| dc.description.abstract | Biomarker analysis plays a decisive role in health care. This role is not only limited to disease diagnosis, but it also involves understanding of biological and physiological functions in order to monitor health, observe the efficiency of treatment, etc. This calls for a need of better methods for clinical analysis in several non-clinical point-of-care (POC) settings, including analysis for patients at home. On the focus of biomarker detection, many sensitive methods are being developed. However, only the methods which are inexpensive, sensitive, automated, and rapid pass as POC systems, which can serve to speed up health improvements, especially in developing countries. One standout among these methods is the electrochemical sensor. Due to its inexpensive instrumentation with well-developed miniaturization strategies, electrochemistry has proven to be ideal for POC settings. This dissertation focuses on the development of DNA-based electrochemical methods toward an effective drop-and-read quantification of multiple classes of biomarkers, namely small molecules, oligonucleotides, and proteins.
In Chapter 1 we discuss conventional electrochemical DNA-based biomarker detection methods and related developments for POC and real-time systems.
In Chapter 2, the electrochemical proximity assay (ECPA), a sensitive protein detection and potential POC method developed in our lab is discussed. We have completed studies that can support the development and quick expansion to quantify biologically relevant molecules. ECPA was applied to leptin and insulin quantification, and insulin was successfully detected in the presence of undiluted, un-spiked human serum. We further highlight the importance of understanding that ECPA sensing complex is made of six components undergoing non-covalent binding (DNA hybridization and antigen-antibody interactions) on the surface of the electrode, which is kinetically and thermodynamically different from complex formation in solution. In addition to that, square-wave voltammetry (SWV), a relatively complicated but very sensitive electrochemical technique is employed.
Chapter 3 focuses on the understanding of surface hybridization, and its effects on electrochemical kinetics by way of the distance between the redox moiety and the electrode. Most importantly, we offer a different outlook on interpreting SWV signal and the efficiency of SWV frequency, which is useful for any electrochemical DNA-based sensor.
In Chapter 4, we developed an electrochemical oligonucleotide quantification method, specifically designed for short length nucleotides (~20 nt) in hopes of developing a mix-and-read quantification for micro-RNA (miRNA). We have developed a system for fine tuning or optimizing electrochemical DNA sensor by carefully controlling hybridization energies, SWV frequency, and temperature. In addition to this, we used polydimethylsiloxane (PDMS) electrochemical cells fabricated from 3D-printed polylactic acid molds to reduce the volume of our sample and reagents (10 µL).
Chapter 5 depicts the development of a generalizable drop-and-read quantification method which is interchangeable between protein and small molecule. A change in the rate of diffusion in the redox moiety is used for quantification, and protein is used as an anchor (large molecule weight) which measurably slows diffusion. By exploiting enzymatic DNA ligation, a DNA nanostructure was built on the surface of the electrode, providing control over the orientation of various tags. In this work, we placed the redox moiety in close proximity to an anchor recognition unit, which helps in aiding signal change by the anchor. As a proof of concept, streptavidin and biotin were quantified. Digoxigenin and anti-digoxigenin antibody complexes also show promising results.
Finally, chapter 6 summarizes this dissertation and provides an insight on future projects stemming from this work. Concepts toward the development of fully surface-confined, real-time electrochemical sensors of a wide range of analytes are proposed, and proposals to improve the sensitivity of our nanostructure are also outlined. | en_US |
