Figure 5.

Automated multi-step malaria assay in a folding card format. A) Card design and reagent locations for the assay. The card design is the same as in Figure 4. The sample (PfHRP2 antigen spiked into fetal bovine serum) was mixed with anti-PfHRP2-gold label (gold-Ab). Gold enhancement (GE) reagent was used to create the amplified gold signal. Each fluid was added to the appropriate pad, and the card was activated by folding it closed. Detection zone: 0.5 μL of 1 mg/mL anti-PfHRP2 antibody spotted on nitrocellulose and dried in a desiccator. Sample/gold: 5.5 μL of 1 μg/mL PfHRP2 antigen in FBS mixed with 5.5 μL anti-PfHRP2-gold in Tris-buffered saline with 1% BSA. Wash fluids: 11 μL of Tris-buffered saline with 0.1% Tween-20 (TBST). GE reagent: 11 μL of commercial reagent. B) Illustration of the amplified sandwich immunoassay. Like ELISA, this amplified chemistry can only be carried out by multiple timed steps. C) Photos of the signal at two time points during the assay. The first image represents signal from gold nanoparticles (endpoint of a typical LFT); the second image shows darkened signal after the GE and final wash step. Non-uniform test line darkness across the strip appears during the first delivery step (sample/gold); since delivery of the first fluid was typically uniform (as in Figure 4), test line non-uniformity was likely caused by the capture line itself. This non-uniformity is typical of strips patterned by our reagent spotting system due to misalignment (strips are cut first, then patterned). D) Time course of darkness at the detection zone. Approximate time periods for delivery of each fluid are indicated by background colors (arrival times approximately 2, 6, 13, and 27 minutes). Normalized test line darkness was calculated by inverting pixel values (larger numbers correspond to darker areas) and normalizing to the full intensity range (zero: darkness of the wetted strip, one: maximum darkness detectable by the camera).