Temperature-Independent, Portable, and Rapid Field Detection of Analytes

A device for conducting an assay for the presence of a disease state in a subject, the disease characterized by the presence of an targeted analyte in a sample from the subject, the device comprising:

• (a) a substrate;
• (b) at least three lateral-flow paths, each flow path formed by a porous fluid conducting medium on the substrate, wherein each fluid conducting medium includes a porous stratum overlying a hydrophobic layer;
• each flow path having an exterior end and an interior end;
• (c) a detection region, formed by a porous fluid retaining medium on the substrate;
• wherein the interior ends of the flow paths are fluidly coupled to the detection region and the detection region therefore lies at a junction of the flow paths, the fluid retaining medium including the same porous stratum overlying the same hydrophobic layer;
• and
• (d) first, second, and third load spots formed on the substrate, each load spot being at a corresponding one of the exterior ends of the flow paths and configured to receive an aqueous fluid;
• wherein the first load spot is configured to receive a sample from the subject, wherein the sample is to be assayed for presence of the targeted analyte;
• wherein the detection region includes a first mixture of a first silk fibroin and a detection reagent including a first immunoreactant, the first mixture forming a first silk film in the detection region;
• wherein the second load spot includes a second mixture of a second silk fibroin and a secondary detection immunoreactant that is released when the second load spot receives an aliquot of an aqueous compound, the second mixture forming a second silk film in the second load spot;
• and
• wherein the third load spot includes a third mixture of a third silk fibroin and a color reagent that is released when the third load spot receives the aliquot of an aqueous compound, and that causes a change in color in the detection region in the presence of a combination of the targeted analyte, if contained in the sample, the primary and secondary detection immunoreactants, and the color reagent, the third mixture forming a third silk film in the third load spot;
• so that when the first load spot receives a sample from the subject that contains the targeted analyte, and the second and third load spots are sequentially provided with aliquots of the aqueous compound, the components at each of the load spots sequentially react at the detection region after traversing the corresponding lateral-flow paths to produce the change in color as an indicator of the presence of the targeted analyte in the sample of the subject.