A research team at the Living Systems Institute of the University of Exeter has established a method for real-time monitoring of the properties and structure of individual molecules using light.
Using this breakthrough method, the researchers were able to momentarily bridge the molecules together to introduce a central lens into their dynamics.
It is difficult to study the structure of individual molecules and their characteristics such as chirality. In the latest study, headed by Professor Frank Vollmer, the researchers were able to watch the reactions at the nanoscale, which otherwise would not have been accessible.
The primary way disulfide bonds are created and reorganized in a protein—or thiol/disulfide exchange—has not been fully explored yet at equilibrium at the single-molecule level, partly because this phenomenon cannot be optically determined in the case of large samples.
But light can form resonances by circulating around tiny-sized glass spheres. The captured light can then continuously communicate with its surrounding environment. When gold nanoparticles are attached to the sphere, light is enhanced and spatially limited down to the size of amino acids and viruses.
The ensuing optoplasmonic coupling helps detect biomolecules that come close to the nanoparticles, while attaching to the gold, detaching, and interacting in many different ways.
While this method is quite sensitive, it lacks specificity. For example, molecules as fundamental as atomic ions can be identified and specific dynamics can be distinguished, but one cannot essentially differentiate them.
It took some time before we could narrow down how to reliably sample individual molecules. Forward and backward reaction rates at equilibrium are counterbalanced and, to certain extent, we sought to lift the veil over these subtle dynamics.
Serge Vincent, PhD Student, University of Exeter
Reaction pathways controlled by disulfide bonds can limit the interactions to single thiol-sensing sites present on the nanoparticles. The method’s high fidelity establishes accurate probing of the properties of molecules experiencing the reaction.
When linkers are placed on the gold surface, communications with the thiolated species are separated depending on their charge and also on the cycling itself.
Sensor signals exhibit distinct patterns in relation to the presence or absence of a reducing agent. If it is present, the signal will rotate in a controlled manner, and if it is absent, the oscillations turn out to be stochastic.
For every reaction, the dimer state or the monomer state of the leaving group can be determined.
Remarkably, interactions between the optoplasmonic resonance and single molecules cause the former to change in frequency and/or shift in linewidth.
In a majority of cases, such a result indicates a plasmon-vibrational coupling that may help detect individual molecules, ultimately accomplishing characterization.
This excellent work by my PhD student, Serge Vincent, paves the way for many future single-molecule analysis techniques that we have only been dreaming about. It is a crucial step for our project ULTRACHIRAL. ULTRACHIRAL seeks to develop breakthroughs in how we use light to analyse chiral molecules.
Frank Vollmer, Professor, University of Exeter
Vincent, S., et al. (2020) Optoplasmonic characterisation of reversible disulfide interactions at single thiol sites in the attomolar regime. Nature Communications. doi.org/10.1038/s41467-020-15822-8.