title: Label-free measurement of amyloid elongation by suspended microchannel resonators.
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.
authors.affiliation: Max Planck Institute for Biophysical Chemistry , Am Fassberg 11, 37077 Göttingen, Germany.
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.forename: Mario Matteo
authors.initials: MM
authors.lastname: Modena
authors.forename: Mitja
authors.initials: M
authors.lastname: Platen
authors.forename: Iwan Alexander Taco
authors.initials: IA
authors.lastname: Schaap
authors.forename: Thomas Peter
authors.initials: TP
authors.lastname: Burg
mesh_terms: D000682:Amyloid / Q000737:chemistry*; D004867:Equipment Design; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry; D007328:Insulin / Q000737:chemistry*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D066329:Protein Aggregates
chemical_list: D000682:Amyloid; D055767:Immobilized Proteins; D007328:Insulin; D066329:Protein Aggregates