Coupling of Optical tweezers with Tip-enhanced Raman Spectroscopy for single-molecule investigation of supramolecular systems

Project Information

TweeTERS

Grant agreement ID: 892667

DOI

10.3030/892667

Project closed

EC signature date 20 March 2020

Start date 1 October 2020

End date 1 April 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 160 932,48

EU contribution

€ 160 932,48

Coordinated by

FUNDACION IMDEA NANOCIENCIA
Spain

Periodic Reporting for period 1 - TweeTERS (Coupling of Optical tweezers with Tip-enhanced Raman Spectroscopy for single-molecule investigation of supramolecular systems)

Reporting period: 2020-10-01 to 2022-09-30

Summary of the context and overall objectives of the project

Synthetic supramolecular systems consist of an ensemble of molecules held together by non-covalent interactions. Among such forces, hydrogen bonds are particularly relevant in fundamental biological and chemical processes. From the fundamental point of view, understanding non-covalent interactions is paramount to disentangle the chemistry of biological systems. One of the main challenges in the field nowadays is the investigation of single-molecule events under non-equilibrium conditions. To this end, optical trapping (OT) experiments have been successfully implemented to determine the mechanical properties and operational dynamics of synthetic supramolecular systems in the single-molecule regime and under aqueous environments. This is achieved by exerting calibrated forces on systems immobilized between optically trapped particles and measuring the corresponding displacements in real time. For example, characteristic H-bond strength have been determined in situ for single host-guest pairs as well as real-time shuttling of molecular shuttles using optical trapping.
Despite the essential information that optical force microscopy experiments provide, there is still a lack of quantitative chemical information. Raman spectroscopy is one of the most powerful tools in analytical chemistry since it can access the vibrational spectrum of samples in a non-invasive way, from which the specific composition of a sample, its conformation and interaction between species can be determined. Consequently, Raman is an ideal tool to provide complementary chemical characterization in OT experiments, particularly in biologically relevant aqueous environments. However, due to the small scattering cross-sections of the Raman process, nearfield approaches using metallic nanoantennas have to be used to access the Raman spectrum of a single molecule. In tip-enhanced Raman spectroscopy (TERS) the excitation light is coupled to the apex of nanometric metallic tip, resulting in a highly enhanced and confined nearfield around the tip apex. The Raman signal from molecules within the nearfield is enhanced by several orders of magnitude with respect to the farfield signal, boosting the sensitivity of the process to the single-molecule limit.
By combining state-of-the-art technologies in the fields of OT and nearfield Raman spectroscopy, the main objective of TweeTERS is to create a hybrid tool that can disentangle the relation between mechanical, conformational and chemical properties of individual synthetic supramolecular systems and the non-covalent interactions governing their behavior, with single-molecule sensitivity and spatial resolution in the range of 5-10nm. On the one hand, we will be able to follow in real-time the formation and breaking of individual non-covalent interactions both kinetically and chemically tackling open questions in the field of supramolecular chemistry from a completely new approach. On the other hand, this unique novel instrument will merge two state-of-art single molecule techniques, resulting in a versatile setup with applicability far beyond a single research field and topic pushing the limits of current technology in the single molecule regime.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The setup development has been one of the main activities of the grant, which is at the moment in the final stage of construction. The final setup design consists of a dual trap geometry, i.e a molecule is embedded between optically trapped beads, used to apply forces in the pN range and measure the corresponding molecular elongation. At the same time, a visible laser is focused at the apex of a metallic tip, which is used as nanometric Raman probe. A single objective (used for focusing the 3 beams) is fixed next to a fluidic cell (with the tip mounted steadily on it). The sample cell can be freely moved with nanometric precision (3D piezo stage) with respect to the objective, allowing the positioning of the tip apex at the exact focus of the Raman light. At the same time, the two optically trapped beads with a molecule embedded between them can be independently moved around the tip by changing the incidence angle of the OT beams into the objective using steering mirrors. Currently, all required elements are purchased and are being installed. The project long-term viability is ensured through a collaboration with the company supplying the optical twezeers as well as the investment of the host institution.

Moreover, we have worked on the investigation of supramolecular systems and their interactions by means of optical tweezers. We have studied the shuttling dynamics of rotaxane-based molecular shuttles as a function of salt concentration in the environment. We have found that increasing NaCl concentration, the coexistence force at which the macrocycle resides equally in both stations increases. This result implies that the hydrogen bonding between macrocycle and stations weakens at lower concentration of ions. Energy landscapes and kinetic rates are calculated for each salt concentration, showing the change in the energy barriers as a function of applied force. Moreover, we were able to observe in situ the change in dynamics upon increase/decrease of salt concentration, in an impressive live experiment. These results open the door to the systematic extension of OT to the field of supramolecular chemistry and a publication describing them is currently under preparation.

Finally, the fellow has been involved in several side-projects and collaborations, related with different aspects of the TweeTERS project. Within the enhanced-Raman spectroscopy field, we have participated in the spectroscopic characterization of nanomaterials (TMDCs, carbon-based materials and heterostructures of those). In the field of optical tweezers, we are currently working on a project where we use OT hoping results of a molecular shuttle to demonstrate experimentally the principle of microscopic reversibility, expanding the application of the technique to the fundamental understanding of chemical principles.

The project has been very productive scientifically, with 5 publications in high impact journals, and 4 additional ones in peer-review or preparation phases. Furthermore, it has been presented in 5 conferences.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

TweeTERS has been consolidated as a new research line at IMDEA nanoscience. In the long-term, we are planning to use the setup for the investigation of different supramolecular systems other than artificial molecular shuttles (for example, host-guest systems) and mechanically-interlocked nanotubes. The capabilities of the OT-TERS instrument will allow us to understand the role of mechanical bonds in the conformation of those systems in the single molecule regime. The developed instrument has potential beyond the field of supramolecular chemistry and is expected to boost the applicability of optical tweezers to different areas of science in biology, chemistry and physics.
Regarding the innovation capacity of the TweeTERS project, the work performed during this grant included the purchase of a state-of-art optical trapping system from the company Lumicks (Netherlands), which is being customized to include simultaneous tip-enhanced Raman acquisition. Once finalized, it will add a unique functionality to the conventional optical trapping instruments (simultaneous acquisition of vibrational spectra in the single molecule limit) with potential for new users and market opportunities.

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