Malakoutikhah Morteza, Kauppi Laura, Mäntylä Kalle, Härmä Harri, Kopra Kari
Department of Chemistry, University of Turku, Henrikinkatu 2, 20500, Turku, Finland; Alimetrics Research Ltd., Koskelontie 19B, 02920, Espoo, Finland.
Department of Chemistry, University of Turku, Henrikinkatu 2, 20500, Turku, Finland.
Anal Chim Acta. 2025 Oct 1;1369:344366. doi: 10.1016/j.aca.2025.344366. Epub 2025 Jun 25.
The detection of amyloid fibrils is critical in production, storage and therapeutic use of insulin due to impact on efficacy and potential cytotoxicity after injection. Monitoring insulin aggregation, particularly at early stages, offers a valuable insight to aid the design of stable and effective insulin analogs, and addressing challenges in diabetes management. Despite numerous methods and probes developed this far, the detection of insulin fibers at nanomolar concentrations has remained a challenge. Moreover, as rapid-acting or slow-acting engineered insulin analogs are constantly developed, simple and sensitive methodologies also for monitoring structural transition of hexameric TR insulin forms are needed.
To address limitations in methodologies for insulin research, we developed an intramolecular Förster Resonance Energy Transfer (FRET) based peptide-probe, named as the FRET-Probe, for the detection of insulin fibers and hexamer TR transition changes at nanomolar concentrations. Using a comprehensive panel of insulin concentrations and therapeutically available insulin formulations, we highlight the sensitivity of the FRET-Probe in insulin fibril detection at early stages. In a comparative study with thioflavin T (ThT), we demonstrated 15-fold improved sensitivity of the FRET-Probe, and its ability for early insulin fiber detection. In addition, we demonstrate the ability of the FRET-Probe to differentiate between insulin hexameric forms (T, TR, and R), in the presence of anionic ligands and phenol derivatives. Thus, the FRET-Probe provides an unprecedented tool for characterizing structural dynamics using a luminescent external probe.
The FRET-Probe provides a simple and sensitive method for insulin fibril detection, enabling significantly improved detection of especially early insulin aggregation events, in comparison to ThT. The FRET-Probe also provides valuable insights into insulin analog stability and function, enabling insulin hexamer conformational measurements in real-time. The FRET-Probe can give comprehensive perspective on insulin behavior in varying conditions, thus supporting the insulin engineering and formulation processes.
由于淀粉样纤维对胰岛素的疗效有影响且注射后可能具有细胞毒性，因此在胰岛素的生产、储存和治疗应用中，淀粉样纤维的检测至关重要。监测胰岛素聚集，尤其是在早期阶段，对于辅助设计稳定有效的胰岛素类似物以及应对糖尿病管理中的挑战具有重要意义。尽管到目前为止已经开发了许多方法和探针，但在纳摩尔浓度下检测胰岛素纤维仍然是一项挑战。此外，随着速效或长效工程胰岛素类似物的不断开发，还需要简单且灵敏的方法来监测六聚体TR胰岛素形式的结构转变。
为了解决胰岛素研究方法中的局限性，我们开发了一种基于分子内Förster共振能量转移（FRET）的肽探针，命名为FRET探针，用于在纳摩尔浓度下检测胰岛素纤维和六聚体TR转变变化。通过使用一系列胰岛素浓度和治疗可用的胰岛素制剂，我们突出了FRET探针在早期胰岛素纤维检测中的灵敏度。在与硫黄素T（ThT）的比较研究中，我们证明FRET探针的灵敏度提高了15倍，并且具有早期检测胰岛素纤维的能力。此外，我们证明了FRET探针在存在阴离子配体和酚类衍生物的情况下区分胰岛素六聚体形式（T、TR和R）的能力。因此，FRET探针提供了一种前所未有的工具，可使用发光外部探针来表征结构动力学。
与ThT相比，FRET探针为胰岛素纤维检测提供了一种简单且灵敏的方法，能够显著改善对尤其是早期胰岛素聚集事件的检测。FRET探针还为胰岛素类似物的稳定性和功能提供了有价值的见解，能够实时进行胰岛素六聚体构象测量。FRET探针可以全面了解胰岛素在不同条件下的行为，从而支持胰岛素工程和制剂工艺。
