Microfluidic diffusional sizing at the single molecule level

As the need for single-molecule techniques grows, commensurate with the desire to study complex samples and the heterogeneity of systems, this project presents the single-molecule microfluidic diffusional sizing (sm-MDS) platform and demonstrates its capabilities at performing diffusional sizing at picomolar sensitivity. Microfluidic diffusional sizing (MDS) is a robust, in-solution method to measure hydrody- namic radii (Rhyd) of particles. The range of MDS is very much suited to measuring the size of individual proteins and higher-order protein assemblies, and MDS has indeed been applied to good effect to study proteins and the strength of their interactions. However, conven- tional MDS is inherently an ensemble level method - it is performed in the widefield optical regime, and it is fluorescence intensity data that is used for analysis and calculation, which ultimately gives an ensemble-averaged measurement. This limits its detection sensitivity to the nanomolar regime, and limits its capability to probe heterogenous mixtures. Here, single-molecule microfluidic diffusional sizing has been achieved for the first time by a combination of confocal illumination and digital single molecule counting in the data analysis process. Operating in the single-molecule regime significantly improves the detection limit of platform, allowing the measurement of Rhyd of particles in solution at concentrations down to 1 pM. Consequently, this allows access to the picomolar range of dissociation constants, KD, in a surface-free way. Picomolar KD corresponds to high-affinity binding and is of significant interest in many fields, but is challenging to measure in free solution - the capability of sm-MDS to do so is thus a notable addition to the toolbox for characterising high-affinity binding. Crucially, picomolar sensitivity also opens up the method to studying heterogenous sample mixtures by consideration of single molecules instead of requiring a deconvolution of aggregated intensity, and this is applied in the first instance to a mixture of monomers and oligomers of the protein α-synuclein to demonstrate.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Fan, Jieyuan

Advisors dc:contributor.advisor

• Lee, steven
• Knowles, tuomas

• Microfluidics
• Single molecule
• Proteins
• Diffusional sizing
• Protein affinity
• Kd binding affinity
• Picomolar detection
• Confocal
• Microscopy

Licence

• https://www.repository.cam.ac.uk/bitstreams/41a3de06-cdad-4c04-8922-d54bc8d448f7/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.121998

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/390448