Assay Development for the Point of Care Detection of Small Molecules for Diagnostic: Two Biomarkers, L-Kynurenine and Ethanolamine, as Examples

Abstract

Diagnostic assays for the detection of small molecules play a key role in modern medicine, as they enable the precise identification of biomarkers, metabolites, drugs, and environmental toxins in biological samples such as blood, urine, or tissue. Traditionally, techniques like mass spectrometry and chromatography are used, providing in-depth insights into disease processes, pharmacokinetics, and patients’ responses to therapeutic interventions due to their high sensitivity and specificity. However, these methods are often complex, expensive, and require specialized equipment and expertise. This dissertation focuses on the development of mass spectrometry-independent diagnostic assays for the detection of small molecules. The aim was to overcome the limitations of traditional methods and explore the potential of label-free detection techniques in various diagnostic applications. These new methods could form the basis for clinical tests that enable broader availability and application in routine diagnostics. The dissertation is divided into three parts: The first part describes the development of a fluorescence-based, competitive antibody binding assay for L-Kynurenine, a potential biomarker for kidney transplant failure. A fluorescein-labeled kynurenine derivative was synthesized and its binding to specific antibodies in a bead assay was demonstrated, offering a promising approach for a kynurenine point of care test in saliva. The second part focuses on implementing a nanopore platform for the label-free quantification of small molecules, using ethanolamine as a model combound. A strand displacement assay with an ethanolamine-binding aptamer enabled indirect detection via commercially available protein nanopores in the micromolar range. Both assays rely on the binding of the target molecule to magnetic beads and employ innovative detection methods that have not been described yet. The third part adresses the fabrication process of solid-state nanopores, comparing their stability, sensitivity, and variability with those of protein nanopores, investigating the challenges and advantages of using solid-state nanopores for the detection of small molecules. In summary, this dissertation contributes to the advancement of diagnostic tests by presenting novel, label-free detection methods for small molecules, which hold great potential for future clinical applications.