Quantitative and equipment-free paper-based agglutination assay of bacterial cells.

Background: point-of-care (POC) tests are useful for bedside/home applications, emergencies, frequent follow-ups, and resource-limited areas. Limited quantitative and equipment-free POC assays have been reported. This study aims to develop, validate, and apply a simple, quantitative, paper-based POC assay. Methods: wax-channeled paper treated with specific anti-Brucella and anti-Salmonella antibodies was used for distance-based chromatographic elution of stained bacterial cell agglutinations. Results: a qualitative paper-based agglutination POC test was developed using color intensity, tail appearance, and "+/-" signs that clearly distinguish the positive and negative results. The optimization of the test for paper type, microfluidic channel design, antibody and bacterial cell concentrations, and elution methods was carried out. Quantitative assay transformation was successfully developed using the color intensity of the original reaction zone, intensity of elution tail, and distance-based migration that correspond to bacterial agglutination size. The migration distance of eluted bacterial agglutination bands corresponds to the target concentration with good linearity and minimal variability. Reporting of colored band migration with numbers using microfluidic patterns was used to enhance non-technical end-user applications. A distance-based POC assay prototype was then successfully used for the accurate detection of known and unknown samples in comparison with standard assays. Conclusions: the migration distance of an eluted stained bacterial agglutination correlated with anti-bacterial antibody concentrations. A simple, cheap, quantitative, and equipment-free paper-based POC assay of bacterial cell agglutination was developed. This test can be used for simple "+/-" results, thermometer-like quantification, or text reporting with numbers corresponding to target concentrations. The assay has extended applications to different human disease biomarkers.

Development of Novel Paper-Based Assay for Direct Serum Separation.

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.