Heparin-Bead Extraction Enhanced Fluorogenic Aptamer-Thrombin Composite Reporter Enables Sensitive and Rapid Detection of Functional Antithrombin.

ABSTRACT

Antithrombin (AT) deficiency in the extracorporeal circulation during cardiac surgery leads to uncontrolled inflammation and vascular damage in patients. AT levels decrease in sepsis, major trauma, extracorporeal membrane oxygenation, and eclampsia. Monitoring plasma AT levels facilitates the accurate restoration of AT to baseline values through precise supplementation. Traditional methods of chromogenic assay and enzyme-linked immunosorbent assay (ELISA) kits encounter challenges, such as interference, inconsistency, and delayed response times, making real-time, reliable antithrombin monitoring a clinical gap. To address this critical need, we develop a heparin-bead extraction enhanced fluoroGenic aptamer-thrombin composite reporter (HExGATOR) for the rapid, sensitive, and precise detection of functional AT in plasma. Our design employs thrombin-binding aptamers and a fluorescence "turn on" technology such that a signal is produced upon the interaction of AT with the otherwise "turned off" aptamer-thrombin complex. The prominent signal-background interference originating from plasma is remarkably diminished by using a heparin-bead solid-phase extraction of AT. We achieved highly sensitive and rapid detection of AT in 5 to 20 min with a limit of detection of 15.11 nM. This approach offers a promising alternative to traditional AT tests in clinical settings, potentially facilitating personalized anticoagulant therapy.