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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.

This paper describes an efficient and high throughput method for fabricating three-dimensional (3D) paper-based microfluidic devices. The method avoids tedious alignment and assembly steps and eliminates a major bottleneck that has hindered the development of these types of devices. A single researcher now can prepare hundreds of devices within 1 h.

This article describes the use of poly(carbamate) oligomers that depolymerize from head-to-tail as phase-switching reagents for increasing the sensitivity of quantitative point-of-care assays that are based on measurements of time. The poly(carbamate) oligomers selectively react with hydrogen peroxide (a model analyte) and provide sensitivity by depolymerizing in the presence of the analyte to convert from water-insoluble oligomers to water-soluble products. This switching reaction enables a sample to wick through a three-dimensional paper-based microfluidic device, where the flow-through time reflects the quantity of the analyte in the sample. Oligomers as short as octamers enable quantitative detection to low nanomolar concentrations of the analyte.

This Communication describes a prototype quantitative paper-based assay that simultaneously measures the levels of Pb(2+) and Hg(2+) in water. The assay requires only measurements of time to yield a quantitative readout, and the results are independent of sample volume, humidity, and sample viscosity.