullary thyroid carcinoma Ratiometric assay 



This work was supported by National Natural Science Foundation of China (21775122, 21775123, 51473136, 21575116, 21705115), and Nature Science Foundation of Chongqing City (cstc2018jcyjAX0693) China.

Compliance with ethical standards

Conflict of interest

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

Supplementary material

604_2019_3934_MOESM1_ESM.docx (403 kb)
ESM 1 (DOCX 402 kb)


  1. 1.
    Naot D, Musson DS, Cornish J (2019) The activity of peptides of the calcitonin family in bone. Physiol Rev 99:781–805CrossRefGoogle Scholar
  2. 2.
    Rigoldi F, Metrangolo P, Redaelli A, Gautieri A (2017) Nanostructure and stability of calcitonin amyloids. J Biol Chem 292:7348–7357CrossRefGoogle Scholar
  3. 3.
    Dopp DH, Cheney B (1962) Calcitonin–a hormone from the parathyroid which lowers the calcium-level of the blood. Nature 193:381–382CrossRefGoogle Scholar
  4. 4.
    Cao FH, Gamble AB, Onagi H, Howes J, Hennessy JE, Gu C, Morgan JAM, Easton CJ (2017) Detection of biosynthetic precursors, discovery of glycosylated forms, and homeostasis of calcitonin in human cancer cells. Anal Chem 89:6992–6999CrossRefGoogle Scholar
  5. 5.
    Ou GP, Chen QL, Liu WL, Li XH, Chen SL, Wu XP, Chen H (2018) Study on the degradation rule of calcitonin in vitro in patients with medullary thyroid carcinoma. Biochem Biophys Res Commun 507:106–109CrossRefGoogle Scholar
  6. 6.
    Lu XW, Wu Y, Zhang DH (2016) The application value of combined detection of serum lactic acid, calcitonin and C reactive protein in the diagnosis of senile pneumonia. Chin J Pharm Econ 12:74Google Scholar
  7. 7.
    Snider RH, Moore CF, Silva OL, Becker KL (1978) Radioimmunoassay of calcitonin in normal human urine. Anal Chem 50:449–454CrossRefGoogle Scholar
  8. 8.
    TAS C, Mansoor S, Banga AK, Prausnitz MR (2012) Development and validation of a rapid isocratic rp-HPLC method for the quantification of salmon calcitonin. Turk J Pharm Sci 9:323–334Google Scholar
  9. 9.
    Sha DS, Zhuge BZ, Lin F (2017) Detection of calcitonin in medullary thyroid carcinoma by an electrochemical sensor. Int J Electrochem Sci 12:10129–10139CrossRefGoogle Scholar
  10. 10.
    Alarfaj NA, El-Tohamy MF (2017) A label-free electrochemical immunosensor based on gold nanoparticles and graphene oxide for the detection of tumor marker calcitonin. New J Chem 41:11029–11035CrossRefGoogle Scholar
  11. 11.
    Zhao J, Lei YM, Chai YQ, Yuan R, Zhuo Y (2016) Novel electrochemiluminescence of perylene derivative and its application to mercury ion detection based on a dual amplification strategy. Biosens Bioelectron 86:720–727CrossRefGoogle Scholar
  12. 12.
    Zuo FM, Zhang H, Xie J, Chen SH, Yuan R (2018) A sensitive ratiometric electrochemiluminescence biosensor for hypoxanthine detection by in situ generation and consumption of coreactants. Electrochim Acta 271:173–179CrossRefGoogle Scholar
  13. 13.
    Wang HM, Fang Y, Yuan PX, Wang AJ, Luo XL, Feng JJ (2019) Construction of ultrasensitive label-free aptasensor for thrombin detection using palladium nanocones boosted electrochemiluminescence system. Electrochim Acta 310:195–202CrossRefGoogle Scholar
  14. 14.
    Zhang TT, Zhao HM, Fan GF, Li YX, Li L, Quan X (2016) Electrolytic exfoliation synthesis of boron doped graphene quantum dots: a new luminescent material for electrochemiluminescence detection of oncogene microRNA-20a. Electrochim Acta 190:1150–1158CrossRefGoogle Scholar
  15. 15.
    Wang YZ, Zhao W, Dai PP, Lu HJ, Xu JJ, Pan J, Chen HY (2016) Spatial-resolved electrochemiluminescence ratiometry based on bipolar electrode for bioanalysis. Biosens Bioelectron 86:683–689CrossRefGoogle Scholar
  16. 16.
    Zhang HR, Xu JJ, Chen HY (2013) Electrochemiluminescence ratiometry: a new approach to DNA biosensing. Anal Chem 85:5321–5325CrossRefGoogle Scholar
  17. 17.
    Lu HJ, Zhao W, Xu JJ, Chen HY (2018) Visual electrochemiluminescence ratiometry on bipolar electrode for bioanalysis. Biosens Bioelectron 102:624–630CrossRefGoogle Scholar
  18. 18.
    Ong WJ, Tan LL, Ng YH, Yong ST, Chai SP (2016) Graphitic carbon nitride (g-C3N4)-based photocatalysts for artificial photosynthesis and environmental remediation: are we a step closer to achieving sustainability? Chem Rev 116:7159–7329CrossRefGoogle Scholar
  19. 19.
    Yuan F, Gu TT, Li XQ, Wang GL (2016) Split photoelectrochemistry for the immunoassay of α-fetoprotein based on graphitic carbon nitride. J Electroanal Chem 783:226–232CrossRefGoogle Scholar
  20. 20.
    Cao SH, Chen H, Jiang F, Hu ZX, Wang X (2018) Construction of acetaldehyde-modified g-C3N4 ultrathin nanosheets via ethylene glycol-assisted liquid exfoliation for selective fluorescence sensing of Ag+. ACS Appl Mater Interfaces 10:44624–44633CrossRefGoogle Scholar
  21. 21.
    Chen WL, Yao X, Zhou XC, Zhao K, Deng AP, Li JG (2018) Electrochemiluminescence based competitive immunoassay for Sudan I by using gold-functionalized graphitic carbon nitride and au/cu alloy nanoflowers. Microchim Acta 185(5):275CrossRefGoogle Scholar
  22. 22.
    Xiao YT, Tian GH, Li W, Xie Y, Jiang BJ, Tian CG, Zhao DY, Fu HG (2019) Molecule self-assembly synthesis of porous few-layer carbon nitride for highly efficient photoredox catalysis. J Am Chem Soc 141:2508–2515CrossRefGoogle Scholar
  23. 23.
    Tian JQ, Liu Q, Asiri AM, Al-Youbi AO, Sun XP (2013) Ultrathin graphitic carbon nitride nanosheet: a highly efficient fluorosensor for rapid, ultrasensitive detection of Cu2+. Anal Chem 85:5595–5599CrossRefGoogle Scholar
  24. 24.
    Cheng CM, Huang Y, Tian XQ, Zheng BZ, Li Y, Yuan HY, Xiao D, Xie SP, Choi MMF (2012) Electrogenerated chemiluminescence behavior of graphite-like carbon nitride and its application in selective sensing Cu2+. Anal Chem 84:4754–4759CrossRefGoogle Scholar
  25. 25.
    Feng QM, Shen YZ, Li MX, Zhang ZL, Zhao W, Xu JJ, Chen HY (2016) Dual-wavelength electrochemiluminescence ratiometry based on resonance energy transfer between au nanoparticles functionalized g-C3N4 nanosheet and Ru(bpy)32+ for microRNA detection. Anal Chem 88:937–944CrossRefGoogle Scholar
  26. 26.
    Liu D, Hu FX, Zhang H, Zhang C, Chen SH (2019) ECL biosensor for sensitive detection of soybean agglutinin based on AuPt@C60 nanoflowers enhanced N-(aminobutyl)-N-(ethylisoluminol). J Electrochem Soc 166:B49–B55CrossRefGoogle Scholar
  27. 27.
    Zhang H, Zhang C, Liu D, Zuo FM, Chen SH, Yuan R, Xu WJ (2018) A ratiometric electrochemiluminescent biosensor for con a detecting based on competition of dissolved oxygen. Biosens Bioelectron 120:40–46CrossRefGoogle Scholar
  28. 28.
    Lu QY, Zhang JJ, Liu XF, Wu YY, Yuan R, Chen SH (2014) Enhanced electrochemiluminescence sensor for detecting dopamine based on gold nanoflower@graphitic carbon nitride polymer nanosheet–polyaniline hybrids. Analyst 139:6556–6562CrossRefGoogle Scholar
  29. 29.
    Chen LC, Zeng XT, Si P, Chen YM, Chi YW, Kim DH, Chen GN (2014) Gold nanoparticle-graphite-like C3N4 nanosheet nanohybrids used for electrochemiluminescent immunosensor. Anal Chem 86:4188–4195CrossRefGoogle Scholar
  30. 30.
    Ye F, Zhao BT, Ran R, Shao ZP (2015) A polyaniline-coated mechanochemically synthesized tin oxide/graphene nanocomposite for high-power and high-energy lithium-ion batteries. J Power Sources 290:61–70CrossRefGoogle Scholar
  31. 31.
    Fan Y, Tan XR, Ou X, Lu QY, Chen SH, Wei SP (2016) A novel “on-off” electrochemiluminescence sensor for the detection of concanavalin a based on Ag-doped g-C3N4. Electrochim Acta 202:90–99CrossRefGoogle Scholar
  32. 32.
    Fan Y, Tan XR, Liu XF, Ou X, Chen SH, Wei SP (2015) A novel non-enzymatic electrochemiluminescence sensor for the detection of glucose based on the competitive reaction between glucose and phenoxy dextran for concanavalin a binding sites. Electrochim Acta 180:471–478CrossRefGoogle Scholar
  33. 33.
    Koçdan D, Basan H (2013) A novel spectrofluorimetric method for the determination of calcitonin in ampules through derivatization with fluorescamine. Luminescence 28:961–966CrossRefGoogle Scholar
  34. 34.
    Shah RB, Siddiqui A, Shah G (2003) A validated HPLC assay for simultaneous analysis of salmon calcitonin and duck ovomucoid. Pharmazie 58:620–622PubMedGoogle Scholar
  35. 35.
    Basuyau JP, Mallet E, Leroy M, Brunelle P (2004) Reference intervals for serum calcitonin in men, women, and children. Clin Chem 50:1828–1830CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Cong Zhang
    • 1
  • Di Liu
    • 1
  • Han Zhang
    • 1
  • Xingrong Tan
    • 2
  • Shihong Chen
    • 1
    Email author
  1. 1.Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, College of Chemistry and Chemical EngineeringSouthwest UniversityChongqingChina
  2. 2.Department of Endocrinology9th People’s Hospital of ChongqingChongqingPeople’s Republic of China

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