Rapid Microfluidic Paper Assay for Detecting and Quantifying Antithrombin III

Abstract

For cardiopulmonary bypass, antithrombin III (ATIII) is one of the most important proteins for adequate anticoagulation. Without sufficient levels of ATIII, there becomes a risk of not only forming clots in our circuit but creating embolic strokes in our patients. This issue arises in patients who have received heparin prior to surgery, in newborns with immature organ systems, and those with unmanaged diabetics mellitus. However, due to the cost of drug supplementation and the length of current quantifying methods available, anesthesiologists and perfusionists can be hesitant to give ATIII when heparin resistance is indicated or trending. We propose a novel, point-of-care ATIII assay with the ability to quantify ATIII levels in a patient’s whole blood sample with a faster turnaround time than current testing methods. This platform features a competitive immunoassay model using a paper microfluidic chip and microscope to detect the target protein. Antithrombin III antibodies were covalently conjugated to fluorescent particles allowing for their detection under a blue-light microscope. The chip is first preloaded with diluted ATIII in distilled water (DIW), followed by the conjugated antibodies, and finally the diluted blood sample. The ATIII and antibody complexes are visualized using a blue-light microscope and a smartphone platform to image. The average number of particles for each channel and each concentration is determined using ImageJ allowing for a correlation between the antithrombin concentration and the pixels observed. Whole blood ATIII concentrations ranging from .29 ng/ml to 3 ng/ml were tested and their correlating average pixels were determined. This assay demonstrates preliminary success with detecting ATIII; however, quantification is yet to be determined. Next steps include obtaining an IRB for fresh clinical samples allowing for better assay optimization and to increase our sample size. Overall, we aim to provide the groundwork for a new point-of-care test that quantifies and detects ATIII in a user-friendly and cost-effective manner.