Photoreversible DNA nanoswitch-based eluent-free strategy for the direct and effective isolation of highly-active thrombin from whole blood

This study proposes an eluent-free isolation strategy for the direct isolation of thrombin from whole blood via tandem temperature/pH dual-responsive polyether sulfone monolith and photoreversible DNA nanoswitch-functionalized metal-organic framework (MOF) aerogel. Temperature/pH dual-responsive microgel immobilized on polyether sulfone monolith was adopted to remove the matrix complexity of blood sample via size/charge screening effect. Photoreversible DNA nanoswitches, comprising thrombin aptamer, aptamer complementary ssDNA (cDNA) and the azobenzene-modified ssDNA (control DNA), were functionalized on MOF aerogel to offer efficient capturing of thrombin under irradiation of ultraviolet light (365 nm), driven by electrostatic and hydrogen bond interactions. The release of captured thrombin was easily achieved by changing the complementary behaviors of DNA strands via blue light (450 nm) irradiation. Thrombin with purity higher than 95 % can be directly obtained from whole blood using this tandem isolation procedure. Fibrin production and substrate chromogenic tests showed that the released thrombin possessed high biological activity. The photoreversible thrombin capturing-release strategy is merited with eluent-free, avoiding the loss of activity of thrombin in chemical circumstances and undesired dilution, providing a robust guarantee for subsequent application.