A Novel Electrochemical Biosensor: Sensitive and Quantitative Detection of Circulating Tumor DNA

Abstract

The detection of circulating tumor DNA (ctDNA) with high sensitivity and specificity is crucial for the early diagnosis and monitoring of tumors, as well as for drug therapy. In this study, a simple and highly sensitive biosensor was specifically designed for the identification of targeted ctDNA. For the first time, a three-dimensional polyvinylidene fluoride-graphene oxide-chitosan (PVDF/CS/GO) nanofiber mesh was fabricated on a polydimethylsiloxane (PDMS) micropillar substrate using electrospinning technology, and the nanofibers were functionalized with peptide nucleic acids probe-gold nanoparticle (PNA-AuNP) complexes, which served as affinity molecules for detecting the methylation of the E542K variant of the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA) gene in the peripheral blood of cancer patients. Additionally, an anti-5-Methylcytosine monoclonal antibody - multi-walled carbon nanotubes-COOH complex (Anti-5-mC-MWCNTs-COOH) complex was incubated to result in significantly amplified electrochemical signals for the accurate quantification of the E542K variant of the PIK3CA gene. Detectable signal responses were observed only when both molecules were simultaneously present, greatly enhancing the accuracy of the analysis. The biosensor exhibits high capture sensitivity for the methylation level of the E542K variant of the PIK3CA gene across a concentration range of 50 to 10 000 fmol/L, with the lowest detection limit of 10 fmol/L. The ctDNA nanobiosensor has been shown to be both feasible and valuable for quantifying ctDNA concentrations in clinical blood samples. Consequently, this 3D nanofiber biosensor shows significant potential for clinical applications in cancer diagnosis and personalized medical treatments.

Keywords

nanobiosensor / polyvinylidene fluoride–graphene–chitosan nanofibers / carboxylated multi-walled carbon nanotubes / liquid biopsy / circulating tumour DNA

References

[1] Bhagat P, Upadhyay LSB. A Review Towards Sustainable Analyte Detection: Biomimetic Inspiration in Biosensor Technology[J]. Journal of Biotechnology, 2025, 398: 51-65

[2] Mobed A, Malehmir S, Ahmad Alipour A, et al.. Biosensors, Modern Technology for the Detection of Cancer-Associated Bacteria[J]. Biotechnology Letters, 2022, 44(5–6): 683-701

[3] Pollard TD, Ong JJ, Goyanes A, et al.. Electrochemical Biosensors: a Nexus for Precision Medicine[J]. Drug Discovery Today, 2021, 26(1): 69-79

[4] Lin C, Liu X, Zheng B, et al.. Liquid Biopsy, ctDNA Diagnosis through NGS[J]. Life, 2021, 11(9): 890-901

[5] Perakis S, Speicher MR. Emerging Concepts in Liquid Biopsies[J]. BMC Medicine, 2017, 15(1): 75-86

[6] El Hejjioui B, Bouguenouch L, Melhouf MA, et al.. Clinical Evidence of Circulating Tumor DNA Application in Aggressive Breast Cancer[J]. Diagnostics, 2023, 13(3): 470-485

[7] Pinzani P, D’argenio V, Del Re M, et al.. Updates on Liquid Biopsy: Current Trends and Future Perspectives for Clinical Application in Solid Tumors[J]. Clinical Chemistry and Laboratory Medicine (CCLM), 2021, 59(7): 1 181-1 200

[8] Mendaza S, Guerrero-setas D, Moneral-santesteban I, et al.. A DNA Methylation-Based Gene Signature Can Predict Triple-Negative Breast Cancer Diagnosis[J]. Biomedicines, 2021, 9(10): 1 394-1 407

[9] Sant M, Bernat-peguera A, Felip E, et al.. Role of ctDNA in Breast Cancer[J]. Cancers, 2022, 14(2): 310-323

[10] Openshaw MR, Page K, Fernandez-garcia D, et al.. The Role of ctDNA Detection and the Potential of the Liquid Biopsy for Breast Cancer Monitoring[J]. Expert Review of Molecular Diagnostics, 2016, 16(7): 751-755

[11] Wang K, Peng Z, Lin X, et al.. Electrochemical Biosensors for Circulating Tumor DNA Detection[J]. Biosensors, 2022, 12(8): 649-664

[12] Mallick S, Duttaroy AK, Dutta S. The PIK3CA Gene and Its Pivotal Role in Tumor Tropism of Triple-Negative Breast Cancer[J]. Translational Oncology, 2024, 50: 102 140-102 148

[13] Alqahtani A, Ayesh HSK, Halawani H. PIK3CA Gene Mutations in Solid Malignancies: Association with Clinicopathological Parameters and Prognosis[J]. Cancers, 2019, 12(1): 93-110

[14] Wang H, Tang R, Jiang L, et al.. The Role of PIK3CA Gene Mutations in Colorectal Cancer and the Selection of Treatment Strategies[J]. Frontiers in Pharmacology, 2024, 15: 1 494 802-1 494 815

[15] Pessoa LS, Heringer M, Ferrer VP. ctDNA as a Cancer Biomarker: A Broad Overview[J]. Critical Reviews in Oncology/Hematology, 2020, 155: 103 109-103 124

[16] Kok P-S, Lee K, Lord S, et al.. Clinical Utility of Plasma EGFR Mutation Detection with Quantitative PCR in Advanced Lung Cancer: A meta-analysis[J]. Lung Cancer, 2021, 154: 113-117

[17] Balasan N, Kharrat F, Di Lorenzo G, et al.. Sensitive Detection of Gynecological Cancer Recurrence Using Circulating Tumor DNA and Digital PCR: A Comparative Study with Serum Biochemical Markers[J]. International Journal of Molecular Sciences, 2024, 25(22): 11 997-12 009

[18] O’leary B, Hrebien S, Beaney M, et al.. Comparison of BEAMing and Droplet Digital PCR for Circulating Tumor DNA Analysis[J]. Clinical Chemistry, 2019, 65(11): 1 405-1 413

[19] Nikanjam M, Kato S, Kurzrock R. Liquid Biopsy: Current Technology and Clinical Applications[J]. J. Hematol. Oncol., 2022, 15(1): 131-144

[20] Min L, Chen J, Yu M, et al.. Using Circulating Tumor DNA as a Novel Biomarker to Screen and Diagnose Colorectal Cancer: A Meta-Analysis[J]. Journal of Clinical Medicine, 2023, 12(2): 408-424

[21] Nishiyama A, Nakanishi M. Navigating the DNA Methylation Landscape of Cancer[J]. Trends in Genetics, 2021, 37(11): 1 012-1 027

[22] Chen C, He R, Zhang Z, et al.. Dual-Recognition-Based Determination of ctDNA via the Clamping Function of Peptide Nucleic Acid and Terminal Protection of Small-Molecule-Linked DNA[J]. The Analyst, 2020, 145(23): 7 603-7 608

[23] Wu X, Xiao T, Luo Z, et al.. A Micro-/Nano-Chip and Quantum Dots-Based 3D Cytosensor for Quantitative Analysis of Circulating Tumor Cells[J]. Journal of Nanobiotechnology, 2018, 16(1): 7 603-7 608

[24] Qi M, Ruan M, Liang J, et al.. Three-Dimensional PLGA Nanofiber-Based Microchip for High-Efficiency Cancer Cell Capture[J]. Materials, 2023, 16(8): 65-73

[25] Samanman S, Numnuam A, Limbut W, et al.. Highly-Sensitive Label-Free Electrochemical Carcinoembryonic Antigen Immunosensor Based on a Novel Au Nanoparticles–Graphene–Chitosan Nanocomposite Cryogel Electrode[J]. Analytica Chimica Acta, 2015, 853: 521-532

[26] Nguyen AH, Sim SJ. Nanoplasmonic Biosensor: Detection and Amplification of Dual Bio-Signatures of Circulating Tumor DNA[J]. Biosensors and Bioelectronics, 2015, 67: 443-449

[27] Nie Y, Wang P, Ma Q, et al.. Confined Gold Single Atoms–MXene Heterostructure-Based Electrochemiluminescence Functional Material and Its Sensing Application[J]. Analytical Chemistry, 2022, 94(31): 11 016-11 022

RIGHTS & PERMISSIONS

Wuhan University of Technology and Springer-Verlag GmbH Germany, Part of Springer Nature