Optoplasmonic characterisation of reversible disulfide interactions at single thiol sites in the attomolar regime

Serge Vincent,Frank Vollmer,Sivaraman Subramanian

doi:10.1038/s41467-020-15822-8

Serge Vincent, Frank Vollmer + Show 1 more

Open Access

https://doi.org/10.1038/s41467-020-15822-8

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Journal: Nature Communications	Publication Date: Apr 27, 2020
Citations: 36	License type: open-access

Affiliation: University of Exeter

Abstract

Probing individual chemical reactions is key to mapping reaction pathways. Trace analysis of sub-kDa reactants and products is obfuscated by labels, however, as reaction kinetics are inevitably perturbed. The thiol-disulfide exchange reaction is of specific interest as it has many applications in nanotechnology and in nature. Redox cycling of single thiols and disulfides has been unresolvable due to a number of technological limitations, such as an inability to discriminate the leaving group. Here, we demonstrate detection of single-molecule thiol-disulfide exchange using a label-free optoplasmonic sensor. We quantify repeated reactions between sub-kDa thiolated species in real time and at concentrations down to 100’s of attomolar. A unique sensing modality is featured in our measurements, enabling the observation of single disulfide reaction kinetics and pathways on a plasmonic nanoparticle surface. Our technique paves the way towards characterising molecules in terms of their charge, oxidation state, and chirality via optoplasmonics.

Highlights

Probing individual chemical reactions is key to mapping reaction pathways
A gold NP surface serves as an effective detection area for biomolecular characterisation on an optoplasmonic sensor
Thiol and amine based immobilisation has been explored on our optoplasmonic sensor[33]

Summary

Introduction

Probing individual chemical reactions is key to mapping reaction pathways. Trace analysis of sub-kDa reactants and products is obfuscated by labels, as reaction kinetics are inevitably perturbed. The thiol-disulfide exchange reaction is of specific interest as it has many applications in nanotechnology and in nature. We demonstrate detection of single-molecule thiol-disulfide exchange using a label-free optoplasmonic sensor. We quantify repeated reactions between sub-kDa thiolated species in real time and at concentrations down to 100’s of attomolar. A unique sensing modality is featured in our measurements, enabling the observation of single disulfide reaction kinetics and pathways on a plasmonic nanoparticle surface. The application of fluorescent optical methods to investigate a single molecule’s reaction pathway is often non-trivial. Disulfide bonds are a fulcrum for cell biochemistry Reactions that form these links usually occur post-translation, stabilising folding and providing structure for a number of proteins[9,10,11]. Thiol/disulfide equilibria can be quantified in bulk, often at the expense of high kinetic reactivity and the need for fluorescent or absorptive reagents to measure the exchange[14]

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