Improving DNA Monolayer-based Electrochemical Sensors through Simplified Data Analysis, Enhanced Sensitivity, and Temporal Response Customization

Abstract

Biomarkers are quantifiable indicators of a biological state. Detection and quantification of biomarkers are essential for disease diagnosis, monitoring disease progression, and assessment of treatment efficacy. However, the standard methods utilized for biomarker detection—such as enzyme-linked immunosorbent assays (ELISA), polymerase chain reaction (PCR), and mass spectrometry (MS)—offer high sensitivity but often require expensive instrumentation and trained personnel to operate. Moreover, the time-consuming and labor-intensive nature of these standard methods limits their applicability as point-of-care diagnostic tools. Recently, DNA monolayer-based electrochemical sensors have gained significant attention due to their high sensitivity, specificity, and rapid label-free detection. Their ease of miniaturization, ability to function in turbid biofluids, and real-time monitoring capabilities make these sensors promising candidates for point-of-care diagnosis. The Easley research group has previously reported several DNA monolayer-based electrochemical sensors, including the bowtie sensor, and the electrochemical proximity assays. These sensors are easy to synthesize, cost effective, and can be modified to detect different classes of clinically relevant molecules. Methylene blue (MB) conjugated to a single-stranded DNA is used as a signaling unit in these sensors, which undergoes electron transfer with the gold electrodes upon voltametric interrogation. The aim of this dissertation is to improve these DNA monolayer-based sensors and facilitate their translation toward real-world applications through advances in sensor design, enhancing sensitivity, enabling multiplexing, simplifying the instrumentation, and improved data interpretation. Chapter 1: Introduces the basic principles of sensor architecture and discusses the evolution of DNA monolayer-based electrochemical sensors over the last few decades. Chapter 2: Discusses the development of a MATLAB-based graphical user interface (GUI) for automated data analysis and data visualization. The program is highly efficient for baseline approximation and storing both faradaic and nonfaradaic currents separately. The batch processing ability of the program is also validated. Chapter 3: Introduces a modified redox probe (MB5) for DNA monolayer-based sensors. The applicability of the redox probe is tested across three different sensor assemblies to enhance sensitivity and improve the stability and reproducibility of the sensor responses. An equilibrium binding model is also introduced for calibration-free quantification of estradiol using the bowtie sensor assembly with the modified redox probe. Chapter 4: Temporal response of the bowtie sensor and a short strand DNA sensor were tuned to achieve significant faradaic current when nonfaradaic current decays away. We hypothesized that the MB5 probe, when placed furthest away from the surface, can retain the faradaic current long enough to allow nonfaradaic current to approach zero. Sensor response at this stage will only contain faradaic current. Hence, no digital or hardware separation or subtraction of nonfaradaic current will be required. Strategies to perform multiplexing or ratiometric sensing have also been discussed. Chapter 5: Presents the adaptation of a fluorescence-based antibody detection assay to an electrochemical platform. Effect of the flexible polyethylene glycol linkers in the sensor architecture were utilized to improve the limit of detection (LOD). The assay applicability was tested in 90% human serum in the presence of increased ionic strength. Chapter 6: Summarizes this dissertation and outlines potential directions for future research.