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Nanoscale Vibrational Spectroscopy of Sensitive 2D Molecular Materials

Periodic Reporting for period 3 - 2DNanoSpec (Nanoscale Vibrational Spectroscopy of Sensitive 2D Molecular Materials)

Reporting period: 2020-09-01 to 2022-02-28

Two-dimensional (2D) materials are the materials that are only a few, or sometimes one atom thin, i.e. the thinnest one could possibly imagine (e.g. graphene). There is no doubt that 2D materials will shape the world of tomorrow. Production technology has dramatically improved with time, but analytical methods tend to fail in characterizing their nanometer-size structure; obtaining information from such a small quantity of matter is very hard. Tip-enhanced Raman spectroscopy (TERS) offers a solution to this problem. It combines scanning probe microscopy, which is able to provide topographical information with lateral resolutions below 1 nm, with Raman spectrometry, which gives details about the chemical nature of the sample with a similar resolution. During TERS imaging, the samples are exposed to very intense electromagnetic fields that could damage them, one of the main limitations for widespread application of TERS to this area. In this project, our main goal is to understand and control the sample degradation mechanism resulting from the exposure to the intense electromagnetic field and expand the application of TERS to “delicate” samples, such as 2D organic polymers or biological samples. Despite being very challenging, applying TERS to biological membranes represents a pioneering investigation that we hope could lead to a breakthrough in our understanding of how cell membranes are organized.
Initially, much work went into set-up of equipment and data processing pipelines. This is now (April 2022) finished. Our set-up includes TERS set-ups with different scanning probe microscopy feedback (AFM, STM), different illumination and collection geometries (top / side / bottom), and an electrochemical TERS setup for performing experiments in liquid. We have also installed a nitrogen gas box chamber on our TERS system to reduce the contamination of the sample and degradation of the STM-TERS tip during the measurement, this has also helped in extending the lifetime of the TERS tip, hence we can perform a high-resolution TERS map without loss of enhancement of the tip.

In terms of sample preparation, much work was done to mitigate contaminants in the lipid membrane studies. Artificial membranes formed on a Langmuir-Blodgett trough and transfer of lipid monolayers onto template stripped gold, for STM-TERS measurement has been optimized. We have performed isotherm studies of the cationic antimicrobial peptide indolicidin and its interaction with DOPC. We have measured confocal Raman spectra of the peptide and identified the Raman bands of interest. Deposition of biomimetic and bacterial cell membranes (bilayers) on gold via vesicle fusion was optimized. We have achieved STM-TERS imaging of a DPPC self-assembled monolayer on gold, and are working on high-resolution chemical imaging of biphasic lipid systems consisting of DPPC and deuterated (d31) SM.

For the investigation of soft samples like polymers and biological samples, we studied the correlation between TERS signal and AFM feedback mode in AFM-TERS experiments. Counterintuitively, tapping mode yielded a systematically higher Raman signal intensity, despite the weaker probe-sample interaction. We interpreted this result as a consequence of the lower molecular perturbation generated in tapping mode. Conversely, the strong probe-sample interaction in contact mode can alter the structure and orientation of the sample, leading to a lower signal. These findings are of key relevance for the development of TERS of delicate samples.

For providing a better understanding of TER spectra, we simulate Raman spectra of molecular systems, allowing the assignment of the complex TERS and Raman spectra. We have written a robust software code for processing and analyzing the TERS data, and are continuously updating it, adding high-level data analysis routines to extract the maximum amount of information from our data. Furthermore, it also helps us in understanding and dealing with the large amount of data generated in our experiments.

For typical metal-organic coordination systems in biological samples, we achieved the molecular-scale chemical imaging of the molecular specificity, orientation, and spatial distribution of model coordination complexes using TERS. We were able to visualize the soft coordination species spectroscopically on the scale of a single molecule under ambient conditions, which has laid the foundation for further in situ molecular-scale TERS imaging of the biological dynamics on surfaces.

A lot of work was done to improve the performance of the bottom-illumination AFM-TERS system, starting with the instrument realignment up to designing a data processing software. This system was used in a comprehensive study of polystyrene (PS)-poly(methyl methacrylate) (PMMA) polymer blend film in which it is well known that the polymers phase separate laterally. However, we also detected a vertical phase separation of polymers within 20 nm from the sample surface, which was not detected before. The same AFM-TERS system was also used to study pancreatic cancer cells (BxPC3 cell line). High resolution of this instrument allowed us to reveal segregation of lipids and proteins and distinguish between cell domains rich in different proteins, which we couldn't detect using the confocal Raman spectroscopy. These studies demonstrate TERS potential to study soft molecules at nanoscale under ambient conditions.
Progress: TERS is a very powerful nano-spectroscopic technique. Not only it allows imaging of thin materials at ~10 nm spatial resolution, but it also yields chemical information. This makes it very useful in studying 2D materials which are very promising and useful in fields such as electronics, computer science, catalysis, biology, etc. We have studied reversible photoisomerization of molecular switches using TERS which are very promising materials for molecular electronics and high-density data storage. We have also elucidated the mechanism of sample degradation under the TERS tip which is very useful in optimizing the future TERS experiments of fragile samples, such as peptides, proteins, biological membranes, etc. We investigated the sample-probe interaction regimes in AFM-TERS in order to understand which mode should be the preferred one for the characterization of soft samples.

Expected Results: 1. To obtain a better theoretical understanding of TER spectra of complex biological model systems, using chemometrics and data analysis, and to better understand the TER response of 2D materials. 2. Achieve TERS imaging of model biological systems (lipid monolayers, bilayers) using STM and AFM-TERS respectively. 3. Obtain stable TERS maps of biphasic lipid membrane systems, to differentiate between the different lipid components. 4. See the interaction of peptides (such as indolicidin) and pore forming molecules (such as amphotericin B) in a lipid bilayer, and chemically map their surrounding lipid environment to better understand their mechanism of action. 5. Perform TERS studies of biomolecules in the native-like environment, e.g. physiological fluids. Our ultimate aim is to do TERS imaging of living cells, and we are confident that we are moving in the right direction towards achieving it.
Schematic representation of a TERS experiment