Modulating the ion-transfer electrochemistry of perfluorooctanoate with serum albumin and ß-cyclodextrin.

Per- and polyfluoroalkyl substances (PFAS) are durable synthetic pollutants that persist in the environment and resist biodegradation. Ion-transfer electrochemistry at aqueous-organic interfaces is a simple strategy for the detection of ionised PFAS. Herein, we investigate the modulation of the ion transfer voltammetry of perfluorooctanoate (PFOA) at liquid-liquid micro-interface arrays by aqueous phase bovine serum albumin (BSA) or ß-cyclodextrin (ß-CD) and examine the determination of association constants for these binding interactions. By tracking the ion transfer current due to ionised, uncomplexed PFOA as a function of BSA or ß-CD concentration, titration curves are produced. Fitting of a binding isotherm to these data provides the association constants. The association constant of PFOA with the BSA determined in this way was ca. 105 M-1 assuming a 1 : 1 binding. Likewise, the association constant for PFOA with ß-CD was ca. 104 M-1 for a 1 : 1 ß-CD-PFOA complex. Finally, the simultaneous effect of both BSA and ß-CD on the ion transfer voltammetry of PFOA was studied, showing clearly that PFOA bound to BSA is released (de-complexed) upon addition of ß-CD. The results presented here show ion transfer voltammetry as a simple strategy for the study of molecular and biomolecular binding of ionised PFAS and is potentially useful in understanding the affinity of different PFAS with aqueous phase binding agents such as proteins and carbohydrates.

Structural Changes in Insulin at a Soft Electrochemical Interface.

Understanding the interaction of proteins at interfaces, which occurs at or within cell membranes and lipoprotein vesicles, is central to our understanding of protein function. Therefore, new experimental approaches to understand how protein structure is influenced by protein-interface interactions are important. Herein we build on our previous work exploring electrochemistry at the interface between two immiscible electrolyte solutions (ITIES) to investigate changes in protein secondary structure that are modulated by protein-interface interactions. The ITIES provides an experimental framework to drive protein adsorption at an interface, allowing subsequent spectroscopic analysis (e.g., Fourier transform infrared spectroscopy) to monitor changes in protein structure. Here, we reveal that the interaction between insulin and the interface destabilizes native insulin secondary structure, promoting formation of α helix secondary structures. These structural alterations result from protein-interface rather than protein-protein interactions at the interface. Although this is an emerging approach, our results provide a foundation highlighting the value of the ITIES as a tool to study protein structure and interactions at interfaces. Such knowledge may be useful to elucidate protein function within biological systems or to aid sensor development.

Mapping sub-cellular protein aggregates and lipid inclusions using synchrotron ATR-FTIR microspectroscopy.

Visualising direct biochemical markers of cell physiology and disease pathology at the sub-cellular level is an ongoing challenge in the biological sciences. A suite of microscopies exists to either visualise sub-cellular architecture or to indirectly view biochemical markers (e.g. histochemistry), but further technique developments and innovations are required to increase the range of biochemical parameters that can be imaged directly, in situ, within cells and tissue. Here, we report our continued advancements in the application of synchrotron radiation attenuated total reflectance Fourier transform infrared (SR-ATR-FTIR) microspectroscopy to study sub-cellular biochemistry. Our recent applications demonstrate the much needed capability to map or image directly sub-cellular protein aggregates within degenerating neurons as well as lipid inclusions within bacterial cells. We also characterise the effect of spectral acquisition parameters on speed of data collection and the associated trade-offs between a realistic experimental time frame and spectral/image quality. Specifically, the study highlights that the choice of 8 cm-1 spectral resolutions provide a suitable trade-off between spectral quality and collection time, enabling identification of important spectroscopic markers, while increasing image acquisition by ∼30% (relative to 4 cm-1 spectral resolution). Further, this study explores coupling a focal plane array detector with SR-ATR-FTIR, revealing a modest time improvement in image acquisition time (factor of 2.8). Such information continues to lay the foundation for these spectroscopic methods to be readily available for, and adopted by, the biological science community to facilitate new interdisciplinary endeavours to unravel complex biochemical questions and expand emerging areas of study.