Addressing criticalities in the INFOGEST static in vitro digestion protocol for oleogel analysis.

The interest on the digestive fate of oleogels, i.e., substitutes for solid fats rich in liquid oil, have pushed researchers to use the widely adopted INFOGEST protocol for static in vitro digestion. However, this protocol was originally designed to simulate the digestibility of conventional foods and to accommodate the large fraction of oil in oleogels, researchers have deliberately modified the INFOGEST protocol, inadvertently leading to results difficult to be compared. In this study, we highlighted possible problems that may arise during oleogel simulated digestion such as under- or overestimation of oleogel lipolysis. The effect of oleogel amount, oleogelator type and concentration, and shear applied during digestion on the rate and extent of oleogel digestion was studied. The release of fatty acids during the application of INFOGEST protocol was monitored using the pH-stat method and compared to those analyzed by HPLC-ELSD. Oleogels' structural information was obtained using brightfield, polarized, and fluorescence microscopy, and DSC. We determined that lipolysis of ethylcellulose oleogels follow the "interaction with enzymes and bile salts" pattern, whereas that of wax oleogels follow the "disintegration of oleogel and interaction with enzymes and bile salts". We also observed that the chemical composition of wax, crystal morphology, and crystal distribution do not alter the lipolysis of oil entrapped inside the wax crystals. We finally recommended a few minimal but fundamental modifications to the INFOGEST protocol to achieve more reliable results from the static in vitro digestion of oleogels and possibly other lipid-based systems.

Novel Electrochemically Switchable, Flexible, Microporous Cloth that Selectively Captures, Releases, and Concentrates Intact Extracellular Vesicles.

There is a significant and growing research interest in the isolation of extracellular vesicles (EVs) from large volumes of biological samples and their subsequent concentration into clean and small volumes of buffers, especially for applications in medical diagnostics. Materials that are easily incorporated into simple sampling devices and which allow the release of EVs without the need for auxiliary and hence contaminating reagents are particularly in demand. Herein, we report on the design and fabrication of a flexible, microporous, electrochemically switchable cloth that addresses the key challenges in diagnostic applications of EVs. We demonstrate the utility of our electrochemically switchable substrate for the fast, selective, nondestructive, and efficient capture and subsequent release of EVs. The substrate consists of an electrospun cloth, infused with a conducting polymer and decorated with gold particles. Utilizing gold-sulfur covalent bonding, the electrospun substrates may be functionalized with SH-terminated aptamer probes selective to EV surface proteins. We demonstrate that EVs derived from primary human dermal fibroblast (HDFa) and breast cancer (MCF-7) cell lines are selectively captured with low nonspecific adsorption using an aptamer specific to the CD63 protein expressed on the EV membranes. The specific aptamer-EV interactions enable easy removal of the nonspecifically bound material through washing steps. The conducting polymer component of the cloth provides a means for efficient (>92%) and fast (<5 min) electrochemical release of clean and intact captured EVs by cathodic cleavage of the Au-S bond. We demonstrate successful capture of diluted EVs from a large volume sample and their release into a small volume of clean phosphate-buffered saline buffer. The developed cloth can easily be incorporated into different designs for separation systems and would be adaptable to other biological entities including cells and other EVs. Furthermore, the capture/release capability holds great promise for liquid biopsies if used to targeted disease-specific markers.