Development of Novel Paper-Based Assay for Direct Serum Separation

[Image: see text] Background: Many conventional laboratory tests require serum separation using a clot activator/gel tube, followed by centrifugation in an equipped laboratory. The aim of this study is development of novel, equipment-free, paper-based assay for direct and efficient serum separation. Methods: Fresh blood was directly applied to wax-channeled filter paper treated with clotting activator/s and then observed for serum separation. The purity, efficiency, recovery, reproducibility, and applicability of the assay were validated after optimization. Results: Serum was successfully separated using activated partial thromboplastin time (APTT) reagent and calcium chloride-treated wax-channeled filter paper within 2 min. The assay was optimized using different coagulation activators, paper types, blood collection methods, and incubation conditions. Confirmation of serum separation from cellular components was achieved by direct visualization of the yellow serum band, microscopic imaging of the pure serum band, and absence of blood cells in recovered serum samples. Successful clotting was evaluated by the absence of clotting of recovered serum by prolonged prothrombin time and APTT, absence of fibrin degradation products, and absence of Staphylococcus aureus-induced coagulation. Absence of hemolysis was confirmed by undetectable hemoglobin from recovered serum bands. The applicability of serum separated in paper was tested directly by positive color change on paper using bicinchoninic acid protein reagent, on recovered serum samples treated with Biuret and Bradford reagents in tubes, or measurement of thyroid-stimulating hormone and urea compared to standard serum samples. Serum was separated using the paper-based assay from 40 voluntary donors and from the same donor for 15 days to confirm reproducibility. Dryness of coagulants in paper prevents serum separation that can be re-stored by a re-wetting step. Conclusions: Paper-based serum separation allows for development of sample-to-answer paper-based point-of-care tests or simple and direct blood sampling for routine diagnostic tests.