Rapid antithrombin assay from human blood plasma utilizing smartphone-based flow observation on paper chips

Antithrombin (AT) is a protein that plays a critical role in regulating the coagulation cascade. Currently available methods for detecting AT levels are not portable, rapid, or simple enough to be used in urgent and clinical settings. This work presents a novel method for detecting AT levels from human blood through the smartphone-based capillary flow observation on paper chips. Assays were initially conducted with AT-spiked phosphate-buffered saline (PBS), demonstrating high linearity (R² = 0.988) between flow velocity and the logarithm of AT concentration (ng/ mL), with a linear range of up to 12 ng/mL. Assays were also conducted on diluted human plasma samples, showing increasing, plateaued, and decreasing regions over the AT concentrations, respectively. A high linearity (R² = 0.973) was observed within the linear range up to 19 ng/mL. This behavior mirrors the classic Heidelberger-Kendall antibody-antigen precipitation assay, characterized by a bell-shaped curve, despite substantial differences in assay mechanisms and a concentration range that is orders of magnitude lower. To validate this similarity, we directly measured the extent of particle immunoagglutination on the paper chips using a smartphone-based fluorescence microscope and confirmed the same bell-shaped curve. These results indicate that capillary flow velocity is positively correlated with the extent of particle immunoagglutination. Using multiple human blood samples (n = 14; each subject was tested three times), the smartphone-based flow velocity AT assay was compared with ELISA, and a strong correlation was observed. The assay was essentially near-real-time, as only the first 35 frames (1.17 seconds) of data were required, provided that the antibody-conjugated particles were pre-loaded on the paper chip before the assay. It requires only a paper chip and a smartphone, and involves only one pipetting step for sample loading. This assay could one day address the critical need for determining AT deficiency and support clinicians in managing patients’ anticoagulation status.