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Label-Free Measurements of Amyloid Formation by Suspended Microchannel Resonators

dc.contributor.advisorBurg, Thomas Dr.
dc.contributor.authorWang, Yu
dc.date.accessioned2014-12-12T09:25:42Z
dc.date.available2014-12-12T09:25:42Z
dc.date.issued2014-12-12
dc.identifier.urihttp://hdl.handle.net/11858/00-1735-0000-0023-995D-E
dc.identifier.urihttp://dx.doi.org/10.53846/goediss-4822
dc.language.isoengde
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/3.0/
dc.subject.ddc530de
dc.titleLabel-Free Measurements of Amyloid Formation by Suspended Microchannel Resonatorsde
dc.typedoctoralThesisde
dc.contributor.refereeSchmidt, Christoph F. Prof. Dr.
dc.date.examination2014-01-15
dc.subject.gokPhysik (PPN621336750)de
dc.description.abstractengAmyloid deposition in tissues is a hallmark of many severe diseases including Alzheimer’s and Parkinson’s disease. The formation of amyloid fibrils involves several intermediate steps, and revealing the kinetics of each step is of great importance for a deeper understanding of the underlying mechanisms. However, conventional colorimetric or fluorescent assays, such as Thioflavin T, are not well suited for precise measurements of aggregation kinetics, as these dyes are only sensitive to specific states of the aggregation and their binding may interfere with the process. Biosensor techniques are superior in this regard since the measurement is label-free. However, established methods such as Surface Plasmon Resonance (SPR) and Quarz Crystal Microbalance (QCM) have several limitations, including large sample consumption, low sensitivity, and complex data analysis. This thesis presents, for the first time, the label-free kinetic measurement of amyloid formation by mass using Suspended Microchannel Resonators (SMR). SMR devices achieve low sample consumption at high mass resolution and provide a direct readout of the buoyant mass of aggregates. As a prerequisite for quantitative measurements of amyloid elongation kinetics by SMR, a new method for the reliable immobilization of seed fibrils inside the device was first established. The method allowed the reproducible deposition of ensembles of insulin amyloid fibrils inside the 3 m tall detector channels without clogging. To this end, a robust vapor-based method for the deposition of aminosilane layers in closed microfluidic systems has been developed, and a novel quantitative fluorescence imaging method was introduced to optimize the density, quality and stability of these layers in situ. In the surface-based SMR measurements, the reproducible seed fibril immobilization has been confirmed by having consistent surface fibril density of 734 +/- 89 molecules per square micrometers. After prevention of non-specific protein adsorption and validation of the stability, the fibril elongation rates and the absolute rate constants can be readily determined. For 1 mg/mL monomer concentration, the results show that the binding of a monomer to a fibril occurs every 4.9 +/- 0.5 s, corresponding to the rate constant of (1.2 +/- 0.1) x10<sup>3</sup> M<sup>−1</sup> s <sup>−1</sup>  at 37 ° C. Further on-rate measurements depending on monomer concentration and temperature provide insights into the mechanism and thermodynamics of the process. A two-step "dock-lock" elongation model fits well with the measurements. The determined enthalpy and the entropic contribution of the activation are in agreement with literature data and indicate in general an enthalpically unfavorable but entropically favorable process. In the last part of the thesis, effects of shear flow and additives such as metal ion salts, natural products and small organic compounds, have been studied by measuring the fibril elongation rates. In contrast, dissociation experiments using the surface-based SMR technique have been applied to monitor the degradation of the immobilized fibrils. Both approaches are demonstrated to be promising platforms for screening potential inhibitors and therapeutic drugs for amyloid-related diseases.de
dc.contributor.coRefereeBurg, Thomas Dr.
dc.subject.engMicrofluidicsde
dc.subject.engLab-on-a-chipde
dc.subject.engSurface chemistryde
dc.subject.engAmyloid formationde
dc.subject.engProtein immobilizationde
dc.subject.engProtein aggregationde
dc.subject.engAggregation kinetics and thermodynamicsde
dc.subject.engLabel-free measurementsde
dc.subject.engin vitro diagnosticsde
dc.subject.engHigh-throughputde
dc.subject.engFluorescence microscopyde
dc.identifier.urnurn:nbn:de:gbv:7-11858/00-1735-0000-0023-995D-E-5
dc.affiliation.instituteFakultät für Physikde
dc.identifier.ppn812644735


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