This document summarizes a study that developed a low-cost method for uniformly patterning proteins on nitrocellulose using a novel stamping technique. The method uses a glass fiber pad soaked in an antibody solution as a "stamp" that is pressed onto a nitrocellulose strip. This produced a more even distribution compared to direct pipetting, as assessed using protein staining and gold nanoparticle labeling. Varying the stamp contact time from 1 to 10 minutes did not significantly change the level of antibody transferred. This new stamping method shows potential for patterning lateral flow tests in a reproducible and affordable manner.

1. Figure 1. (A) Images of protein-stained
nitrocellulose after stamping (top) and pipetting
(bottom). (B) Images of the gold-labeled
nitrocellulose after stamping (top) and pipetting
(bottom). (C) Plot of intensity vs. distance
along the direction of flow after labeling with
gold nanoparticles (from B). Error bars are the
standard deviation. (D) Plot of the average
intensity of the strip after labeling with gold
nanoparticles for different stamp contact times.
Error bars are the standard deviation (N=3).
Low Cost Method for Patterning Proteins onto Porous Materials
Jessalyn Imdieke and Elain Fu
Oregon State University
Introduction: Conventional laboratory-based testing is the current standard of health care. However,
conventional laboratory tests often require trained technicians and instrumentation (with requirements for
maintenance and electricity), and can add up to considerable cost. These restrictions greatly impact the
availability of point-of-care options for patients in low-resource settings where timely access to a laboratory may
be unavailable. Lateral flow tests (LFTs) provide a great solution for testing in low-resource settings as they are
rapid, user friendly, inexpensive, disposable, and don’t require pumps or electricity. However, LFTs usually only
provide a qualitative output. LFTs with semi-qualitative output have been demonstrated, but require controlled
patterning over an extended area with costly liquid dispensing instrumentation. A goal of this research is to
develop and characterize a low-cost method for patterning an extended capture region of a lateral flow sandwich
immunoassay using a stamping method. As a first step towards that goal, we have demonstrated uniform
patterning of proteins over an extended region of nitrocellulose using a novel stamping method.
Materials and Methods: The substrate was nitrocellulose (Millipore) and the “stamp” was composed of glass
fiber (Ahlstrom). The patterning solution was composed of a 1:99 ratio of (i) biotinylated goat anti-mouse IgG
antibody representing the specific antibody to the target analyte, and (ii) unconjugated goat anti-mouse IgG
antibody representing a non-specific antibody. The total IgG concentration was 1.5 mg/mL. A three-piece folding
card was used to pattern and evaluate the patterning method. The top piece of the folding card held the Ahlstrom
glass fiber pad that, when folded down, made contact with the nitrocellulose substrate. Antibody solution was
applied to fill the glass fiber pad (capacity 15 µL) and the card was closed to bring the wetted glass fiber pad into
contact with the nitrocellulose for 1 or 10 minutes. This contact allowed the fluid containing the antibodies to be
transferred from the glass fiber to the nitrocellulose (capacity 3.4 µL) - like a stamp transferring ink to paper. For
comparison, 3.4 µL of the antibody solution was hand pipetted directly onto one end of the nitrocellulose and
allowed to fill the nitrocellulose laterally. The nitrocellulose was stored in a desiccator overnight. The distribution
of the specific antibody was visualized using streptavidin attached to a gold nanoparticle label (Arista Biologicals)
or by protein staining (Pierce). Image data was acquired and analyzed using custom MATLAB code.
Results and Discussion: The stamping method produced a relatively uniform distribution of antibody along the
nitrocellulose in the direction of flow, while hand-pipetting the same concentration of antibody solution directly
into the nitrocellulose produced a non-uniform distribution (with no detectable binding downstream) as assessed
by protein staining (Figure 1A). Similar results were observed after gold labeling (Figure 1B) and analysis of the
binding profiles (Figure 1C). When antibodies are stamped onto the nitrocellulose, the antibody solution is in
contact with the nitrocellulose across the entire strip, whereas in pipetting, the contact is localized. This is critical
for antibody patterning because antibodies bind rapidly to nitrocellulose. Thus, the simultaneous contact of the
patterning solution across the surface of the nitrocellulose is important to achieve a uniform distribution. The
effect of contact time in the stamping method was investigated and the results indicated that times of 1 minute and
10 minutes produced similar levels of antibody transfer to the nitrocellulose (Figure 1D).
Conclusions: We have demonstrated a novel protein patterning method for porous materials. Next steps include
optimization of the method for reproducibility and use of the method for the controlled patterning of different
concentrations of protein.