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Sensitive electrochemical detection of polymorphisms in IL6 and TGFβ1 genes from ovarian cancer DNA patients using EcoRI and DNA hairpin-modified gold electrodes

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Abstract

Two electrochemical bioplatforms were prepared based on thiolated hairpin DNA probes tethered to AuNP-modified screen-printed electrodes to detect T > G and T > C polymorphisms, namely rs1880269 and rs1800469, present the interleukin-6 (IL6) and transforming growth factor β1 (TGFβ1) genes. The electrochemical readout was ensured by the detection of the double-stranded DNA using methylene blue as a redox probe after treatment by EcoRI restrictase. The main parameters influencing the analytical response such as the thiolated DNA probe concentration, incubation time with electrode, DNA hybridization time, EcoRI enzyme load, and its cleavage time were optimized based on the current intensity and signal-to-blank (S/B) ratio as selection criteria. Using spiked buffer solutions, the IL6 and TGFβ1 E-bioplatforms display wide ranges of linearity (1 × 102–1 × 108 fM and 5 × 101–1 × 105 fM, respectively) and limits of detection (47.9 fM and 16.6 fM, respectively). The two bioelectrodes have also good discrimination toward 1-mismatched, two mismatched, and non-complementary sequences, when they were used 30-fold higher than the target sequences. More importantly, the two bioplatforms successfully detected the single nucleotide polymorphisms (SNPs) in scarcely diluted genomic DNA, collected from 52 donors, and showed they can reliably distinguish between heterozygous (TG and TC genotypes) and homozygous (GG and CC genotypes) patients with  respect to the control subjects (TT genotype), where the differences are statistically highly significant (p-value < 0.0001). Thus, the designed devices could be used to conduct large cohort studies targeting these mutations or extended to other SNPs.

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Funding

The authors acknowledge the financial support of the Tunisian PRF program (NanofastResponse, grant ref.: PRF2017D4P1 and SmartBioSens, grant ref.: PRFCOV19–D2P2).

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Authors and Affiliations

  1. Sensors and Biosensors Group, Analytical Chemistry and Electrochemistry Lab (LR99ES15), Chemistry Department, Faculty of Science of Tunis, University of Tunis El Manar, 2092, Tunis El Manar, Tunisia

    Maroua Meftah, Sabrine Baachaoui & Noureddine Raouafi

  2. Laboratory of Mycology, Pathologies and Biomarkers (LR16ES05), Biology Department, Faculty of Science of Tunis, University of Tunis El Manar, 2092, Tunis El Manar, Tunisia

    Azza Habel & Basma Yaacoubi-Loueslati

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  1. Maroua Meftah
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Meftah, M., Habel, A., Baachaoui, S. et al. Sensitive electrochemical detection of polymorphisms in IL6 and TGFβ1 genes from ovarian cancer DNA patients using EcoRI and DNA hairpin-modified gold electrodes. Microchim Acta 190, 15 (2023). https://doi.org/10.1007/s00604-022-05595-w

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