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• W2031219710 abstract "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 atomic force microscopy (AFM) and transmission electron microscopy (TEM), were covalently immobilized inside microchannels embedded within a micromechanical resonator, and the elongation of these fibrils under a 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) × 10(3) M(-1) s(-1). Rate saturation occurred at higher concentrations. The nonlinear on-rate for monomer concentrations from 0 to 6 mg/mL and for temperatures from 20 to 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." @default.
• W2031219710 created "2016-06-24" @default.
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• W2031219710 date "2015-01-14" @default.
• W2031219710 modified "2023-10-18" @default.
• W2031219710 title "Label-Free Measurement of Amyloid Elongation by Suspended Microchannel Resonators" @default.
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• W2031219710 doi "https://doi.org/10.1021/ac503845f" @default.
• W2031219710 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/25539393" @default.
• W2031219710 hasPublicationYear "2015" @default.
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