Point-of-care assay platform for quantifying active enzymes to femtomolar levels using measurements of time as the readout

This Article describes a strategy for quantifying active enzyme analytes in a paper-based device by measuring the time for a reference region in the paper to turn green relative to an assay region. The assay requires a single step by the user, yet accounts for variations in sample volume, assay temperature, humidity, and contaminants in a sample that would otherwise prevent a quantitative measurement. The assay is capable of measuring enzymes in the low to mid femtomolar range with measurement times that range from ∼30 s to ∼15 min (lower measurement times correspond to lower quantities of the analyte). Different targets can be selected in the assay by changing a small molecule reagent within the paper-based device, and the sensitivity and dynamic range of the assays can be tuned easily by changing the composition and quantity of a signal amplification reagent or by modifying the configuration of the paper-based microfluidic device. By tuning these parameters, limits-of-detection for assays can be adjusted over an analyte concentration range of low femtomolar to low nanomolar, with dynamic ranges for the assays of at least 1 order of magnitude. Furthermore, the assay strategy is compatible with complex fluids such as serum.