Integrated separation of blood plasma from whole blood for microfluidic paper-based analytical devices

TLDR

Many clinical diagnostics rely on plasma, and recent efforts have focused on developing microfluidic paper‑based analytical devices for point‑of‑care testing in resource‑limited settings. This study introduces a method that uses red blood cell agglutination to separate plasma from finger‑prick whole blood directly on paper and integrates the separation with a colorimetric assay in a single μPAD. The μPAD is fabricated by printing wax barriers on chromatography paper, functionalized with agglutinating antibodies at the center and assay reagents at the periphery, so that a drop of whole blood yields plasma that wicks to the readout zones and produces a color change digitized by a portable scanner. The device achieves sufficient plasma purity and yield, with RBCs remaining agglutinated centrally, plasma reacting to produce a visible color change, and the method proving suitable for fully integrated μPADs using small whole blood samples.

Abstract

Many diagnostic tests in a conventional clinical laboratory are performed on blood plasma because changes in its composition often reflect the current status of pathological processes throughout the body. Recently, a significant research effort has been invested into the development of microfluidic paper-based analytical devices (μPADs) implementing these conventional laboratory tests for point-of-care diagnostics in resource-limited settings. This paper describes the use of red blood cell (RBC) agglutination for separating plasma from finger-prick volumes of whole blood directly in paper, and demonstrates the utility of this approach by integrating plasma separation and a colorimetric assay in a single μPAD. The μPAD was fabricated by printing its pattern onto chromatography paper with a solid ink (wax) printer and melting the ink to create hydrophobic barriers spanning through the entire thickness of the paper substrate. The μPAD was functionalized by spotting agglutinating antibodies onto the plasma separation zone in the center and the reagents of the colorimetric assay onto the test readout zones on the periphery of the device. To operate the μPAD, a drop of whole blood was placed directly onto the plasma separation zone of the device. RBCs in the whole blood sample agglutinated and remained in the central zone, while separated plasma wicked through the paper substrate into the test readout zones where analyte in plasma reacted with the reagents of the colorimetric assay to produce a visible color change. The color change was digitized with a portable scanner and converted to concentration values using a calibration curve. The purity and yield of separated plasma was sufficient for successful operation of the μPAD. This approach to plasma separation based on RBC agglutination will be particularly useful for designing fully integrated μPADs operating directly on small samples of whole blood.