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Development of an Impedimetric Immunosensor for Specific Detection of Snake Venom

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Abstract

Serotherapy is the only approved method to treat victims of snakebites; therefore, it is very important to identify the type of venom implicated in the accident to ensure the most specific antivenom is administered to the patient. In Brazil, the majority of snakebite accidents are from snakes of Bothrops genus. In this work, we developed an immunosensor capable to recognize specifically venoms from Bothrops snakes based on the electrochemical impedance spectroscopy technique. For this, Crofer 22 APU steel was used as transducer substrate functionalized with antibothropic antibodies. The immunosensor was incubated with different concentrations of venoms from Bothrops, Crotalus, and Micrurus in order to evaluate its specificity. The formation of the antigen-antibody immunocomplex at the surface of the transducer substrate increased the leakage resistance in a concentration-dependent manner when the device was exposed to the bothropic venom, while no considerable variation of this parameter could be observed for the venoms of the heterologous genera. In addition to the observed specificity, the sensor proved reusable, since the immersion in an elution buffer permitted many regeneration and charge cycles without considerable loss of activity. The results strongly indicated that the developed immunosensor stands as a promising device to aid in snakebites diagnosis.

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References

  1. Kasturiratne, A., Wickremasinghe, A. R., Silva, N., Gunawardena, N. K., Pathmeswaran, A., Premaratna, R., Savioli, L., Lalloo, D. G., & Silva, H. J. (2008). The global burden of snakebite: a literature analysis and modelling based on regional estimates of envenoming and deaths. PLoS Medicine, 5(11). https://doi.org/10.1371/journal.pmed.0050218.

    Article  Google Scholar 

  2. Williams, D., Gutiérrez, J. M., Harrison, R., Warrel, D. A., White, J., Winkel, K. D., & Gopalakrishnakone, P. (2010). The Global Snake Bite Initiative: an antidote for snake bite. Lancet, 375, 89–91. https://doi.org/10.1016/S0140-6736(09)61159-4.

    Article  Google Scholar 

  3. FUNED, Fundação Ezequiel Dias (2014) Cartilha Animais peçonhentos. www.funed.mg.gov.br/wp-content/uploads/2010/03/cartilha.pdf. Accessed 15 December 2017.

  4. Warrel, D. A. (2010). Guidelines for the management of snake-bites. WHO Library Cataloguing-in-Publication data. http://www.who.int/snakebites/resources/9789290223774/en/. Acessed 20 Feb 2018.

  5. Hifumi, T., Sakai, A., Kondo, Y., Yamamoto, A., Morine, N., Ato, M., Shibayama, K., Umezawa, K., Kiriu, N., Kato, H., Koido, Y., Inoue, J., Kawakita, K., & Kuroda, Y. (2015). Venomous snake bites: clinical diagnosis and treatment. Journal of Intensive Care, 3, 16. https://doi.org/10.1186/s40560-015-0081-8.

    Article  Google Scholar 

  6. WHO (2018). Expert Committee on Biological Standardization: sixty-eighth report. Geneva: World Health Organization (WHO Technical Report Series, No. 1011). Licence: CC BY-NC-SA 3.0 IGO.

  7. Makwana, H. D., Thakor, A. V., & Madaria, M. (2015). A case report: a very rarely occurring snakebite. Int J Res Med Sci, 3(11), 3435–3439. https://doi.org/10.18203/2320-6012.ijrms20151208.

    Article  Google Scholar 

  8. Theakston, E. D. G., & Laing, G. D. (2014). Diagnosis of snakebite and the importance of immunological tests in venom research. Toxins, 6, 1667–1695. https://doi.org/10.3390/toxins6051667.

    Article  Google Scholar 

  9. Cockrell, M., Swanson, K., Sanders, A., Prater, S., Wenckstern, T., & Mick, J. (2017). Safe handling of snakes in an ED setting. Journal of Emergency Nursing, 43(1), 21–23. https://doi.org/10.1016/j.jen.2016.07.009.

    Article  Google Scholar 

  10. Heneine, L. G. D., Catty, D., & Theakston, R. (1990). Development of a species-specific ELISA for Brazilian pit-viper venoms. Brazilian Journal of Medical and Biological Research, 23, 585–588.

    Google Scholar 

  11. Heneine, L. G. D., & Catty, D. (1993). Species-specific detection of venom antigens from snakes of the Bothrops and Lachesis genera. Toxicon, 31(5), 591–603. https://doi.org/10.1016/0041-0101(93)90114-X.

    Article  Google Scholar 

  12. Dong, L. V., Selvanayagam, Z. E., Gopalakrishnakone, P., & Eng, K. H. (2002). A new avidin-biotin optical immunoassay for the detection of beta-bungarotoxin and application in diagnosis of experimental snake envenomation. Journal of Immunological Methods, 260, 125–136. https://doi.org/10.1016/S0022-1759(01)00527-0.

    Article  Google Scholar 

  13. Dong, L. V., Eng, K. H., Quyen, L. K., & Gopalakrishnakone, P. (2004). Optical immunoassay for snake venom detection. Biosensors & Bioelectronics, 19, 1285–1294. https://doi.org/10.1016/j.bios.2003.11.020.

    Article  Google Scholar 

  14. Gao, R., Zhang, Y., & Gopalakrishnakone, P. (2008). Single-bead-based immunofluorescence assay for snake venom detection. Biotechnology Progress, 24(1), 245–249. https://doi.org/10.1021/bp070099e.

    Article  Google Scholar 

  15. Hartono, D., Lai, S. L., Yang, K. L., & Yung, L. Y. (2009). A liquid crystal-based sensor for real-time and label-free identification of phospholipase-like toxins and their inhibitors. Biosensors & Bioelectronics, 24, 2289–2293. https://doi.org/10.1016/j.bios.2008.

    Article  Google Scholar 

  16. Pimenta-Martins, M. G. R., Furtado, R. F., Dutra, R. F., Heneine, L. G. D., Dias, R. S., Borges, M. F., & Alves, C. R. (2014). Immunosensor based on ink printed electrode for staphylococcal enterotoxin detection. Rev Uni Vale do Rio Verde, 12, 895–904. https://doi.org/10.1016/j.mimet.2012.05.016.

    Article  Google Scholar 

  17. Liang, Q., Yamashita, T., Yamamoto-Ikemoto, R., & Yokoyama, H. (2018). Flame-oxidized stainless-steel anode as a probe in bioelectrochemical system-based biosensors to monitor the biochemical oxygen demand of wastewater. Sensors, 18(2). https://doi.org/10.3390/s18020607.

    Article  Google Scholar 

  18. Rezaei, B., Shams-Ghahfarokhi, L., Havakeshian, E., & Ensafi, A. A. (2016). An electrochemical biosensor based on nanoporous stainless steel modified by gold and palladium nanoparticles for simultaneous determination of levodopa and uric acid. Talanta, 158, 42–50. https://doi.org/10.1016/j.talanta.2016.04.061.

    Article  Google Scholar 

  19. Elshafey, R., Tlili, C., Abulrob, A., Tavares, A. C., & Zourob, M. (2013). Label-free impedimetric immunosensor for ultrasensitive detection of cancer marker murine double minute 2 in brain tissue. Biosensors & Bioelectronics, 39, 220–225. https://doi.org/10.1016/j.bios.2012.07.049.

    Article  Google Scholar 

  20. Farka, Z., Juřik, T., Pastucha, M., Kovář, D., Lacina, K., & Skládal, P. (2016). Rapid Immunosensing of Salmonella typhimurium using electrochemical impedance spectroscopy: the effect of sample treatment. Electroanal, 28, 1803–1809. https://doi.org/10.1002/elan.201600093.

    Article  Google Scholar 

  21. Mavrogiannis, M., Fu, X., Desmond, M., McLarnon, R., & Gagnon, Z. R. (2017). Monitoring microfluidic interfacial flows using impedance spectroscopy. Sensors and Actuators B: Chemical, 239, 218–225. https://doi.org/10.1016/j.snb.2016.07.123.

    Article  Google Scholar 

  22. Rocha, C. G., Ferreira, A. A. P., & Yamanaka, H. (2016). Label-free impedimetric immunosensor for detection of the textile azo dye Disperse Red 1 in treated water. Sensors and Actuators B: Chemical, 236, 52–59. https://doi.org/10.1016/j.snb.2016.05.040.

    Article  Google Scholar 

  23. Wong, L. C. C., Jolly, P., & Estrela, P. (2017). Development of a sensitive multiplexed open circuit potential system for the detection of prostate cancer biomarkers. BioNanoScience, 8(2), 701–706. https://doi.org/10.1007/s12668-017-0408-0.

    Article  Google Scholar 

  24. VDM Metals (2010). VDM Crofer 22 APU. Material Data Sheet No. 4046. https://www.vdmmetals.com/fileadmin/user_upload/Downloads/Data_Sheets/Data_Sheet_VDM_Crofer_22_APU.pdf. Acessed 20 Feb 2018.

  25. Vashist, S. K., Lam, E., Hrapovic, S., Male, K. B., & Luong, J. H. T. (2014). Immobilization of antibodies and enzymes on 3-aminopropyltriethoxysilane-functionalized bioanalytical platforms for biosensors and diagnostics. Chemical Reviews, 114, 11083–11130. https://doi.org/10.1021/cr5000943.

    Article  Google Scholar 

  26. Jijie, R., Kahlouche, K., Barras, A., Yamakawa, N., Bouckaert, J., Gharbi, T., Szunerits, S., & Boukherroub, R. (2018). Reduced graphene oxide/polyethylenimine based immunosensor for the selective and sensitive electrochemical detection of uropathogenic Escherichia coli. Sensors and Actuators B: Chemical, 26, 255–263. https://doi.org/10.1016/j.SNB2017.12.169.

    Article  Google Scholar 

  27. Das, A., & Sangaranarayanan, M. V. (2018). A sensitive electrochemical detection of progesterone using tin-nanorods modified glassy carbon electrodes: voltammetric and computational studies. Sensors and Actuators B: Chemical, 256, 775–789. https://doi.org/10.1016/j.snb.2017.10.008.

    Article  Google Scholar 

  28. Raghava, R., & Srivastava, S. (2015). Core–shell gold–silver nanoparticles based impedimetric immunosensor for cancer antigen CA125. Sensors and Actuators B: Chemical, 220, 557–564. https://doi.org/10.1016/j.snb.2015.05.108.

    Article  Google Scholar 

  29. Canbaz, M. Ç., & Sezgintürk, M. K. (2014). Fabrication of a highly sensitive disposable immunosensor based on indium tin oxide substrates for cancer biomarker detection. Analytical Biochemistry, 446, 9–18. https://doi.org/10.1016/j.ab.2013.10.014.

    Article  Google Scholar 

  30. Rezaei, B., Havakeshian, E., & Ensafi, A. A. (2013). Stainless steel modified with an aminosilane layer and gold nanoparticles as a novel disposable substrate for impedimetric immunosensors. Biosensors & Bioelectronics, 48, 61–66. https://doi.org/10.1016/j.bios.2013.03.061.

    Article  Google Scholar 

  31. Fomo, G., Waryo, T. T., Sunday, C. E., Baleg, A. A., Baker, P. G., & Iwuoha, E. I. (2015). Aptameric recognition-modulated electroactivity of poly(4-styrenesolfonic acid)-doped polyaniline films for single-shot detection of tetrodotoxin. Sensors, 15, 22547–22560. https://doi.org/10.3390/s150922547.

    Article  Google Scholar 

  32. Jorcin, J., Orazem, M. E., Pébère, N., & Tribollet, B. (2006). CPE analysis by local electrochemical impedance spectroscopy. Electrochimica Acta, 51, 1473–1479. https://doi.org/10.1016/j.electacta.2005.02.128.

    Article  Google Scholar 

  33. Posseckardt, J., Schirmer, C., Kick, A., Rebatschek, K., Lamz, T., & Mertig, M. (2018). Monitoring of Saccharomyces cerevisiae viability by non-Faradaic impedance spectroscopy using interdigitated screen-printed platinum electrodes. Sensors and Actuators B: Chemical, 255, 3417–3424. https://doi.org/10.1016/j.SNB2017.09.171.

    Article  Google Scholar 

  34. Valiūnienė, A., Rekertaitė, A. I., Ramanavičienė, A., Mikoliūnaitė, L., & Ramanavičius, A. (2017). Fast Fourier transformation electrochemical impedance spectroscopy for the investigation of inactivation of glucose biosensor based on graphite electrode modified by Prussian blue, polypyrrole and glucose oxidase. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 532, 165–171. https://doi.org/10.1016/j.colsurfa.2017.05.048.

    Article  Google Scholar 

  35. Jose, B., Spain, E., Adamson, K., Carthy, E., Boyle, D., & Forster, R. J. (2017). Probing the interaction of a glycoprotein IIb/IIIa receptor antagonist with bound platelets using electrochemical impedance. Electrochemistry Communications, 75, 69–72. https://doi.org/10.1016/j.elecom.2016.12.012.

    Article  Google Scholar 

  36. Lasia, A. (1999). Electrochemical impedance spectroscopic and its applications. In B. E. Conway, J. O.’. M. Bockris, & R. E. White (Eds.), (p. 143). New York: Kluwer Academic/Plenum Publishers.

    Google Scholar 

  37. Jinlong, L., Hongyun, L., & Tongxiang, L. (2016). Investigation of microstructure and corrosion behavior of burnished aluminum alloy by TEM, EWF, XPS and EIS techniques. Materials Research Bulletin, 83, 148–154. https://doi.org/10.1016/j.materresbull.2016.05.013.

    Article  Google Scholar 

  38. Schönleber, M., Klotz, D., & Ivers-Tiffée, E. (2014). A method for improving the robustness of linear Kramers-Kronig validity tests. Electrochimica Acta, 131, 20–27. https://doi.org/10.1016/j.electacta.2014.01.034.

    Article  Google Scholar 

  39. Shukla, P. K., Orazem, M. E., & Crisalle, O. D. (2004). Validation of the measurement model concept for error structure identification. Electrochimica Acta, 49, 2881–2889. https://doi.org/10.1016/j.electacta.2004.01.047.

    Article  Google Scholar 

  40. MacDonald, M. A., & Andreas, H. A. (2014). Method for equivalent circuit determination for electrochemical impedance spectroscopy data of protein adsorption on solid surfaces. Electrochimica Acta, 129, 290–299. https://doi.org/10.1016/j.electacta.2014.02.046.

    Article  Google Scholar 

  41. Fasmin, F., & Srinivasan, R. (2015). Detection of nonlinearities in electrochemical impedance spectra by Kramers–Kronig transforms. Journal of Solid State Electrochemistry, 19(6), 1833–1847. https://doi.org/10.1007/s10008-015-2824-9.

    Article  Google Scholar 

  42. Pulido, Y. F., Blanco, C., Anseán, D., García, V. M., Ferrero, F., & Valledor, M. (2017). Determination of suitable parameters for battery analysis by electrochemical impedance spectroscopy. Measur, 106, 1–11. https://doi.org/10.1016/j.measurement.2017.04.022.

    Article  Google Scholar 

  43. Brug, G. J., Van Den Eeden, A. L. G., Sluyters-Rebach, M., & Sluyters, J. H. (1984). The analysis of electrode impedances complicated by the presence of a constant phase element. Journal of Electroanalytical Chemistry and Interfacial Electrochemistry, 176(1–2), 275–295. https://doi.org/10.1016/S0022-0728(84)80324-1.

    Article  Google Scholar 

  44. Afkhami, A., Hashemi, P., Bagheri, H., Salimian, J., Ahmadi, A., & Madrakian, T. (2017). Impedimetric immunosensor for the label-free and direct detection of botulinum neurotoxin serotype A using Au nanoparticles/graphene-chitosan composite. Biosensors & Bioelectronics, 93, 124–131. https://doi.org/10.1016/j.bios.2016.09.059.

    Article  Google Scholar 

  45. Fusco, G., Gallo, F., Tortolini, C., Bollella, P., Ietto, F., Mico, A., D’annibale, A., Antiochia, R., Favero, G., & Mazzei, F. (2017). AuNPs-functionalized PANABA-MWCNTs nanocomposite-based impedimetric immunosensor for 2,4-dichlorophenoxy acetic acid detection. Biosensors & Bioelectronics, 93, 52–56. https://doi.org/10.1016/j.bios.2016.10.016.

    Article  Google Scholar 

  46. Akter, R., Jeong, B., Lee, Y., Choi, J., & Rahman, M. A. (2017). Femtomolar detection of cardiac troponin I using a novel label-free and reagent-free dendrimer enhanced impedimetric immunosensor. Biosensors & Bioelectronics, 91, 637–643. https://doi.org/10.1016/j.bios.2017.01.021.

    Article  Google Scholar 

  47. Donmez, S., Arslan, F., Sari, N., Özkan, E. H., & Arslan, H. (2017). Glucose biosensor based on immobilization of glucose oxidase on a carbon paste electrode modified with microsphere-attached l-glycine. Biotechnology and Applied Biochemistry, 64(5), 745–753. https://doi.org/10.1002/bab.1533.

    Article  Google Scholar 

  48. Balasubramanian, P., Balamurugan, T. S. T., Chen, S. M., & Chen, T. W. (2017). Facile synthesis of orthorhombic strontium copper oxide microflowers for highly sensitive nonenzymatic detection of glucose in human blood. Journal of the Taiwan Institute of Chemical Engineers, 81, 182–189. https://doi.org/10.1016/j.jtice.2017.10.040.

    Article  Google Scholar 

  49. Barroso, T. G., Martins, R. C., Fernandes, E., Cardoso, S., Rivas, J., & Freitas, P. P. (2018). Detection of BCG bacteria using a magnetoresistive biosensor: a step towards a fully electronic platform for tuberculosis point-of-care detection. Biosensors & Bioelectronics, 100, 259–265. https://doi.org/10.1016/j.bios.2017.09.004.

    Article  Google Scholar 

  50. Khan, M. S., Dighe, K., Wang, Z., Srivastava, I., Daza, E., Schwartz-Duval, A. S., Ghannam, J., Misra, S. K., & Pan, D. (2018). Detection of prostate specific antigen (PSA) in human saliva using an ultra-sensitive nanocomposite of graphene nanoplatelets with diblock-co-polymers and Au electrodes. Analyst, 143(5), 1094–1103. https://doi.org/10.1039/c7an01932g.

    Article  Google Scholar 

  51. Hung, D. Z., Liau, M. Y., & Lin-Shiau, S. Y. (2003). The clinical significance of venom detection in patients of cobra snakebite. Toxicon, 41, 409–415. https://doi.org/10.1016/S0041-0101(02)00336-7.

    Article  Google Scholar 

  52. Dong, L. V., Quyen, L. K., Eng, K. H., & Gopalakrishnakone, P. (2003). Immunogenicity of venoms from four common snakes in the South of Vietnam and development of ELISA kit for venom detection. Journal of Immunological Methods, 282(1–2), 13–31. https://doi.org/10.1016/S0022-1759(03)00277-1.

    Article  Google Scholar 

  53. Núñez Rangel, V., Fernández Culma, M., Rey-Suárez, P., & Pereañez, J. A. (2012). Development of a sensitive enzyme immunoassay (ELISA) for specific identification of Lachesis acrochorda venom. Journal of Venomous Animals and Toxins Including Tropical Diseases, 18(2), 173–179. https://doi.org/10.1590/S1678-91992012000200007.

    Article  Google Scholar 

  54. Sanhajariya, S., Duffull, S. B., & Isbister, G. K. (2018). Pharmacokinectics of snake venom. Toxins, 10, 73. https://doi.org/10.3390/toxins10020073.

    Article  Google Scholar 

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Acknowledgements

The authors of this research acknowledge Fundação Ezequiel Dias (FUNED) for providing the snake venoms utilized in this work.

Funding

This research received financial support from CAPES, CNPq, FAPEMIG, and UFMG.

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Authors and Affiliations

  1. Department of Chemical Engineering, Universidade Federal de Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil

    Ricardo Adriano Dorledo de Faria & Vanessa de Freitas Cunha Lins

  2. Department of Applied Immunology, Fundação Ezequiel Dias, Belo Horizonte, MG, 30510-010, Brazil

    Giancarlo Ubaldo Nappi & Luiz Guilherme Dias Heneine

  3. Department of Chemistry, Universidade Federal de Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil

    Tulio Matencio

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  1. Ricardo Adriano Dorledo de Faria
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de Faria, R.A.D., Lins, V.F.C., Nappi, G.U. et al. Development of an Impedimetric Immunosensor for Specific Detection of Snake Venom. BioNanoSci. 8, 988–996 (2018). https://doi.org/10.1007/s12668-018-0559-7

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