With paper thicknesses commonly ranging from to m

With paper thicknesses commonly ranging from 50 to 400μm, light transmission measurements offer enhanced accuracy and sensitivity by interrogating the cumulative density of light absorbing particles throughout the thickness of the paper [7]. Further, sensitivity may be tuned by layering sheets of paper to create different thicknesses [6]. However, user-friendly methods to quantify colorimetric results in μ-PADs using light transmission have not been demonstrated due to several technical challenges concerning the paper substrate and sample matrix. Foremost, dry white paper, depending on its thickness and density, strongly reflects light resulting in less than 25% of incident light being transmitted through the paper [1]. This limits the possible range of light intensity differences that can be directly caused by concentration changes in the μ-PADs. To enhance the percent of incident light that is transmitted through paper, the paper may be wetted. Previous groups have saturated the μ-PAD with vegetable oil, however, this may be cumbersome in a point-of-care setting [6]. Further, because light transmission directly depends on the dampness of the paper, the μ-PAD must be kept uniformly wet over the time period of the assay [3]. This is difficult because the small sample volumes quickly evaporate from the large surface area of the paper and because paper as a substrate is variable. Across a single sheet of paper, thickness and fiber density may vary up to 5%, altering light transmission and wetting [1]. In addition to the intrinsic properties of paper, the hematological traits of biological samples may cause difference in their light nobiletin capacity and viscosity. These variations can cause disparities in the degree of wetting, the capillary flow rate, and ultimately the light transmission through the μ-PAD. This paper details the engineering of our transmittance-based reader system and presents evidence of its function through rapid assessment of liver function using quantification of alanine aminotransferase (ALT) concentrations in human serum. Section 2 describes the manufacturing of the reader-compatible μ-PAD, the engineering theory, components and function of the reader, and the alignment and calibration techniques employed for measurements. Section 3 uses the μ-PAD and reader for quantification of ALT in human serum samples and compares the sensitivity of the system to a scanner-based method. Sections 4 and 5 discuss results and provide conclusions and future directions.
Materials and methods
Results Because dry paper transmits light poorly, it was critical to measure light transmission at the read zone when the paper was fully wetted. This increased the possible range of light intensity differences that could be directly caused by changes in the buildup of blue-dye complex at the read zone. Further, the read zone had to be kept uniformly wet over the time period of the assay because light transmission directly depends on the dampness of the paper. Previously, Ellerbee et al. measured light transmission in colorimetric paper by placing the paper assays in plastic sleeves and saturating them in vegetable oil [6]. Because this method is unwieldy for the field and may introduce errors, we designed a μ-PAD that was pre-sealed on the front and back with plastic laminate. To enhance user-friendliness, a small sample port was left open on the front side for easy sample addition (Fig. 1a). To explore the effects of evaporation on our μ-PAD, we added 12.5μL of human serum (Valley Biomedical, VA, USA) to the sample port of assays and tracked their change in weight for 15min – the normal duration of our enzymatic assay. In testing conditions of 21°C and 15% relative humidity, our assays lost an average of 0.3μL of fluid each minute (Fig. 3a). As this is a significant (36%) decrease in fluid volume over the time period of the assay, we examined if this evaporation from the open sample port area affected light transmission at the read zones. For each assay, we added 12.5μL of serum and measured the change in light transmittance at the read zone for 15min. In contrast to the evaporation measurements, the light transmittance at the read zone changed less than 1% over the 15-min period (Fig. 3b). Together, these measurements indicate that sealing the read zone area with laminate prevented evaporation from this specific area and maintained its light transmitting properties.