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.
Point of Care