Nanoliter-droplet thermophoresis for biomedical applications

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