Microchimica Acta

, Volume 182, Issue 11–12, pp 2045–2053

Aptamer-antibody sandwich assay for cytochrome c employing an MWCNT platform and electrochemical impedance

Original Paper

Abstract

We report on a sensitive aptamer-antibody interaction-based assay for cytochrome c (Cyt c) using electrochemical impedance. 4-Amino benzoic acid is used for the oriented immobilization of aminated aptamers onto multi-walled carbon nanotubes on the surface of a screen-printed electrode via electrochemical grafting. Impedance was measured in a solution containing the redox system ferro/ferricyanide. The change in interfacial charge transfer resistance (Rct) experienced by the redox marker was recorded to confirm the formation of a complex between aptamer and the target (Cyt c). A biotinylated antibody against cytochrome c was then used in a sandwich type of assay. The addition of streptavidin conjugated to gold nanoparticles and signal enhancement by treatment with silver led to a further increase in Rct. Under optimized conditions, a detection limit as low as 12 pM was obtained. Cross-reactivity against other serum proteins including fibrinogen, BSA and immunoglobulin G demonstrated improved selectivity.

Graphical Abstract

Sensitive and selective assay for cytochrome c protein using aptamer linked to multi-walled carbon nanotube screen printed electrode via diazonium electrochemical grafting and specific biotinylated antibody to improve selectivity. Detection can be based on electrochemical impedance spectroscopy, or using a streptavidin-gold nanoparticle conjugate.

Keywords

Aptamer Sandwich Cytochrome c SEM Gold-nanoparticles electrochemical impedance spectroscopy 

Supplementary material

604_2015_1540_MOESM1_ESM.doc (78 kb)
ESM 1(DOC 78 kb)

References

  1. 1.
    Alleyne T, Joseph J, Sampson V (2001) Cytochrome-c detection: a diagnostic marker for myocardial infarction. Appl Biochem Biotechnol 90(2):97–105CrossRefGoogle Scholar
  2. 2.
    Clark SL, Remcho VT (2002) Aptamers as analytical reagents. Electrophoresis 23(9):1335–1340CrossRefGoogle Scholar
  3. 3.
    Radi A, Acero Sánchez JL, Baldrich E, O’Sullivan CK (2005) Reusable impedimetric aptasensor. Anal Chem 77(19):6320–6323CrossRefGoogle Scholar
  4. 4.
    Biesecker G, Dihel L, Enney K, Bendele RA (1999) Derivation of RNA aptamer inhibitors of human complement C5. Immunopharmacology 42(1–3):219–230CrossRefGoogle Scholar
  5. 5.
    Hicke BJ, Marion C, Chang YF, Gould T, Lynott CK, Parma D, Schmidt PG, Warren S (2001) Tenascin-C aptamers are generated using tumor cells and purified protein. J Biol Chem 276(52):48644–48654CrossRefGoogle Scholar
  6. 6.
    Cox JC, Ellington AD (2001) Automated selection of anti-protein aptamers. Bioorg Med Chem 9(10):2525–2531CrossRefGoogle Scholar
  7. 7.
    Cai H, Lee TM-H, Hsing IM (2006) Label-free protein recognition using an aptamer-based impedance measurement assay. Sensors Actuators B Chem 114(1):433–437CrossRefGoogle Scholar
  8. 8.
    Jayasena SD (1999) Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clin Chem 45(9):1628–1650Google Scholar
  9. 9.
    Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346(6287):818–822CrossRefGoogle Scholar
  10. 10.
    Tuerk C, Gold L (1990) Systemic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249(4968):505–510CrossRefGoogle Scholar
  11. 11.
    Tombelli S, Minunni M, Mascini M (2005) Analytical applications of aptamers. Biosens Bioelectron 20(12):2424–2434CrossRefGoogle Scholar
  12. 12.
    Zhang L, Cui P, Zhang B, Gao F (2013) Aptamer-based turn-on detection of thrombin in biological fluids based on efficient phosphorescence energy transfer from Mn-doped ZnS quantum dots to carbon nanodots. Chem Eur J 19(28):9242–9250CrossRefGoogle Scholar
  13. 13.
    Chang M, Kwon M, Kim S, Yunn NO, Kim D, Ryu SH, Lee JB (2014) Aptamer-based single-molecule imaging of insulin receptors in living cells. J Biomed Opt 19(5):051204CrossRefGoogle Scholar
  14. 14.
    Loo AH, Bonanni A, Pumera M (2012) Impedimetric thrombin aptasensor based on chemically modified graphenes. Nanoscale 4(1):143–147CrossRefGoogle Scholar
  15. 15.
    Ho MY, D’Souza N, Migliorato P (2012) Electrochemical aptamer-based sandwich assays for the detection of explosives. Anal Chem 84(10):4245–4247CrossRefGoogle Scholar
  16. 16.
    Lei P, Tang H, Ding S, Ding X, Zhu D, Shen B, Chang Q, Yan Y (2015) Determination of the invA gene of Salmonella using surface plasmon resonance along with streptavidin aptamer amplification. Microchim Acta 182:289–296CrossRefGoogle Scholar
  17. 17.
    Shen B, Li J, Cheng W, Yan Y, Tang R, Li Y, Ju H, Ding S (2015) Electrochemical aptasensor for highly sensitive determination of cocaine using a supramolecular aptamer and rolling circle amplification. Microchim Acta 182:361–367CrossRefGoogle Scholar
  18. 18.
    McDonald JR (1987) Impedance spectroscopy. Wiley, New YorkGoogle Scholar
  19. 19.
    Bonanni A, Ambrosi A, Pumera M (2012) On oxygen-containing groups in chemically modified graphenes. Chem-Eur J 18(15):4541–4548CrossRefGoogle Scholar
  20. 20.
    Bonanni A, Ambrosi A, Pumera M (2012) Nucleic acid functionalized graphene for biosensing. Chem-Eur J 18(6):1668–1673CrossRefGoogle Scholar
  21. 21.
    Ocaña C, del Valle M (2014) A comparison of four protocols for the immobilization of an aptamer on graphite composite electrode. Microchim Acta 181:355–363CrossRefGoogle Scholar
  22. 22.
    Bardea A, Patolsky F, Dagan A, Willner I (1999) Sensing and amplification of oligonucleotide-DNA interactions by means of impedance spectroscopy: a route to a Tay-Sachs sensor. Chem Comm 1:21–22CrossRefGoogle Scholar
  23. 23.
    Loo AH, Bonanni A, Ambrosi A, Poh HL, Pumera M (2012) Impedimetric immunoglobulin G immunosensor based on chemically modified graphenes. Nanoscale 4(3):921–925CrossRefGoogle Scholar
  24. 24.
    Moreno-Guzmán M, Ojeda I, Villalonga R, González-Cortés A, Yáñez-Sedeño P, Pingarrón JM (2012) Ultrasensitive detection of adrenocorticotropin hormone (ACTH) using disposable phenylboronic-modified electrochemical immunosensors. Biosens Bioelectron 35(1):82–86CrossRefGoogle Scholar
  25. 25.
    Zhang Y, Li Y, Wu W, Jiang Y, Hu B (2014) Chitosan coated on the layers’ glucose oxidase immobilized on cysteamine/Au electrode for use as glucose biosensor. Biosens Bioelectron 60:271–276CrossRefGoogle Scholar
  26. 26.
    Bonanni A, Esplandiu MJ, del Valle M (2010) Impedimetric genosensing of DNA polymorphism correlated to cystic fibrosis: a comparison among different protocols and electrode surfaces. Biosens Bioelectron 26(4):1245–1251CrossRefGoogle Scholar
  27. 27.
    Bonanni A, Pumera M, Miyahara Y (2010) Rapid, sensitive, and label-free impedimetric detection of a single-nucleotide polymorphism correlated to kidney disease. Anal Chem 82(9):3772–3779CrossRefGoogle Scholar
  28. 28.
    Blin F, Koutsoukos P, Klepetsianis P, Forsyth M (2007) The corrosion inhibition mechanism of new rare earth cinnamate compounds—electrochemical studies. Electrochim Acta 52(21):6212–6220CrossRefGoogle Scholar
  29. 29.
    Liao Y-M, Feng Z-D, Chen Z-L (2007) In situ tracing the process of human enamel demineralization by electrochemical impedance spectroscopy (EIS). J Dent 35(5):425–430CrossRefGoogle Scholar
  30. 30.
    Bonanni A, del Valle M (2010) Use of nanomaterials for impedimetric DNA sensors: a review. Anal Chim Acta 678(1):7–17CrossRefGoogle Scholar
  31. 31.
    Deng C, Chen J, Nie Z, Wang M, Chu X, Chen X, Xiao X, Lei C, Yao S (2008) Impedimetric aptasensor with femtomolar sensitivity based on the enlargement of surface-charged gold nanoparticles. Anal Chem 81(2):739–745CrossRefGoogle Scholar
  32. 32.
    Zheng J, Feng W, Lin L, Zhang F, Cheng G, He P, Fang Y (2007) A new amplification strategy for ultrasensitive electrochemical aptasensor with network-like thiocyanuric acid/gold nanoparticles. Biosens Bioelectron 23(3):341–347CrossRefGoogle Scholar
  33. 33.
    Bonanni A, Esplandiu MJ, del Valle M (2008) Signal amplification for impedimetric genosensing using gold-streptavidin nanoparticles. Electrochim Acta 53(11):4022–4029CrossRefGoogle Scholar
  34. 34.
    Bonanni A, Esplandiu MJ, Pividori MI, Alegret S, del Valle M (2006) Impedimetric genosensors for the detection of DNA hybridization. Anal Bioanal Chem 385(7):1195–1201CrossRefGoogle Scholar
  35. 35.
    Cai H, Wang YQ, He PG, Fang YH (2002) Electrochemical detection of DNA hybridization based on silver-enhanced gold nanoparticle label. Anal Chim Acta 469(2):165–172CrossRefGoogle Scholar
  36. 36.
    Hanaee H, Ghourchian H, Ziaee AA (2007) Nanoparticle-based electrochemical detection of hepatitis B virus using stripping chronopotentiometry. Anal Biochem 370(2):195–200CrossRefGoogle Scholar
  37. 37.
    Loo FC, Ng SP, Wu C-ML, Kong SK (2014) An aptasensor using DNA aptamer and white light common-path SPR spectral interferometry to detect cytochrome-c for anti-cancer drug screening. Sensors Actuators B: Chem 198:416–423CrossRefGoogle Scholar
  38. 38.
    Lau IPM, Ngan EKS, Loo JFC, Suen YK, Ho HP, Kong SK (2010) Aptamer-based bio-barcode assay for the detection of cytochrome-c released from apoptotic cells. Biochem Biophys Res Commun 395:560–564CrossRefGoogle Scholar
  39. 39.
    Liu JM, Yan XP (2011) Ultrasensitive, selective and simultaneous detection of cytochrome c and insulin based on immunoassay and aptamer-based bioassay in combination with Au/Ag nanoparticle tagging and ICP-MS detection. J Anal At Spectrom 26:1191–1197CrossRefGoogle Scholar
  40. 40.
    Ocaña C, Arcay E, Del Valle M (2014) Label-free impedimetric aptasensor based on epoxy-graphite electrode for the recognition of cytochrome c. Sensors Actuators B Chem 191:860–865CrossRefGoogle Scholar
  41. 41.
    Pandiaraj M, Benjamin AR, Madasamy T, Vairamani K, Arya A, Sethy NK (2014) A cost-effective volume miniaturized and microcontroller based cytochrome c assay. Sensors Actuators A Phys 220:290–297CrossRefGoogle Scholar
  42. 42.
    Pandiaraj M, Madasamy T, Gollavilli PN, Balamurugan M, Kotamraju S, Rao (2013) Nanomaterial-based electrochemical biosensors for cytochrome c using cytochrome c reductase. Bioelectrochemistry 91:1–7CrossRefGoogle Scholar
  43. 43.
    Sakaida I, Kimura T, Yamasaki T, Fukumoto Y, Watanabe K, Aoyama M, Okita K (2005) Cytochrome c is a possible new marker for fulminant hepatitis in humans. J Gastroenterol 40(2):179–185CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2015

Authors and Affiliations

  • Cristina Ocaña
    • 1
  • Sonja Lukic
    • 2
  • Manel del Valle
    • 1
  1. 1.Sensors and Biosensors Group, Department of ChemistryUniversitat Autònoma de BarcelonaBarcelonaSpain
  2. 2.Institute of Analytical Chemistry, Chemo- and BiosensorsUniversity of RegensburgRegensburgGermany

Personalised recommendations