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Label-free capacitive assaying of biomarkers for molecular diagnostics


The label-free analysis of biomarkers offers important advantages in developing point-of-care (PoC) biosensors. In contrast to label-based methodologies, such as ELISA, label-free analysis enables direct detection of targets without additional steps and labeled reagents. Nonetheless, label-free approaches require high sensitivity to detect the intrinsic features of a biomarker and low levels of nonspecific signals. Electrochemical capacitance, \(C_{\bar \mu }\), is a feature of electroactive nanoscale films that can be measured using electrochemical impedance spectroscopy. \(C_{\bar \mu }\) is promising as an electrochemical transducing signal for the development of high-sensitivity, reagentless and label-free molecular diagnostic assays. We used a proprietary ferrocene (Fc)-tagged peptide that is able to self-assemble onto gold electrodes (thicknesses <2 nm) to which any biological receptor can be coupled. When coupled with biological receptors (e.g., a monoclonal antibody), \(C_{\bar \mu }\) exhibited by the redox-tagged peptide changes as a function of the target concentration. We provide herein the steps for the qualitative and quantitative detection of dengue non-structural protein 1 (NS1) biomarker. Detection of NS1 can be used to diagnose dengue virus infection, which causes epidemics each year in tropical and subtropical regions of the world. Including the pre-treatment of the electrode surface, the analysis takes ~25 h. This time can be reduced to minutes if the electrode surface is fabricated separately, demonstrating that \(C_{\bar \mu }\) is promising for PoC applications. We hope this protocol will serve as a reference point for researchers and companies that intend to further develop capacitive devices for molecular diagnostic assays.

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Fig. 1: Schematic representation of electrode-electrolyte interfaces.
Fig. 2: Representative electrochemical characterization of the Fc-tagged peptide self-assembled monolayer (SAM).
Fig. 3: Capacitance Nyquist plots of the qualitative NS1 assay obtained after 30 min of incubation of the electrode.
Fig. 4: Electrochemical capacitive results of the quantitative NS1 assay.
Fig. 5: Experimental setup for coating silver wire with silver chloride.
Fig. 6: Experimental setup and representative gold characterization.
Fig. 7

Data availability

Data are available from the authors upon request.


  1. Ray, S., Mehta, G. & Srivastava, S. Label-free detection techniques for protein microarrays: prospects, merits and challenges. Proteomics 10, 731–748 (2010).

    Article  CAS  Google Scholar 

  2. Lisdat, F. & Schäfer, D. The use of electrochemical impedance spectroscopy for biosensing. Anal. Bioanal. Chem. 391, 1555–1567 (2008).

    Article  CAS  Google Scholar 

  3. Garrote, B. L., Santos, A. & Bueno, P. R. Perspectives on and precautions for the uses of electric spectroscopic methods in label-free biosensing applications. ACS Sens 4, 2216–2227 (2019).

    Article  CAS  Google Scholar 

  4. Bueno, P. R. Common principles of molecular electronics and nanoscale electrochemistry. Anal. Chem. 90, 7095–7106 (2018).

    Article  CAS  Google Scholar 

  5. Bueno, P. R. Nanoscale origins of super-capacitance phenomena. J. Power Sources 414, 420–434 (2019).

    Article  CAS  Google Scholar 

  6. Bueno, P. R., Fernandes, F. C. B. & Davis, J. J. Quantum capacitance as a reagentless molecular sensing element. Nanoscale 9, 15362–15370 (2017).

    Article  CAS  Google Scholar 

  7. Fernandes, F. C. B., Patil, A. V., Bueno, P. R. & Davis, J. J. Optimized diagnostic assays based on redox tagged bioreceptive interfaces. Anal. Chem. 87, 12137–12144 (2015).

    Article  Google Scholar 

  8. Cecchetto, J., Fernandes, F. C. B., Lopes, R. & Bueno, P. R. The capacitive sensing of NS1 Flavivirus biomarker. Biosens. Bioelectron. 87, 949–956 (2017).

    Article  CAS  Google Scholar 

  9. Piccoli, J. et al. Redox capacitive assaying of C-reactive protein at a peptide supported aptamer interface. Anal. Chem. 90, 3005–3008 (2018).

    Article  CAS  Google Scholar 

  10. Oliveira, R. M. B., Fernandes, F. C. B. & Bueno, P. R. Pseudocapacitance phenomena and applications in biosensing devices. Electrochim. Acta 306, 175–184 (2019).

    Article  CAS  Google Scholar 

  11. Miranda, D. A. & Bueno, P. R. Density functional theory and an experimentally-designed energy functional of electron density. Phys. Chem. Chem. Phys. 18, 25984–25992 (2016).

    Article  CAS  Google Scholar 

  12. Bueno, P. R. Nanoscale Electrochemistry of Molecular Contacts (Springer, 2018).

  13. Garrote, B. L., Fernandes, F. C. B., Cilli, E. M. & Bueno, P. R. Field effect in molecule-gated switches and the role of target-to-receptor size ratio in biosensor sensitivity. Biosens. Bioelectron. 127, 215–220 (2019).

    Article  CAS  Google Scholar 

  14. Bueno, P. R., Benites, T. A. & Davis, J. J. The mesoscopic electrochemistry of molecular junctions. Sci. Rep. 6, 18400 (2016).

    Article  CAS  Google Scholar 

  15. Fernandes, F. C. B., Góes, M. S., Davis, J. J. & Bueno, P. R. Label free redox capacitive biosensing. Biosens. Bioelectron. 50, 437–440 (2013).

    Article  CAS  Google Scholar 

  16. Santos, A., Piccoli, J. P., Santos-Filho, N. A., Cilli, E. M. & Bueno, P. R. Redox-tagged peptide for capacitive diagnostic assays. Biosens. Bioelectron. 68, 281–287 (2015).

    Article  CAS  Google Scholar 

  17. Fernandes, F. C. B., Santos, A., Martins, D. C., Góes, M. S. & Bueno, P. R. Comparing label free electrochemical impedimetric and capacitive biosensing architectures. Biosens. Bioelectron. 57, 96–102 (2014).

    Article  CAS  Google Scholar 

  18. Lehr, J., Fernandes, F. C. B., Bueno, P. R. & Davis, J. J. Label-free capacitive diagnostics: exploiting local redox probe state occupancy. Anal. Chem. 86, 2559–2564 (2014).

    Article  CAS  Google Scholar 

  19. Baradoke, A., Hein, R., Li, X. & Davis, J. J. Reagentless redox capacitive assaying of C-reactive protein at a polyaniline interface. Anal. Chem. 92, 3508–3511 (2020).

    Article  CAS  Google Scholar 

  20. Fernandes, F. C. B. & Bueno, P. R. Optimized electrochemical biosensor for human prostatic acid phosphatase. Sens. Actuators B Chem. 253, 1106–1112 (2017).

    Article  CAS  Google Scholar 

  21. Santos, A., Bueno, P. R. & Davis, J. J. A dual marker label free electrochemical assay for Flavivirus dengue diagnosis. Biosens. Bioelectron. 100, 519–525 (2018).

    Article  CAS  Google Scholar 

  22. Ben Aissa, S., Mars, A., Catanante, G., Marty, J. L. & Raouafi, N. Design of a redox-active surface for ultrasensitive redox capacitive aptasensing of aflatoxin M1 in milk. Talanta 195, 525–532 (2019).

    Article  Google Scholar 

  23. Cecchetto, J., Santos, A., Mondini, A., Cilli, E. M. & Bueno, P. R. Serological point-of-care and label-free capacitive diagnosis of dengue virus infection. Biosens. Bioelectron. 151, 111972 (2020).

    Article  CAS  Google Scholar 

  24. Nunes, P. C. G. et al. 30 years of fatal dengue cases in Brazil: a review. BMC Public Health 19, 329 (2019).

    Article  Google Scholar 

  25. Carvalhal, R. F., Freire, R. S. & Kubota, L. T. Polycrystalline gold electrodes: a comparative study of pretreatment procedures used for cleaning and thiol self-assembly monolayer formation. Electroanalysis 17, 1251–1259 (2005).

    Article  CAS  Google Scholar 

  26. Ron, H. & Rubinstein, I. Self-assembled monolayers on oxidized metals. 3. Alkylthiol and dialkyl disulfide assembly on gold under electrochemical conditions. J. Am. Chem. Soc. 120, 13444–13452 (1998).

    Article  CAS  Google Scholar 

  27. Wang, J. et al. Shape-dependent electrocatalytic activity of monodispersed gold nanocrystals toward glucose oxidation. Chem. Commun. (Camb.) 47, 6894–6894 (2011).

    Article  CAS  Google Scholar 

  28. Hermanson, G. T. Bioconjugate reagents. in Bioconjugate Techniques 2nd edn, 214–233 (Academic Press, 2008).

  29. Hosseini, S., Vázquez-Villegas, P., Rito-Palomares, M. & Martinez-Chapa, S. O. Advantages, disadvantages, and modifications of conventional ELISA. in Enzyme-linked Immunosorbent Assay (ELISA): From A to Z 67–115 (Springer, 2018).

  30. Maurer, J. J. Rapid detection and limitations of molecular techniques. Ann. Rev. Food Sci. Technol. 2, 259–279 (2011).

    Article  CAS  Google Scholar 

  31. Chang, B.-Y. & Park, S.-M. Electrochemical impedance spectroscopy. Ann. Rev. Anal. Chem. 3, 207–229 (2010).

    Article  CAS  Google Scholar 

  32. Trilling, A. K., Beekwilder, J. & Zuilhof, H. Antibody orientation on biosensor surfaces: a minireview. Analyst 138, 1619–1627 (2013).

    Article  CAS  Google Scholar 

  33. Trasatti, S. & Petrii, O. A. Real surface area measurements in electrochemistry. Pure Appl. Chem. 63, 711–734 (1991).

    Article  CAS  Google Scholar 

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B.L.G. and A.S. acknowledge the support of FAPESP for their scholarship (2018/26273-7 and 2016/17185-1, respectively), and P.R.B. acknowledges the support of FAPESP for the financial support (2017/24839-0 and 2017/02974-3). P.R.B. also acknowledges the individual support by CNPq provided to his head of research activities at São Paulo State University.

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Correspondence to Paulo R. Bueno.

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Key references using this protocol

Cecchetto, J. et. al. Biosens. Bioelectron. 151, 111972 (2020):

Piccoli, J. et. al. Anal. Chem. 90, 3005–3008 (2018):

Oliveira, R. M. B., Fernandes, F. C. B. & Bueno, P. R. Electrochim. Acta 306, 175–184 (2019):

Baradoke, A. et. al. Anal. Chem. 92, 3508–3511 (2020):

Ben Aissa, S. et. al. Talanta 195, 525–532 (2019):

Key data used in this protocol

Cecchetto, J. et. al. Biosens. Bioelectron. 151, 111972 (2020):

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Garrote, B.L., Santos, A. & Bueno, P.R. Label-free capacitive assaying of biomarkers for molecular diagnostics. Nat Protoc 15, 3879–3893 (2020).

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