{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/390448"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/390448","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Microfluidic diffusional sizing at the single molecule level","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Fan, Jieyuan"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Lee, steven","Knowles, tuomas"],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024-04-15","date_published":"2024-04-15","updated_at":"2026-07-22T22:24:11Z","subjects":["Microfluidics","Single molecule","Proteins","Diffusional sizing","Protein affinity","Kd binding affinity","Picomolar detection","Confocal","Microscopy"],"languages":["eng"],"rights":[],"rights_urls":["https://www.repository.cam.ac.uk/bitstreams/41a3de06-cdad-4c04-8922-d54bc8d448f7/download","http://purl.org/NET/rdflicense/allrightsreserved"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.121998","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Lee, steven","Knowles, tuomas"]},{"key":"dc:creator","label":"Author","values":["Fan, Jieyuan"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2024-04-15"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/390448"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Microfluidics","Single molecule","Proteins","Diffusional sizing","Protein affinity","Kd binding affinity","Picomolar detection","Confocal","Microscopy"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.repository.cam.ac.uk/bitstreams/41a3de06-cdad-4c04-8922-d54bc8d448f7/download","http://purl.org/NET/rdflicense/allrightsreserved"]},{"key":"dc:rights.embargodate","label":"Dc Rights Embargodate","values":["2026-10-06"]},{"key":"dc:rights.embargotype","label":"Dc Rights Embargotype","values":["embargo"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.121998"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/7cda5647-bad5-442c-9f25-56f6c3a28164/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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. 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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. 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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. 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