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

## TL;DR

A smartphone-based paper chip test for antithrombin levels in blood plasma is developed, offering a portable and rapid alternative to current methods.

## Contribution

A novel, portable, and rapid smartphone-based assay for antithrombin detection using paper chips and capillary flow observation is introduced.

## Key findings

- High linearity (R² = 0.988) was observed between flow velocity and AT concentration in buffer solutions.
- The assay showed a bell-shaped curve in plasma samples, similar to traditional antibody-antigen assays.
- Strong correlation with ELISA was confirmed using multiple human blood samples.

## Abstract

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.

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, and simple enough to be used in urgent and clinical settings. This work presents a novel method for detecting AT levels in human blood using smartphone-based capillary flow observation on paper chips. Experiments were conducted with AT dissolved in buffer, showing high linearity between the capillary flow velocity and AT concentration. Experiments were also repeated with diluted human blood plasma, showing the expected increasing, plateaued, and decreasing regions over AT concentration. We hypothesized that capillary flow velocity is positively correlated with the extent of particle immunoagglutination, a hypothesis confirmed by microscopic images and subsequent quantification of particle immunoagglutination directly from the paper chips. The assay was performed near real-time, required only a paper chip (pre-loaded with antibody-conjugated particles) and a smartphone, and involved a single pipetting step for sample loading. This assay could one day address the critical need to determine AT deficiency and aid clinicians in decision-making when managing their patients’ anticoagulation status.