Carbon dots as a versatile tool to monitor insulin aggregation

Abstract

The possibility to monitor peptide and protein aggregation is of paramount importance in the so-called conformational diseases, as the understanding of many physiological pathways, as well as pathological processes involved in the development of such diseases, depends very much on the actual possibility to monitor biomolecule oligomeric distribution and aggregation. In this work, we report a novel experimental method to monitor protein aggregation, based on the change of the fluorescent properties of carbon dots upon protein binding. The results obtained in the case of insulin with this newly proposed experimental approach are compared with those obtained with other common experimental techniques normally used for the same purpose (circular dichroism, DLS, PICUP and ThT fluorescence). The greatest advantage of the hereby presented methodology over all the other experimental methods considered is the possibility to monitor the initial stages of insulin aggregation under the different experimental conditions sampled and the absence of possible disturbances and/or molecular probes during the aggregation process. Graphical abstract

Detection of insulin oligomeric forms by a novel surface plasmon resonance‐diffusion coefficient based approach

“…DLS offers one of the most straightforward means of estimating protein size (Lorber et al, 2012; Stetefeld et al, 2016) and has proven its capability and efficiency in assessing the size distribution of naturally occurring insulin assemblies (Banerjee & Das, 2012; Sklepari et al, 2016). Furthermore, it has proven effective in monitoring the development of larger oligomeric particles that arise from the aggregation of insulin (Chen et al, 2021; Zingale et al, 2023a). Depending on the conditions of the solution, insulin can be found in various forms, including hexamers, tetramers, dimers, and monomers.…”

“…Understanding the biomolecular mechanisms triggering the misfolding and the subsequent aggregation of proteins is of paramount importance for tackling many diseases, including diabetes and Alzheimer's disease, among others (Laneri et al, 2022). Notably, insulin is a protein deeply involved in the development and management of diabetes and, for this reason, its structure (Weiss, 2009), folding (Hua, 2010; Liu et al, 2018), protease susceptibility (Farris et al, 2003; Tundo et al, 2017), oligomerization and fibrillation (Nettleton et al, 2000; Zingale et al, 2023a), as well as interactions with different metal ions also involved in diabetes (Carpenter & Wilcox, 2014; Gavrilova et al, 2014; Kant et al, 2021; Lisi et al, 2014), have been widely investigated with a variety of experimental approaches. Moreover, molecules capable of inhibiting and/or monitoring insulin aggregation have also been recently studied (Frankær et al, 2017; Lisi et al, 2014), as well as how insulin aggregation is affected by metal ions (Ben‐Shushan & Miller, 2021; Frankær et al, 2017; Patriati et al, 2021; Pounot et al, 2021).…”