Integration of DNA-Based Electrochemical Sensors with Microfluidic Technology to Enhance Biosensing

Abstract

Pathogen surveillance and monitoring is the first line of defense in avoiding diseases and adverse outcomes. Point of care (POC) diagnostic devices have made huge strides to achieve that, however, advancements are still required in order to expand the use of portable devices in environmental, food, and clinical diagnostics. In this work, we address critical challenges in biosensing and pathogen detection through three innovative approaches: (i) enhancing the understanding of the impact of nanostructures in DNA hybridization kinetics, (ii) developing a rapid real-time detection system for Legionella pneumophila using functional nucleic acids as biorecognition elements and DNA barcodes as detection barcodes, and (iii) applying biomimicry in microfluidic designs for uniform velocity and DNA hybridization in multiplexing. We first designed a wash and reagent free in situ electrochemical assay to investigate the role of planar and nanostructured surfaces on real-time DNA hybridization kinetics in buffer and complex media (blood, urine, and saliva). We then conducted continuous measurements to understand how these surface modifications influence electroactive DNA hybridization on the surface under a wide range of probe densities (low, medium, high) and target concentrations (0.01-1 µM). The results show that the effectiveness of nanostructures in enhancing electrochemical sensing depends on the probe/target concentration regime and the medium used in biosensing. Specifically, nanostructures were most beneficial in certain target concentration ranges (0.1-1 µM), with enhancing biosensing in all complex media compared to planar surfaces. We then utilized these nanostructures in engineering a rapid and accurate system for the detection of L. pneumophila in cooling tower water - a key factor in preventing Legionnaires' disease. To overcome the limitations of existing technologies (cell culture, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR)), we designed an RNA-cleaving DNAzyme (RCD) electrochemical assay coupled with magnetic beads, fully housed within microfluidics. This system allows for real-time monitoring by programming RCDs to release an electroactive DNA barcode upon encountering L. pneumophila targets. The barcode is detected by an integrated sensor, achieving a limit of detection of 1.4 × 10³ CFU/mL in buffer and 1.9 × 10³ CFU/mL in cooling tower water in 3 hours. This system meets regulatory requirements and enables precise identification of L. pneumophila among other waterborne bacteria and L. non-pneumophila species. Finally, we leveraged biomimicry to design microchannel systems inspired by the efficient transport mechanisms found in human spinal vertebrae and leaf veins network. By replicating and scaling these natural structures, we developed the bio-inspired microfluidic designs that optimize flow uniformity and DNA capture in Silico. Our optimized designs achieved a coefficient of variation for flow velocity of 0.89% for spine-inspired and 0.86% for leaf veins-inspired microchannels compared to 14.68 % and 59.81 % for the unoptimized designs. Additionally, these designs were compared with a simple branched design for uniform DNA capture, using the kinetics parameters extracted from our first objective. The bio-inspired designs demonstrated high DNA capture uniformity, achieving stabilization up to 10 times faster under varying conditions than a simple branched design. Ultimately, this work offers significant advancements in optimizing three crucial aspects of POC diagnostics i) surface reaction kinetics, by studying and identifying the conditions best suited for planar and nanostructured surfaces in both buffer and complex media, ii) mass transport, by investigating flow effects on biorecognition and detection, and determining the optimal conditions for biosensing, ii) and electrochemical biosensing and microfluidics integration and design, by utilizing the optimized parameters for nanostructured surface and develop a rapid, continuous, and real-time microsystem for L. pneumophila detection meeting the regulatory standards. For the second generation of this microsystem, the two bio-inspired designs will enable multiplexed detection of various pathogens. These contributions collectively are pivotal to the development of next generation POC diagnostics, with broad applications in environmental, clinical, and food safety monitoring.