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要旨:
Protein aggregation is a widely studied phenomenon that is associated with many human diseases and with the degradation of biotechnological products. Here we establish a new label-free method for characterizing the aggregation kinetics of proteins into amyloid fibrils by Suspended Microchannel Resonators (SMR). SMR devices are unique in their ability to provide mass based measurements under reaction-limited conditions in a 10 pL volume. To demonstrate the method, insulin seed fibrils of defined length, characterized by AFM and TEM, were covalently immobilized inside microchannels embedded within a micromechanical resonator, and the elongation of these fibrils under continuous flow of monomer solution (rate ~1 nL/s) was measured by monitoring the resonance frequency shift. The kinetics for concentrations below ~0.6 mg/mL fits well with an irreversible bimolecular binding model with the rate constant kon = (1.2 ± 0.1) x 103 M-1 s-1. Rate saturation occurred at higher concentrations. The non-linear on-rate for monomer concentrations from 0 - 6 mg/mL and for temperatures from 20 – 42°C fit well globally with an energy landscape model characterized by a single activation barrier. Finally, elongation rates were studied under different solution conditions and in the presence of a small molecule inhibitor of amyloid growth. Due to the low volume requirements, high precision, and speed of SMR measurements, the method may become a valuable new tool in the screening for inhibitors and the study of fundamental biophysical mechanisms of protein aggregation processes.
