Highly Sensitive Two-Dimensional Paper Network Incorporating Biotin-Streptavidin for the Detection of Malaria

Abstract

Recently, two-dimensional paper networks have been developed to enable multistep assays to be performed in a lateral flow format. These devices have been used to perform simple enzyme linked immunoassays on paper. However, these devices have yet to incorporate more complex immunoassays, including the use of streptavidin-biotin detection strategies. Here we present a modified two-dimensional paper network capable of consecutively delivering six reagents. The device requires only a single user step and delivers (i) the sample, (ii) the biotinylated detection antibody, (iii) streptavidin horseradish peroxidase, (iv) a wash buffer, (v) a colorimetric substrate, and (vi) a final wash buffer. To demonstrate the utility of this approach we designed an assay to detect the malaria protein Pf HRP2. Using this platform, we were able to achieve a limit-of-detection equivalent to that of a traditional 96-well plate sandwich ELISA. In addition to improvements in the limit-of-detection, the inclusion of streptavidin-biotin simplifies the development of similar tests for other targets.

Figure 1

Device diagram. (A) Device prior to folding with key components labeled. (B) Device folded to initiate flow with reagents and test area

Figure 2

Flow profile comparison between devices. Food coloring was used to compare the flow between two device designs. The linear device showed consistently sequential reagent delivery, while the leg device showed parallel reagent flow at the 25, 50 and 80-minute time points

Figure 3

Results of Pf HRP2 2DPN assays. Representative scanned image for the linear device of (A) 0.5 ng/mL Pf HRP2. (B) 0.1 ng/mL and (C) 0 ng/mL after 90 minutes. Representative scanned images for the leg device are shown in (D), (E) and (F) for the same concentrations. (G) Signal and background regions-of-interest. The solid black box corresponds to the signal region-of-interest and the dotted black box to the background region-of-interest. (H) A plot of the signal-to-background ratio for the leg 2DPN for concentrations ranging from 0 to 1 ng/mL Pf HRP2 at 60 and 90 minutes after assay initiation. Each data point corresponds to the average of three separate tests. There is no significant difference in mean SBR between any antigen concentration at 60 or 90 minutes. (J) Presents the same plot for the linear 2DPN. At 60 minutes, there is no significant difference between 0.1 ng/mL and 0 ng/mL or between 0.5 ng/mL and 0.25 ng/mL. The mean SBR is significantly different between all other antigen concentrations. At 90 minutes, the mean SBR is significantly different between all antigen concentrations.