Skip to main content
SpringerLink
Log in

Ratiometric electrochemical immunosensor for simultaneous detection of C-myc and Bcl-2 based on multi-role alloy composites

  • Original Paper
  • Published:
Microchimica Acta Aims and scope Submit manuscript

Abstract

A ratiometric electrochemical immunosensor is proposed for simultaneous detection of cellular-myelocytomatosis oncoprotein (C-myc) and B-cell lymphoma 2 (Bcl-2) via the potential-resolved strategy. It relied on multi-role co-loaded alloy composites (CLACs) and poly(3,4-ethylenedioxythiophene) (PEDOT)-graphene oxide (GO)-multiwalled carbon nanotubes (MWCNTs) (PGM) modified electrodes. CLACs with good catalytic and enzyme-like properties were synthesized in one step by loading tetramethylbenzidine (TMB) or methylene blue (MB) into Pt-Pd alloy and used as label materials. After immunological reactions, CLACs showed distinguishable dual differential pulse voltammetry signals at  − 0.26 V and 0.38 V, corresponding to C-myc and Bcl-2, and the PGM had an electrochemical signal at 1.2 V, which could be used as a reference signal to construct a ratiometric sensor. CLACs had a satisfactory synergistic effect with the PGM, and eventually achieved quadruple signal amplification. Thus, benefiting from multiple magnification and ratiometric self-calibration functions, sensitive detections of C-myc and Bcl-2 were achieved, with detection limits as low as 0.5 and 2.5 pg mL−1, respectively. Additionally, when the designed method was applied to blood samples from lymphoma patients, results consistent with the ELISA kit were obtained. This will open avenues for constructing multiple protein detection sensors.

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

Scheme 1
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Data availability

All data generated or analyzed during this study are included in this article and the supporting information. The additional file is available.

References

  1. Tan AYS, Lo NW, Cheng F, Zhang M, Tan MT, Manickam S (2022) 2D carbon materials based photoelectrochemical biosensors for detection of cancer antigens. Biosen Bioelectron 219:114811. https://doi.org/10.1016/j.bios.2022.114811

    Article  CAS  Google Scholar 

  2. Abdelhak A, Foschi M, Abu-Rumeileh S, Yue JK, D’Anna L, Huss A (2022) Blood GFAP as an emerging biomarker in brain and spinal cord disorders. Nat Rev Neurol 18:158-172. https://doi.org/10.1038/s41582-021-00616-3

  3. Song H, Wang F, Zhao Y, Gao R, He Y, Yan Q (2022) Spatially-directed magnetic molecularly imprinted polymers with good anti-interference for simultaneous enrichment and detection of dual disease-related bio-indicators. Nanoscale 14:11343–11352. https://doi.org/10.1039/D2NR03356A

    Article  CAS  PubMed  Google Scholar 

  4. Sun M, Lu P, Yu C, Feng F, Li Q, Zhan J, Yao L (2022) Force-coded strategy for the simultaneous detection of multiple tumor-related proteins. Anal chem 94:8992–8998. https://doi.org/10.1021/acs.analchem.2c04183

    Article  CAS  PubMed  Google Scholar 

  5. Pothipor C, Bamrungsap S, Jakmunee J, Ounnunkad K (2022) A gold nanoparticle-dye/poly (3-aminobenzylamine)/two dimensional MoSe2/graphene oxide electrode towards label-free electrochemical biosensor for simultaneous dual-mode detection of cancer antigen 15–3 and microRNA-21. Colloid Surface B 210:112260. https://doi.org/10.1016/j.colsurfb.2021.112260

    Article  CAS  Google Scholar 

  6. Singh N, Huang L, Wang DB, Shao N, Zhang XE (2020) Simultaneous detection of a cluster of differentiation markers on leukemia-derived exosomes by multiplex immuno-polymerase chain reaction via capillary electrophoresis analysis. Anal Chem 92:10569–10577. https://doi.org/10.1021/acs.analchem.0c01464

    Article  CAS  PubMed  Google Scholar 

  7. Liu X, Li Y, He L, Feng Y, Tan H, Chen X (2021) Simultaneous detection of multiple neuroendocrine tumor markers in patient serum with an ultrasensitive and antifouling electrochemical immunosensor. Biosen Bioelectron 194:113603. https://doi.org/10.1016/j.bios.2021.113603

    Article  CAS  Google Scholar 

  8. Mei W, Zhou Y, Xia L, Liu X, Huang W, Wang H, Zou LY, Wang Q, Yang XH, Wang KM (2023) DNA tetrahedron-based valency controlled signal probes for tunable protein detection. ACS Sensors 8:381–387. https://doi.org/10.1021/acssensors.2c02476

    Article  CAS  PubMed  Google Scholar 

  9. Cohen L, Walt DR (2018) Highly sensitive and multiplexed protein measurements. Chem Rev 119:293–321. https://doi.org/10.1021/acs.chemrev.8b00257

    Article  CAS  PubMed  Google Scholar 

  10. Klebes A, Kittel AS, Verboket RD, Stetten F, Früh SM (2022) Multianalyte lateral flow immunoassay for simultaneous detection of protein-based inflammation biomarkers and pathogen DNA. Sensor Actuat B-Chem 355:131283. https://doi.org/10.1016/j.snb.2021.131283

    Article  CAS  Google Scholar 

  11. Ao L, Liao T, Huang L, Lin S, Xu K, Ma J, Qiu SR, Wang XY, Zhang QQ (2022) Sensitive and simultaneous detection of multi-index lung cancer biomarkers by an NIR-II fluorescence lateral-flow immunoassay platform. Chem Eng J 436:135204. https://doi.org/10.1016/j.cej.2022.135204

    Article  CAS  Google Scholar 

  12. Wang J, Tan Z, Zhu C, Xu L, Xia XH, Wang C (2023) Ultrasensitive multiplex imaging of cell surface proteins via core-shell surface-enhanced Raman scattering nanoprobes. ACS Sensors 8(3):1348–1356. https://doi.org/10.1021/acssensors.3c00100

    Article  CAS  PubMed  Google Scholar 

  13. Timilsina SS, Jolly P, Durr N, Yafia M, Ingber DE (2021) Enabling multiplexed electrochemical detection of biomarkers with high sensitivity in complex biological samples. Acc Chem Res 54:3529–3539. https://doi.org/10.1021/acs.accounts.1c00382

    Article  CAS  PubMed  Google Scholar 

  14. Zeng R, Qiu M, Wan Q, Huang Z, Liu X, Tang DP, Knopp D (2022) Smartphone-based electrochemical immunoassay for point-of-care detection of SARS-CoV-2 nucleocapsid protein. Anal Chem 94:15155–15161. https://doi.org/10.1021/acs.analchem.2c03606

    Article  CAS  PubMed  Google Scholar 

  15. Li N, Nan C, Mei X, Sun Y, Feng H, Li Y (2020) Electrochemical sensor based on dual-template molecularly imprinted polymer and nanoporous gold leaf modified electrode for simultaneous determination of dopamine and uric acid. Microchim Acta 187:1–10. https://doi.org/10.1007/s00604-020-04413-5

    Article  CAS  Google Scholar 

  16. Meng X, Xu Y, Ma B, Ma Z, Han H (2022) Anti-fouling materials decorated immunoprobe and electrochemical sensing interface to improve immunoassay. Chem Eng J 450:137954. https://doi.org/10.1016/j.cej.2022.137954

    Article  CAS  Google Scholar 

  17. Lv W, Ye H, Yuan Z, Liu X, Chen X, Yang W (2020) Recent advances in electrochemiluminescence-based simultaneous detection of multiple targets. TrAC Trend Anal Chem 123:115767. https://doi.org/10.1016/j.trac.2019.115767

    Article  CAS  Google Scholar 

  18. Kuntamung K, Jakmunee J, Ounnunkad K (2021) A label-free multiplex electrochemical biosensor for the detection of three breast cancer biomarker proteins employing dye/metal ion-loaded and antibody-conjugated polyethyleneimine-gold nanoparticles. J Mater Chem B 9:6576–6585. https://doi.org/10.1039/D1TB00940K

    Article  CAS  PubMed  Google Scholar 

  19. Zupančič U, Jolly P, Estrela P, Moschou D, Ingber DE (2021) Graphene enabled low-noise surface chemistry for multiplexed sepsis biomarker detection in whole blood. Adv Funct Mater 31:2010638. https://doi.org/10.1002/adfm.202010638

    Article  CAS  Google Scholar 

  20. Zhao J, Du J, Luo J, Chen S, Yuan R (2020) A novel potential-resolved electrochemiluminescence immunosensor for the simultaneous determination of brain natriuretic peptide and cardiac troponin I. Sensor Actuat B-Chem 311:127934. https://doi.org/10.1016/j.snb.2020.127934

    Article  CAS  Google Scholar 

  21. Zhang X, Zhi H, Wang F, Zhu M, Meng H, Wan P, Feng L (2022) Target-responsive smart nanomaterials via an Au–S binding encapsulation strategy for electrochemical/colorimetric dual-mode paper-based analytical devices. Anal Chem 94:2569–2577. https://doi.org/10.1021/acs.analchem.1c04537

    Article  CAS  PubMed  Google Scholar 

  22. Chang L, Wu H, Chen R, Sun X, Yang Y, Huang C, Ding SJ, Liu CJ, Chen W (2023) Microporous PdCuB nanotag-based electrochemical aptasensor with Au@CuCl2 nanowires interface for ultrasensitive detection of PD-L1-positive exosomes in the serum of lung cancer patients. J Nanobiotechnol 21:1–11. https://doi.org/10.1186/s12951-023-01845-y

    Article  CAS  Google Scholar 

  23. Xu J, Zeng R, Huang L, Qiu Z, Tang DP (2022) Dual-signaling photoelectrochemical biosensor based on biocatalysis-induced vulcanization of Bi2MoO6 nanosheets. Anal Chem 94:11441–11448. https://doi.org/10.1021/acs.analchem.2c02848

    Article  CAS  PubMed  Google Scholar 

  24. Yu Z, Gong H, Xu J, Li Y, Zeng Y, Liu X, Tang DP (2022) Exploiting photoelectric activities and piezoelectric properties of NaNbO3 semiconductors for point-of-care immunoassay. Anal Chem 94:3418–3426. https://doi.org/10.1021/acs.analchem.2c00066

    Article  CAS  PubMed  Google Scholar 

  25. Luo K, Zhao C, Luo Y, Pan C, Li J (2022) Electrochemical sensor for the simultaneous detection of CA72-4 and CA19-9 tumor markers using dual recognition via glycosyl imprinting and lectin-specific binding for accurate diagnosis of gastric cancer. Biosen Bioelectron 216:114672. https://doi.org/10.1016/j.bios.2022.114672

    Article  CAS  Google Scholar 

  26. Zhao X, Wang J, Chen H, Xu H, Bai L, Wang W, Yuan B (2019) A multiple signal amplification based on PEI and rGO nanocomposite for simultaneous multiple electrochemical immunoassay. Sensor Actuat B-Chem 301:127071. https://doi.org/10.1016/j.snb.2019.127071

    Article  CAS  Google Scholar 

  27. Shu J, Tang DP (2019) Recent advances in photoelectrochemical sensing: from engineered photoactive materials to sensing devices and detection modes. Anal Chem 92(1):363–377. https://doi.org/10.1021/acs.analchem.9b04199

    Article  CAS  PubMed  Google Scholar 

  28. Shu J, Tang DP (2017) Current advances in quantum-dots-based photoelectrochemical immunoassays. Chem-Asian J 12(21):2780–2789. https://doi.org/10.1002/asia.201701229

    Article  CAS  PubMed  Google Scholar 

  29. Zhou S, Wang Y, Zhu JJ (2016) Simultaneous detection of tumor cell apoptosis regulators Bcl-2 and Bax through a dual-signal-marked electrochemical immunosensor. ACS Appl Mater Inter 8(12):7674–7682. https://doi.org/10.1021/acsami.6b01010

    Article  CAS  Google Scholar 

  30. Kalyoncu D, Buyuksunetci YT, Anık Ü (2019) Development of a sandwich immunosensor for concurrent detection of carcinoembryonic antigen (CEA), vascular endothelial growth factor (VEGF) and α-fetoprotein (AFP) biomarkers. Mater Sci Eng C 101:88–91. https://doi.org/10.1016/j.msec.2019.03.079

    Article  CAS  Google Scholar 

  31. Wang N, Zhao X, Chen H, Bai L, Xu H, Wang W, Yang L (2020) Fabrication of novel electrochemical immunosensor by mussel-inspired chemistry and surface-initiated PET-ATRP for the simultaneous detection of CEA and AFP. React Funct Polym 154:104632. https://doi.org/10.1016/j.reactfunctpolym.2020.104632

    Article  CAS  Google Scholar 

  32. Yang H, Bao J, Huo D, Zeng Y, Wang X, Samalo M, Hou C (2021) Au doped poly-thionine and poly-m-Cresol purple: synthesis and their application in simultaneously electrochemical detection of two lung cancer markers CEA and CYFRA21-1. Talanta 224:121816. https://doi.org/10.1016/j.talanta.2020.121816

    Article  CAS  PubMed  Google Scholar 

  33. Li L, Wei Y, Zhang S, Chen X, Shao T, Feng D (2021) Electrochemical immunosensor based on metal ions functionalized CNSs@Au NPs nanocomposites as signal amplifier for simultaneous detection of triple tumor markers. Electroanal Chem 880:114882. https://doi.org/10.1016/j.jelechem.2020.114882

    Article  CAS  Google Scholar 

  34. Wang N, Wang J, Zhao X, Chen H, Xu H, Bai L, Yuan B (2021) Highly sensitive electrochemical immunosensor for the simultaneous detection of multiple tumor markers for signal amplification. Talanta 226:122133. https://doi.org/10.1016/j.talanta.2021.122133

    Article  CAS  PubMed  Google Scholar 

  35. Xiao Y, Cai Y, Tang H, Xiao X (2017) De novo CD5-positive primary cardiac diffuse large B-cell lymphoma coexpressing C-myc and BCL2 in an immunocompetent adult. Eur Hear J 38:1937–1937. https://doi.org/10.1093/eurheartj/ehw523

    Article  Google Scholar 

  36. Zhang Y, Wang H, Ren C, Yu H, Fang W, Zhang N, Hou Q (2019) Correlation between C-MYC, BCL-2, and BCL-6 protein expression and gene translocation as biomarkers in diagnosis and prognosis of diffuse large B-cell lymphoma. Front Pharmacol 9:1497. https://doi.org/10.3389/fphar.2018.01497

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Elbadawy M, Usui T, Yamawaki H, Sasaki K (2019) Emerging roles of C-Myc in cancer stem cell-related signaling and resistance to cancer chemotherapy: a potential therapeutic target against colorectal cancer. Inter J Mol Sci 20:2340. https://doi.org/10.3390/ijms20092340

    Article  CAS  Google Scholar 

  38. Sarosiek KA, Malumbres R, Nechushtan H, Gentles AJ, Avisar E, Lossos IS (2010) Novel IL-21 signaling pathway up-regulates c-Myc and induces apoptosis of diffuse large B-cell lymphomas. Blood 115:570–580. https://doi.org/10.1182/blood-2009-08-239996

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Chowdhury R, Mollick MMR, Biswas Y, Chattopadhyay D, Rashid MH (2018) Biogenic synthesis of shape-tunable Au-Pd alloy nanoparticles with enhanced catalytic activities. J Alloy Compd 763:399–408. https://doi.org/10.1016/j.jallcom.2018.05.343

    Article  CAS  Google Scholar 

  40. Yao K, Zhao C, Wang N, Li T, Lu W, Wang J (2020) An aqueous synthesis of porous PtPd nanoparticles with reversed bimetallic structures for highly efficient hydrogen generation from ammonia borane hydrolysis. Nanoscale 12:638–647. https://doi.org/10.1039/C9NR07144J

    Article  CAS  PubMed  Google Scholar 

  41. Ando D, Teshima TF, Zurita F, Peng H, Ogura K, Kondo K, Malhotra BD (2022) Filtration-processed biomass nanofiber electrodes for flexible bioelectronics. J Nanobiotechnol 20:1–13. https://doi.org/10.1186/s12951-022-01684-3

    Article  CAS  Google Scholar 

  42. Lyu S, Chen Y, Zhang L, Han S, Lu Y, Chen Y, Wang S (2019) Nanocellulose supported hierarchical structured polyaniline/nanocarbon nanocomposite electrode via layer-by-layer assembly for green flexible supercapacitors. RSC Adv 9:17824–17834. https://doi.org/10.1039/C9RA02449B

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  43. Gupta PK, Son SE, Seong GH (2021) Functionalized ultra-fine bimetallic PtRu alloy nanoparticle with high peroxidase-mimicking activity for rapid and sensitive colorimetric quantification of C-reactive protein. Microchim Acta 188:1–10. https://doi.org/10.1007/s00604-021-04775-4

    Article  CAS  Google Scholar 

  44. Xu X, Ji D, Zhang Y, Gao X, Xu P, Li X, Wen W (2019) Detection of phenylketonuria markers using a ZIF-67 encapsulated PtPd alloy nanoparticle (PtPd@ZIF-67)-based disposable electrochemical microsensor. ACS Appl Mater Inter 11:20734–20742. https://doi.org/10.1021/acsami.9b05431

    Article  CAS  Google Scholar 

  45. Li G, Xie G, Gong C, Chen D, Chen X, Zhang Q, Dong L (2022) Hydrogen-assisted synthesis of Ni-ZIF-derived nickel nanoparticle chains coated with nitrogen-doped graphitic carbon layers as efficient electrocatalysts for non-enzymatic glucose detection. Microchim Acta 189:80–92. https://doi.org/10.1007/s00604-022-05172-1

    Article  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Both Xiaoying Wang and Wei Yuan contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Environmental Medicine and Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, 210009, China

    Xiaoying Wang, Wei Yuan, Yijing Kuang & Xuyuan Chen

  2. Department of Hematology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, 710061, China

    Xiaoning Wang

  3. R&D Center, Nanjing Sanhome Pharmaceutical Co. Ltd., Nanjing, 211135, China

    Xiaoyu Zhang

Authors
  1. Xiaoying Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wei Yuan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yijing Kuang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xuyuan Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiaoning Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xiaoyu Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Xiaoying Wang: conceptualization, methodology, software, writing—original draft. Wei Yuan: conceptualization, methodology, formal analysis, writing—review and editing. Yijing Kuang: methodology, software, visualization. Xuyuan Chen and Xiaoyu Zhang: validation, formal analysis, resources. Xiaoning Wang: validation, resources, funding acquisition. Xiaoying Wang: formal analysis, resources, funding acquisition, project administration. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Xiaoying Wang.

Ethics declarations

Consent for publication

All authors have provided consent for the manuscript to be published.

Research involving human participants and/or animals

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The collections and experiments on the human biological samples were approved by the Ethics Committee of The First Affiliated Hospital of Xi’an Jiaotong University.

Competing interests

The authors declare no competing interests.

Additional information

Publisher's Note

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

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 969 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, X., Yuan, W., Kuang, Y. et al. Ratiometric electrochemical immunosensor for simultaneous detection of C-myc and Bcl-2 based on multi-role alloy composites. Microchim Acta 191, 85 (2024). https://doi.org/10.1007/s00604-023-06161-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s00604-023-06161-8

Keywords

Search

Navigation