Abstract
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.
Highlights
Thrombin is a serine proteolytic enzyme containing two polypeptide chains (A and B) that can be activated by sodium ions
By incorporation of the characteristics of aptamer and stilbene, we developed a kinetic aptasensor based on fluorescence attenuation for thrombin detection
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Summary
Thrombin is a serine proteolytic enzyme containing two polypeptide chains (A and B) that can be activated by sodium ions It is a self-produced biocatalyst by organisms playing important physiological and pathological roles [1]. Inactivation of prothrombin and production of thrombin occur, which promotes the cleavage of fibrinogen in the blood into fibrin and results in a clot. This is mainly a self-protection mechanism of the organism to achieve hemostasis [2]. There is an urgent need to develop new thrombin detection methods that are easy to operate, sensitive, specific, rapid, and cost-effective.
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