Programmable DNA Tetrahedron-AuNP Nanostructures for Sequence-Specific Detection of $Escherichia$ $coli$ DNA

The detection of pathogenic microorganisms in wastewater is essential for safeguarding environmental safety and public health. Conventional diagnostic methods are often time-consuming, require sophisticated instrumentation, and are not suitable for on-site monitoring. To address this gap, we aimed to develop a sensitive, specific, and low-cost DNA-based nanosensor for the rapid detection of microbial contaminants, particularly Escherichia coli. DNA, by virtue of its negatively charged sugar--phosphate backbone, behaves as a hydrophilic polyelectrolyte and exhibits remarkable self-assembling properties, enabling the formation of nanostructures with robust molecular recognition capabilities. Harnessing these features, we synthesized DNA tetrahedra (Td) via a one-pot assembly method using four single-stranded oligonucleotides mixed in equimolar ratios, annealed from 95 C to 4 C in MgCl-containing buffer. The structural integrity of the assembled Td was confirmed by agarose gel electrophoresis, dynamic light scattering (DLS), and atomic force microscopy (AFM). To impart biosensing functionality, the Td, bearing a single-stranded probe specific to E. coli DNA, was functionalized onto gold nanoparticles (AuNPs). The substantial shift toward a more negative zeta potential following Td conjugation, compared with bare AuNPs, supports successful surface functionalization. DNA tetrahedron functionalization increased AuNP size to 236.4 nm, supporting AuS-mediated immobilization. Complementary and partially complementary targets increased particle size to 428.2 and 398.9 nm, respectively, while the non-complementary target remained unchanged. DLS results agreed with colorimetric responses, confirming sequence-specific hybridization and selective AuNP aggregation. This study establishes as a proof-of-concept study demonstrating the feasibility of a DNA tetrahedrongold nanoparticle (TdAuNP) platform for the colorimetric detection of Escherichia coli DNA rather than as a fully validated environmental detection method.The biocompatibility, structural stability, and programmability of DNA nanostructures, coupled with their straightforward colorimetric output, make them ideally suited for field applications in water quality assessment. Overall, our findings highlight the potential of DNA nanotechnology in developing efficient, low-cost, and user-friendly diagnostic devices for environmental monitoring and microbial detection.