Counting-based microfluidic paper-based devices capable of analyzing submicroliter sample volumes

In this paper, we report the development of semiquantitative counting-based lateral flow assay (LFA)-type microfluidic paper-based analytical devices ( $$ PADs) to analyze samples at submicroliter volumes. The ability to use submicroliter sample volumes is a significant advantage for $$ PADs since it enables enhanced multiplexing, reduces cost, and increases user-friendliness since small sample volumes can be collected using methods that do not require trained personnel, such as finger pricking and microneedles. The challenge of accomplishing a semiquantitative test readout using submicroliter sample volumes was overcome with a counting-based approach. In order to use submicroliter sample volumes, we developed a flow strategy with a running liquid to facilitate flow through the assay. The efficacy of the devices was confirmed with glucose and total human immunoglobulin E (IgE) tests using 0.5  $$ l and 1  $$ l of sample solutions, respectively. Semiquantitative results were generated to predict glucose concentrations in the range of 0-12 mmol/l and IgE concentrations in the range of 0-400 ng/ml. The counting-based approach correlates the number of dots that exhibited a color change to the concentration of the analyte, which provides a more user-friendly method as compared with interpreting the intensity of a color change. The devices reported herein are the first counting-based LFA-type $$ PADs capable of semiquantitative testing using submicroliter sample volumes.