DNA-based Proximity Assays for Biomarker Detection Using Fluorescence and Electrochemistry with Improved Performance through Probe Flexibility

Abstract

Detection of biomarkers is important in understanding the mechanisms underlying a disease and developing therapeutics for treatment and cure. Although there are several sensitive and specific analytical techniques revolving around complex instrumentation and labor-intensive workflows for biomolecular detection, there is a demand to develop more simpler and sensitive techniques which are easy to use, even with less technical skill and knowledge. In this dissertation, we present our contributions towards developing simpler assays for biomarker sensing leveraging DNA-based proximity assays. A key contribution of this work is the demonstration that increased probe flexibility can result in better assay performance in several platforms. In chapter 1 of this dissertation, we discuss the basic principles of using DNA strands for biomolecule analysis, where focus is directed towards fluorescence and electrochemical detection platforms. In chapter 2 we present our attempts at developing a proximity-based assay for protein sensing using a green fluorescent protein (GFP) mimicking RNA aptamer, broccoli. The broccoli aptamer was split into two strands and fused to target sensing units, where spontaneous binding to protein of interest was expected to fuse the split RNA and form the fluorophore binding pocket. The change in fluorescence was monitored through thermofluorimetric analysis (TFA). In chapter 3, we introduce new applications of TFA, where (1) we leveraged this technique to develop a DNA-based proximity assay for antibody detection. We also demonstrate the importance of conformational flexibility of DNA probes for enhanced assay performance. To promote hybridization of short DNA strands, ssDNA segments of the designed system were substituted with polyethylene glycol (PEG) linkers, which led to improved assay performance. This simple, mix-and-read assay was shown to function in 90 % human plasma. (2) We used this improved system to study the valency effects of antibody oligonucleotide (AbO) conjugates using TFA, where we were able to clearly distinguish monovalent and multivalent AbOs. Chapter 4 presents an electrochemical detection platform for antibody sensing. We adopted the flexible PEG-linker based system in chapter 3 above and combined it with the previously developed electrochemical proximity assay (ECPA) to achieve improved limits of detection compared to the developed TFA-based assay. We have demonstrated that careful positioning of PEG linkers in the signaling DNA strands improved antibody-dependent signal increase by 4-fold compared to the system without PEG modifications. Furthermore, the developed antibody sensor promoted tethered diffusion of two methylene blue molecules upon antibody addition, which also contributed to improved signal and detection limit. The assay was functional in 90 % human serum, in the presence of increased ionic strength, which aided in counteracting electric double layer effects and improved shielding effect of the DNA backbone, leading to efficient hybridization. In chapter 5, we introduce our efforts in adopting electrochemical sensors developed for 2D gold-on-glass planar electrodes to gold microelectrodes. In this work we mainly focus on optimizing experimental design and conditions to achieve improved electrochemical sensing of biomolecules leveraging the DNA nanostructure introduced by our group. Chapter 6 provides concluding remarks of the work detailed in this dissertation, including future improvements for projects mentioned.