Nanoliter-droplet thermophoresis for biomedical applications (2014)

Specific interactions of biomolecules are central to cellular processes, drug discovery and immunodiagnostics. Such biological binding events are quantifiable via thermophoresis, the directed molecule movement driven by a temperature gradient. Biomolecule thermophoresis can be induced by infrared laser heating and analyzed using fluorescence. The objective of this thesis was to enhance and optimize these all-optical measurements, regarding instrumentation, assay design and biomedical applications. In the first part, a novel measurement device and approach are presented, which cut down sample consumption 50-fold compared to established capillary thermophoresis. Instead of capillaries, analysis was performed in 10 nl-sample droplets transferred into standard 1536-well plates with a non-contact liquid handler (Labcyte). To prevent evaporation, the aqueous sample droplets were stabilized in an oil-surfactant mix. Temperature induced effects in this water-in-oil system were experimentally characterized and the results agreed with numerical simulation. The system’s applicability for biomolecular interaction analysis was confirmed with a DNA aptamer. The achieved miniaturization and the easy-to-handle multi-well plate format promote automated high-throughput screens. Besides aptamers, proteins should also be measurable very well when judging from the application depth of capillary measurements. This versatility of protein investigation via capillary thermophoresis is demonstrated in the second part. Successful experiments were not only conducted in diverse liquids including crude cell lysate, but also for binding partners with a broad range of molecular weight ratios. Affinities between protein and protein, protein and peptide, as well as protein and small molecule were determined with high accuracy. Further flexibility arises from the herein presented label free approach which utilizes protein intrinsic UV fluorescence. It is caused by aromatic amino acids with tryptophan being the major intrinsic fluorophore. This approach exempts from the need to attach a dye, which saves time and excludes labeling artifacts. The wide variety of proteins that can be analyzed with thermophoresis also includes anti-bodies. Two applications of such thermophoretic immunoassays are introduced in the third part. Firstly, the therapeutically interesting antibody MCPR3-7 was assessed. MCPR3-7 binds proteinase 3 (PR3), the major autoimmune target in granulomatosis with polyangiitis. Thermophoresis allowed to quantified MCPR3-7’s affinity and selectivity for different PR3 forms. In addition, it revealed that the antibody interferes with the complexation of PR3 and alpha-1-proteinase inhibitor (alpha-1PI). Secondly, a diagnostic autocompetition assay is described, which directly determines affinity and concentration of disease related biomarkers. It was applied for autoantibodies against the cardiac β1-adrenoceptor found in patients suffering from dilated cardiomyopathy. To detect these autoantibodies, the small peptide COR1 mimicking the adrenoceptor’s dominant epitope served as an artificial antigen. This tracer was labeled with a red-fluorescent dye, which ensured selectivity for measurements directly in untreated human blood serum. The results prove that thermophoresis is a valuable tool to characterize antibodies including those of diagnostic value and those with a therapeutic potential. Taken together, the presented innovations in assay design and the novel nl-droplet approach can be expected to considerably widen the application spectrum of thermophoresis in fundamental research, industrial drug discovery and clinical laboratory diagnostics.