Rapid Fractionation and Characterisation of Alpha-Synuclein Oligomers in Solution

Arter, William E.; Xu, Catherine K.; Castellana-Cruz, Marta; Herling, Therese W.; Krainer, Georg; Saar, Kadi L.; Kumita, Janet R.; Dobson, Christopher M.; Knowles, Tuomas P. J.

doi:10.1101/2020.03.10.985804

Cited by 2 publications

(1 citation statement)

References 33 publications

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“…Prior to its use, the aptamer (100 µM stock in 1X TE buffer) was heated to 70°C and cooled to room temperature to facilitate correct folding. The α-synuclein fibrils were prepared by incubating α-synuclein monomer as described earlier 49,50 and sonicated (10 % power, 30 % cycles for 1 min; Sonopuls HD 2070, Bandelin). Following sonication, the fibrils were spun down and re-suspended in respectively.…”

Section: Methodsmentioning

confidence: 99%

Direct digital sensing of protein biomarkers in solution

Krainer

Saar

Arter

et al. 2020

Preprint

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Highly sensitive detection of proteins is of central importance to biomolecular analysis and diagnostics. Conventional protein sensing assays, such as ELISAs, remain reliant on surface-immobilization of target molecules and multi-step washing protocols for the removal of unbound affinity reagents. These features constrain parameter space in assay design, resulting in fundamental limitations due to the underlying thermodynamics and kinetics of the immunoprobe–analyte interaction. Here, we present a new experimental paradigm for the quantitation of protein analytes through the implementation of an immunosensor assay that operates fully in solution and realizes rapid removal of excess probe prior to detection without the need of washing steps. Our single-step optofluidic approach, termed digital immunosensor assay (DigitISA), is based on microfluidic electrophoretic separation combined with single-molecule laser-induced fluorescence microscopy and enables calibration-free in-solution protein detection and quantification within seconds. Crucially, the solution-based nature of our assay and the resultant possibility to use arbitrarily high probe concentrations combined with its fast operation timescale enables quantitative binding of analyte molecules regardless of the capture probe affinity, opening up the possibility to use relatively weak-binding affinity reagents such as aptamers. We establish and validate the DigitISA platform by probing a biomolecular biotin–streptavidin binding complex and demonstrate its applicability to biomedical analysis by quantifying IgE–aptamer binding. We further use DigitISA to detect the presence of α-synuclein fibrils, a biomarker for Parkinson’s disease, using a low-affinity aptamer at high probe concentration. Taken together, DigitISA presents a fundamentally new route to surface-free specificity, increased sensitivity, and reduced complexity in state-of-the-art protein detection and biomedical analysis.

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Section: Methodsmentioning

confidence: 99%