Test strips were made by wax-printing four layers containing wax-free circular regions on Whatman Grade 1 cellulose paper and melting the wax-printed paper in an oven at 150 °C for 30 s. The final diameter of hydrophilic wells in the top layer and in the following layers were 3 and 2.5 mm, respectively. The wax-free surface was blocked with 5% BSA in 1 × PBS. The four-layer devices were then folded and clamped together with absorbent pad (blot paper). Standard reagent solution was prepared by mixing two parts of 1.6 nM SA-HRP, one part of 800 nM NP-CBD, and one part of 800 nM NP-biotin to give 800 pM SA-HRP, 200 nM NP-CBD, and 200 nM NP-biotin in 1% BSA in 1 × PBS. For one test, 25 μL of human serum was mixed with the same volume of the reagent solution and incubated for 5 min. This sample–reagent mixture (50 μL) was applied to test zone. Following the sample loading, the test zone was washed with 1 × PBS (25 μL). Finally, 10 μL of TMB substrate solution was added to the test zone and imaged after 5 min.

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