Quantifying Analytes in Paper-Based Microfluidic Devices Without Using External Electronic Readers

Point-of-care (POC) and point-of-use assays are critical for identifying and measuring the quantity of analytes in a variety of environments that lack access to laboratory infrastructure. In quantitative versions of these assays, both the duration of the assay and the output signal must be measured. Measurements of time most often are performed using a timer that is external to the platform of the assay. Such measurements are relatively simple and inexpensive, and in some cases, can be integrated into the device itself. In contrast, measurements of signal typically are accomplished using hand-held electrochemical, absorbance, reflectance, transmittance, or fluorescence readers, and as such, these measurements can be complicated, time-consuming, and expensive, particularly in the context of extremely resource-limited environments such as remote villages in the developing world. The World Health Organization has identified the use of external readers as a challenge that must be overcome when creating ideal POC diagnostic assays for use in the developing world. In fact, they have listed “equipment-free” as one of seven necessary attributes for diagnostic tests in these regions. Herein, we describe two complimentary assay strategies that address this issue. By using paper-based microfluidic devices, we show that the level of an analyte can be quantified by simply measuring time: no external electronic reader is required for the quantitative measurement (Figure 1). The methods involve either 1) tracking the time required for a sample to react with and ultimately pass through a hydrophobic detection reagent in a single conduit within a threedimensional (3D) paper-based microfluidic device (Figure 1a) (we call this a digital assay), or 2) counting the number of bars that become colored after a fixed assay period in a related paper-based microfluidic device (Figure 1b; we refer to this as an analog assay). The methods described herein require only a timer, the ability to see color, and/or the