Nanoliter-droplet thermophoresis for biomedical applications

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.