Electrochemical biosensor with custom fluidics for amplification-free, low-picomolar DNA detection

Background: The sensitive and rapid detection of low-concentration DNA sequences in complex biological matrices remains a critical need across biomedical, environmental, and food sectors. While amplification-based techniques like PCR offer high sensitivity, they are constrained by the need for complex instrumentation and skilled personnel, limiting their use in decentralized settings. Electrochemical biosensors are attractive for point-of-care testing due to their speed and portability, but are hindered by probe immobilization variability, sample interference, and labeling requirements. There is a clear need for robust, label-free, and easy-to-use DNA biosensors capable of performing reliably in real-world complex matrices. Results: We present a label-free impedimetric DNA biosensor integrating screen-printed gold electrodes (AuSPEs) with an optimized 3D-printed fluidic cell. This innovative design ensures consistent probe immobilization while significantly improving hybridization efficiency through controlled convective transport, as demonstrated by COMSOL simulations. The platform achieves sensitive detection with a LOD of 0.1 pM (0.6 pg/mL) in PBS buffer using electrochemical impedance spectroscopy (EIS), eliminating requirements for target amplification or extensive sample preparation. The biosensor maintains excellent performance in complex biological media, successfully detecting target sequences in untreated S. cerevisiae culture supernatants with minimal matrix interference. Furthermore, the system demonstrates exceptional discrimination capability, reliably identifying single-base mismatches while exhibiting minimal cross-reactivity with supernatants from non-target microorganisms (E. coli and C. albicans). These results highlight the system's sensitivity, selectivity, and robustness for nucleic acid detection. Significance: This study represents a notable advancement in electrochemical biosensing, introducing a modular, amplification-free platform capable of highly sensitive DNA detection in both buffer solutions and complex biological samples. Its low cost, operational simplicity, and reliable performance in real-world matrices make it a promising candidate for point-of-care applications. This approach offers a practical alternative for molecular diagnostics and monitoring in clinical, environmental, and industrial contexts.