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1. Probing protein aggregation at buried interfaces: distinguishing between adsorbed protein monomers, dimers, and a monomer–dimer mixture in situ

   https://doi.org/10.1039/d1sc04300e  

   Lu, Tieyi; Guo, Wen; Datar, Prathamesh M.; Xin, Yue; Marsh, E. Neil; Chen, Zhan (January 2022, Chemical Science)

   Protein adsorption on surfaces greatly impacts many applications such as biomedical materials, anti-biofouling coatings, bio-separation membranes, biosensors, antibody protein drugs etc. For example, protein drug adsorption on the widely used lubricant silicone oil surface may induce protein aggregation and thus affect the protein drug efficacy. It is therefore important to investigate the molecular behavior of proteins at the silicone oil/solution interface. Such an interfacial study is challenging because the targeted interface is buried. By using sum frequency generation vibrational spectroscopy (SFG) with Hamiltonian local mode approximation method analysis, we studied protein adsorption at the silicone oil/protein solution interface in situ in real time, using bovine serum albumin (BSA) as a model. The results showed that the interface was mainly covered by BSA dimers. The deduced BSA dimer orientation on the silicone oil surface from the SFG study can be explained by the surface distribution of certain amino acids. To confirm the BSA dimer adsorption, we treated adsorbed BSA dimer molecules with dithiothreitol (DTT) to dissociate these dimers. SFG studies on adsorbed BSA after the DTT treatment indicated that the silicone oil surface is covered by BSA dimers and BSA monomers in an approximate 6 : 4 ratio. That is to say, about 25% of the adsorbed BSA dimers were converted to monomers after the DTT treatment. Extensive research has been reported in the literature to determine adsorbed protein dimer formation using ex situ experiments, e.g. , by washing off the adsorbed proteins from the surface then analyzing the washed-off proteins, which may induce substantial errors in the washing process. Dimerization is a crucial initial step for protein aggregation. This research developed a new methodology to investigate protein aggregation at a solid/liquid (or liquid/liquid) interface in situ in real time using BSA dimer as an example, which will greatly impact many research fields and applications involving interfacial biological molecules. 

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2. In-plane orientational motions of the functional groups of molecules at the air/water interface by time-resolved vibrational sum frequency generation

   https://doi.org/10.1063/5.0230223  

   Huang-Fu, Zhi-Chao; Zhang, Tong; Brown, Jesse B; Qian, Yuqin; Fisher, Haley; Rao, Yi (October 2024, The Journal of Chemical Physics)

   The movements of molecules at interfaces and surfaces are restricted by their asymmetric environments, leading to anisotropic orientational motions. In this work, in-plane orientational motions of the –C=O and –CF3 groups of coumarin 153 (C153) at the air/water interface were measured using time-resolved (TR) vibrational sum frequency generation (SFG). The in-plane orientational time constants of the –C=O and –CF3 groups of C153 are found to be 41.5 ± 8.2 and 36.0 ± 4.5 ps. These values are over five-times faster than that of 198 ± 15 ps for the permanent dipole of the whole C153 molecule at the interface, which may indicate that the two groups experience different interfacial friction in the plane. These differences could also be the result of the permanent dipole of C153 being almost five times those of the –C=O and –CF3 groups. The difference in orientational motions reveals the microscopic heterogeneous environment that molecules experience at the interface. While the interfacial dynamics of the two functional groups are similar, our TR-SFG experiments allowed the quantification of the in-plane dynamics of individual functional groups for the first time. Our experimental findings about the interfacial molecular motion have implications for molecular rotations, energy transfer, and charge transfer at material interfaces, photocatalysis interfaces, and biological cell/membrane aqueous interfaces. 

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3. Determination of protein conformation and orientation at buried solid/liquid interfaces

   https://doi.org/10.1039/d2sc06958j  

   Guo, Wen; Lu, Tieyi; Crisci, Ralph; Nagao, Satoshi; Wei, Tao; Chen, Zhan (March 2023, Chemical Science)

   Protein structures at solid/liquid interfaces mediate interfacial protein functions, which are important for many applications. It is difficult to probe interfacial protein structures at buried solid/liquid interfaces in situ at the molecular level. Here, a systematic methodology to determine protein molecular structures (orientation and conformation) at buried solid/liquid interfaces in situ was successfully developed with a combined approach using a nonlinear optical spectroscopic technique – sum frequency generation (SFG) vibrational spectroscopy, isotope labeling, spectra calculation, and computer simulation. With this approach, molecular structures of protein GB1 and its mutant (with two amino acids mutated) were investigated at the polymer/solution interface. Markedly different orientations and similar (but not identical) conformations of the wild-type protein GB1 and its mutant at the interface were detected, due to the varied molecular interfacial interactions. This systematic strategy is general and can be widely used to elucidate protein structures at buried interfaces in situ . 

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4. Choose your own adventure: Picosecond or broadband vibrational sum-frequency generation spectroscopy

   https://doi.org/10.1116/6.0001844  

   Carpenter, Andrew P.; Baio, Joe E. (May 2022, Biointerphases)

   Vibrational sum-frequency generation (VSFG) spectroscopy is a method capable of measuring chemical structure and dynamics within the interfacial region between two bulk phases. At the core of every experimental system is a laser source that influences the experimental capabilities of the VSFG spectrometer. In this article, we discuss the differences between VSFG spectrometers built with picosecond and broadband laser sources as it will impact everything from material costs, experimental build time, experimental capabilities, and more. A focus is placed on the accessibility of the two different SFG systems to newcomers in the SFG field and provides a resource for laboratories considering incorporating VSFG spectroscopy into their research programs. This Tutorial provides a model decision tree to aid newcomers when determining whether the picosecond or femtosecond laser system is sufficient for their research program and navigates through it for a few specific scenarios. 

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5. Surface architectural changes upon sonication revealed with sum frequency generation spectroscopy of perfluoroalkyl carboxylic acids

   https://doi.org/10.1063/5.0305155  

   Jenkins, Lindsey D; Hajab, Hawa; Cyran, Jenée D (March 2026, The Journal of Chemical Physics)

   Understanding the interfacial behavior of molecular films is essential for elucidating key processes in both environmental and industrial systems. In this study, we investigate the interfacial behavior of perfluorotetradecanoic acid using vibrational sum frequency generation (SFG) spectroscopy to directly compare its behavior in different monolayer architectures, i.e., a traditional Langmuir monolayer and a monolayer formed from a sonicated bulk solution. Our results reveal significant differences in the vibrational SFG spectra of the carboxylic acid head group between the two monolayer types. These findings demonstrate that monolayer type can fundamentally alter the interfacial structure of perfluoroalkyl carboxylic acids and highlight the importance of monolayer architecture in interpreting interfacial spectroscopic data. 

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