Both the sample pad and the absorbent (wick) pad are critical components to any lateral flow assay. In this section we will cover how to characteristics of each component and how it relates to selection, as well as treatment of the material to improve assay performance.
The sample pad serves two critical purposes:
When selecting the correct sample pad for your lateral flow assay, the following attributes should be considered:
There is a wide variety of sample pads available and it is recommended you evaluate both cellulose and woven materials. The sample matrix (i.e. blood, saliva, urine, serum, etc.) will have a significant impact on which sample pad you will ultimately use.
The treatment of the sample pad with blocking reagents, protein, detergents, and surfactants is a common practice in lateral flow assay development. Treating the sample pad with an optimized buffer can aid in “normalizing” the samples before reaching the conjugate pad to prevent any negative interactions that may occur from the differences in pH, protein composition, mucins, salt concentrations, and any molecules that may cause non-specific interactions with the antibody system. Treatment buffers can normalize the sample pH and salt concentration, act as blocking agent for any non-specific binding, improve flow, and enhance the reproducibility of the assay by incorporating proteins, surfactants, salts, and/or polymers at the appropriate concentrations. To determine what to include in the sample pad treatment, evaluate what aspect of the sample needs to be “normalized.” For saliva samples, one challenge may be the difference in viscosity of the samples. By incorporating increased salt and surfactant concentrations, the mucins and proteins can be broken down decreasing viscosity and improving flow. However, if the sample is whole blood, the same components may cause hemolysis of the red blood cells and cause unwanted passage of these lysed cells through the membrane.
Sample pad treatment can be performed by immersion, or by spraying uniformly with an automated dispenser (e.g. Isoflow, Kinematic, Biodot). After treating the sample pad, we recommend curing in a forced air convection oven at 37°C for 30-60 minutes, and then allowed to dry overnight in a desiccated environment (<20% relative humidity) at 18-25°C. After overnight drying, the sample pad will be ready to use. Treated sample pads should continue to be stored in a dry environment (<20% relative humidity) at room temperature (18-25°C) as the pads will uptake moisture that can destabilize the reagents dried onto the surface.
The purpose of the absorbent (wick) pad is to increase the total volume of sample that can enter the test strip. The bed volume of any membrane is finite, and having an absorbent pad at the distal end of the test strip can increase the volume of sample that can be ran across the membrane as it acts as a sponge for the additional volume. As such, the presence of a wick pad can contribute to the reduction of non-specific binding and sensitivity. This is accomplished due to the additional volume that can run across the test line washing non-specifically bound material off the test line, and allowing for an increase in totally analyte concentration to reach the test line. Eventually, the sample will diffuse back into the membrane so it is important to characterize the stability window for the test line signal via the assay’s kinetic profile. The correct wick pad selection will minimize the backflow of any excess reagents and provide a wide window for the time of assay readout.
The attributes to consider for the wick pad are the same as that of the sample pad (i.e. thickness/weight, tensile strength, and material), although most wick pads are typically made of cellulose fibers due to their large bed volumes. The wick absorption capacity should be much higher than the sample and running buffer volume of the assay. Wick pad materials can be obtained from companies such as Millipore, Whatman/GE, or Ahlstrom. It is important to note that different materials should be screened for best results. A thicker material may not necessarily perform better or have a higher absorption capacity than a thinner material.