A Fluorescence Kinetic-Based Aptasensor Employing Stilbene Isomerization for Detection of Thrombin

It is important to detect thrombin due to its physiological and pathological roles, where rapid and simple analytical approaches are needed. In this study, an aptasensor based on fluorescence attenuation kinetics for the detection of thrombin is presented, which incorporates the features of stilbene and aptamer. We designed and synthesized an aptasensor by one-step coupling of stilbene compound and aptamer, which employed the adaptive binding of the aptamer with thrombin to cause a change in stilbene fluorescence attenuation kinetics. The sensor realized detection of thrombin by monitoring the variation in apparent fluorescence attenuation rate constant (kapp), which could be further used for probing of enzyme–aptamer binding. In comprehensive studies, the developed aptasensor presented satisfactory performance on repeatability, specificity, and regeneration capacity, which realized rapid sensing (10 s) with a limit of detection (LOD) of 0.205 μM. The strategy was successful across seven variants of thrombin aptasensors, with tunable kapp depending on the SITS (4-Acetamido-4′-isothiocyanato-2,2′-stilbenedisulfonic acid disodium salt hydrate) grafting site. Analyte detection mode was demonstrated in diluted serum, requiring no separation or washing steps. The new sensing mode for thrombin detection paves a way for high-throughput kinetic-based sensors for exploiting aptamers targeted at clinically relevant proteins.

Cooperativity and Avidity in Membrane Binding by C2AB Tandem Domains of Synaptotagmins 1 and 7

Synaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7) contain analogous tandem C2 domains, C2A and C2B, which together serve as a Ca2+ sensor to bind membranes and promote the stabilization of exocytotic fusion pores. Functionally, Syt-1 triggers fast release of neurotransmitters, while Syt-7 is involved in lower-Ca2+ processes such as hormone secretion. Evidence suggests that Syt-1 C2 domains bind membranes cooperatively, penetrating farther into membranes as the C2AB tandem than as individual C2 domains. In contrast, we previously reported that the two C2 domains of Syt-7 bind membranes independently, based in part on measurements of their liposome dissociation kinetics. Here, we have investigated the effects of C2A-C2B interdomain cooperativity with Syt-1 and Syt-7 using directly comparable measurements. We report Ca2+ sensitivities, dissociation kinetics, and membrane insertion using liposomes approximating physiological lipid compositions. Equilibrium Ca2+ titrations confirm that the Syt-7 C2AB tandem has a greater Ca2+ sensitivity of membrane binding than either of its individual domains. Stopped-flow fluorescence kinetic measurements show that Syt-1 C2AB dissociates from liposome membranes much more slowly than either of its isolated C2 domains, suggesting that the two C2 domains of Syt-1 bind membranes cooperatively. In contrast, the dominant population of Syt-7 C2AB has a dissociation rate comparable to its C2A domain, indicating a lack of cooperativity, while only a small subpopulation dissociates at a slower rate. Measurements using an environment-sensitive fluorescent probe indicate that the Syt-7 C2B domain inserts more deeply into membranes as part of the C2AB tandem, similarly to Syt-1. Overall, these measurements are consistent with a model in which the structural linkage of C2A and C2B impacts the membrane-binding geometry of synaptotagmin C2B domains, but imparts little or no cooperativity to Syt-7 membrane binding and dissociation events that are dominated by its C2A domain.