Skip to main content
SpringerLink
Log in

A novel peptide-based electrochemical biosensor for the determination of a metastasis-linked protease in pancreatic cancer cells

  • Paper in Forefront
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Proteases are involved in cancer‚ taking part in immune (dis)regulation, malignant progression and tumour growth. Recently, it has been found that expression levels of one of the members of the serine protease family, trypsin, is upregulated in human cancer cells of several organs, being considered as a specific cancer biomarker. Considering the great attention that electrochemical peptide sensors have nowadays, in this work, we propose a novel electroanalytical strategy for the determination of this important biomolecule. It implies the immobilization of a short synthetic peptide sequence, dually labelled with fluorescein isothiocyanate (FITC) and biotin, onto neutravidin-modified magnetic beads (MBs), followed by the peptide digestion with trypsin. Upon peptide disruption, the modified MBs were incubated with a specific fluorescein Fab fragment antibody labelled with horseradish peroxidase (HRP-antiFITC) and magnetically captured on the surface of a screen-printed carbon electrode (SPCE), where amperometric detection was performed using the hydroquinone (HQ)/HRP/H2O2 system. The biosensor exhibited a good reproducibility of the measurements (RSD 3.4%, n = 10), and specificity against other proteins and proteases commonly found in biological samples. This work reports the first quantitative data so far on trypsin expression in human cell lysates. The developed bioplatform was used for the direct determination of this protease in lysates from pancreatic cancer, cervix carcinoma and kidney cells in only 3 h and 30 min using low amounts (~ 0.1 μg) of raw extracts.

Graphical abstract

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

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

Similar content being viewed by others

References

  1. Eatemadi A, Aiyelabegan HT, Negahdari B, Mazlomi MA, Daraee H, Daraee N, et al. Role of protease and protease inhibitors in cancer pathogenesis and treatment. Biomed Pharmacother. 2017;86:221–31.

    Article  CAS  Google Scholar 

  2. Zhou Z, Liu W, Wang Y, Ding F, Liu X, Zhao Q, et al. A fluorometric and colorimetric method for determination of trypsin by exploiting the gold nanocluster-induced aggregation of hemoglobin-coated gold nanoparticles. Microchim Acta. 2019;186:272. https://doi.org/10.1007/s00604-019-3380-2.

    Article  CAS  Google Scholar 

  3. Nyberg P, Ylipalosaari M, Sorsa T, Salo T. Trypsins and their role in carcinoma growth. Exp Cell Res. 2006;312:1219–28.

    Article  CAS  Google Scholar 

  4. Lin X, Zhu Z, Zhao C, Li S, Liu Q, Liu A, et al. Robust oxidase mimicking activity of protamine-stabilized platinum nanoparticles units and applied for colorimetric sensor of trypsin and inhibitor. Sensors Actuators B Chem. 2019;284:346–53.

    Article  CAS  Google Scholar 

  5. Nakanuma S-I, Tajima H, Okamoto K, Hayashi H, Nakagawara H, Onishi I, et al. Int J Oncol. 2010;36:793–800.

    Article  CAS  Google Scholar 

  6. Peregrina-Sandoval J, del Toro-Arreola S, Oceguera-Villanueva A, Cerda-Camacho F, del Toro-Arreola A, Gonzalez-Ramella O, et al. Trypsin proteolytic activity in cervical cancer and precursor lesions. Int J Clin Exp Pathol. 2017;10(5):5587–93.

    CAS  Google Scholar 

  7. Kong W, Li Q, Xia L, Li X, Sun H, Kong R-M, et al. Photoelectrochemical determination of trypsin by using an indium tin oxide electrode modified with a composite prepared from MoS2 nanosheets and TiO2 nanorods. Microchim Acta. 2019;186:490. https://doi.org/10.1007/s00604-019-3589-0.

    Article  CAS  Google Scholar 

  8. Karasevaa NA, Pluhara B, Beliaevab EA, Ermolaevab TN, Mizaikoff B. Synthesis and application of molecularly imprinted polymers for trypsin piezoelectric sensors. Sensors Actuators B Chem. 2019;280:272–9.

    Article  Google Scholar 

  9. Liu Y, Zhang F, Heb X, Ma P, Huang Y, Tao S, et al. A novel and simple fluorescent sensor based on AgInZnS QDs for the detection of protamine and trypsin and imaging of cells. Sensors Actuators B Chem. 2019;294:263–9.

    Article  CAS  Google Scholar 

  10. Soreide K, Janssen EA, Körner H, Baak JPA. Trypsin in colorectal cancer: molecular biological mechanisms of proliferation, invasion, and metastasis. J Pathol. 2006;209:147–56.

    Article  CAS  Google Scholar 

  11. Yi Q, Liu Q, Gao F, Chen Q, Wang G. Application of an electrochemical immunosensor with a MWCNT/PDAA modified electrode for detection of serum trypsin. Sensors. 2014;14:10203–12 https://www.mdpi.com/1424-8220/14/6/10203. Accessed 27 Nov 2019.

  12. Ertürk G, Hedström M, Mattiasson B. A sensitive and real-time assay of trypsin by using molecular imprinting-based capacitive biosensor. Biosens Bioelectron. 2016;86:557–65.

    Article  Google Scholar 

  13. Liu W, Li H, Wei Y, Dong C. A label-free phosphorescence sensing platform for trypsin based on Mn-ZnS QDs. RSC Adv. 2017;7:26930–4.

    Article  CAS  Google Scholar 

  14. Xia T, Ma Q, Hu T, Su X. A novel magnetic/photoluminescence bifunctional nanohybrid for the determination of trypsin. Talanta. 2017;170:286–90.

    Article  CAS  Google Scholar 

  15. Lin Y, Shen R, Liu N, Yi H, Dai H, Lin J. A highly sensitive peptide-based biosensor using NiCo2O4 nanosheets and g-C3N4 nanocomposite to construct amplified strategy for trypsin detection. Anal Chim Acta. 2018;1035:175–83.

    Article  CAS  Google Scholar 

  16. Worldwide cancer data: Global cancer statistics for the most common cancers. CUP Continuous Update Project: Analysing research on cancer prevention and survival. World Cancer Research Fund International; American Institute for Cancer Research. 2018. https://www.wcrf.org/dietandcancer/cancer-trends/worldwide-cancer-data Accessed 27 Nov 2019.

  17. Poon C-Y, Li Q, Zhang J, Li Z, Dong C, Lee AW-M, et al. FRET-based modified graphene quantum dots for direct trypsin quantification in urine. Anal Chim Acta. 2016;917:64–70.

    Article  CAS  Google Scholar 

  18. Artigas JMG, Faure MRA, Garcia ME, Gimeno AMB. Serum trypsin levels in acute pancreatic and non-pancreatic abdominal conditions. Postgrad Med J. 1981;57:219–22.

    Article  CAS  Google Scholar 

  19. González-Fernández E, Avlonitis N, Murray AF, Mount AR, Bradley M. Methylene blue not ferrocene: optimal reporters for electrochemical detection of protease activity. Biosens Bioelectron. 2016;84:82–8.

    Article  Google Scholar 

  20. Li H, Yang M, Kong D, Jin R, Zhao X, Liu F, et al. Sensitive fluorescence sensor for point-of-care detection of trypsin using glutathione-stabilized gold nanoclusters. Sensors Actuators B Chem. 2019;282:366–72.

    Article  CAS  Google Scholar 

  21. Wu P, Zhao T, Zhang J, Wu L, Hou X. Analyte-activable probe for protease based on cytochrome C-capped Mn:ZnS quantum dots. Anal Chem. 2014;86:10078–83.

    Article  CAS  Google Scholar 

  22. Park S, Kim G, Seo J, Yang H. Ultrasensitive protease sensors using selective affinity binding, selective proteolytic reaction, and proximity-dependent electrochemical reaction. Anal Chem. 2016;88:11995–2000.

    Article  CAS  Google Scholar 

  23. Banis G, Beardslee LA, Ghodssi R. Gelatin-enabled microsensor for pancreatic trypsin sensing. Appl Sci. 2018;8:208 https://www.mdpi.com/2076-3417/8/2/208. Accessed 27 Nov 2019.

  24. Zhou G, Jiang H, Zhou Y, Liu P, Jia Y, Ye C. Peptide-coated palladium nanoparticle for highly sensitive bioanalysis of trypsin in human urine samples. Nanomater Nanotechnol. 2018;8:1–8.

    Article  Google Scholar 

  25. Liang R, Ding J, Gao S, Qin W. Mussel-inspired surface-imprinted sensors for potentiometric label-free detection of biological species. Angew Chem Int Ed. 2017;56:6833–7.

    Article  CAS  Google Scholar 

  26. González-Fernández E, Staderinia M, Avlonitis N, Murray AF, Mount AR, Bradley M. Effect of spacer length on the performance of peptide-based electrochemical biosensors for protease detection. Sensors Actuators B Chem. 2018;255:3040–6.

    Article  Google Scholar 

  27. Karimzadeh A, Hasanzadeh M, Shadjou N, De la Guardia M. Peptide based biosensors. Trends Anal Chem. 2018;107:1–20.

    Article  CAS  Google Scholar 

  28. Liu Q, Wang J, Boyd BJ. Peptide-based biosensors. Talanta. 2015;136:114–27.

    Article  CAS  Google Scholar 

  29. Muñoz-San Martín C, Pedrero M, Manuel de Villena FJ, Garranzo-Asensio M, Rodríguez N, Domínguez G, et al. Disposable amperometric immunosensor for the determination of the E-cadherin tumor suppressorprotein in cancer cells and human tissues. Electroanalysis. 2019;31:309–17.

    Article  Google Scholar 

  30. Barderas R, Desmet J, Timmerman P, Meloen R, Casal JI. Affinity maturation of antibodies assisted by in silico modeling. Proc Natl Acad Sci U S A. 2008;105(26):9029–34.

    Article  CAS  Google Scholar 

  31. Barderas R, Babel I, Díaz-Uriarte R, Moreno V, Suárez A, Bonilla F, et al. An optimized predictor panel for colorectal cancer diagnosis based on the combination of tumor-associated antigens obtained from protein and phage microarrays. J Proteome. 2012;75(15):4647–55.

    Article  CAS  Google Scholar 

  32. Zhao S, Walker DS, Reichert WM. Cooperativity in the binding of avidin to biotin-lipid-doped Langmuir-Blodgett films. Langmuir. 1993;9:3166–73.

    Article  CAS  Google Scholar 

  33. Nguyen TT, Sly KL, Conboy JC. Comparison of the energetics of avidin, streptavidin, neutravidin, and anti-biotin antibody binding to biotinylated lipid bilayer examined by second-harmonic generation. Anal Chem. 2012;84:201–8.

    Article  CAS  Google Scholar 

  34. Eguílaz M, Moreno-Guzmán M, Campuzano S, González-Cortés A, Yáñez-Sedeño P, Pingarrón JM. An electrochemical immunosensor for testosterone using functionalized magnetic beads and screen-printed carbon electrodes. Biosens Bioelectron. 2010;26:517–22.

    Article  Google Scholar 

  35. Gamella M, Campuzano S, Conzuelo F, Reviejo AJ, Pingarrón JM. Amperometric magnetoimmunosensor for direct determination of D-dimer in human serum. Electroanalysis. 2012;24:2235–43.

    Article  CAS  Google Scholar 

Download references

Funding

The financial support of the CTQ2015-64402-C2-1-R (Spanish Ministerio de Economía y Competitividad) Research Project and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) and predoctoral contract from Universidad Complutense de Madrid (C.M.-S.M.) are gratefully acknowledged. R.B. acknowledges the financial support of the PI17CIII/00045 Grant from the AES-ISCIII program. A.M-C. is a recipient of a FPU fellowship from the Ministerio de Educación, Cultura y Deporte.

Author information

Authors and Affiliations

  1. Departamento de Química Analítica, Facultad de CC. Químicas, Universidad Complutense de Madrid, 28040, Madrid, Spain

    Cristina Muñoz-San Martín, María Pedrero, Maria Gamella, Susana Campuzano & José M. Pingarrón

  2. Chronic Disease Programme, UFIEC, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain

    Ana Montero-Calle & Rodrigo Barderas

Authors
  1. Cristina Muñoz-San Martín
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. María Pedrero
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maria Gamella
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ana Montero-Calle
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Rodrigo Barderas
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Susana Campuzano
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. José M. Pingarrón
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Susana Campuzano or José M. Pingarrón.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Disclaimer

The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Published in the topical collection featuring Female Role Models in Analytical Chemistry.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Muñoz-San Martín, C., Pedrero, M., Gamella, M. et al. A novel peptide-based electrochemical biosensor for the determination of a metastasis-linked protease in pancreatic cancer cells. Anal Bioanal Chem 412, 6177–6188 (2020). https://doi.org/10.1007/s00216-020-02418-w

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-020-02418-w

Keywords

Search

Navigation